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Sample records for adeno-associated viral vector

  1. Production and Titering of Recombinant Adeno-associated Viral Vectors

    OpenAIRE

    McClure, Christina; Cole, Katy L. H.; Wulff, Peer; Klugmann, Matthias; Murray, Andrew J.

    2011-01-01

    In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and ...

  2. Production and titering of recombinant adeno-associated viral vectors.

    Science.gov (United States)

    McClure, Christina; Cole, Katy L H; Wulff, Peer; Klugmann, Matthias; Murray, Andrew J

    2011-11-27

    In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

  3. Adeno-associated viral vectors for the treatment of hemophilia

    Science.gov (United States)

    High, Katherine A.; Anguela, Xavier M.

    2016-01-01

    Gene transfer studies for the treatment of hemophilia began more than two decades ago. A large body of pre-clinical work evaluated a variety of vectors and target tissues, but by the start of the new millennium it became evident that adeno-associated viral (AAV)-mediated gene transfer to the liver held great promise as a therapeutic tool. The transition to the clinical arena uncovered a number of unforeseen challenges, mainly in the form of a human-specific immune response against the vector that poses a significant limitation in the application of this technology. While the full nature of this response has not been elucidated, long-term expression of therapeutic levels of factor IX is already a reality for a small number of patients. Extending this success to a greater number of hemophilia B patients remains a major goal of the field, as well as translating this strategy to clinical therapy for hemophilia A. This review summarizes the progress of AAV-mediated gene therapy for the hemophilias, along with its upcoming prospects and challenges. PMID:26614390

  4. Novel adeno-associated viral vectors for retinal gene therapy.

    Science.gov (United States)

    Vandenberghe, L H; Auricchio, A

    2012-02-01

    Vectors derived from adeno-associated virus (AAV) are currently the most promising vehicles for therapeutic gene delivery to the retina. Recently, subretinal administration of AAV2 has been demonstrated to be safe and effective in patients with a rare form of inherited childhood blindness, suggesting that AAV-mediated retinal gene therapy may be successfully extended to other blinding conditions. This is further supported by the great versatility of AAV as a vector platform as there are a large number of AAV variants and many of these have unique transduction characteristics useful for targeting different cell types in the retina including glia, epithelium and many types of neurons. Naturally occurring, rationally designed or in vitro evolved AAV vectors are currently being utilized to transduce several different cell types in the retina and to treat a variety of animal models of retinal disease. The continuous and creative development of AAV vectors provides opportunities to overcome existing challenges in retinal gene therapy such as efficient transfer of genes exceeding AAV's cargo capacity, or the targeting of specific cells within the retina or transduction of photoreceptors following routinely used intravitreal injections. Such developments should ultimately advance the treatment of a wide range of blinding retinal conditions.

  5. Perinatal systemic gene delivery using adeno-associated viral vectors

    Directory of Open Access Journals (Sweden)

    Rajvinder eKarda

    2014-11-01

    Full Text Available Neurodegenerative monogenic diseases can also affect a broad range of tissues and organs throughout the body. An effective treatment would require a systemic approach. The intravenous administration of novel therapies is ideal but is hampered by the inability of such drugs to cross the blood-brain barrier and precludes efficacy in the central nervous system. A number of these early lethal intractable diseases also present devastating irreversible pathology at birth or soon after. Therefore, any therapy would ideally be administered during the perinatal period to prevent, stop or ameliorate disease progression. The concept of perinatal gene therapy has moved a step further towards being a feasible approach to treating such disorders. This has primarily been driven by the recent discoveries that particular serotypes of adeno-associated virus (AAV gene delivery vectors have the ability to cross the blood-brain barrier following intravenous administration. Furthermore, this has been safely demonstrated in perinatal mice and non-human primates. This review focuses on the progress made in using AAV to achieve systemic transduction and what this means for developing perinatal gene therapy for early lethal neurodegenerative diseases.

  6. The Impact of Peptide Insertions on Adeno-Associated Viral Vector Fate

    OpenAIRE

    Uhrig, Silke

    2010-01-01

    Recombinant adeno-associated viral (rAAV) vectors possess a number of attractive properties including low immunogenicity, high stability, longevity of transgene expression and the potential to integrate site-specifically without known side-effects. The major limitation regarding the use of AAV vectors for gene therapy is the broad tissue tropism of AAV following in vivo gene tranfer application. Recently, genetic modification of the AAV capsid by insertion of receptor-specific ligands (AAV ta...

  7. An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

    OpenAIRE

    Vreugdenhil Erno; Garner Keith M; Luijendijk Mieneke CM; Brans Maike AD; Fitzsimons Carlos P; de Backer Marijke WA; Adan Roger AH

    2010-01-01

    Abstract Background This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected...

  8. Adenoviral and adeno-associated viral vector mediated gene transfer in the guinea pig cochlea.

    Science.gov (United States)

    Li Duan, Mao; Bordet, Thierry; Mezzina, Mauro; Kahn, Axel; Ulfendahl, Mats

    2002-07-19

    Peripheral sensorineural hearing loss is a very common inner ear disorder affecting nearly 10% of the population. At present there is no cure for this disorder but gene therapy has been suggested as a potentially effective method for clinical treatment in the future. Thus we investigated the effectiveness of adenoviral (Ad) and adeno-associated viral (AAV) vectors to transduce the cochlea of guinea pigs. After direct injection into the basal turn of the cochlea, we found that both Ad and AAV vectors coding for the reporter genes lacZ or GFP readily transduced spiral ganglion cells. In addition, some transgene expression was detected in the stria vascularis after AAV-GFP injection. Gene expression persisted at least 8 weeks after viral vector injection. Present findings will help to develop future gene therapy protocols in the inner ear by using Ad and AAV coding for neurotrophins such as NT-3, BDNF, GDNF and VEGF.

  9. In vitro selection of viral vectors with modified tropism: the adeno-associated virus display.

    Science.gov (United States)

    Perabo, Luca; Büning, Hildegard; Kofler, David M; Ried, Martin U; Girod, Anne; Wendtner, Clemens M; Enssle, Jörg; Hallek, Michael

    2003-07-01

    Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.

  10. Adeno-associated viral vector transduction of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Stender, Stefan; Murphy, Mary; O'Brien, Tim

    2007-01-01

    Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector...... in human MSCs and to assess whether AAV transduction affects MSC multipotentiality. The results indicated that human MSCs could indeed be transiently transduced in vitro by the AAV2 vector with efficiencies of up to 65%. The percentage of GFP-positive cells peaked at 4 days post-transduction and declined...... rapidly towards 0% after day 8. The level of transgene expression in the GFP-positive population increased 4-fold over a 10,000 fold viral dose increase. This dose-response contrasted with the 200-fold increase observed in similarly transduced 293-cells, indicating a relatively restricted transgene...

  11. A compact dual promoter adeno-associated viral vector for efficient delivery of two genes to dorsal root ganglion neurons

    NARCIS (Netherlands)

    Fagoe, N D; Eggers, R; Verhaagen, J; Mason, M R J

    Adeno-associated viral (AAV) vectors based on serotype 5 are an efficient means to target dorsal root ganglia (DRG) to study gene function in the primary sensory neurons of the peripheral nervous system. In this study, we have developed a compact AAV dual promoter vector composed of the

  12. Adeno-associated viral vector-mediated gene transduction in mesencephalic slice culture.

    Science.gov (United States)

    Nihira, Tomoko; Yasuda, Toru; Hirai, Yukihiko; Shimada, Takashi; Mizuno, Yoshikuni; Mochizuki, Hideki

    2011-09-30

    Adeno-associated viral (AAV) vector is a non-pathogenic vehicle that is suitable for the delivery of foreign genes into non-dividing neuronal cells. This vector has been utilized for in vivo neurological research and in clinical trials of gene therapy for neurodegenerative disorders. Viral vector-mediated gene delivery has the limitation that progressive changes in cellular phenotype cannot be monitored in living animals. To visualize living neurons transduced with foreign genes in vitro, we used cultured mesencephalic tissue harboring living dopaminergic (DA) neurons and examined cellular tropism of serotype-1 and serotype-2 AAV vectors in a culture system. The viability of DA neurons was evaluated using transgenic mice carrying enhanced green fluorescent protein under the control of the rat tyrosine hydroxylase (TH) promoter, which enables the visualization of living DA cells in the substantia nigra. Apoptosis of a subset of neuronal cells was noted within one day of culture. After 7 days, the serotype-1 AAV vector had successfully delivered the foreign gene into neurons and astrocytes, and serotype-2 AAV vector was able to transduce TH-positive DA neurons efficiently. Our method should be useful for in vitro investigations of pathological changes in DA neurons following transduction with foreign genes. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Efficient production of recombinant adeno-associated viral vector, serotype DJ/8, carrying the GFP gene.

    Science.gov (United States)

    Hashimoto, Haruo; Mizushima, Tomoko; Chijiwa, Tsuyoshi; Nakamura, Masato; Suemizu, Hiroshi

    2017-06-15

    The purpose of this study was to establish an efficient method for the preparation of an adeno-associated viral (AAV), serotype DJ/8, carrying the GFP gene (AAV-DJ/8-GFP). We compared the yields of AAV-DJ/8 vector, which were produced by three different combination methods, consisting of two plasmid DNA transfection methods (lipofectamine and calcium phosphate co-precipitation; CaPi) and two virus DNA purification methods (iodixanol and cesium chloride; CsCl). The results showed that the highest yield of AAV-DJ/8-GFP vector was accomplished with the combination method of lipofectamine transfection and iodixanol purification. The viral protein expression levels and the transduction efficacy in HEK293 and CHO cells were not different among four different combination methods for AAV-DJ/8-GFP vectors. We confirmed that the AAV-DJ/8-GFP vector could transduce to human and murine hepatocyte-derived cell lines. These results show that AAV-DJ/8-GFP, purified by the combination of lipofectamine and iodixanol, produces an efficient yield without altering the characteristics of protein expression and AAV gene transduction. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Adeno-Associated Viral Vector-Induced Overexpression of Neuropeptide Y Y2 Receptors in the Hippocampus Suppresses Seizures

    Science.gov (United States)

    Woldbye, David P. D.; Angehagen, Mikael; Gotzsche, Casper R.; Elbrond-Bek, Heidi; Sorensen, Andreas T.; Christiansen, Soren H.; Olesen, Mikkel V.; Nikitidou, Litsa; Hansen, Thomas v. O.; Kanter-Schlifke, Irene; Kokaia, Merab

    2010-01-01

    Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is…

  15. Noninvasive Imaging Reveals Stable Transgene Expression in Mouse Airways After Delivery of a Nonintegrating Recombinant Adeno-Associated Viral Vector

    NARCIS (Netherlands)

    Vidovic, D; Gijsbers, R; Quiles Jimenez, A; Dooley, J; Van Den Haute, C; Van der Perren, A; Liston, A; Baekelandt, V; Debyser, Z; Carlon, MS

    2015-01-01

    Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV

  16. Mycophenolate Mofetil Impairs Transduction of Single-Stranded Adeno-Associated Viral Vectors

    NARCIS (Netherlands)

    Montenegro-Miranda, Paula; ten Bloemendaal, Lysbeth; Kunne, Cindy; de Waart, Dirk R.; Bosma, Piter J.

    2011-01-01

    Adeno-associated virus (AAV) liver-directed gene therapy seems a feasible treatment for Crigler-Najjar syndrome type I, an inherited liver disorder characterized by severe unconjugated hyperbilirubinemia. Transient immunosuppression coupled with vector administration seems needed to overcome host

  17. Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures

    DEFF Research Database (Denmark)

    Woldbye, David Paul Drucker; Ängehagen, Mikael; Gøtzsche, Casper René

    2010-01-01

    Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure...... suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using...... recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2...

  18. Pseudotyped Adeno-associated Viral Vector Tropism and Transduction Efficiencies in Murine Wound Healing

    OpenAIRE

    Keswani, Sundeep G.; Balaji, Swathi; Le, Louis; Leung, Alice; Lim, Foong-Yen; Habli, Mounira; Jones, Helen N.; Wilson, James M.; Crombleholme, Timothy M.

    2012-01-01

    Cell specific gene transfer and sustained transgene expression are goals of cutaneous gene therapy for tissue repair and regeneration. Adeno-associated virus serotype 2 (AAV2/2) mediated gene transfer to the skin results in stable transgene expression in the muscle fascicles of the panniculus carnosus in mice, with minimal gene transfer to the dermal or epidermal elements. We hypothesized that pseudotyped AAV vectors may have a unique and characteristic tropism and transduction efficiency pro...

  19. Intravitreal injection of adeno-associated viral vectors result in the transduction of different types of retinal neurons in neonatal and adult rats: A comparison with lentiviral vectors

    NARCIS (Netherlands)

    Harvey, A.R.; Kamphuis, W.; Eggers, R.; Symons, N.A.; Blits, B.; Niclou, S.; Boer, G. J.; Verhaagen, J.

    2002-01-01

    Replication-deficient viral vectors encoding the marker gene green fluorescent protein (GFP) were injected into the vitreous of newborn, juvenile (P14), and adult rats. We tested two different types of modified virus: adeno-associated viral-2-GFP (AAV-GFP) and lentiviral-GFP vectors (LV-GFP). The

  20. An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

    Science.gov (United States)

    2010-01-01

    Background This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 108 or 1 × 109 genomic copies of AAV1-GFP and 1 × 105 transducing units of LV-dsRed. Results Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. Conclusions This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest. PMID:20626877

  1. An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector.

    Science.gov (United States)

    de Backer, Marijke W A; Fitzsimons, Carlos P; Brans, Maike A D; Luijendijk, Mieneke C M; Garner, Keith M; Vreugdenhil, Erno; Adan, Roger A H

    2010-07-13

    This study compared the transduction efficiencies of an adeno-associated viral (AAV) vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP), with a lentiviral (LV) vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed), to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 x 108 or 1 x 109 genomic copies of AAV1-GFP and 1 x 105 transducing units of LV-dsRed. Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.

  2. An adeno-associated viral vector transduces the rat hypothalamus and amygdala more efficient than a lentiviral vector

    Directory of Open Access Journals (Sweden)

    Vreugdenhil Erno

    2010-07-01

    Full Text Available Abstract Background This study compared the transduction efficiencies of an adeno-associated viral (AAV vector, which was pseudotyped with an AAV1 capsid and encoded the green fluorescent protein (GFP, with a lentiviral (LV vector, which was pseudotyped with a VSV-G envelop and encoded the discosoma red fluorescent protein (dsRed, to investigate which viral vector transduced the lateral hypothalamus or the amygdala more efficiently. The LV-dsRed and AAV1-GFP vector were mixed and injected into the lateral hypothalamus or into the amygdala of adult rats. The titers that were injected were 1 × 108 or 1 × 109 genomic copies of AAV1-GFP and 1 × 105 transducing units of LV-dsRed. Results Immunostaining for GFP and dsRed showed that AAV1-GFP transduced significantly more cells than LV-dsRed in both the lateral hypothalamus and the amygdala. In addition, the number of LV particles that were injected can not easily be increased, while the number of AAV1 particles can be increased easily with a factor 100 to 1000. Both viral vectors appear to predominantly transduce neurons. Conclusions This study showed that AAV1 vectors are better tools to overexpress or knockdown genes in the lateral hypothalamus and amygdala of adult rats, since more cells can be transduced with AAV1 than with LV vectors and the titer of AAV1 vectors can easily be increased to transduce the area of interest.

  3. Corticospinal tract transduction: a comparison of seven adeno-associated viral vector serotypes and a non-integrating lentiviral vector.

    Science.gov (United States)

    Hutson, T H; Verhaagen, J; Yáñez-Muñoz, R J; Moon, L D F

    2012-01-01

    The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.

  4. Differential myocardial gene delivery by recombinant serotype-specific adeno-associated viral vectors.

    Science.gov (United States)

    Du, Lingling; Kido, Masakuni; Lee, Darwin V; Rabinowitz, Joseph E; Samulski, R Jude; Jamieson, Stuart W; Weitzman, Matthew D; Thistlethwaite, Patricia A

    2004-09-01

    Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors.

  5. Biology of Adeno-Associated Viral Vectors in the Central Nervous System

    Directory of Open Access Journals (Sweden)

    Giridhar eMurlidharan

    2014-09-01

    Full Text Available Gene therapy is a promising approach for treating a spectrum of neurological and neurodegenerative disorders by delivering corrective genes to the central nervous system (CNS. In particular, Adeno-Associated Viruses (AAV have emerged as promising tools for clinical gene transfer in a broad range of genetic disorders with neurological manifestations. In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms and transport mechanisms within the CNS. Continued efforts to dissect AAV-host interactions within the brain are likely to aid in the development of improved vectors for CNS-directed gene transfer applications in the clinic.

  6. Optimization of adeno-associated viral vector-mediated gene delivery to the hypothalamus.

    Science.gov (United States)

    de Backer, Marijke W A; Brans, Maike A D; Luijendijk, Mieneke C; Garner, Keith M; Adan, Roger A H

    2010-06-01

    To efficiently deliver genes and short hairpin RNAs to the hypothalamus we aimed to optimize the transduction efficiency of adeno-associated virus (AAV) in the rat hypothalamus. We compared the transduction efficiencies of AAV2 vectors pseudotyped with AAV1, AAV8, and mosaic AAV1/2 and AAV2/8 coats with that of an AAV2 coated vector after injection into the lateral hypothalamus of rats. In addition, we determined the transduction areas and the percentage of neurons infected after injection of various titers and volumes of two AAV1-pseudotyped vectors in the paraventricular hypothalamus (PVN). Successful gene delivery to the hypothalamus was achieved with AAV1-pseudotyped AAV vectors. The optimal approach to transduce an area, with the size of the PVN, was to inject 1 x 10(9) genomic copies of an AAV1-pseudotyped vector in a volume of 1 microl. At a radius of 0.05 mm from the injection site almost all neurons were transduced. In addition, overexpression of AgRP with the optimal approach resulted in an increase in food intake and body weight when compared with AAV-GFP.

  7. The potential of adeno-associated viral vectors for gene delivery to muscle tissue.

    Science.gov (United States)

    Wang, Dan; Zhong, Li; Nahid, M Abu; Gao, Guangping

    2014-03-01

    Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8(+) T-cell response against AAV capsid in humans. In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.

  8. Adeno Associated Viral Vector Delivered RNAi for Gene Therapy of SOD1 Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic.

  9. Highly Efficient Delivery of Adeno-Associated Viral Vectors to the Primate Retina.

    Science.gov (United States)

    Boye, Shannon E; Alexander, John J; Witherspoon, C Douglas; Boye, Sanford L; Peterson, James J; Clark, Mark E; Sandefer, Kristen J; Girkin, Chris A; Hauswirth, William W; Gamlin, Paul D

    2016-08-01

    Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and

  10. Hybrid adeno-associated viral vectors utilizing transposase-mediated somatic integration for stable transgene expression in human cells.

    Directory of Open Access Journals (Sweden)

    Wenli Zhang

    Full Text Available Recombinant adeno-associated viral (AAV vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and long-term transduction in non-dividing cells with negligible side-effects. However, as AAV vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Therefore, to stably transduce cells, we developed a novel AAV/transposase hybrid-vector. To facilitate SB-mediated transposition from the rAAV genome, we established a system in which one AAV vector contains the transposon with the gene of interest and the second vector delivers the hyperactive Sleeping Beauty (SB transposase SB100X. Human cells were infected with the AAV-transposon vector and the transposase was provided in trans either by transient and stable plasmid transfection or by AAV vector transduction. We found that groups which received the hyperactive transposase SB100X showed significantly increased colony forming numbers indicating enhanced integration efficiencies. Furthermore, we found that transgene copy numbers in transduced cells were dose-dependent and that predominantly SB transposase-mediated transposition contributed to stabilization of the transgene. Based on a plasmid rescue strategy and a linear-amplification mediated PCR (LAM-PCR protocol we analysed the SB100X-mediated integration profile after transposition from the AAV vector. A total of 1840 integration events were identified which revealed a close to random integration profile. In summary, we show for the first time that AAV vectors can serve as template for SB transposase mediated somatic integration. We developed the first prototype of this hybrid-vector system which with further improvements may be explored for treatment of diseases which originate from rapidly dividing cells.

  11. Oral-tolerization Prevents Immune Responses and Improves Transgene Persistence Following Gene Transfer Mediated by Adeno-associated Viral Vector.

    Science.gov (United States)

    Hardet, Romain; Chevalier, Benjamin; Dupaty, Léa; Naïmi, Yassine; Riou, Gaëtan; Drouot, Laurent; Jean, Laetitia; Salvetti, Anna; Boyer, Olivier; Adriouch, Sahil

    2016-02-01

    Gene therapy represents a feasible strategy to treat inherited monogenic diseases and intramuscular (i.m.) injection of recombinant adeno-associated viral (AAV) vector is now recognized as a convenient and safe method of gene transfer. However, this approach is hampered by immune responses directed against the vector and against the transgenic protein. We used here to reproduce this situation a mouse model where robust immune responses are induced following injection of an AAV vector coding for an immunogenic transgenic protein. We show that prophylactic oral administration of the immunogenic protein before AAV-mediated gene transfer completely prevented antibody formation and cytotoxic CD8(+) T-cell response. Consistently, prophylactic oral-tolerization considerably improved long-term transgene persistence and expression. Mechanistically, inhibition of the cytotoxic immune response involved abortive proliferation of antigen-specific cytotoxic CD8(+) T cells, upregulation of the PD-1 immunoregulatory molecule, downregulation of the Bcl-2 antiapoptotic factor, and their deletion in the context of AAV-mediated gene transfer. Hence, gene therapy may represent an ideal situation where oral-tolerization can be adopted before or at the same time as vector injection to efficiently prevent deleterious immune responses directed against the transgenic protein.

  12. Transduction of the central nervous system after intracerebroventricular injection of adeno-associated viral vectors in neonatal and juvenile mice.

    Science.gov (United States)

    Gholizadeh, Shervin; Tharmalingam, Sujeenthar; Macaldaz, Margarita E; Hampson, David R

    2013-08-01

    Several neurodevelopmental and neurodegenerative disorders affecting the central nervous system are potentially treatable via viral vector-mediated gene transfer. Adeno-associated viral (AAV) vectors have been used in clinical trials because of their desirable properties including a high degree of safety, efficacy, and stability. Major factors affecting tropism, expression level, and cell type specificity of AAV-mediated transgenes include encapsidation of different AAV serotypes, promoter selection, and the timing of vector administration. In this study, we evaluated the ability of single-stranded AAV2 vectors pseudotyped with viral capsids from serotype 9 (AAV2/9) to transduce the brain and target gene expression to specific cell types after intracerebroventricular injection into mice. Titer-matched AAV2/9 vectors encoding the enhanced green fluorescent protein (eGFP) reporter, driven by the cytomegalovirus (CMV) promoter, or the neuron-specific synapsin-1 promoter, were injected bilaterally into the lateral ventricles of C57/BL6 mice on postnatal day 5 (neonatal) or 21 (juvenile). Brain sections were analyzed 25 days after injection, using immunocytochemistry and confocal microscopy. eGFP immunohistochemistry after neonatal and juvenile administration of viral vectors revealed transduction throughout the brain including the striatum, hippocampus, cerebral cortex, and cerebellum, but with different patterns of cell-specific gene expression. eGFP expression was seen in astrocytes after treatment on postnatal day 5 with vectors carrying the CMV promoter, expanding the usefulness of AAVs for modeling and treating diseases involving glial cell pathology. In contrast, injection of AAV2/9-CMV-eGFP on postnatal day 21 resulted in preferential transduction of neurons. Administration of AAV2/9-eGFP with the synapsin-1 promoter on either postnatal day 5 or 21 resulted in widespread neuronal transduction. These results outline efficient methods and tools for gene delivery

  13. Generation and validation of novel adeno-associated viral vectors for the analysis of Ca2+ homeostasis in motor neurons.

    Science.gov (United States)

    Norante, Rosa Pia; Massimino, Maria Lina; Lorenzon, Paolo; De Mario, Agnese; Peggion, Caterina; Vicario, Mattia; Albiero, Mattia; Sorgato, Maria Catia; Lopreiato, Raffaele; Bertoli, Alessandro

    2017-07-26

    A finely tuned Ca2+ homeostasis in restricted cell domains is of fundamental importance for neurons, where transient Ca2+ oscillations direct the proper coordination of electro-chemical signals and overall neuronal metabolism. Once such a precise regulation is unbalanced, however, neuronal functions and viability are severely compromised. Accordingly, disturbed Ca2+ metabolism has often been claimed as a major contributor to different neurodegenerative disorders, such as amyotrophic lateral sclerosis that is characterised by selective motor neuron (MN) damage. This notion highlights the need for probes for the specific and precise analysis of local Ca2+ dynamics in MNs. Here, we generated and functionally validated adeno-associated viral vectors for the expression of gene-encoded fluorescent Ca2+ indicators targeted to different cell domains, under the transcriptional control of a MN-specific promoter. We demonstrated that the probes are specifically expressed, and allow reliable local Ca2+ measurements, in MNs from murine primary spinal cord cultures, and can also be expressed in spinal cord MNs in vivo, upon systemic administration to newborn mice. Preliminary analyses using these novel vectors have shown larger cytosolic Ca2+ responses following stimulation of AMPA receptors in the cytosol of primary cultured MNs from a murine genetic model of ALS compared to the healthy counterpart.

  14. Characterization of Adeno-Associated Viral Vector-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice.

    Science.gov (United States)

    Greig, Jenny A; Wang, Qiang; Reicherter, Amanda L; Chen, Shu-Jen; Hanlon, Alexandra L; Tipper, Christopher H; Clark, K Reed; Wadsworth, Samuel; Wang, Lili; Wilson, James M

    2017-05-01

    Adeno-associated viral (AAV) vectors are promising vehicles for hemophilia gene therapy, with favorable clinical trial data seen in the treatment of hemophilia B. In an effort to optimize the expression of human coagulation factor VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed with numerous combinations of liver-specific promoter and enhancer elements with a codon-optimized hFVIII transgene. After generating 42 variants of three reduced-size promoters and three small enhancers, transgene cassettes were packaged within a single AAV capsid, AAVrh10, to eliminate performance differences due to the capsid type. Each hFVIII vector was administered to FVIII knockout (KO) mice at a dose of 10(10) genome copies (GC) per mouse. Criteria for distinguishing the performance of the different enhancer/promoter combinations were established prior to the initiation of the studies. These criteria included prominently the level of hFVIII activity (0.12-2.12 IU/mL) and the pattern of development of anti-hFVIII antibodies. In order to evaluate the impact of capsid on hFVIII expression and antibody formation, one of the enhancer and promoter combinations that exhibited high hFVIII immunogenicity was evaluated using AAV8, AAV9, AAVrh10, AAVhu37, and AAVrh64R1 capsids. The capsids subdivided into two groups: those that generated anti-hFVIII antibodies in ≤20% of mice (AAV8 and AAV9), and those that generated anti-hFVIII antibodies in >20% of mice (AAVrh10, AAVhu37, and AAVrh64R1). The results of this study, which entailed extensive vector optimization and in vivo testing, demonstrate the significant impact that transcriptional control elements and capsid can have on vector performance.

  15. Therapeutic liabilities of in vivo viral vector tropism: adeno-associated virus vectors, NMDAR1 antisense, and focal seizure sensitivity.

    Science.gov (United States)

    Haberman, Rebecca; Criswell, Hugh; Snowdy, Stephen; Ming, Zhen; Breese, George; Samulski, R; McCown, Thomas

    2002-10-01

    The N-methyl-D-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1A, patch clamp studies found a significant decrease in maximal NMDA-evoked currents, indicative of a decrease in the number of NMDA receptors. Similarly, infusion of AAV-tTAK-NR1A (1 microl) into the rat temporal cortex significantly decreased NMDAR1-like immunoreactivity in layer V pyramidal cells. When AAV-tTAK-NR1A vectors were infused into the seizure-sensitive site of the rat inferior collicular cortex, the seizure sensitivity increased significantly over a period of 4 weeks. However, collicular infusion of AAV-CMV-NR1A vectors caused the opposite effect, a significant decrease in seizure sensitivity. Subsequent collicular coinfusion of vector encoding green fluorescent protein (GFP) driven by the tetracyclineoff promoter (AAV-tTAK-GFP) and vector encoding beta-galactosidase driven by the CMV promoter (AAV-CMV-LacZ) transduced distinct neuronal populations with only partial overlap. Thus, differing transduction ratios of inhibitory interneurons to primary output neurons likely account for the divergent seizure influences. Although AAV vector-derived NMDAR1 antisense can influence NMDA receptor function both in vitro and in vivo, promoter-related tropic differences dramatically alter the physiological outcome of this receptor-based gene therapy.

  16. Protocol for efficient generation and characterization of adeno-associated viral (AAV) vectors.

    Science.gov (United States)

    Jungmann, Andreas; Leuchs, Barbara; Katus, Hugo A; Rommelaere, Jean; Müller, Oliver J

    2017-09-21

    AAV vectors are a powerful tool for gene transfer approaches. We have established a simple and fast plasmid-based production system for achieving high AAV titers within 6 working days. The same procedure can be used for all serotypes and thus allows direct comparability of different serotypes. In this protocol we describe a step-by-step procedure which results in well-characterized vectors suitable for both in vitro approaches and preclinical studies.

  17. Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system.

    Science.gov (United States)

    Ehlert, Erich M; Eggers, Ruben; Niclou, Simone P; Verhaagen, Joost

    2010-02-18

    After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2). We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

  18. Cellular toxicity following application of adeno-associated viral vector-mediated RNA interference in the nervous system

    Directory of Open Access Journals (Sweden)

    Verhaagen Joost

    2010-02-01

    Full Text Available Abstract Background After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs and several axon guidance molecules, including all members of the secreted (class 3 Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV mediated expression of short hairpin RNAs (shRNAs to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1 and Neuropilin 2 (Npn-2. Results We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. Conclusions RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.

  19. Adeno-associated viral vector serotype 5 poorly transduces liver in rat models.

    Directory of Open Access Journals (Sweden)

    Paula S Montenegro-Miranda

    Full Text Available Preclinical studies in mice and non-human primates showed that AAV serotype 5 provides efficient liver transduction and as such seems a promising vector for liver directed gene therapy. An advantage of AAV5 compared to serotype 8 already shown to provide efficient correction in a phase 1 trial in patients suffering from hemophilia B, is its lower seroprevalence in the general population. Our goal is liver directed gene therapy for Crigler-Najjar syndrome type I, inherited severe unconjugated hyperbilirubinemia caused by UGT1A1 deficiency. In a relevant animal model, the Gunn rat, we compared the efficacy of AAV 5 and 8 to that of AAV1 previously shown to be effective. Ferrying a construct driving hepatocyte specific expression of UGT1A1, both AAV8 and AAV1 provided an efficient correction of hyperbilirubinemia. In contrast to these two and to other animal models AAV5 failed to provide any correction. To clarify whether this unexpected finding was due to the rat model used or due to a problem with AAV5, the efficacy of this serotype was compared in a mouse and two additional rat strains. Administration of an AAV5 vector expressing luciferase under the control of a liver specific promoter confirmed that this serotype poorly performed in rat liver, rendering it not suitable for proof of concept studies in this species.

  20. Tropism and toxicity of adeno-associated viral vector serotypes 1,2,5,6,7,8,9 in rat neurons and glia in vitro

    OpenAIRE

    Howard, Douglas B.; Powers, Kathleen; Wang, Yun; Harvey, Brandon K.

    2007-01-01

    Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction...

  1. Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results.

    LENUS (Irish Health Repository)

    Flotte, Terence R

    2011-10-01

    Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes\\/kg (n=3 subjects\\/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20 μg\\/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.

  2. Comparative analysis of adeno-associated viral vector serotypes 1, 2, 5, 7, and 8 in mouse brain.

    Science.gov (United States)

    Taymans, Jean-Marc; Vandenberghe, Luk H; Haute, Chris Van Den; Thiry, Irina; Deroose, Christophe M; Mortelmans, Luc; Wilson, James M; Debyser, Zeger; Baekelandt, Veerle

    2007-03-01

    Recombinant adeno-associated virus serotype 2 (rAAV2) vectors have been shown to deliver genes effectively to neurons in the brain, retina, and spinal cord. The characterization of new AAV serotypes revealed different patterns of transduction in a diverse array of tissues (Gao, G., Vandenberghe, L.H., and Wilson, J.M. [2005]. Curr. Gene Ther. 5, 285-297). Here, we extensively compare the neural tropism of human-derived rAAVs (types 2/1, 2, and 2/5) with nonhuman primate-derived rAAVs (types 2/7 and 2/8) in adult mouse brain. Mice were injected with rAAV type 2/1, 2, 2/5, 2/7, or 2/8 via the caudate-putamen and substantia nigra. Intrahippocampal injections were also performed for rAAV2/7 and rAAV2/8. In all regions injected, the vectors transduced neurons almost exclusively. Retrograde transduction of all rAAV pseudotypes was also observed in particular CNS areas. At high titers, all rAAV pseudotypes transduced comparable brain volumes in all targeted regions except for rAAV2, which transduced much smaller brain volumes. A dose-range comparison of intrastriatally injected rAAV types 2/5, 2/7, and 2/8 highlighted that the transduction efficiency, as determined by transduced volume and biophotonic imaging of green fluorescent protein expression intensity, was significantly higher for rAAV2/5 and rAAV2/7 compared with rAAV2/8 at low titers, whereas all three serotypes performed equally well at higher doses. These results demonstrate the use and efficiency of both human- and nonhuman primate-derived rAAV vectors for disease modeling and their potential for gene therapy.

  3. Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

    DEFF Research Database (Denmark)

    Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

    2010-01-01

    as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP......Viral vector-mediated gene transfer has emerged as a powerful means to target transgene expression in the central nervous system. Here we characterized the efficacy of serotypes 1 and 5 recombinant adeno-associated virus (rAAV) vectors encoding green fluorescent protein (GFP) after stereotaxic...... to study effects of genetic manipulation in this non-primate large animal species. Finally, we generated an atlas of the Göttingen minipig brain for guiding future studies in this large animal species....

  4. Adeno-associated viral vector-mediated gene transfer of brain-derived neurotrophic factor reverses atrophy of rubrospinal neurons following both acute and chronic spinal cord injury.

    Science.gov (United States)

    Ruitenberg, Marc J; Blits, Bas; Dijkhuizen, Paul A; te Beek, Erik T; Bakker, Arne; van Heerikhuize, Joop J; Pool, Chris W; Hermens, Wim T J; Boer, Gerard J; Verhaagen, Joost

    2004-03-01

    Rubrospinal neurons (RSNs) undergo marked atrophy after cervical axotomy. This progressive atrophy may impair the regenerative capacity of RSNs in response to repair strategies that are targeted to promote rubrospinal tract regeneration. Here, we investigated whether we could achieve long-term rescue of RSNs from lesion-induced atrophy by adeno-associated viral (AAV) vector-mediated gene transfer of brain-derived neurotrophic factor (BDNF). We show for the first time that AAV vectors can be used for the persistent transduction of highly atrophic neurons in the red nucleus (RN) for up to 18 months after injury. Furthermore, BDNF gene transfer into the RN following spinal axotomy resulted in counteraction of atrophy in both the acute and chronic stage after injury. These novel findings demonstrate that a gene therapeutic approach can be used to reverse atrophy of lesioned CNS neurons for an extended period of time.

  5. Antibody neutralization poses a barrier to intravitreal adeno-associated viral vector gene delivery to non-human primates.

    Science.gov (United States)

    Kotterman, M A; Yin, L; Strazzeri, J M; Flannery, J G; Merigan, W H; Schaffer, D V

    2015-02-01

    Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that preexisting immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, NAB levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of preexisting NAB titers in the serum of monkeys correlated strongly (R=0.76) with weak, decaying or no transgene expression following intravitreal administration of AAV

  6. Adeno-associated viral gene delivery in neurodegenerative disease.

    Science.gov (United States)

    Morgenstern, Peter F; Marongiu, Roberta; Musatov, Sergei A; Kaplitt, Michael G

    2011-01-01

    The advent of viral gene therapy technology has contributed greatly to the study of a variety of medical conditions, and there is increasing promise for clinical translation of gene therapy into human treatments. Adeno-associated viral (AAV) vectors provide one of the more promising approaches to gene delivery, and have been used extensively over the last 20 years. Derived from nonpathogenic parvoviruses, these vectors allow for stable and robust expression of desired transgenes in vitro and in vivo. AAV vectors efficiently and stably transduce neurons, with some strains targeting neurons exclusively in the brain. Thus, AAV vectors are particularly useful for neurodegenerative diseases, which have led to numerous preclinical studies and several human trials of gene therapy in patients with Parkinson's disease, Alzheimer's disease, and pediatric neurogenetic disorders. Here, we describe an efficient and reliable method for the production and purification of AAV serotype 2 vectors for both in vitro and in vivo applications.

  7. Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery

    Directory of Open Access Journals (Sweden)

    Harrison C Brown

    2014-01-01

    Full Text Available Clinical data support the feasibility and safety of adeno-associated viral (AAV vectors in gene therapy applications. Despite several clinical trials of AAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include (i the size of the factor VIII (fVIII transgene, (ii humoral immune responses to fVIII, (iii inefficient biosynthesis of human fVIII, and (iv AAV vector immunity. Through bioengineering approaches, a novel fVIII molecule, designated ET3, was developed and shown to improve biosynthetic efficiency 10- to 100-fold. In this study, the utility of ET3 was assessed in the context of liver-directed, AAV-mediated gene transfer into hemophilia A mice. Due to the large size of the expression cassette, AAV-ET3 genomes packaged into viral particles as partial genome fragments. Despite this potential limitation, a single peripheral vein administration of AAV-ET3 into immune-competent hemophilia A mice resulted in correction of the fVIII deficiency at lower vector doses than previously reported for similarly oversized AAV-fVIII vectors. Therefore, ET3 appears to improve vector potency and mitigate at least one of the critical barriers to AAV-based clinical gene therapy for hemophilia A.

  8. Global diffuse distribution in the brain and efficient gene delivery to the dorsal root ganglia by intrathecal injection of adeno-associated viral vector serotype 1.

    Science.gov (United States)

    Iwamoto, Naotaka; Watanabe, Atsushi; Yamamoto, Motoko; Miyake, Noriko; Kurai, Toshiyuki; Teramoto, Akira; Shimada, Takashi

    2009-06-01

    The success of gene therapy for inherited neurodegenerative diseases such as metachromatic leukodystrophy (MLD) depends on the development of efficient gene delivery throughout the brain guarded by the blood-brain barrier and achieves distribution of the deficient enzyme throughout the brain. Direct injection of viral vector into the brain parenchyma is too invasive and may not be sufficient to treat the entire brain. As an alternative approach, we examined the feasibility of intrathecal (IT) injection of adeno-associated viral vector serotype 1 (AAV1). AAV1 vector expressing arylsulfatase A (ASA) and green fluorescence protein (GFP) was intrathecally injected into ASA knockout MLD model mice. Expression of GFP was assessed by fluorescence microscopy and immunohistochemical methods, whereas the concentration of ASA was determined by a quantitative enzyme-linked immunosorbent assay. Broad distribution of GFP expression was seen throughout the brain after IT injection of AAV1 vector. In addition, a large number of nerve fibers in the dorsal spinal cord and many neural cell bodies in the dorsal root ganglia were efficiently transduced. Widespread distribution of ASA activity and a significant reduction of sulfatide content were confirmed in treated MLD model mice. IT injection of AAV1 vector is a useful and non-invasive method for widespread gene delivery to the brain and dorsal root ganglia. (c) 2009 John Wiley & Sons, Ltd.

  9. Transduction of striatum and cortex tissues by adeno-associated viral vectors produced by herpes simplex virus- and baculovirus-based methods.

    Science.gov (United States)

    Zhang, H Steve; Kim, Eunmi; Lee, Slgirim; Ahn, Ik-Sung; Jang, Jae-Hyung

    2012-01-01

    Recombinant adeno-associated virus (AAV) vectors can be engineered to carry genetic material encoding therapeutic gene products that have demonstrated significant clinical promise. These viral vectors are typically produced in mammalian cells by the transient transfection of two or three plasmids encoding the AAV rep and cap genes, the adenovirus helper gene, and a gene of interest. Although this method can produce high-quality AAV vectors when used with multiple purification protocols, one critical limitation is the difficulty in scaling-up manufacturing, which poses a significant hurdle to the broad clinical utilization of AAV vectors. To address this challenge, recombinant herpes simplex virus type I (rHSV-1)- and recombinant baculovirus (rBac)-based methods have been established recently. These methods are more amenable to large-scale production of AAV vectors than methods using the transient transfection of mammalian cells. To investigate potential applications of AAV vectors produced by rHSV-1- or rBac-based platforms, the in vivo transduction of rHSV-1- or rBac-produced AAV serotype 2 (AAV2) vectors within the rat brain were examined by comparing them with vectors generated by the conventional transfection method. Injection of rHSV-1- or rBac-produced AAV vectors into rat striatum and cortex tissues revealed no differences in cellular tropism (i.e., predominantly neuronal targeting) or anteroposterior spread compared with AAV2 vectors produced by transient transfection. This report represents a step towards validating AAV vectors produced by the rHSV-1- and the rBac-based systems as promising tools, especially for delivering therapeutic molecules to the central nervous system. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex.

    Science.gov (United States)

    Watakabe, Akiya; Ohtsuka, Masanari; Kinoshita, Masaharu; Takaji, Masafumi; Isa, Kaoru; Mizukami, Hiroaki; Ozawa, Keiya; Isa, Tadashi; Yamamori, Tetsuo

    2015-04-01

    Here we investigated the transduction characteristics of adeno-associated viral vector (AAV) serotypes 1, 2, 5, 8 and 9 in the marmoset cerebral cortex. Using three constructs that each has hrGFP under ubiquitous (CMV), or neuron-specific (CaMKII and Synapsin I (SynI)) promoters, we investigated (1) the extent of viral spread, (2) cell type tropism, and (3) neuronal transduction efficiency of each serotype. AAV2 was clearly distinct from other serotypes in small spreading and neuronal tropism. We did not observe significant differences in viral spread among other serotypes. Regarding the cell tropism, AAV1, 5, 8 and 9 exhibited mostly glial expression for CMV construct. However, when the CaMKII construct was tested, cortical neurons were efficiently transduced (>∼70% in layer 3) by all serotypes, suggesting that glial expression obscured neuronal expression for CMV construct. For both SynI and CaMKII constructs, we observed generally high-level expression in large pyramidal cells especially in layer 5, as well as in parvalbumin-positive interneurons. The expression from the CaMKII construct was more uniformly observed in excitatory cells compared with SynI construct. Injection of the same viral preparations in mouse and macaque cortex resulted in essentially the same result with some species-specific differences. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  11. Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord.

    Science.gov (United States)

    Eaton, M J; Blits, B; Ruitenberg, M J; Verhaagen, J; Oudega, M

    2002-10-01

    Changing the levels of neurotrophins in the spinal cord micro-environment after nervous system injury has been proposed to recover normal function, such that behavioral response to peripheral stimuli does not lead to chronic pain. We have investigated the effects of recombinant adeno-associated viral (rAAV)-mediated over-expression of brain-derived neurotrophic factor (BDNF) in the spinal cord on chronic neuropathic pain after unilateral chronic constriction injury (CCI) of the sciatic nerve. The rAAV-BDNF vector was injected into the dorsal horn at the thirteenth thoracic spinal cord vertebra (L(1) level) 1 week after CCI. Allodynia and hyperalgesia induced by CCI in the hindpaws were permanently reversed, beginning 1 week after vector injection, compared with a similar injection of a control rAAV-GFP vector (green fluorescent protein) or saline. In situ hybridization for BDNF demonstrated that both dorsal and ventral lumbar spinal neurons contained an intense signal for BDNF mRNA, at 1 to 8 weeks after vector injection. There was no similar BDNF mRNA over-expression associated with either injections of saline or rAAV-GFP. These data suggest that chronic neuropathic pain is sensitive to early spinal BDNF levels after partial nerve injury and that rAAV-mediated gene transfer could potentially be used to reverse chronic pain after nervous system injuries in humans.

  12. Adeno-associated viral vector-mediated neurotrophin gene transfer in the injured adult rat spinal cord improves hind-limb function.

    Science.gov (United States)

    Blits, B; Oudega, M; Boer, G J; Bartlett Bunge, M; Verhaagen, J

    2003-01-01

    To foster axonal growth from a Schwann cell bridge into the caudal spinal cord, spinal cells caudal to the implant were transduced with adeno-associated viral (AAV) vectors encoding for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (AAV-NT-3). Control rats received AAV vectors encoding for green fluorescent protein or saline. AAV-BDNF- and AAV-NT-3-transduced 293 human kidney cells produced and secreted BDNF or NT-3, respectively, in vitro. The secreted neurotrophins were biologically active; they both promoted outgrowth of sensory neurites in vitro. In vivo, transgene expression was observed predominantly in neurons for at least 16 weeks after injection. Compared with controls, a modest though significant improvement in hind-limb function was found in rats that received AAV-BDNF and AAV-NT-3. Retrograde tracing demonstrated that twice as many neurons with processes extending toward the Schwann cell graft were present in the second lumbar cord segment of AAV-BDNF- and AAV-NT-3-injected animals compared with controls. We found no evidence, however, for growth of regenerated axons from the Schwann cell implant into the caudal cord. Our results suggest that AAV vector-mediated overexpression of BDNF and NT-3 in the cord caudal to a Schwann cell bridge modified the local lumbar axonal circuitry, which was beneficial for locomotor function.

  13. Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells.

    Science.gov (United States)

    Penaud-Budloo, Magalie; Lecomte, Emilie; Guy-Duché, Aurélien; Saleun, Sylvie; Roulet, Alain; Lopez-Roques, Céline; Tournaire, Benoît; Cogné, Benjamin; Léger, Adrien; Blouin, Véronique; Lindenbaum, Pierre; Moullier, Philippe; Ayuso, Eduard

    2017-06-01

    Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (≤0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.

  14. Tropism and toxicity of adeno-associated viral vector serotypes 1, 2, 5, 6, 7, 8, and 9 in rat neurons and glia in vitro.

    Science.gov (United States)

    Howard, Douglas B; Powers, Kathleen; Wang, Yun; Harvey, Brandon K

    2008-03-01

    Recombinant adeno-associated viral (rAAV) vectors are frequently used for gene delivery to the central nervous system and are capable of transducing neurons and glia in vitro. In this study, seven serotypes of a rAAV vector expressing green fluorescent protein (GFP) were characterized for tropism and toxicity in primary cortical cells derived from embryonic rat brain. At 2 days after transduction, serotypes 1 and 5 through 8 expressed GFP predominately in glia, but by 6 days post-transduction expression was neuronal except for AAV5. AAV2 and 9 produced minimal GFP expression. Using cell viability assays, toxicity was observed at higher multiplicities of infection (MOI) for all serotypes except AAV2 and 9. The toxicity of AAV1 and 5-8 affected mostly glia as indicated by a loss of glial-marker immunoreactivity. A frameshift mutation in the GFP gene reduced overall toxicity for serotypes 1, 5 and 6, but not 7 and 8 suggesting that the toxicity was not solely due to the overexpression of GFP. Collectively, a differential tropism and toxicity was observed among the AAV serotypes on primary cortical cultures with an overall preferential glial transduction and toxicity.

  15. Characterization of a rat model of Huntington's disease based on targeted expression of mutant huntingtin in the forebrain using adeno-associated viral vectors.

    Science.gov (United States)

    Gabery, Sanaz; Sajjad, Muhammad U; Hult, Sofia; Soylu, Rana; Kirik, Deniz; Petersén, Åsa

    2012-09-01

    Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (htt) gene. Neuropathology is most severe in the striatum and cerebral cortex. As mutant htt is ubiquitously expressed, it has not been possible to establish clear structure-to-function relationships for the clinical aspects. In the present study, we have injected recombinant adeno-associated viral vectors of serotype 5 (rAAV5) expressing an 853-amino-acid fragment of htt with either 79 (mutant) or 18 (wild-type) glutamines (Q) in the dorsal striatum of neonatal rats to achieve expression of htt in the forebrain. Rats were followed for 6 months and compared with control rats. Neuropathological assessment showed long-term expression of the green fluorescent protein (GFP) transgene (used as a marker protein) and accumulation of htt inclusions in the cerebral cortex with the rAAV5-htt-79Q vectors. We estimated that around 10% of NeuN-positive cells in the cerebral cortex and 2% of DARPP-32 neurons in the striatum were targeted with the GFP-expressing vector. Formation of intracellular htt inclusions was not associated with neuronal loss, gliosis or microglia activation and did not lead to altered motor activity or changes in body weight. However, the same mutant htt vector caused orexin loss in the hypothalamus - another area known to be affected in HD. In conclusion, our results demonstrate that widespread forebrain expression of mutant htt can be achieved using rAAV5-vectors and suggest that this technique can be further explored to study region-specific effects of mutant htt or other disease-causing genes in the brain. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  16. Determination of Anti-Adeno-Associated Viral Vector Neutralizing Antibodies in Patients With Heart Failure in the Cardiovascular Foundation of Colombia (ANVIAS): Study Protocol.

    Science.gov (United States)

    Rincon, Melvin Y; Prada, Carlos E; Lopez, Marcos; Castillo, Victor; Echeverria, Luis Eduardo; Serrano, Norma

    2016-06-09

    Recent progress in the pathophysiology of heart failure (HF) has led to the development of new therapeutic options such as gene therapy and the use of adeno-associated viral (AAV) vectors. Despite the promising results in early clinical trials of gene therapy for HF, various obstacles have been faced, such as the presence of neutralizing antibodies (NAbs) against the capsid vectors. NAb activity limits vector transduction levels and therefore diminishes the final therapeutic response. Recent studies evaluating the prevalence of NAbs in various populations found considerable geographic variability for each AAV serotype. However, the levels of NAbs in Latin American populations are unknown, becoming a limiting factor to conducting AAV vector therapeutic trials in this population. The goal of this study is to determine for the first time, the prevalence of anti-AAV NAbs for the serotypes 1, 2, and 9 in HF patients from the city of Bucaramanga, Colombia, using the in vitro transduction inhibition assay. We will conduct a cross-sectional study with patients who periodically attend the HF clinic of the Cardiovascular Foundation of Colombia and healthy volunteers matched for age and sex. For all participants, we will evaluate the NAb levels against serotypes AAV1, AAV2, and AAV9. We will determine NAb levels using the in vitro transduction inhibition assay. In addition, participants will answer a survey to evaluate their epidemiological and socioeconomic variables. Participation in the study will be voluntary and all participants will sign an informed consent document before any intervention. The project is in the first phase: elaboration of case report forms and the informed consent form, and design of the recruitment strategy. Patient recruitment is expected to begin in the spring of 2016. We expect to have preliminary results, including the titer of the viral vectors, multiplicity of infections that we will use for each serotype, and the general validation of the assay

  17. Retinal Gene Therapy for Choroideremia: In Vitro Testing for Gene Augmentation Using an Adeno-Associated Viral (AAV) Vector.

    Science.gov (United States)

    Patrício, Maria I; MacLaren, Robert E

    2018-01-01

    As gene therapy of choroideremia is becoming a clinical reality, there is a need for reliable and sensitive assays to determine the expression of exogenously delivered Rab Escort Protein-1 (REP1), in particular to test new gene therapy vectors and as a quality control screen for clinical vector stocks. Here we describe an in vitro protocol to test transgene expression following AAV2/2-REP1 transduction of a human cell line. Gene augmentation can be confirmed by western blot and quantification of the fold-increase of human REP1 levels over untransduced controls.

  18. Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene.

    Science.gov (United States)

    Rashnonejad, Afrooz; Chermahini, Gholamhossein Amini; Li, Shaoyong; Ozkinay, Ferda; Gao, Guangping

    2016-01-01

    Recombinant AAV (rAAV) vectors are a suitable vector for gene therapy studies because of desired characteristics such as low immunogenicity, transfection of non-dividing and dividing cells, and long-term expression of the transgene. In this study, the large-scale production of single stranded (ss) and self-complementary (sc) AAV9 carrying the human survival motor neuron (SMN) gene (AAV9-SMN) suitable for in vivo gene therapy studies of SMA was described. SMN cDNA has been cloned into pAAV-CB6-PI and pAAVsc-CB6-PI with and without its specific UTRs, respectively. Both plasmids bear CMV enhancer/beta-actin (CB) promoter, CMV IE enhancer, and polyadenylation signal sequences. 2.5 μg of constructed pAAV-CB6-PI-SMN and pAAVsc-CB6-PI-SMN cause to, respectively, 4.853- and 2.321-fold increases in SMN protein levels in transfected cells compared to untransfected cells. Ss and scAAV9-SMN vectors were also produced from these plasmids by transient transfection of HEK293 cells using CaCl2 solution. The silver staining and electron microscopy analysis demonstrated good quality of both isolated vectors, ssAAV9-SMN and scAAV9-SMN, with the titers of 2.00E+13 and 1.00E+13 GC/ml. The results of this study show that, the plasmid containing UTR elements causes to twice more SMN gene expression in transfected cells. The quality control results show that both produced ss and scAAV9-SMN are suitable for in vivo studies.

  19. Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

    Science.gov (United States)

    Hall, Hélène; Jewett, Michael; Landeck, Natalie; Nilsson, Nathalie; Schagerlöf, Ulrika; Leanza, Giampiero; Kirik, Deniz

    2013-01-01

    Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

  20. Characterization of cognitive deficits in rats overexpressing human alpha-synuclein in the ventral tegmental area and medial septum using recombinant adeno-associated viral vectors.

    Directory of Open Access Journals (Sweden)

    Hélène Hall

    Full Text Available Intraneuronal inclusions containing alpha-synuclein (a-syn constitute one of the pathological hallmarks of Parkinson's disease (PD and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration.

  1. Adeno-associated viral vector 2.9 thymosin ß4 application attenuates rejection after heart transplantation: results of a preclinical study in the pig.

    Science.gov (United States)

    Postrach, Johannes; Schmidt, Maximilian; Thormann, Michael; Thein, Eckart; Burdorf, Lars; Reichart, Bruno; Sotlar, Karl; Walz, Christoph; Faber, Claudius; Bauer, Andreas; Schmoeckel, Michael; Kupatt, Christian; Hinkel, Rabea

    2014-10-27

    Graft survival is the most important factor for morbidity and mortality in cardiac transplantation. Improved immunosuppression significantly reduced early graft rejection. However, acute rejection may predispose to chronic rejection. Targeting both phases of the recipient's immune-reactivity by means of long-acting recombinant adeno-associated viral vectors (AAVs) encoding anti-inflammatory and cardioprotective factors appears to be a promising therapeutic approach. We investigate thymosin ß4 (Tß4) possessing anti-inflammatory and prosurvival abilities, as a means for pretransplant gene therapy. Heterotopic, abdominal transplantation of cardiac allografts into landrace or into Munich mini pigs (n=5 per group) was performed. Transplants were transduced with AAV2.9 before transplantation by means of in situ perfusion of the donor organ. Vascuar endothelial growth factor and AAV2.9.Tß4 or AAV2.9.LacZ were added to the autologous blood used for perfusing the grafts for a period of 45 min. Immunosuppression was applied for 10 days after the operation. Transgene expression, capillary density, graft function, survival, and rejection were assessed. The AAV2.9 transduction induced robust overexpression of the transgene. In addition, Tß4 ameliorated inflammation, necrosis, vascular reaction (acute rejection) and in parallel improved capillary density. In addition, graft survival was significantly prolonged (10±3 days AAV2.9.LacZ vs. 31±4 days AAV2.9.Tß4). In the mini pig model, regional myocardial function of the grafts was improved by Tß4 transduction compared to LacZ (9.1%±0.9% subendocardial segment shortening in AAV2.9.LacZ vs. 15.8%±2.3% in AAV2.9.Tß4). In situ AAV2.9-mediated gene transfer of thymosin β4 attenuated graft rejection in a heterotopic heart transplantation model. Perioperative cardioprotection by means of gene therapy might improve graft survival in cardiac allotransplantation.

  2. [Inhibition of infectious bursal disease virus replication in chicken embryos by miRNAs delivered by recombinant avian adeno-associated viral vector].

    Science.gov (United States)

    Shen, Pengpeng; Wang, Yongjuan; Sun, Huaichang; Zhang, Xinyu; Xia, Xiaoli

    2011-02-01

    We studied the inhibition of infectious bursal disease virus (IBDV) replication in chicken embryos by recombinant avian adeno-associated virus (AAAV)-delivered VP1- and VP2-specific microRNAs (miRNAs). We co-transfected AAV-293 cells with the VP1- or VP2 gene-specific miRNA expression vector pAITR-RFPmiVP1 or AITR-RFPmiVP2E, AAAV packaging vector pcDNA-ARC and adenovirus helper vector pHelper, resulting in recombinant virus rAAAV-RFPmiVP1 or rAAAV-RFPmiVP2E. We also generated the recombinant viruses rAAAV-RFP (without miRNA expression cassette) and rAAAV-RFPmiVP2con (expressing control miRNA) using the same method as the control purpose. Electron microscopy showed that the recombinant viruses had a typical morphology of AAV. We confirmed the presence of miRNA expression cassette in the recombinant viral genomes by using PCR. Our poly (A)-tailed RT-PCR showed correct expression of the miRNAs in the rAAAV-transduced DF-1 cells. We inoculated the recombinant viruses individually into 8-day-old SPF chicken embryos and then challenged them using Lukert strain IBDV on day 2 after inoculation. Our IBDV titration assay showed that the 50% tissue culture infectious dose (TCID50) of rAAAV-RFP- or rAAAV-RFPmiVP2con-inoculated group was 8.0 log10, whereas the TCID50 of rAAAV-RFPmiVP1-inoculated group decreased to 1.0 and 0.8 log10 on day 3 and 6 after challenge, respectively. Similarly, the TCID50 of rAAAV-RFPmiVP2E-inoculated group decreased to 1.5 and 2.0 log10, respectively. These data suggest that rAAAV can transduce efficiently chicken embryos and the expressed VP1- and VP2-specific miRNAs can inhibit the replication of IBDV efficiently.

  3. Improved Adeno-associated Viral Gene Transfer to Murine Glioma

    OpenAIRE

    Zolotukhin, I; Luo, D.; Gorbatyuk, OS; Hoffman, BE; Warrington, KH; Herzog, RW; Harrison, JK; Cao, O

    2013-01-01

    Glioblastoma (GBM) is a deadly primary brain tumor. Current treatment, consisting of surgical removal of the tumor mass followed by chemotherapy and/or radiotherapy, does not significantly prolong survival. Gene therapies for GBM are being developed in clinical trials, for example using adenoviral vectors. While adeno-associated virus (AAV) represents an alternative vector system, limited gene transfer to glioma cells has hampered its use. Here, we evaluated newly emerged variants of AAV caps...

  4. Adeno-associated viral vector (AAV)-mediated gene transfer in the red nucleus of the adult rat brain: comparative analysis of the transduction properties of seven AAV serotypes and lentiviral vectors.

    Science.gov (United States)

    Blits, Bas; Derks, Sanne; Twisk, Jaap; Ehlert, Erich; Prins, Jolanda; Verhaagen, Joost

    2010-01-15

    Recombinant adeno-associated viral vectors (AAVs) are very promising gene transfer tools for the nervous system. We have compared the efficiency of gene expression of seven AAV serotypes in young adult rats following a single injection in a major nucleus of the mid brain, the red nucleus, which is the origin of the rubrospinal tract. AAV serotypes 1-6 and 8 and a lentiviral vector (LV) were used, all encoding green fluorescent protein (GFP) under control of the cytomegalovirus (CMV) promoter. AAV vectors were titer matched at 5x10(11) genomic copies (GC)/ml and 1mul was injected into the red nucleus. The proportion of transduced neurons in the red nucleus was determined at 1 and 4 weeks post-injection. AAV1 would be the vector of choice if the aim would be to overexpress a transgene at high level for a longer period of time. AAV5 and AAV8 would be the preferred serotype if onset of expression is should be somewhat delayed. The use of lentiviral vectors should be considered when transduction of both glial cells and neurons is required. Serotypes 3 and 4 did not transduce red nucleus neurons. AAV1, AAV6 and LV would be the vectors of choice if the aim of the experiment would be to rapidly express a transgene. The current data are important for the design of experiments that aim to study the effects of transgene products on the regenerative capacity of injured red nucleus neurons.

  5. Behavioral recovery in a primate model of Parkinson's disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes.

    Science.gov (United States)

    Muramatsu, Shin-Ichi; Fujimoto, Ken-Ichi; Ikeguchi, Kunihiko; Shizuma, Nami; Kawasaki, Katsuyoshi; Ono, Fumiko; Shen, Yang; Wang, Lijun; Mizukami, Hiroaki; Kume, Akihiro; Matsumura, Masaru; Nagatsu, Ikuko; Urano, Fumi; Ichinose, Hiroshi; Nagatsu, Toshiharu; Terao, Keiji; Nakano, Imaharu; Ozawa, Keiya

    2002-02-10

    One potential strategy for gene therapy of Parkinson's disease (PD) is the local production of dopamine (DA) in the striatum induced by restoring DA-synthesizing enzymes. In addition to tyrosine hydroxylase (TH) and aromatic-L-amino-acid decarboxylase (AADC), GTP cyclohydrolase I (GCH) is necessary for efficient DA production. Using adeno-associated virus (AAV) vectors, we previously demonstrated that expression of these three enzymes in the striatum resulted in long-term behavioral recovery in rat models of PD. We here extend the preclinical exploration to primate models of PD. Mixtures of three separate AAV vectors expressing TH, AADC, and GCH, respectively, were stereotaxically injected into the unilateral putamen of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkeys. Coexpression of the enzymes in the unilateral putamen resulted in remarkable improvement in manual dexterity on the contralateral to the AAV-TH/-AADC/-GCH-injected side. Behavioral recovery persisted during the observation period (four monkeys: 48 days, 65 days, 50 days, and >10 months, each). TH-immunoreactive (TH-IR), AADC-IR, and GCH-IR cells were present in a large region of the putamen. Microdialysis demonstrated that concentrations of DA in the AAV-TH/-AADC/-GCH-injected putamen were increased compared with the control side. Our results show that AAV vectors efficiently introduce DA-synthesizing enzyme genes into the striatum of primates with restoration of motor functions. This triple transduction method may offer a potential therapeutic strategy for PD.

  6. Efficacy of recombinant adeno-associated viral vectors serotypes 1, 2, and 5 for the transduction of pancreatic and colon carcinoma cells.

    Science.gov (United States)

    Teschendorf, Christian; Emons, Barbara; Muzyczka, Nicholas; Graeven, Ullrich; Schmiegel, Wolff

    2010-06-01

    The development of efficient and specific vector systems remains a central issue in gene therapy. Several different adeno-associated virus (AAV) serotypes have so far been characterized so far which show different tissue tropisms. The vectors used here contained AAV2 transgene cassette containing green fluorescent protein (GFP) in AAV1, AAV2, or AAV5 capsids, producing the recombinant pseudotypes rAAV2/1, rAAV2/2, and rAAV2/5. The transduction efficiency of the different pseudotyped AAV vectors was tested in vitro in pancreatic and colon cancer cells lines (HT-29, BXPC3, and Hs766T). For all three serotypes, the percentage of GFP-positive cells was below 10% at multiplicities of infection (MOI) 100 rAAV vectors when used alone for infection. However, transduction efficiency for rAAV vectors increased dramatically when the cells were co-infected with wild-type adenovirus (wtAd). The percentage of GFP-positive cells ranged from 19.8-65.3% for AAV2/1 and 16.9-70.2% for AAV2/5, respectively. It was highest for rAAV2/2, at 40.9-88.4%. Variation between the cell lines was observed, with BXPC3 scoring the highest transduction rates and HT-29 the lowest. This study indicates that vectors based on distinct AAV serotypes 1, 2, and 5 all transduce pancreatic and colon cell lines poorly when used alone. Co-infection with wtAd increase transduction rates dramatically indicating that slow second-strand synthesis is a reason for the poor transduction efficiency. Due to the poor transduction rates, none of the rAAV serotypes tested here seem to be feasible for the treatment of malignant tumors.

  7. In Utero Transfer of Adeno-Associated Viral Vectors Produces Long-Term Factor IX Levels in a Cynomolgus Macaque Model.

    Science.gov (United States)

    Mattar, Citra N Z; Gil-Farina, Irene; Rosales, Cecilia; Johana, Nuryanti; Tan, Yvonne Yi Wan; McIntosh, Jenny; Kaeppel, Christine; Waddington, Simon N; Biswas, Arijit; Choolani, Mahesh; Schmidt, Manfred; Nathwani, Amit C; Chan, Jerry K Y

    2017-08-02

    The safe correction of an inherited bleeding disorder in utero prior to the onset of organ damage is highly desirable. Here, we report long-term transgene expression over more than 6 years without toxicity following a single intrauterine gene transfer (IUGT) at 0.9G using recombinant adeno-associated vector (AAV)-human factor IX (hFIX) in the non-human primate model we have previously described. Four of six treated animals monitored for around 74 months expressed hFIX at therapeutic levels (3.9%-120.0%). Long-term expression was 6-fold higher in males and with AAV8 compared to AAV5, mediated almost completely at this stage by random genome-wide hepatic proviral integrations, with no evidence of hotspots. Post-natal AAV challenge without immunosuppression was evaluated in two animals exhibiting chronic low transgene expression. The brief neutralizing immune reaction elicited had no adverse effect and, although expression was not improved at the dose administered, no clinical toxicity was observed. This long-term surveillance thus confirms the safety of late-gestation AAV-hFIX transfer and demonstrates that postnatal re-administration can be performed without immunosuppression, although it requires dose optimization for the desired expression. Nevertheless, eventual vector genotoxicity and the possibility of germline transmission will require lifelong monitoring and further evaluation of the reproductive function of treated animals. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Sensorimotor Cortex Injection of Adeno-Associated Viral Vector Mediates Knockout of PTEN in Neurons of the Brain and Spinal Cord of Mice.

    Science.gov (United States)

    Yang, Ping; Qin, Yu; Zhang, Wen; Bian, Zhiqun; Wang, Ruiqi

    2015-12-01

    Cre/loxP-mediated inactivation of phosphatase and tensin homolog (PTEN) is proposed to be a promising therapeutic agent for promoting CNS and PNS regeneration. And adeno-associated virus (AAV) vector has been developed as an attractive gene delivery system with proven safety. In the present study, we investigated Cre/loxP-mediated knockout of PTEN in the sensorimotor cortex, hippocampus, and spinal cord in PTEN floxed mice by immunohistological analysis of PI3K/AKT/mTOR expression in neurons of the sensorimotor cortex, hippocampus, and spinal cord after sensorimotor cortex injection of AAV-Cre. Two weeks after injection of AAV-Cre, the sensorimotor cortex, hippocampus, and spinal cord were dissected and examined the expression of downstream molecules pAKT and pS6 of PI3K/AKT signaling pathway. The results showed that remote delivery of AAV-Cre through sensorimotor cortex injection mediated PTEN knockout in neurons of the sensorimotor cortex, hippocampus, and spinal cord. We propose sensorimotor cortex injection of AAV may provide a potential strategy of gene therapy for the CNS diseases.

  9. Rapid, long-term labeling of cells in the developing and adult rodent visual cortex using double-stranded adeno-associated viral vectors.

    Science.gov (United States)

    Lowery, Rebecca L; Zhang, Yu; Kelly, Emily A; Lamantia, Cassandra E; Harvey, Brandon K; Majewska, Ania K

    2009-09-01

    Chronic in vivo imaging studies of the brain require a labeling method that is fast, long-lasting, efficient, nontoxic, and cell-type specific. Over the last decade, adeno-associated virus (AAV) has been used to stably express fluorescent proteins in neurons in vivo. However, AAV's main limitation for many studies (such as those of neuronal development) is the necessity of second-strand DNA synthesis, which delays peak transgene expression. The development of double-stranded AAV (dsAAV) vectors has overcome this limitation, allowing rapid transgene expression. Here, we have injected different serotypes (1, 2, 6, 7, 8, and 9) of a dsAAV vector carrying the green fluorescent protein (GFP) gene into the developing and adult mouse visual cortex and characterized its expression. We observed labeling of both neurons and astrocytes with serotype-specific tropism. dsAAV-GFP labeling showed high levels of neuronal GFP expression as early as 2 days postinjection and as long as a month, surpassing conventional AAV's onset of expression and matching its longevity. Neurons labeled with dsAAV-GFP appeared structurally and electrophysiologically identical to nonlabeled neurons, suggesting that dsAAV-GFP is neither cytotoxic nor alters normal neuronal function. We also demonstrated that dsAAV-labeled cells can be imaged with subcellular resolution in vivo over multiple days. We conclude that dsAAV is an excellent vector for rapid labeling and long-term in vivo imaging studies of astrocytes and neurons on the single cell level within the developing and adult visual cortex.

  10. Amelioration of chronic neuropathic pain after partial nerve injury by adeno-associated viral (AAV) vector-mediated over-expression of BDNF in the rat spinal cord

    NARCIS (Netherlands)

    Eaton, M.J.; Blits, B.; Ruitenberg, M.J.; Verhaagen, J.; Oudega, M.

    2002-01-01

    Changing the levels of neurotrophins in the spinal cord micro-environment after nervous system injury has been proposed to recover normal function, such that behavioral response to peripheral stimuli does not lead to chronic pain. We have investigated the effects of recombinant adeno-associated

  11. Adeno-associated virus (AAV) vectors in cancer gene therapy.

    Science.gov (United States)

    Santiago-Ortiz, Jorge L; Schaffer, David V

    2016-10-28

    Gene delivery vectors based on adeno-associated virus (AAV) have been utilized in a large number of gene therapy clinical trials, which have demonstrated their strong safety profile and increasingly their therapeutic efficacy for treating monogenic diseases. For cancer applications, AAV vectors have been harnessed for delivery of an extensive repertoire of transgenes to preclinical models and, more recently, clinical trials involving certain cancers. This review describes the applications of AAV vectors to cancer models and presents developments in vector engineering and payload design aimed at tailoring AAV vectors for transduction and treatment of cancer cells. We also discuss the current status of AAV clinical development in oncology and future directions for AAV in this field. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Adeno-Associated Virus Vectors (AAV Expressing Phenylalanine Hydroxylase (PAH

    Directory of Open Access Journals (Sweden)

    Ayşegül Akbay Yarpuzlu

    2009-06-01

    Full Text Available Recent articles have appeared in the literature reporting use of adeno-associated virus vectors (AAV expressing phenylalanine hydroxylase in animal trials and suggesting its use in treatment of phenylketonuria (PKU as a form of gene therapy However, agents used in gene therapy to deliver genes are not site-specific and DNA is may be put in the wrong place, causing damage to the organism. The adverse immunogenicity of AAVs also needs to be reconsidered. This letter is written to discuss present unreadiness for Phase 1 clinical trials of gene therapy of PKU. Turk Jem 2009; 13: 18-9

  13. Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors.

    Science.gov (United States)

    Kothari, P; De, B P; He, B; Chen, A; Chiuchiolo, M J; Kim, D; Nikolopoulou, A; Amor-Coarasa, A; Dyke, J P; Voss, H U; Kaminsky, S M; Foley, C P; Vallabhajosula, S; Hu, B; DiMagno, S G; Sondhi, D; Crystal, R G; Babich, J W; Ballon, D

    2017-01-06

    Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.

  14. Novel Cytotoxic Vectors Based on Adeno-Associated Virus

    Directory of Open Access Journals (Sweden)

    Johannes Kohlschütter

    2010-12-01

    Full Text Available Vectors based on adeno-associated virus (AAV are promising tools for gene therapy. The production of strongly toxic vectors, for example for cancer-directed gene transfer, is often unfeasible due to uncontrolled expression of toxic genes in vector-producing cells. Using an approach based on transcriptional repression, we have created novel AAV vectors carrying the genes coding for diphtheria toxin A (DTA and the pro-apoptotic PUMA protein. The DTA vector had a significant toxic effect on a panel of tumor cell lines, and abrogation of protein synthesis could be shown. The PUMA vector had a toxic effect on HeLa and RPMI 8226 cells, and sensitized transduced cells to doxorubicin. To permit targeted gene transfer, we incorporated the DTA gene into a genetically modified AAV-2 capsid previously developed by our group that mediates enhanced transduction of murine breast cancer cells in vitro. This vector had a stronger cytotoxic effect on breast cancer cells than DTA vectors with wildtype AAV capsid or vectors with a random capsid modification. The vector production and application system presented here allows for easy exchange of promotors, transgenes and capsid specificity for certain target cells. It will therefore be of great possible value in a broad range of applications in cytotoxic gene therapy and significantly broadens the spectrum of available tools for AAV-based gene therapy.

  15. Improved Adeno-associated Viral Gene Transfer to Murine Glioma.

    Science.gov (United States)

    Zolotukhin, I; Luo, D; Gorbatyuk, Os; Hoffman, Be; Warrington, Kh; Herzog, Rw; Harrison, Jk; Cao, O

    2013-04-29

    Glioblastoma (GBM) is a deadly primary brain tumor. Current treatment, consisting of surgical removal of the tumor mass followed by chemotherapy and/or radiotherapy, does not significantly prolong survival. Gene therapies for GBM are being developed in clinical trials, for example using adenoviral vectors. While adeno-associated virus (AAV) represents an alternative vector system, limited gene transfer to glioma cells has hampered its use. Here, we evaluated newly emerged variants of AAV capsid for gene delivery to murine glioma. We tested a mutant AAV2 capsid devoid of 3 surface-exposed tyrosine residues, AAV2 (Y444-500-730F), and a "shuffed" capsid (ShH19, containing sequences from several serotypes) that had previously been selected for enhanced glial gene delivery. AAV2 (Y-F) and ShH19 showed improved transduction of murine glioma GL261 cells in vitro by 2- to 6-fold, respectively, over AAV2. While AAV2 gene transfer to GL261 cells in established tumors in brains of syngeneic mice was undetectable, intratumoral injection of AAV2 (Y-F) or ShH19 resulted in local transduction of approximately 10% of tumor cells. In addition, gene transfer to neurons adjacent to the tumor was observed, while microglia were rarely transduced. Use of self-complementary vectors further increased transduction of glioma cells. Together, the data demonstrate the potential for improved AAV-based gene therapy for glioma using recently developed capsid variants.

  16. Adeno-associated virus (AAV) as a vehicle for therapeutic gene delivery: improvements in vector design and viral production enhance potential to prolong graft survival in pancreatic islet cell transplantation for the reversal of type 1 diabetes.

    Science.gov (United States)

    Kapturczak, M H; Flotte, T; Atkinson, M A

    2001-05-01

    Most viral gene delivery syslems utilized to date have demonstrated significant limitations in practicality and safety due to the level and duration of recombinant transgene expression as well as their induction of host immunogenicity to vector proteins. Recombinant adeno-associated virus (rAAV) vectors appear to offer a vehicle for safe, long-term therapeutic gene transfer; factors afforded through the propensity of rAAV to establish long-term latency without deleterious effects on the host cell and the relative non-immunogenicity of the virus or viral expressed transgenes. The principal historical limitation of this vector system, efficiency of rAAV-mediated transduction, has recently observed a dramatic increase as the titer, purity, and production capacity of rAAV preparations have improved. In terms of systems that could benefit from such improvements, rAAV gene therapy to enhance solid organ transplantation would appear an obvious choice with islet transplantation forming a promising candidate due to the ability to perform viral transductions ex vivo. Currently, islet transplantation can be used to treat type 1 diabetes yet persisting alloimmune and autoimmune responses represent major obstacles to the clinical success for this procedure. The delivery of transgenes capable of interfering with antigenic recognition and/or cell death [e.g., Fas ligand (FasL), Bcl-2, Bcl-XL] as well as imparting tolerance/immunoregulation [e.g., interleukin(IL)-4, IL-10, transforming growth factor (TGF)-beta], or cytoprotection [e.g., heme oxygenase-1 (HO-1), catalase, manganese superoxide dismutase (MnSOD)] may prevent recurrent type 1 diabetes in islet transplantation and offer a promising form of immunotherapy. Research investigations utilizing such systems may also provide information vital to understanding the immunoregulatory mechanisms critical to the development of both alloimmune and autoimmune islet cell rejection mechanisms and recurrent type 1 diabetes.

  17. Adenoviral and adeno-associated viral transfer of genes to the peripheral nervous system

    Science.gov (United States)

    Glatzel, Markus; Flechsig, Eckhard; Navarro, Beatriz; Klein, Michael A.; Paterna, Jean C.; Büeler, Hansruedi; Aguzzi, Adriano

    2000-01-01

    Targeted expression of foreign genes to the peripheral nervous system is interesting for many applications, including gene therapy of neuromuscular diseases, neuroanatomical studies, and elucidation of mechanisms of axonal flow. Here we describe a microneurosurgical technique for injection of replication-defective viral vectors into dorsal root ganglia (DRG). Adenovirus- and adeno-associated virus-based vectors with transcriptional competence for DRG neurons led to expression of the gene of interest throughout the first neuron of the sensory system, from the distal portions of the respective sensory nerve to the ipsilateral nucleus gracilis and cuneatus, which contains the synapses to the spinothalamic tracts. Use of Rag-1 ablated mice, which lack all B and T lymphocytes, allowed for sustained expression for periods exceeding 100 days. In immunocompetent mice, long-term (52 days) expression was achieved with similar efficiency by using adeno-associated viral vectors. DRG injection was vastly superior to intraneural injection into the sciatic nerve, which mainly transduced Schwann cells in the vicinity of the site of inoculation site but only inefficiently transduced nerve fibers, whereas i.m. injection did not lead to any significant expression of the reporter gene in nerve fibers. The versatile and efficient transduction of genes of interest should enable a wide variety of functional studies of peripheral nervous system pathophysiology. PMID:10618437

  18. Intramyocardial Injection of Recombinant Adeno-Associated Viral Vector Coexpressing PR39/Adrenomedullin Enhances Angiogenesis and Reduces Apoptosis in a Rat Myocardial Infarction Model

    Directory of Open Access Journals (Sweden)

    Rui An

    2017-01-01

    Full Text Available Cotransfer of angiogenic and antiapoptotic genes could be the basis of new gene therapy strategies for myocardial infarction. In this study, rAAV-PR39-ADM, coexpressing antimicrobial peptide (PR39 and adrenomedullin (ADM, was designed with the mediation of recombinant adeno-associated virus. In vitro, CRL-1730 cells were divided into four groups, namely, the sham group, the AAV-null group, the NS (normal saline group, and the PR39-ADM group. Immunocytochemistry analysis, CCK-8 assays, Matrigel assays, and apoptotic analysis were performed; in vivo, myocardial infarction model was established through ligation of the left coronary artery on rats, and treatment groups corresponded to those used in vitro. Myocardial injury, cardiac performance, and the extent of myocardial apoptosis were assessed. Results suggested that rAAV-PR39-ADM administration after myocardial infarction improved cell viability and cardiac function, attenuated apoptosis and myocardial injury, and promoted angiogenesis. Subsequently, levels of 6×His, HIF-1α, VEGF, p-Akt, Akt, ADM, Bcl-2, and Bax were measured by western blot. rAAV-PR39-ADM increased p-Akt, HIF-1α, and VEGF levels and induced higher Bcl-2 expression and lower Bax expression. In conclusion, our results demonstrate that rAAV-PR39-ADM mitigates myocardial injury by promoting angiogenesis and reducing apoptosis. This study suggests a potential novel gene therapy-based method that could be used clinically for myocardial infarction.

  19. Enhanced transduction of colonic cell lines in vitro and the inflamed colon in mice by viral vectors, derived from adeno-associated virus serotype 2, using virus-microbead conjugates bearing lectin

    Directory of Open Access Journals (Sweden)

    Sano Takeshi

    2007-11-01

    Full Text Available Abstract Background Virus-mediated delivery of therapeutic transgenes to the inflamed colon holds a great potential to serve as an effective therapeutic strategy for inflammatory bowel disease, since local, long-term expression of the encoded therapeutic proteins in the colorectal system is potentially achievable. Viral vectors, derived from adeno-associated virus (AAV, should be very useful for such therapeutic strategies, particularly because they can establish long-term expression of transgenes. However, few studies have been carried out to investigate the ability of AAV-based vectors to transduce the inflamed colon. Results AAV, derived from adeno-associated virus serotype 2 (AAV2, showed a limited ability to transduce colonic cell lines in vitro when used in free form. No appreciable enhancement of the transduction efficiency was seen when AAV2 particles were attached stably to the surfaces of microbeads and delivered to target cells in the form of AAV2-microbead conjugates. However, the transduction efficiency of these colonic cell lines was enhanced substantially when a lectin, concanavalin A (Con A, was co-attached to the microbead surfaces, to which AAV2 particles had been conjugated. This considerable infectivity enhancement of AAV2-microbead conjugates by the co-attachment of Con A may be derived from the fact that Con A binds to α-D-mannosyl moieties that are commonly and abundantly present in cell-surface carbohydrate chains, allowing the conjugates to associate stably with target cells. Intracolonical administration of free AAV2 or AAV2-microbead conjugates without Con A into a mouse colitis model by enema showed very poor transduction of the colonic tissue. In contrast, the delivery of AAV2 in the form of AAV2-microbead conjugates bearing Con A resulted in efficient transduction of the inflamed colon. Conclusion AAV2-microbead conjugates bearing Con A can serve as efficient gene transfer agents both for poorly permissive colonic

  20. Generation of Targeted Adeno-Associated Virus (AAV) Vectors for Human Gene Therapy.

    Science.gov (United States)

    Liu, Yarong; Siriwon, Natnaree; Rohrs, Jennifer A; Wang, Pin

    2015-01-01

    Adeno-associated virus (AAV) vectors are promising human gene delivery vehicles due to their ability to establish long-term gene expression in a wide variety of target tissues; however, the broad native viral tropism raises concerns over the feasibility and safety of their systemic administration. To overcome this issue, much effort has been made to redirect AAVs toward specific tissues. This review presents several design strategies that have been applied to generate AAVs that target specific tissues and cells while inhibiting the transduction of non-target tissues. Multiple methods of vector capsid engineering have shown promise in vitro, including indirect targeting by adaptor systems and direct targeting by the insertion of antibodies or receptor-specific small peptide motifs. Other strategies, including creating mosaic or chimeric capsids and directed evolution, have also been used to successfully retarget AAV vectors. This research will further expand the clinical applications of AAV vectors by enhancing the control over tissue-specific gene delivery.

  1. Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation combined with intraspinal adeno-associated viral vector-mediated expression of glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor.

    Science.gov (United States)

    Blits, Bas; Carlstedt, Thomas P; Ruitenberg, Marc Jan; de Winter, Fred; Hermens, Wim T J M C; Dijkhuizen, Paul A; Claasens, Jill W C; Eggers, Ruben; van der Sluis, Ronald; Tenenbaum, Liliane; Boer, Gerard J; Verhaagen, Joost

    2004-10-01

    Following avulsion of a spinal ventral root, motoneurons that project through the avulsed root are axotomized. Avulsion between, for example, L2 and L6 leads to denervation of hind limb muscles. Reimplantation of an avulsed root directed to the motoneuron pool resulted in re-ingrowth of some motor axons. However, most motoneurons display retrograde atrophy and subsequently die. Two neurotrophic factors, glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), promote the survival of motoneurons after injury. The long-term delivery of these neurotrophic factors to the motoneurons in the ventral horn of the spinal cord is problematic. One strategy to improve the outcome of the neurosurgical reinsertion of the ventral root following avulsion would involve gene transfer with adeno-associated viral (AAV) vectors encoding these neurotrophic factors near the denervated motoneuron pool. Here, we show that AAV-mediated overexpression of GDNF and BDNF in the spinal cord persisted for at least 16 weeks. At both 1 and 4 months post-lesion AAV-BDNF- and -GDNF-treated animals showed an increased survival of motoneurons, the effect being more prominent at 1 month. AAV vector-mediated overexpression of neurotrophins also promoted the formation of a network of motoneuron fibers in the ventral horn at the avulsed side, but motoneurons failed to extent axons into the reinserted L4 root towards the sciatic nerve nor to improve functional recovery of the hind limbs. This suggests that high levels of neurotrophic factors in the ventral horn promote sprouting, but prevent directional growth of axons of a higher number of surviving motoneurons into the implanted root.

  2. Overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors

    National Research Council Canada - National Science Library

    Schuster, Benjamin S; Kim, Anthony J; Kays, Joshua C; Kanzawa, Mia M; Guggino, William B; Boyle, Michael P; Rowe, Steven M; Muzyczka, Nicholas; Suk, Jung Soo; Hanes, Justin

    2014-01-01

    .... We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials...

  3. Creation of a cardiotropic adeno-associated virus: the story of viral directed evolution

    Directory of Open Access Journals (Sweden)

    Yang Lin

    2013-02-01

    Full Text Available Abstract Adeno-associated virus (AAV is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolution of viral vectors. We further attempted to evolve the AAV using DNA shuffling and in vivo biopanning in a mouse model. An AAVM41 mutant was characterized, which was found to have improved transduction efficiency and specificity in myocardium, an attribute unknown for any natural AAV serotypes. This review focuses on the development of AAV vector for cardiac gene transfer, the history of directed evolution of viral vectors, and our creation of a cardiotropic AAV, which might have implications for the future design and application of viral vectors.

  4. DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.

    Science.gov (United States)

    Koerber, James T; Jang, Jae-Hyung; Schaffer, David V

    2008-10-01

    Adeno-associated virus (AAV) vectors are extremely effective gene-delivery vehicles for a broad range of applications. However, the therapeutic efficacy of these and other vectors is currently limited by barriers to safe, efficient gene delivery, including pre-existing antiviral immunity, and infection of off-target cells. Recently, we have implemented directed evolution of AAV, involving the generation of randomly mutagenized viral libraries based on serotype 2 and high-throughput selection, to engineer enhanced viral vectors. Here, we significantly extend this capability by performing high-efficiency in vitro recombination to create a large (10(7)), diverse library of random chimeras of numerous parent AAV serotypes (AAV1, 2, 4-6, 8, and 9). In order to analyze the extent to which such highly chimeric viruses can be viable, we selected the library for efficient viral packaging and infection, and successfully recovered numerous novel chimeras. These new viruses exhibited a broad range of cell tropism both in vitro and in vivo and enhanced resistance to human intravenous immunoglobulin (IVIG), highlighting numerous functional differences between these chimeras and their parent serotypes. Thus, directed evolution can potentially yield unlimited numbers of new AAV variants with novel gene-delivery properties, and subsequent analysis of these variants can further extend basic knowledge of AAV biology.

  5. Regulation of gene expression in adeno-associated virus vectors in the brain.

    Science.gov (United States)

    Haberman, Rebecca P; McCown, Thomas J

    2002-10-01

    Regulated adeno-associated virus (AAV) vectors have broad utility in both experimental and applied gene therapy, and to date, several regulation systems have exhibited a capability to control gene expression from viral vectors over two orders of magnitude. The tetracycline responsive system has been the most used in AAV, although other regulation systems such as RU486- and rapamycin-responsive systems are reasonable options. AAV vectors influence how regulation systems function by several mechanisms, leading to increased background gene expression and restricted induction. Methods to reduce background expression continue to be explored and systems not yet tried in AAV may prove quite functional. Although regulated promoters are often assumed to exhibit ubiquitous expression, the tropism of different neuronal subtypes can be altered dramatically by changing promoters in recombinant AAV vectors. Differences in promoter-directed tropism have significant consequences for proper expression of gene products as well as the utility of dual vector regulation. Thus regulated vector systems must be carefully optimized for each application. Copyright 2002 Elsevier Science (USA)

  6. Comparison of nonviral transfection and adeno-associated viral transduction on cardiomyocytes.

    Science.gov (United States)

    Djurovic, Srdjan; Iversen, Nina; Jeansson, Stig; Hoover, Frank; Christensen, Geir

    2004-09-01

    Cardiomyocytes are terminally differentiated cells that to date have been characterized as poor targets for nonviral gene transfer. This study was therefore designed to determine the optimal nonviral gene transfer parameters in cell cultures of neonatal rat cardiomyocytes and to compare them with the efficiency of gene transfer using adeno-associated viral vectors (AAV). Transfection efficiency was measured by quantitative chloramphenicol acetyltransferase type I (CAT)-enzyme-linked immunosorbent assay and beta-galactosidase staining, based on overexpression of reporter genes (CAT and LacZ). The efficiency of CAT/LacZ overexpression was assessed using the following techniques: (1) liposomal reagents, such as: FuGENE 6, LipofectAMINE 2000, LipofectAMINE PLUS, GenePORTER, Metafectene, and LipoGen; (2) electroporation and nucleofector techniques; and (3) an AAV2 vector harboring a lacZ reporter gene. Toxicity was monitored by total protein measurement and by analyzing cell metabolism. On average, Lipofectamine 2000 was the most effective nonviral method examined yielding consistently high transfection rates (8.1% beta-galactosidase-positive cells) combined with low toxicity. Electroporation also resulted in high transfection values (7.5%); however, cellular toxicity was higher than that of Lipofectamine 2000. Finally, transduction with AAV2 vectors provided the highest levels of transduction (88.1%) with no cellular toxicity. We conclude that although transduction with AAV is more efficient (88.1%), transfections with nonviral techniques, when optimized, may provide a useful alternative for overexpression of therapeutic genes in neonatal cardiomyocytes.

  7. Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats

    NARCIS (Netherlands)

    Seppen, Jurgen; Bakker, Conny; de Jong, Berry; Kunne, Cindy; van den Oever, Karin; Vandenberghe, Kristin; de Waart, Rudi; Twisk, Jaap; Bosma, Piter

    2006-01-01

    Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have

  8. Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints

    NARCIS (Netherlands)

    Apparailly, F.; Khoury, M.; Vervoordeldonk, M. J. B.; Adriaansen, J.; Gicquel, E.; Perez, N.; Riviere, C.; Louis-Plence, P.; Noel, D.; Danos, O.; Douar, A.-M.; Tak, P. P.; Jorgensen, C.

    2005-01-01

    The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis ( RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues.

  9. Optimization of design and production strategies for novel adeno-associated viral display peptide libraries.

    Science.gov (United States)

    Körbelin, J; Hunger, A; Alawi, M; Sieber, T; Binder, M; Trepel, M

    2017-08-01

    Libraries displaying random peptides on the surface of adeno-associated virus (AAV) are powerful tools for the generation of target-specific gene therapy vectors. However, for unknown reasons the success rate of AAV library screenings is variable and the influence of the production procedure has not been thoroughly evaluated. During library screenings, the capsid variants with the most favorable tropism are enriched over several selection rounds on a target of choice and identified by subsequent sequencing of the encapsidated viral genomes encoding the library capsids with targeting peptide insertions. Thus, a high capsid-genome correlation is crucial to obtain the correct information about the selected capsid variants. Producing AAV libraries by a two-step protocol with pseudotyped library transfer shuttles has been proposed as one way to ensure such a correlation. Here we show that AAV2 libraries produced by such a protocol via transfer shuttles display an unexpected additional bias in the amino-acid composition which confers increased heparin affinity and thus similarity to wildtype AAV2 tropism. This bias may fundamentally impair the intended use of AAV libraries, discouraging the use of transfer shuttles for the production of AAV libraries in the future.

  10. Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion.

    Science.gov (United States)

    Haberman, Rebecca P; Samulski, R Jude; McCown, Thomas J

    2003-08-01

    Seizure disorders present an attractive gene therapy target, particularly because viral vectors such as adeno-associated virus (AAV) and lentivirus can stably transduce neurons. When we targeted the N-methyl-D-aspartic acid (NMDA) excitatory amino acid receptor with an AAV-delivered antisense oligonucleotide, however, the promoter determined whether focal seizure sensitivity was significantly attenuated or facilitated. One potential means to circumvent this liability would be to express an inhibitory neuroactive peptide and constitutively secrete the peptide from the transduced cell. The neuropeptide galanin can modulate seizure activity in vivo, and the laminar protein fibronectin is usually secreted through a constitutive pathway. Initially, inclusion of the fibronectin secretory signal sequence (FIB) in an AAV vector caused significant gene product secretion in vitro. More importantly, the combination of this secretory signal with the coding sequence for the active galanin peptide significantly attenuated in vivo focal seizure sensitivity, even with different promoters, and prevented kainic acid-induced hilar cell death. Thus, neuroactive peptide expression and local secretion provides a new gene therapy platform for the treatment of neurological disorders.

  11. [Protective effect of adeno-associated viral vector-mediated expression of human brain-derived neurotrophic factor in rat neurons against beta-amyloid-induced Alzheimer's disease in vitro].

    Science.gov (United States)

    Liu, Zhao-hui; Ma, Dong-liang; Jin, Hui; Ma, Yan-bing; Hu, Hai-tao

    2006-10-01

    To achieve expression of human brain-derived neurotrophic factor (hBDNF) mediated by recombinant adeno-associated virus (rAAV) and explore the mechanism of its neuroprotective effects in rat neurons against beta-amyloid-induced Alzheimer's disease. Using molecular cloning technique, rAAV vector containing hBDNF gene (AAV-hBDNF) was constructed to transfect SD rat hippocampal neurons exposed to beta-amyloid treatment. The changes in cell apoptosis were observed by MTT assay and flow cytometry, and the expression of hBDNF and Bcl-2 protein were determined by immunocytochemical staining. Laser scanning confocal microscopy (LSCM) was used to observe the changes of [Ca(2+)](i). The cultured rat hippocampal neurons were effectively transfected with AAV-hBDNF and expression of BDNF protein was obviously increased. hBNDF expression showed significant protective effects against beta-amyloid-induced neuronal damage, and the expression of Bcl-2 protein was increased significantly and the balance of [Ca(2+)](i) was maintained in BDNF-treated cells with beta-amyloid exposure. hBDNF expression can effectively protect cultured rat hippocampal cells from beta-amyloid-induced apoptosis through inhibiting the intracellular calcium overload and increasing the expression of Bcl-2 protein.

  12. A simplified purification protocol for recombinant adeno-associated virus vectors

    Directory of Open Access Journals (Sweden)

    Mark Potter

    2014-01-01

    Full Text Available We describe a new rapid, low cost, and scalable method for purification of various recombinant adeno-associated viruses (rAAVs from the lysates of producer cells of either mammalian or insect origin. The method takes advantage of two general biochemical properties of all characterized AAV serotypes: (i low isoelectric point of a capsid and (ii relative biological stability of the viral particle in the acidic environment. A simple and rapid clarification of cell lysate toremove the bulk of proteins and DNA is accomplished by utilizing inexpensive off-the-shelf reagents such as sodium citrate and citric acid. After the low-speed centrifugation step, the supernatant is subjected to cation exchange chromatography via sulfopropyl (SP column. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and Good Manufacturing Practice (GMP grade biochemical purity. The protocol is validated for rAAV serotypes 2, 8, and 9. The described method makes rAAV vector technology readily available for the low budget research laboratories and could be easily adapted for a large scale GMP production format.

  13. Recombinant adeno-associated virus vectors in the treatment of rare diseases.

    Science.gov (United States)

    Hastie, Eric; Samulski, R Jude

    An estimated 25 million Americans are living with rare diseases. Adeno-associated virus (AAV)-mediated gene therapy is an emerging therapeutic option for the more than 7,000 identified rare diseases. This paper highlights the benefits of AAV therapy compared to conventional small molecules, discusses current pre-clinical and clinical applications of AAV-mediated gene therapy, and offers insights into cutting edge research that will shape the future of AAV for broad therapeutic use. In this review the biology of AAV and our ability to generate disease-specific variants is summarized. Limitations of current therapy are reviewed, with an emphasis on immune detection of virus, viral tropism and tissue targeting, and limitations of gene expression. Information for this review was found using PubMed and clinicaltrials.gov. Currently the scope of clinical trials of AAV gene therapy is concentrated in an array of phase I/II safety trials with less than two dozen rare diseases featured. Pre-clinical, translational studies are expanding in number as developments within the last decade have made generation of improved AAV vectors available to more researchers. Further, one bottleneck that is being overcome is the availability of disease models, which will allow for improved preclinical testing and advancement of AAV to more clinical applications.

  14. Genetic Vaccines for Anthrax Based on Recombinant Adeno-associated Virus Vectors

    OpenAIRE

    Liu, Te-Hui; Oscherwitz, Jon; Schnepp, Bruce; Jacobs, Jana; Yu, Fen; Cease, Kemp B; Johnson, Philip R.

    2008-01-01

    Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction of toxin-neutralizing antibodies in rabbits. Codon-optimized segments enc...

  15. Reducing the risk of adeno-associated virus (AAV) vector mobilization with AAV type 5 vectors.

    Science.gov (United States)

    Hewitt, F Curtis; Li, Chengwen; Gray, Steven J; Cockrell, Shelley; Washburn, Michael; Samulski, R Jude

    2009-04-01

    Current adeno-associated virus (AAV) gene therapy vectors package a transgene flanked by the terminal repeats (TRs) of AAV type 2 (AAV2). Although these vectors are replication deficient, wild-type (wt) AAV2 prevalent in the human population could lead to replication and packaging of a type 2 TR (TR2)-flanked transgene in trans during superinfection by a helper virus, leading to "mobilization" of the vector genome from treated cells. More importantly, it appears likely that the majority of currently characterized AAV serotypes as well as the majority of new novel isolates are capable of rescuing and replicating AAV2 vector templates. To investigate this possibility, we flanked a green fluorescent protein transgene with type 2 and, the most divergent AAV serotype, type 5 TRs (TR2 or TR5). Consistent with AAV clades, AAV5 specifically replicated TR5 vectors, while AAV2 and AAV6 replicated TR2-flanked vectors. To exploit this specificity, we created a TR5 vector production system for Cap1 to Cap5. Next, we showed that persisting recombinant AAV genomes flanked by TR2s or TR5s were mobilized in vitro after addition of the cognate AAV Rep (as well as Rep6 for TR2) and adenoviral helper. Finally, we showed that a cell line containing a stably integrated wt AAV2 genome resulted in mobilization of a TR2-flanked vector but not a TR5-flanked vector upon adenoviral superinfection. Based on these data and the relative prevalence of wt AAV serotypes in the population, we propose that TR5 vectors have a significantly lower risk of mobilization and should be considered for clinical use.

  16. Adeno-Associated Virus Vector-Mediated Transgene Integration into Neurons and Other Nondividing Cell Targets

    OpenAIRE

    WU, Ping; Phillips, M. Ian; Bui, John; Terwilliger, Ernest F.

    1998-01-01

    The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration...

  17. Retinal gene delivery by adeno-associated virus (AAV) vectors: Strategies and applications.

    Science.gov (United States)

    Schön, Christian; Biel, Martin; Michalakis, Stylianos

    2015-09-01

    Adeno-associated virus (AAV) vectors are the most widely used vehicle systems for neuronal gene transfer. This popularity is based on the non-pathogenic nature of AAVs and their versatility making them a multifunctional vector system for basic research and clinical applications. AAVs are successfully applied in clinical and pre-clinical gene therapy studies for inherited retinal disorders. Their excellent transduction profile and efficiency also boosted the use of AAV vectors in basic research. The AAV vector system can be easily modified and adjusted at multiple levels to allow for optimized and specific gene expression in target cells. Here, we will provide an overview on the AAV vector system and its applications focusing on gene transfer into retinal cells. Furthermore, we will outline and discuss strategies for the optimization of AAV gene transfer by modifications to the AAV vector expression cassette, the AAV capsid or the routes of vector administration. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Adeno-associated virus vector-mediated transgene integration into neurons and other nondividing cell targets.

    Science.gov (United States)

    Wu, P; Phillips, M I; Bui, J; Terwilliger, E F

    1998-07-01

    The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.

  19. Transduction of pancreatic islets with pseudotyped adeno-associated virus: effect of viral capsid and genome conversion.

    Science.gov (United States)

    Zhang, Nan; Clément, Nathalie; Chen, Dongmei; Fu, Shuang; Zhang, Haojiang; Rebollo, Patricia; Linden, R Michael; Bromberg, Jonathan S

    2005-09-15

    Recombinant adeno-associated viral (rAAV) vectors currently show promise for islet gene therapy. In the presence of complementing AAV2 Rep proteins, AAV2 genomes can be packaged with other serotype capsids to assemble infectious virions. During transduction, the ssDNA to dsDNA conversion is one of the major rate-limiting steps that contribute to the slow onset of transgene expression. Using pseudotyping strategy, we produced double-stranded (dsAAV) and single-stranded (ssAAV) rAAV2 genomes carrying the GFP reporter gene packaged into AAV1, AAV2, and AAV5 capsids. The ability of cross-packaged AAV1, AAV2, and AAV5 at the same genome containing particle (gcp) concentration to transduce murine and human pancreatic islets was evaluated by GFP positive cell percentage. Transgenic expression was also determined by transplant transduced human islet into SCID mice. Pseudotyped rAAV2/1 based vectors transduced murine islets at greater efficiency than either rAAV2/2 or rAAV2/5 vectors. For human islets transduction, the rAAV2/2 vector was more efficient than rAAV2/1 or rAAV2/5 vectors. rAAV2/2 transduced human islets more efficiently than murine islets, while rAAV2/1 transducted murine islets more efficiently than human islets. dsAAV, which do not require second strand synthesis and thus are potentially more efficient, evidenced 5 fold higher transduction ability than ssAAV vectors. Pseudotyped rAAV transduced islet grafts maintained normal function, expressed transgenic product persistently in vivo, and reversed diabetes. The transduction efficiency of rAAV vectors was dependent on the cross-packaged capsid. The vector capsids permit species-specific transduction. For human islets, dsAAV2/2 vectors may be the most efficient vector for clinical development.

  20. Adeno-associated viral-mediated insulin-like growth factor delivery protects motor neurons in vitro.

    Science.gov (United States)

    Vincent, Andrea M; Feldman, Eva L; Song, Debbie K; Jung, Verena; Schild, Andreas; Zhang, Wei; Imperiale, Michael J; Boulis, Nicholas M

    2004-01-01

    Recent work has demonstrated that adeno-associated viral (AAV) vector-mediated delivery of the insulin-like growth factor (IGF-I) gene through retrograde axonal transport can prolong survival and delay disease onset in the superoxide dismutase mutant mouse model of motor neuron (MN) disease. The present experiment examines IGF-I gene transfer in vitro. Adenoviral and AAV vectors for IGF-I infect neurons triggering expression and secretion of biologically active IGF-I. AAV-mediated IGF-I expression in SH-SY5Y neurons protects both cells expressing the transgene, and bystanders without transgene expression from glutamate-induced apoptosis. Similarly, AAV-mediated IGF-I delivery in primary E15 MN culture provides a titer-dependent neuroprotection from glutamate-induced DNA fragmentation. Both infected and noninfected neurons are equally protected. These observations argue that vector-mediated IGF-I gene transfer induces secretion of active IGF-I that acts through direct effects on spinal cord MNs. This mechanism may explain the therapeutic effects observed in vivo despite relatively low affinity AAV spinal cord uptake.

  1. Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats.

    Science.gov (United States)

    Seppen, Jurgen; Bakker, Conny; de Jong, Berry; Kunne, Cindy; van den Oever, Karin; Vandenberghe, Kristin; de Waart, Rudi; Twisk, Jaap; Bosma, Piter

    2006-06-01

    Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.

  2. Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors

    Science.gov (United States)

    CHEN, SIFENG; KAPTURCZAK, MATTHIAS; LOILER, SCOTT A.; ZOLOTUKHIN, SERGEI; GLUSHAKOVA, OLENA Y.; MADSEN, KIRSTEN M.; SAMULSKI, RICHARD J.; HAUSWIRTH, WILLIAM W.; CAMPBELL-THOMPSON, MARTHA; BERNS, KENNETH I.; FLOTTE, TERENCE R.; ATKINSON, MARK A.; TISHER, C. CRAIG

    2006-01-01

    Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human α1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding β-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies. OVERVIEW SUMMARY Gene delivery to the vasculature has significant potential as a therapeutic strategy for several cardiovascular disorders including atherosclerosis, hypertension, angiogenesis, and chronic vascular rejection of transplanted organs. However, limited advances have been made in achieving successful vascular endothelial cell gene transfer. The results of the present study demonstrate the superior efficacy of recombinant adeno-associated virus (rAAV) serotype 1 and 5 vectors in comparison to the traditionally used rAAV serotype 2 in transduction of primary vascular endothelial and smooth muscle cells in vitro. Our results have identified sialic acid residues for rAAV1 transduction in endothelial

  3. Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system

    Directory of Open Access Journals (Sweden)

    Pierre-Olivier Buclez

    2016-01-01

    Full Text Available Recombinant adeno-associated viruses (rAAV are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology.

  4. Preferential labeling of inhibitory and excitatory cortical neurons by endogenous tropism of adeno-associated virus and lentivirus vectors.

    Science.gov (United States)

    Nathanson, J L; Yanagawa, Y; Obata, K; Callaway, E M

    2009-06-30

    Despite increasingly widespread use of recombinant adeno-associated virus (AAV) and lentiviral (LV) vectors for transduction of neurons in a wide range of brain structures and species, the diversity of cell types within a given brain structure is rarely considered. For example, the ability of a vector to transduce neurons within a brain structure is often assumed to indicate that all neuron types within the structure are transduced. We have characterized the transduction of mouse somatosensory cortical neuron types by recombinant AAV pseudotyped with serotype 1 capsid (rAAV2/1) and by recombinant lentivirus pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein. Both vectors used human synapsin (hSyn) promoter driving DsRed-Express. We demonstrate that high titer rAAV2/1-hSyn efficiently transduces both cortical excitatory and inhibitory neuronal populations, but use of lower titers exposes a strong preference for transduction of cortical inhibitory neurons and layer 5 pyramidal neurons. In contrast, we find that VSV-G-LV-hSyn principally labels excitatory cortical neurons at the highest viral titer generated. These findings demonstrate that endogenous tropism of rAAV2/1 and VSV-G-LV can be used to obtain preferential gene expression in mouse somatosensory cortical inhibitory and excitatory neuron populations, respectively.

  5. Optimization of adeno-associated virus vector-mediated gene transfer to the respiratory tract.

    Science.gov (United States)

    Kurosaki, F; Uchibori, R; Mato, N; Sehara, Y; Saga, Y; Urabe, M; Mizukami, H; Sugiyama, Y; Kume, A

    2017-05-01

    An efficient adeno-associated virus (AAV) vector was constructed for the treatment of respiratory diseases. AAV serotypes, promoters and routes of administration potentially influencing the efficiency of gene transfer to airway cells were examined in the present study. Among the nine AAV serotypes (AAV1-9) screened in vitro and four serotypes (AAV1, 2, 6, 9) evaluated in vivo, AAV6 showed the strongest transgene expression. As for promoters, the cytomegalovirus (CMV) early enhancer/chicken β-actin (CAG) promoter resulted in more robust transduction than the CMV promoter. Regarding delivery routes, intratracheal administration resulted in strong transgene expression in the lung, whereas the intravenous and intranasal administration routes yielded negligible expression. The combination of the AAV6 capsid and CAG promoter resulted in sustained expression, and the intratracheally administered AAV6-CAG vector transduced bronchial cells and pericytes in the lung. These results suggest that AAV6-CAG vectors are more promising than the previously preferred AAV2 vectors for airway transduction, particularly when administered into the trachea. The present study offers an optimized strategy for AAV-mediated gene therapy for lung diseases, such as cystic fibrosis and pulmonary fibrosis.

  6. Recombination and population mosaic of a multifunctional viral gene, adeno-associated virus cap.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Takeuchi

    Full Text Available Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred.

  7. A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Müller cells.

    Directory of Open Access Journals (Sweden)

    Ryan R Klimczak

    2009-10-01

    Full Text Available The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV. Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection.We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6, capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60% and AAV6.Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina.

  8. A novel adeno-associated viral variant for efficient and selective intravitreal transduction of rat Müller cells.

    Science.gov (United States)

    Klimczak, Ryan R; Koerber, James T; Dalkara, Deniz; Flannery, John G; Schaffer, David V

    2009-10-14

    The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection. We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6. Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina.

  9. Adeno-associated virus pseudotype 5 vector improves gene transfer in arthritic joints.

    Science.gov (United States)

    Apparailly, F; Khoury, M; Vervoordeldonk, M J B; Adriaansen, J; Gicquel, E; Perez, N; Riviere, C; Louis-Plence, P; Noel, D; Danos, O; Douar, A-M; Tak, P P; Jorgensen, C

    2005-04-01

    The potential for gene delivery to joints, using recombinant adeno-associated virus (rAAV) vectors for the treatment of rheumatoid arthritis (RA), has received much attention. Different serotypes have different virion shell proteins and, as a consequence, vary in their tropism for diverse tissues. The aim of this study was to compare the transduction efficiency of different AAV serotypes encoding murine secreted alkaline phosphatase (mSEAP) or Escherichia coli beta-galactosidase for intraarticular gene delivery in an experimental model of arthritis. The vectors contained AAV2 terminal repeats flanking the reporter gene in an AAV1, AAV2, or AAV5 capsid, producing the pseudotypes rAAV-2/1, rAAV-2/2, and rAAV-2/5. Left knee joints of mice with collagen-induced arthritis were injected and transgene expression was analyzed by chemiluminescence or direct in situ staining of frozen sections. We show for the first time that intraarticular gene transfer with AAV- 2/5 was far more efficient than with the other serotypes tested. Transgene expression was detectable as early as 7 days after injection, reached a maximum at 21 days, and was stably expressed for at least 130 days, whereas AAV-2/1- and AAV-2/2-mediated expression levels were barely detectable. These findings provide a practical application for future local AAV-mediated gene therapy trials in RA.

  10. Directed evolution of adeno-associated virus (AAV) as vector for muscle gene therapy.

    Science.gov (United States)

    Yang, Lin; Li, Juan; Xiao, Xiao

    2011-01-01

    Adeno-associated virus (AAV) is emerging as a vector of choice for muscle gene therapy because of its effective and stable transduction in striated muscles. AAV naturally evolve into multiple serotypes with diverse capsid gene sequences that are apparently the determinants of their tissue tropism and infectivity. Certain AAV serotypes show robust gene transfer upon direct intramuscular injection, while others are effective in crossing the endothelial barrier to reach muscle when delivered intravenously. Muscular dystrophy gene therapy requires efficient body-wide muscle gene transfer. However, preferential liver transduction by nearly all natural AAV serotypes could be an undesirable feature for muscle-directed applications, especially by means of systemic gene delivery. Here we describe a method of in vitro evolution and in vivo selection of AAV capsids that target striated muscles and detarget the liver. Using DNA shuffling technology, we have generated a capsid gene library by in vitro scrambling and shuffling the capsid genes of natural AAV1 to AAV9. To minimize the bias and limitation of in vitro screening on culture cells, we performed direct in vivo panning in adult mice after intravenous injection of the shuffled capsid library that packaged their own coding sequences. The AAV variants enriched in the heart and muscle are retrieved by capsid gene PCR and subsequently characterized for their tissue tropisms. This directed evolution and in vivo selection method should be useful in generating novel gene therapy vectors for muscle and heart and other tissues.

  11. K137R mutation on adeno-associated viral capsids had minimal effect on enhancing gene delivery in vivo.

    Science.gov (United States)

    Qiao, Chunping; Li, Chengwen; Zhao, Chunxia; Li, Jianbin; Bian, Tao; Grieger, Joshua; Li, Juan; Samulski, R Jude; Xiao, Xiao

    2014-02-01

    The adeno-associated viral (AAV) vector has emerged as an attractive vector for gene therapy applications. Development of AAV vectors with enhanced gene transduction efficiency is important to ease the burden of AAV production and minimize potential immune responses. Rational mutations on AAV capsids have gained attention as a simple method of enhancing AAV transduction efficiency. A single-amino acid mutation, K137R, on AAV1 and AAV8 was recently reported to increase liver transgene expression by 5-10-fold. To determine whether the same mutation on other AAV serotypes would result in similar gene enhancement effects, K137R mutants were generated on AAV7, AAV8, and AAV9, and their effects were evaluated in vivo. Two reporter genes were utilized: the nuclear LacZ gene driven by the cytomegalovirus promoter and the luciferase gene driven by the CB promoter. Surprisingly, we found no difference in luciferase gene expression in the liver or other tissues using either the wild-type AAV8 capsid or AAV8-K137R. LacZ gene expression in the liver by AAV8-K137R was about onefold higher than that of wild-type AAV8. However, no difference was found in other tissues, such as skeletal muscle and cardiac muscle. In addition, no difference was found in transgene expression with either AAV7-K137R or AAV9-K137R mutants. Our results indicated that the K137R mutation on AAV7, AAV8, and AAV9 had minimal to no effect on transduction efficiency in vivo.

  12. Widespread dispersion of adeno-associated virus serotype 1 and adeno-associated virus serotype 6 vectors in the rat central nervous system and in human glioblastoma multiforme xenografts.

    Science.gov (United States)

    Huszthy, Peter C; Svendsen, Agnete; Wilson, James M; Kotin, Robert M; Lønning, Per Eystein; Bjerkvig, Rolf; Hoover, Frank

    2005-03-01

    The transduction patterns of recombinant adeno-associated virus serotype 1 (AAV1) and serotype 6 (AAV6) vectors were assessed in human glioblastoma multiforme (GBM) cell lines, in human GBM biopsy spheroids, and in tumor xenografts growing in nude rat brains. All the cell lines tested (A172, D37, GaMg, HF66, and U373Mg) were found to be permissive to both AAV1 and AAV6 vectors, and thus displayed a transduction pattern similar to AAV2 vectors. For every cell line tested, the transduction efficiency displayed by AAV2 vectors was better than by isogenic and isopromoter AAV1 vectors. Transduction efficiency was dependent on the viral particle number used, suggesting that the receptors for these vectors are widely distributed in GBM tissues. Interestingly, AAV1, AAV2, and AAV6 vectors were able to infect and transduce the same cells when added simultaneously to monolayer cultures. Infection of human GBM biopsy spheroids with AAV1 and AAV6 vectors resulted in transgene expression both at the surface layers and in the core of the spheroids. Following injection of AAV1 and AAV6 vectors into human GBM biopsy xenografts growing in nude rat brains, reporter gene expression was seen both in the periphery as well as in the central regions of the tumors. When injected into the normal rat brain, both AAV1 and AAV6 vectors were found to transduce several central nervous system (CNS) regions. The presented results suggest a potential therapeutic role for AAV1 and AAV6 vectors in gene therapy for GBM and also for other CNS malignancies.

  13. Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors.

    Science.gov (United States)

    Chen, Sifeng; Kapturczak, Matthias; Loiler, Scott A; Zolotukhin, Sergei; Glushakova, Olena Y; Madsen, Kirsten M; Samulski, Richard J; Hauswirth, William W; Campbell-Thompson, Martha; Berns, Kenneth I; Flotte, Terence R; Atkinson, Mark A; Tisher, C Craig; Agarwal, Anupam

    2005-02-01

    Recombinant adeno-associated virus (rAAV) has become an attractive tool for gene therapy because of its ability to transduce both dividing and nondividing cells, elicit a limited immune response, and the capacity for imparting long-term transgene expression. Previous studies have utilized rAAV serotype 2 predominantly and found that transduction of vascular cells is relatively inefficient. The purpose of the present study was to evaluate the transduction efficiency of rAAV serotypes 1 through 5 in human and rat aortic endothelial cells (HAEC and RAEC). rAAV vectors with AAV2 inverted terminal repeats containing the human alpha1-antitrypsin (hAAT) gene were transcapsidated using helper plasmids to provide viral capsids for the AAV1 through 5 serotypes. True type rAAV2 and 5 vectors encoding beta-galactosidase or green fluorescence protein were also studied. Infection with rAAV1 resulted in the most efficient transduction in both HAEC and RAEC compared to other serotypes (p Transduction with rAAV1 was completely inhibited by removal of sialic acid with sialidase, while heparin had no effect. These studies are the first demonstration that sialic acid residues are required for rAAV1 transduction in endothelial cells. Transduction of rat aortic segments ex vivo and in vivo demonstrated significant transgene expression in endothelial and smooth muscle cells with rAAV1 and 5 serotype vectors, in comparison to rAAV2. These results suggest the unique potential of rAAV1 and rAAV5-based vectors for vascular-targeted gene-based therapeutic strategies.

  14. Impact of Heparan Sulfate Binding on Transduction of Retina by Recombinant Adeno-Associated Virus Vectors

    Science.gov (United States)

    Boye, Sanford L.; Bennett, Antonette; Scalabrino, Miranda L.; McCullough, K. Tyler; Van Vliet, Kim; Choudhury, Shreyasi; Ruan, Qing; Peterson, James

    2016-01-01

    ABSTRACT Adeno-associated viruses (AAVs) currently are being developed to efficiently transduce the retina following noninvasive, intravitreal (Ivt) injection. However, a major barrier encountered by intravitreally delivered AAVs is the inner limiting membrane (ILM), a basement membrane rich in heparan sulfate (HS) proteoglycan. The goal of this study was to determine the impact of HS binding on retinal transduction by Ivt-delivered AAVs. The heparin affinities of AAV2-based tyrosine-to-phenylalanine (Y-F) and threonine-to-valine (T-V) capsid mutants, designed to avoid proteasomal degradation during cellular trafficking, were established. In addition, the impact of grafting HS binding residues onto AAV1, AAV5, and AAV8(Y733F) as well as ablation of HS binding by AAV2-based vectors on retinal transduction was investigated. Finally, the potential relationship between thermal stability of AAV2-based capsids and Ivt-mediated transduction was explored. The results show that the Y-F and T-V AAV2 capsid mutants bind heparin but with slightly reduced affinity relative to that of AAV2. The grafting of HS binding increased Ivt transduction by AAV1 but not by AAV5 or AAV8(Y733F). The substitution of any canonical HS binding residues ablated Ivt-mediated transduction by AAV2-based vectors. However, these same HS variant vectors displayed efficient retinal transduction when delivered subretinally. Notably, a variant devoid of canonical HS binding residues, AAV2(4pMut)ΔHS, was remarkably efficient at transducing photoreceptors. The disparate AAV phenotypes indicate that HS binding, while critical for AAV2-based vectors, is not the sole determinant for transduction via the Ivt route. Finally, Y-F and T-V mutations alter capsid stability, with a potential relationship existing between stability and improvements in retinal transduction by Ivt injection. IMPORTANCE AAV has emerged as the vector of choice for gene delivery to the retina, with attention focused on developing vectors

  15. Efficient production of an avian adeno-associated virus vector using insect cell/baculovirus expression system.

    Science.gov (United States)

    Wang, Anping; Wang, Yongjuan; Wu, Shuang; Zuo, Weiyong; Guo, Changming; Hong, Weiming; Zhu, Shanyuan

    2017-02-01

    Recombinant avian adeno-associated virus (rAAAV) is a promising gene transfer vector for avian cells. Although rAAAV can be produced by co-transfection of HEK293 cells with three plasmids, both scalability and productivity of the transient transfection method can not meet the demand for large-scale in vivo experiments. In this study, a scalable rAAAV production method was established by using insect cell/baculovirus expression system. Three recombinant baculoviruses, namely BacARep, BacAVP and BacAGFP, were generated by transfection of Sf9 cells with the three plasmids expressing AAAV Rep genes, modified VP gene or the inverted terminal repeats-flanked green fluorescent protein (GFP) gene. After demonstration of the correct expression of AAAV genes, rAAAV-GFP was produced by triple infection of insect cells or triple transfection of HEK293 cells for comparison purpose. Electron microscopy revealed the formation of typical AAAV particles in the insect cells. Western blotting showed the correct assembly of rAAAV particles with a VP protein ratio similar to that of AAAV. Quantitative PCR showed that the insect cell-produced rAAAV yield was almost 25-fold higher than that produced by HEK293 cells. Fluorescent microscopy showed that the insect cell-produced rAAAV could transfer GFP reporter gene into two avian cell types with similar transfer efficiency to that of HEK293 cell-produced rAAAV. These data suggest that insect cell/baculovirus expression system could be used for scalable production of rAAAV, and the viral vector produced could be used as the gene transfer vehicle for avian cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Systemic cancer gene therapy using adeno-associated virus type 1 vector expressing MDA-7/IL24.

    Science.gov (United States)

    Tahara, Ichiro; Miyake, Koichi; Hanawa, Hideki; Kurai, Toshiyuki; Hirai, Yukihiko; Ishizaki, Masamichi; Uchida, Eiji; Tajiri, Takashi; Shimada, Takashi

    2007-10-01

    Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL24), selectively induces apoptosis in cancer cells without harming normal cells. It also exerts immunomodulatory and antiangiogenic effects, as well as potent antitumor bystander effects, making it an ideal candidate for a new anticancer gene therapy. Here, we examined the feasibility of adeno-associated virus type 1 (AAV1) vector-mediated systemic gene therapy using mda-7/IL24. In vitro studies showed that medium conditioned by AAV1-mda7-transducedC2C12 cells induces tumor cell-specific apoptosis and inhibits angiogenesis in a human umbilical vein endothelial cell tube formation assay. To assess the in vivo effects of AAV1-mediated systemic delivery of MDA-7/IL24, we generated a subcutaneous tumor model by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice. A single intravenous injection of AAV1-mda7 (2.0 x 10(11) viral genomes) significantly inhibited tumor growth. In addition, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical analyses showed significant induction of tumor-cell-specific apoptosis and reduction of microvessel formation within the tumors, and there was a significant increase in survival among the AAV1-mda7-treated mice. These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treatment for cancer. Thus, AAV1 vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.

  17. Infectious Clones and Vectors Derived from Adeno-Associated Virus (AAV) Serotypes Other Than AAV Type 2

    OpenAIRE

    Rutledge, Elizabeth A.; Halbert, Christine L.; Russell, David W.

    1998-01-01

    Adeno-associated viruses (AAVs) are single-stranded dependent parvoviruses being developed as transducing vectors. Although at least five serotypes exist (AAV types 1 to 5 [AAV1 to -5]), only AAV2, AAV3, and AAV4 have been sequenced, and the vectors in use were almost all derived from AAV2. Here we report the cloning and sequencing of a second AAV3 genome and a new AAV serotype designated AAV6 that is related to AAV1. AAV2, AAV3, and AAV6 were 82% identical at the nucleotide sequence level, a...

  18. Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Shuohao [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kawabe, Yoshinori; Ito, Akira [Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Kamihira, Masamichi, E-mail: kamihira@chem-eng.kyushu-u.ac.jp [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

  19. Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo.

    Science.gov (United States)

    Shevtsova, Z; Malik, J M I; Michel, U; Bähr, M; Kügler, S

    2005-01-01

    The brain parenchyma consists of several different cell types, such as neurones, astrocytes, microglia, oligodendroglia and epithelial cells, which are morphologically and functionally intermingled in highly complex three-dimensional structures. These different cell types are also present in cultures of brain cells prepared to serve as model systems of CNS physiology. Gene transfer, either in a therapeutic attempt or in basic research, is a fascinating and promising tool to manipulate both the complex physiology of the brain and that of isolated neuronal cells. Viral vectors based on the parvovirus, adeno-associated virus (AAV), have emerged as powerful transgene delivery vehicles. Here we describe highly efficient targeting of AAV vectors to either neurones or astrocytes in cultured primary brain cell cultures. We also show that transcriptional targeting can be achieved by the use of small promoters, significantly boosting the transgene capacity of the recombinant viral genome. However, we also demonstrate that successful targeting of a vector in vitro does not necessarily imply that the same targeting works in the adult brain. Cross-packaging the AAV-2 genome in capsids of other serotypes adds additional benefits to this vector system. In the brain, the serotype-5 capsid allows for drastically increased spread of the recombinant vector as compared to the serotype-2 capsid. Finally, we emphasize the optimal targeting approach, in which the natural tropism of a vector for a specific cell type is employed. Taken together, these data demonstrate the flexibility which AAV-based vector systems offer in physiological research.

  20. Gene therapy with adeno-associated virus vector 5-human factor IX in adults with hemophilia B

    DEFF Research Database (Denmark)

    Miesbach, Wolfgang; Meijer, Karina; Coppens, Michiel

    2018-01-01

    Hemophilia B gene therapy aims to ameliorate bleeding risk and provide endogenous factor IX (FIX) activity/synthesis through a single treatment, eliminating the requirement for FIX concentrate. AMT-060 combines an adeno-associated virus-5 (AAV5) vector with a liver-specific promoter driving...... expression of a codon-optimized wild-type human FIX gene. This multi-national, open-label study included ten adults with hemophilia B (FIX ≤2% of normal) and severe-bleeding phenotype. No participants tested positive for AAV5-neutralizing antibodies using a green-fluorescent protein-based assay and all 10...

  1. A high-capacity, capsid-modified hybrid adenovirus/adeno-associated virus vector for stable transduction of human hematopoietic cells.

    Science.gov (United States)

    Shayakhmetov, Dmitry M; Carlson, Cheryl A; Stecher, Hartmut; Li, Qiliang; Stamatoyannopoulos, George; Lieber, André

    2002-02-01

    To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, DeltaAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The DeltaAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity DeltaAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenovirus vectors. The resultant vector genome contained an 11.6-kb expression cassette including the human gamma-globin gene and the HS2 and HS3 elements of the beta-globin locus control region. The expression cassette was flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs). Infection with DeltaAd5/35.AAV allowed for stable transgene expression in a hematopoietic cell line after integration into the host genome through the AAV ITR(s). This new vector exhibits advantages over existing integrating vectors, including an increased insert capacity and tropism for hematopoietic cells. It has the potential for stable ex vivo transduction of hematopoietic stem cells in order to treat sickle cell disease.

  2. Rational design and engineering of a modified adeno-associated virus (AAV1)-based vector system for enhanced retrograde gene delivery.

    Science.gov (United States)

    Davis, Adam S; Federici, Thais; Ray, William C; Boulis, Nicholas M; OʼConnor, Deirdre; Clark, K Reed; Bartlett, Jeffrey S

    2015-02-01

    After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.

  3. Differential myofiber-type transduction preference of adeno-associated virus serotypes 6 and 9

    NARCIS (Netherlands)

    Riaz, Muhammad; Raz, Yotam; Moloney, E.; van Putten, Maaike; Krom, Yvonne D; van der Maarel, Silvere M; Verhaagen, J.; Raz, Vered

    2015-01-01

    BACKGROUND: Gene therapy strategies are promising therapeutic options for monogenic muscular dystrophies, with several currently underways. The adeno-associated viral (AAV) vector is among the most effective gene delivery systems. However, transduction efficiency in skeletal muscles varies between

  4. Differential myofiber-type transduction preference of adeno-associated virus serotypes 6 and 9

    NARCIS (Netherlands)

    Riaz, M.; Raz, Y.; Moloney, E.B.; Putten, M.; Krom, Y.D.; van der Maarel, S.M.; Verhaagen, J.; Raz, V.

    2015-01-01

    Background: Gene therapy strategies are promising therapeutic options for monogenic muscular dystrophies, with several currently underways. The adeno-associated viral (AAV) vector is among the most effective gene delivery systems. However, transduction efficiency in skeletal muscles varies between

  5. Adeno-associated virus (AAV) vectors in gene therapy: immune challenges and strategies to circumvent them.

    Science.gov (United States)

    Hareendran, Sangeetha; Balakrishnan, Balaji; Sen, Dwaipayan; Kumar, Sanjay; Srivastava, Alok; Jayandharan, Giridhara R

    2013-11-01

    AAV-based gene transfer protocols have shown remarkable success when directed to immune-privileged sites such as for retinal disorders like Lebers congenital amaurosis. In contrast, AAV-mediated gene transfer into liver or muscle tissue for diseases such as hemophilia B, α1 anti-trypsin deficiency and muscular dystrophy has demonstrated a decline in gene transfer efficacy over time. It is now known that in humans, AAV triggers specific pathways that recruit immune sensors. These factors initiate an immediate reaction against either the viral capsid or the vector encoded protein as part of innate immune response or to produce a more specific adaptive response that generates immunological memory. The vector-transduced cells are then rapidly destroyed due to this immune activation. However, unlike other viral vectors, AAV is not immunogenic in murine models. Its immunogenicity becomes apparent only in large animal models and human subjects. Moreover, humans are natural hosts to AAV and exhibit a high seroprevalence against AAV vectors. This limits the widespread application of AAV vectors into patients with pre-existing neutralising antibodies or memory T cells. To address these issues, various strategies are being tested. Alternate serotype vectors (AAV1-10), efficient expression cassettes, specific tissue targeting, immune-suppression and engineered capsid variants are some approaches proposed to minimise this immune stimulation. In this review, we have summarised the nature of the immune response documented against AAV in various pre-clinical and clinical settings and have further discussed the strategies to evade them. Copyright © 2013 John Wiley & Sons, Ltd.

  6. Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes.

    Science.gov (United States)

    Okada, Takashi; Nonaka-Sarukawa, Mutsuko; Uchibori, Ryosuke; Kinoshita, Kazue; Hayashita-Kinoh, Hiromi; Nitahara-Kasahara, Yuko; Takeda, Shin'ichi; Ozawa, Keiya

    2009-09-01

    In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.

  7. Gene Therapy Vectors Based on Adeno-Associated Virus Type 1

    OpenAIRE

    Xiao, Weidong; Chirmule, Narendra; Berta, Scott C.; McCullough, Beth; Gao, Guangping; Wilson, James M.

    1999-01-01

    The complete sequence of adeno-associated virus type 1 (AAV-1) was defined. Its genome of 4,718 nucleotides demonstrates high homology with those of other AAV serotypes, including AAV-6, which appears to have arisen from homologous recombination between AAV-1 and AAV-2. Analysis of sera from nonhuman and human primates for neutralizing antibodies (NAB) against AAV-1 and AAV-2 revealed the following. (i) NAB to AAV-1 are more common than NAB to AAV-2 in nonhuman primates, while the reverse is ...

  8. Germline viral “fossils” guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus

    Science.gov (United States)

    Smith, Richard H.; Hallwirth, Claus V.; Westerman, Michael; Hetherington, Nicola A.; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B.; Koonin, Eugene V.; Agbandje-McKenna, Mavis; Kotin, Robert M.; Alexander, Ian E.

    2016-01-01

    Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV “fossils” provide novel capsid sequences for use in translational research and clinical applications. PMID:27377618

  9. Pre-Clinical Assessment of Immune Responses to Adeno-Associated Virus (AAV) Vectors.

    Science.gov (United States)

    Basner-Tschakarjan, Etiena; Bijjiga, Enoch; Martino, Ashley T

    2014-01-01

    Transitioning to human trials from pre-clinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL) responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity and explored methods for bypassing these responses. Many efforts toward measuring innate immunity have utilized Toll-like receptor deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T-cells as well as cytotoxicity toward AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T-cell proliferation, but actual transgene level reduction and parameters of cytotoxicity toward transduced target cells have only been shown in one model. The model utilized adoptive transfer of capsid-specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells as well as modulating the response pharmacologically has also

  10. Preclinical Assessment of Immune Responses to AAV (adeno-associated virus Vectors

    Directory of Open Access Journals (Sweden)

    Etiena eBasner-Tschakarjan

    2014-02-01

    Full Text Available Transitioning to human trials from preclinical models resulted in the emergence of inhibitory AAV vector immune responses which has become a hurdle for sustained correction. Early animal studies did not predict the full range of host immunity to the AAV vector in human studies. While pre-existing antibody titers against AAV vectors has been a lingering concern, cytotoxic T-cell (CTL responses against the input capsid can prevent long-term therapy in humans. These discoveries spawned more thorough profiling of immune response to rAAV in pre-clinical models, which have assessed both innate and adaptive immunity, and explored methods for bypassing these responses. Many efforts towards measuring innate immunity have utilized Toll-Like Receptor (TLR deficient models and have focused on differential responses to viral capsid and genome. From adaptive studies, it is clear that humoral responses are relevant for initial vector transduction efficiency while cellular responses impact long-term outcomes of gene transfer. Measuring humoral responses to AAV vectors has utilized in vitro neutralizing antibody (NAb assays and transfer of seropositive serum to immunodeficient mice. Overcoming antibodies using CD20 inhibitors, plasmapheresis, altering route of delivery and using different capsids have been explored. CTL responses were measured using in vitro and in vivo models. In in vitro assays expansion of antigen-specific T cells as well as cytotoxicity towards AAV transduced cells can be shown. Many groups have successfully mimicked antigen-specific T cell proliferation, but actual transgene level reduction and parameters of cytotoxicity towards transduced target cells has only been shown in one model. The model utilized adoptive transfer of capsid specific in vitro expanded T-cells isolated from immunized mice with LPS as an adjuvant. Finally, the development of immune tolerance to AAV vectors by enriching regulatory T-cells has also been explored as well

  11. Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing.

    Science.gov (United States)

    Vandenberghe, Luk H; Xiao, Ru; Lock, Martin; Lin, Jianping; Korn, Michael; Wilson, James M

    2010-10-01

    Vectors based on adeno-associated virus (AAV) are the subject of increasing interest as research tools and agents for in vivo gene therapy. A current limitation on the technology is the versatile and scalable manufacturing of vector. On the basis of experience with AAV2-based vectors, which remain strongly cell associated, AAV vector particles are commonly harvested from cell lysates, and must be extensively purified for use. We report here that vectors based on other AAV serotypes, including AAV1, AAV8, and AAV9, are found in abundance in, and can be harvested from, the medium of production cultures carried out with or without serum. For AAV2, this difference in compartmentalization is largely due to the affinity of the AAV2 particle for heparin, because an AAV2 variant in which the heparin-binding motif has been ablated gives higher yields and is efficiently released from cells. Vector particles isolated from the culture medium appear to be functionally equivalent to those purified from cell lysates in terms of transduction efficiency in vitro and in vivo, immunogenicity, and tissue tropism. Our findings will directly lead to methods for increasing vector yields and simplifying production processes for AAV vectors, which should facilitate laboratory-scale preparation and large-scale manufacture.

  12. Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids.

    Directory of Open Access Journals (Sweden)

    Nagesh Pulicherla

    Full Text Available The capsid proteins of adeno-associated viruses (AAV have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.

  13. Efficient Gene Suppression in Dorsal Root Ganglia and Spinal Cord Using Adeno-Associated Virus Vectors Encoding Short-Hairpin RNA.

    Science.gov (United States)

    Enomoto, Mitsuhiro; Hirai, Takashi; Kaburagi, Hidetoshi; Yokota, Takanori

    2016-01-01

    RNA interference is a powerful tool used to induce loss-of-function phenotypes through post-transcriptional gene silencing. Small interfering RNA (siRNA) molecules have been used to target the central nervous system (CNS) and are expected to have clinical utility against refractory neurodegenerative diseases. However, siRNA is characterized by low transduction efficiency, insufficient inhibition of gene expression, and short duration of therapeutic effects, and is thus not ideal for treatment of neural tissues and diseases. To address these problems, viral delivery of short-hairpin RNA (shRNA) expression cassettes that support more efficient and long-lasting transduction into target tissues is expected to be a promising delivery tool. Various types of gene therapy vectors have been developed, such as adenovirus, adeno-associated virus (AAV), herpes simplex virus and lentivirus; however, AAV is particularly advantageous because of its relative lack of immunogenicity and lack of chromosomal integration. In human clinical trials, recombinant AAV vectors are relatively safe and well-tolerated. In particular, serotype 9 of AAV (AAV9) vectors show the highest tropism for neural tissue and can cross the blood-brain barrier, and we have shown that intrathecal delivery of AAV9 yields relatively high gene transduction into dorsal root ganglia or spinal cord. This chapter describes how to successfully use AAV vectors encoding shRNA in vivo, particularly for RNA interference in the central and peripheral nervous system.

  14. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Directory of Open Access Journals (Sweden)

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  15. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Science.gov (United States)

    Balakrishnan, Balaji; Sen, Dwaipayan; Hareendran, Sangeetha; Roshini, Vaani; David, Sachin; Srivastava, Alok; Jayandharan, Giridhara R

    2013-01-01

    The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12-48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  16. Simple downstream process based on detergent treatment improves yield and in vivo transduction efficacy of adeno-associated virus vectors

    Directory of Open Access Journals (Sweden)

    Gabriella Dias Florencio

    2015-01-01

    Full Text Available Recombinant adeno-associated viruses (rAAV are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability.

  17. Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab'gamma)2 antibody.

    Science.gov (United States)

    Bartlett, J S; Kleinschmidt, J; Boucher, R C; Samulski, R J

    1999-02-01

    We have developed a system for the targeted delivery of adeno-associated virus (AAV) vectors. Targeting is achieved via a bispecific F(ab')2 antibody that mediates a novel interaction between the AAV vector and a specific cell surface receptor expressed on human megakaryocytes. Targeted AAV vectors were able to transduce megakaryocyte cell lines, DAMI and MO7e, which were nonpermissive for normal AAV infection, 70-fold above background and at levels equivalent to permissive K562 cells. Transduction was shown to occur through the specific interaction of the AAV vector-bispecific F(ab')2 complex and cell-associated targeting receptor. Importantly, targeting appeared both selective and restrictive as the endogenous tropism of the AAV vector was significantly reduced. Binding and internalization through the alternative receptor did not alter subsequent steps (escape from endosomes, migration to nucleus, or uncoating) required to successfully transduce target cells. These results demonstrate that AAV vectors can be targeted to a specific cell population and that transduction can be achieved by circumventing the normal virus receptor.

  18. Specific and efficient transduction of Cochlear inner hair cells with recombinant adeno-associated virus type 3 vector.

    Science.gov (United States)

    Liu, Yuhe; Okada, Takashi; Sheykholeslami, Kianoush; Shimazaki, Kuniko; Nomoto, Tatsuya; Muramatsu, Shin-Ichi; Kanazawa, Takeharu; Takeuchi, Koichi; Ajalli, Rahim; Mizukami, Hiroaki; Kume, Akihiro; Ichimura, Keiichi; Ozawa, Keiya

    2005-10-01

    Recombinant adeno-associated virus (AAV) vectors are of interest for cochlear gene therapy because of their ability to mediate the efficient transfer and long-term stable expression of therapeutic genes in a wide variety of postmitotic tissues with minimal vector-related cytotoxicity. In the present study, seven AAV serotypes (AAV1-5, 7, 8) were used to construct vectors. The expression of EGFP by the chicken beta-actin promoter associated with the cytomegalovirus immediate-early enhancer in cochlear cells showed that each of these serotypes successfully targets distinct cochlear cell types. In contrast to the other serotypes, the AAV3 vector specifically transduced cochlear inner hair cells with high efficiency in vivo, while the AAV1, 2, 5, 7, and 8 vectors also transduced these and other cell types, including spiral ganglion and spiral ligament cells. There was no loss of cochlear function with respect to evoked auditory brain-stem responses over the range of frequencies tested after the injection of AAV vectors. These findings are of value for further molecular studies of cochlear inner hair cells and for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutations in these cells.

  19. Rescue of avian adeno-associated virus from a recombinant plasmid containing deletions in the viral inverted terminal repeats.

    Science.gov (United States)

    Wang, Jianye; Zhu, Liqian; Zhu, Jun; Zhang, Xinjun; Tao, Jie; Duan, Qiangde; Zhu, Guoqiang

    2012-01-01

    We have previously reported the complete genome sequence of avian adeno-associated virus (AAAV) strain YZ-1, isolated from healthy chickens in China. In this study, we describe the successful rescue of infectious virions from a recombinant plasmid containing the genome of YZ-1 with deletions in the viral inverted terminal repeats (ITRs). The complete genome of YZ-1 was cloned into a bacterial plasmid by a modified "A-T" cloning method. Six recombinant plasmids were selected for further experiments. Sequence analysis indicated that the six clones shared identical internal sequences except for the various deletions within ITRs at either end of the cloned genome. The recombinant plasmid pYZ525, harboring a YZ-1 genome with a 96-nt deletion at the 5' end, was used to transfect CEL or HEK293 cells in the presence of the CELO virus or a helper plasmid, and rescued virions were obtained by both of the methods despite the presence of the deletions. Here, for the first time, we provide evidence that a certain number of nt deletions in the ITRs are not lethal for the rescue of viable AAAV from recombinant plasmids. This study provides insight into the unique biology of AAAV and the mechanism of viral replication.

  20. Non-viral adeno-associated virus-based platform for stable expression of antibody combination therapeutics.

    Science.gov (United States)

    Wilmes, Gwendolyn M; Carey, Kimberly L; Hicks, Stuart W; Russell, Hugh H; Stevenson, Jesse A; Kocjan, Paulina; Lutz, Stephen R; Quesenberry, Rachel S; Shulga-Morskoy, Sergey V; Lewis, Megan E; Clark, Ethan; Medik, Violetta; Cooper, Anthony B; Reczek, Elizabeth E

    2014-01-01

    Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures.

  1. Partial correction of sensitivity to oxidant stress in Friedreich ataxia patient fibroblasts by frataxin-encoding adeno-associated virus and lentivirus vectors.

    Science.gov (United States)

    Fleming, Jane; Spinoulas, Afroditi; Zheng, Maolin; Cunningham, Sharon C; Ginn, Samantha L; McQuilty, Robert C; Rowe, Peter B; Alexander, Ian E

    2005-08-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of a number of debilitating inherited and acquired neurological conditions. The lack of effective treatments for many such conditions provides a strong rationale for exploring novel therapeutic approaches, including gene therapy. Friedreich ataxia (FRDA), a sensory neuropathy, is a progressive neurodegenerative disease associated with a loss of large sensory neurons from the dorsal root ganglia. Because a mouse model for this well-characterized disease has been generated, we elected to use FRDA as a model disease. In previous studies we achieved efficient and sustained delivery of a reporter gene to PNS sensory neurons, using recombinant adeno-associated viral (AAV) and lentiviral (LV) vectors. In the current study, AAV and LV vectors encoding the human frataxin cDNA were constructed and assessed for frataxin expression and function in primary FRDA patient fibroblast cell lines. FRDA fibroblasts have been shown to exhibit subtle biochemical changes, including increased mitochondrial iron and sensitivity to oxidant stress. Despite the inherent difficulty in working with primary cells, transduction of patient fibroblasts with either vector resulted in the expression of appropriately localized frataxin and partial reversal of phenotype.

  2. Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector.

    LENUS (Irish Health Repository)

    Luo, Xiaoyan

    2011-11-01

    Glycogen storage disease type Ia (GSD-Ia) is caused by the deficiency of glucose-6-phosphatase (G6Pase). Long-term complications of GSD-Ia include life-threatening hypoglycemia and proteinuria progressing to renal failure. A double-stranded (ds) adeno-associated virus serotype 2 (AAV2) vector encoding human G6Pase was pseudotyped with four serotypes, AAV2, AAV7, AAV8, and AAV9, and we evaluated efficacy in 12-day-old G6pase (-\\/-) mice. Hypoglycemia during fasting (plasma glucose <100 mg\\/dl) was prevented for >6 months by the dsAAV2\\/7, dsAAV2\\/8, and dsAAV2\\/9 vectors. Prolonged fasting for 8 hours revealed normalization of blood glucose following dsAAV2\\/9 vector administration at the higher dose. The glycogen content of kidney was reduced by >65% with both the dsAAV2\\/7 and dsAAV2\\/9 vectors, and renal glycogen content was stably reduced between 7 and 12 months of age for the dsAAV2\\/9 vector-treated mice. Every vector-treated group had significantly reduced glycogen content in the liver, in comparison with untreated G6pase (-\\/-) mice. G6Pase was expressed in many renal epithelial cells of with the dsAAV2\\/9 vector for up to 12 months. Albuminuria and renal fibrosis were reduced by the dsAAV2\\/9 vector. Hepatorenal correction in G6pase (-\\/-) mice demonstrates the potential of AAV vectors for the correction of inherited diseases of metabolism.

  3. Adeno-associated Virus Vectors Efficiently Transduce Mouse and Rabbit Sensory Neurons Coinfected with Herpes Simplex Virus 1 following Peripheral Inoculation.

    Science.gov (United States)

    Watson, Zachary L; Ertel, Monica K; Lewin, Alfred S; Tuli, Sonal S; Schultz, Gregory S; Neumann, Donna M; Bloom, David C

    2016-09-01

    Following infection of epithelial tissues, herpes simplex virus 1 (HSV-1) virions travel via axonal transport to sensory ganglia and establish a lifelong latent infection within neurons. Recent studies have revealed that, following intraganglionic or intrathecal injection, recombinant adeno-associated virus (rAAV) vectors can also infect sensory neurons and are capable of stable, long-term transgene expression. We sought to determine if application of rAAV to peripheral nerve termini at the epithelial surface would allow rAAV to traffic to sensory ganglia in a manner similar to that seen with HSV. We hypothesized that footpad or ocular inoculation with rAAV8 would result in transduction of dorsal root ganglia (DRG) or trigeminal ganglia (TG), respectively. To test this, we inoculated the footpads of mice with various amounts of rAAV as well as rAAV capsid mutants. We demonstrated that this method of inoculation can achieve a transduction rate of >90% of the sensory neurons in the DRG that innervate the footpad. Similarly, we showed that corneal inoculation with rAAV vectors in the rabbit efficiently transduced >70% of the TG neurons in the optic tract. Finally, we demonstrated that coinfection of mouse footpads or rabbit eyes with rAAV vectors and HSV-1 resulted in colocalization in nearly all of the HSV-1-positive neurons. These results suggest that rAAV is a useful tool for the study of HSV-1 infection and may provide a means to deliver therapeutic cargos for the treatment of HSV infections or of dysfunctions of sensory ganglia. Adeno-associated virus (AAV) has been shown to transduce dorsal root ganglion sensory neurons following direct intraganglionic sciatic nerve injection and intraperitoneal and intravenous injection as well as intrathecal injection. We sought to determine if rAAV vectors would be delivered to the same sensory neurons that herpes simplex virus (HSV-1) infects when applied peripherally at an epithelial surface that had been treated to expose

  4. Gene transfer to the gastrointestinal tract after peroral administration of recombinant adeno-associated virus type 2 vectors.

    Science.gov (United States)

    Shao, Guohong; Greathouse, Kristin; Huang, Qin; Wang, Chiou-Miin; Sferra, Thomas J

    2006-08-01

    The transfer of exogenous genetic material to cells within the gastrointestinal (GI) tract has many potential therapeutic applications. An attractive feature of the GI tract for gene transfer is its accessibility through the orogastric route. In this study, we evaluated the stability of recombinant adeno-associated virus type 2 (rAAV2) vectors within the GI tract and whether rAAV2-mediated gene transfer could be increased through manipulation of the intraluminal environment. The stability of rAAV2 vectors carrying beta-galactosidase and enhanced green fluorescence protein transgenes was determined in the presence of hydrochloric acid, pepsin, trypsin, chymotrypsin gastric fluid and intestinal fluid and after in vivo administration. For in vivo experiments, the rAAV2 vector carrying the beta-galactosidase transgene was administered perorally to FVB/NJ mice. Groups of mice received the vector alone or in combination with sodium bicarbonate and aprotinin. Gene transfer to the stomach and small intestine was evaluated by polymerase chain reaction and histochemical assays. The stability of rAAV2 was reduced by hydrochloric acid, trypsin, chymotrypsin, gastric fluid and intestinal fluid. The vector was not stable within the lumen of the GI tract. Gastric acid neutralization with sodium bicarbonate and protease inhibition with aprotinin increased the in vivo stability of the vector and the level of gene transfer to the stomach and all regions of the small bowel. In both groups of mice (vector alone and vector plus sodium bicarbonate and aprotinin), transgene-derived protein expression (beta-galactosidase) was below the level of detection of the histochemical assay. Recombinant AAV2 are adversely affected by physiological conditions within the proximal GI tract. Gastric acid neutralization and inhibition of intestinal protease activity improved rAAV2 stability and increased the level of gene transfer within the GI tract. Despite these changes, transduction of the GI tract

  5. [Construction of adeno-associated virus vector containing ANG-1 gene and its expression in pig mesenchymal stem cells].

    Science.gov (United States)

    ZHU, Cheng-chu; CHEN, Shi-lin; LIU, Yu-qing; TANG, Li-jiang; BAO, Wei-guang

    2009-07-01

    To construct recombinant adeno-associated virus (rAAV) vector containing angiopoietin-1 (ANG-1) gene and to express the ANG-1 in targeting cells. ANG-1 cDNA was obtained from human spleen by RT-PCR and was inserted into AAV vectors to form rAAV ANG-1, the virus stocks in high titer were harvested. The rAAVANG-1 and rAAV GFP were transferred into pig mesenchymal stem cells and the expression of ANG-1 was detected by Western blot. The cloned ANG-1 cDNA was 1515bp in length which was in accordance with that reported previously. Titration of rAAVANG-1 stock was 9 X 10(11)v.g/ml. The expression of ANG-1 gene was detected in transfected cells. Forty-eight hours after rAAV GFP was transfected into mesenchymal stem cells, 55% cells expressed GFP. The constructed rAAV ANG-1 vector has successfully transfered and expressed in pig mesenchymal stem cells.

  6. Enhanced selective gene delivery to neural stem cells in vivo by an adeno-associated viral variant.

    Science.gov (United States)

    Kotterman, Melissa A; Vazin, Tandis; Schaffer, David V

    2015-05-15

    Neural stem cells (NSCs) are defined by their ability to self-renew and to differentiate into mature neuronal and glial cell types. NSCs are the subject of intense investigation, owing to their crucial roles in neural development and adult brain function and because they present potential targets for gene and cell replacement therapies following injury or disease. Approaches to specifically genetically perturb or modulate NSC function would be valuable for either motivation. Unfortunately, most gene delivery vectors are incapable of efficient or specific gene delivery to NSCs in vivo. Vectors based on adeno-associated virus (AAV) present a number of advantages and have proven increasingly successful in clinical trials. However, natural AAV variants are inefficient in transducing NSCs. We previously engineered a novel AAV variant (AAV r3.45) capable of efficient transduction of adult NSCs in vitro. Here, to build upon the initial promise of this variant, we investigated its in vitro and in vivo infectivity. AAV r3.45 was more selective for NSCs than mature neurons in a human embryonic stem cell-derived culture containing a mixture of cell types, including NSCs and neurons. It was capable of more efficient and selective transduction of rat and mouse NSCs in vivo than natural AAV serotypes following intracranial vector administration. Delivery of constitutively active β-catenin yielded insights into mechanisms by which this key regulator modulates NSC function, indicating that this engineered AAV variant can be harnessed for preferential modulation of adult NSCs in the hippocampus. The capacity to rapidly genetically modify these cells might greatly accelerate in vivo investigations of adult neurogenesis. © 2015. Published by The Company of Biologists Ltd.

  7. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors.

    Science.gov (United States)

    Gray, Steven J; Foti, Stacey B; Schwartz, Joel W; Bachaboina, Lavanya; Taylor-Blake, Bonnie; Coleman, Jennifer; Ehlers, Michael D; Zylka, Mark J; McCown, Thomas J; Samulski, R Jude

    2011-09-01

    With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken β-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.

  8. A NEW RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV)-BASED RANDOM PEPTIDE DISPLAY LIBRARY SYSTEM: INFECTION-DEFECTIVE AAV1.9-3 AS A NOVEL DETARGETED PLATFORM FOR VECTOR EVOLUTION*

    OpenAIRE

    Adachi, Kei; Nakai, Hiroyuki

    2010-01-01

    Directed evolution through genetic engineering of viral capsids followed by selection has emerged as a powerful means to create novel recombinant adeno-associated virus (rAAV) vectors with desired tropism and enhanced properties. One of the most effective approaches uses rAAV-based random peptide display libraries. Here we report a novel system based on an infection-defective rAAV1.9-3 as a platform for random peptide display, and show that biopanning of the libraries in vitro effectively ide...

  9. Improved adeno-associated virus (AAV) serotype 1 and 5 vectors for gene therapy

    Science.gov (United States)

    Sen, Dwaipayan; Balakrishnan, Balaji; Gabriel, Nishanth; Agrawal, Prachi; Roshini, Vaani; Samuel, Rekha; Srivastava, Alok; Jayandharan, Giridhara R.

    2013-01-01

    Despite significant advancements with recombinant AAV2 or AAV8 vectors for liver directed gene therapy in humans, it is well-recognized that host and vector-related immune challenges need to be overcome for long-term gene transfer. To overcome these limitations, alternate AAV serotypes (1–10) are being rigorously evaluated. AAV5 is the most divergent (55% similarity vs. other serotypes) and like AAV1 vector is known to transduce liver efficiently. AAV1 and AAV5 vectors are also immunologically distinct by virtue of their low seroprevalence and minimal cross reactivity against pre-existing AAV2 neutralizing antibodies. Here, we demonstrate that targeted bio-engineering of these vectors, augment their gene expression in murine hepatocytes in vivo (up to 16-fold). These studies demonstrate the feasibility of the use of these novel AAV1 and AAV5 vectors for potential gene therapy of diseases like hemophilia. PMID:23665951

  10. Gene Transfer of Heme Oxygenase-1 Using an Adeno-Associated Virus Serotype 6 Vector Prolongs Cardiac Allograft Survival

    Directory of Open Access Journals (Sweden)

    Jacqueline M. Evans

    2012-01-01

    Full Text Available Introduction. Allograft survival can be prolonged by overexpression of cytoprotective genes such as heme oxygenase-1 (HO-1. Modifications in vector design and delivery have provided new opportunities to safely and effectively administer HO-1 into the heart prior to transplantation to improve long-term graft outcome. Methods. HO-1 was delivered to the donor heart using an adeno-associated virus vector (AAV with a pseudotype 6 capsid and vascular endothelial growth factor (VEGF to enhance myocardial tropism and microvascular permeability. Survival of mouse cardiac allografts, fully or partially mismatched at the MHC, was determined with and without cyclosporine A. Intragraft cytokine gene expression was examined by PCR. Results. The use of AAV6 to deliver HO-1 to the donor heart, combined with immunosuppression, prolonged allograft survival by 55.3% when donor and recipient were completely mismatched at the MHC and by 94.6% if partially mismatched. The combination of gene therapy and immunosuppression was more beneficial than treatment with either AAV6-HO-1 or CsA alone. IL-17a, b, e and f were induced in the heart at rejection. Conclusions. Pretreatment of cardiac allografts with AAV6-HO-1 plus cyclosporine A prolonged graft survival. HO-1 gene therapy represents a beneficial adjunct to immunosuppressive therapy in cardiac transplantation.

  11. Attenuation of vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon vector

    Science.gov (United States)

    Wollmann, Guido; Paglino, Justin C.; Maloney, Patrick R; Ahmadi, Sebastian A; van den Pol, Anthony N

    2015-01-01

    Vesicular stomatitis virus (VSV) shows promise as vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-β-D-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-β protected mouse brain cells from VSV, as did a combination of ribavirin and chloroquine. Intracranial AAV-mIFN-β protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-β moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-β; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain. PMID:25462341

  12. Attenuation of vesicular stomatitis virus infection of brain using antiviral drugs and an adeno-associated virus-interferon vector.

    Science.gov (United States)

    Wollmann, Guido; Paglino, Justin C; Maloney, Patrick R; Ahmadi, Sebastian A; van den Pol, Anthony N

    2015-01-15

    Vesicular stomatitis virus (VSV) shows promise as a vaccine-vector and oncolytic virus. However, reports of neurotoxicity of VSV remain a concern. We compared 12 antiviral compounds to control infection of VSV-CT9-M51 and VSV-rp30 using murine and human brain cultures, and in vivo mouse models. Inhibition of replication, cytotoxicity and infectivity was strongest with ribavirin and IFN-α and to some extent with mycophenolic acid, chloroquine, and adenine 9-β-d-arabinofuranoside. To generate continuous IFN exposure, we made an adeno-associated virus vector expressing murine IFN; AAV-mIFN-β protected mouse brain cells from VSV, as did a combination of IFN, ribavirin and chloroquine. Intracranial AAV-mIFN-β protected the brain against VSV-CT9-M51. In SCID mice bearing human glioblastoma, AAV-mIFN-β moderately enhanced survival. VSV-CT9-M51 doubled median survival when administered after AAV-mIFN-β; some surviving mice showed complete tumor destruction. Together, these data suggest that AAV-IFN or IFN with ribavirin and chloroquine provide an optimal anti-virus combination against VSV in the brain. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Basic fibroblast growth factor enhances transduction, distribution, and axonal transport of adeno-associated virus type 2 vector in rat brain.

    Science.gov (United States)

    Hadaczek, Piotr; Mirek, Hanna; Bringas, John; Cunningham, Janet; Bankiewicz, Krys

    2004-05-01

    The ubiquitous expression of cell surface heparan sulfate proteoglycan, a binding receptor for adeno-associated virus type 2 (AAV-2), may account for the broad host range of this vector. Because the fibroblast growth factor receptor type 1 has been postulated to be a coreceptor for successful AAV-2 entry into host cells, we designed a strategy to investigate whether coadministration of this virus with basic fibroblast growth factor (bFGF) can enhance AAV-2-mediated gene delivery. We injected AAV-2-thymidine kinase (AAV-2-TK) vector into rat striata and checked whether coinjection with bFGF enhanced transduction and/or enlarged the area of transgene expression. Immunostaining confirmed the tropism of AAV-2-TK for neurons. The previous injection (7 days before vector delivery) of bFGF had no major impact on vector distribution area. However, when the vector was coinjected with bFGF, the right striatum showed an average viral transduction volume of 5 mm(3), which was more than 4-fold larger when compared with the left side (AAV-2-TK plus phosphate-buffered saline). This result clearly indicates that simultaneous injection of bFGF with AAV-2-TK can greatly enhance the volume of transduced tissue, probably by way of a competitive block of AAV-2-binding sites within the striatum. Robust TK immunoreactivity was also observed in the globus pallidus, which receives anterograde projections from the striatum. We propose that postsynaptic transport of recombinant particles was likely responsible for the distribution of TK in the globus pallidus on both bFGF-treated and untreated sides. In summary, we found that bFGF acts as an adjuvant for distribution of AAV-2 in rat brain.

  14. Long-term sex-biased correction of circulating propionic acidemia disease markers by adeno-associated virus vectors.

    Science.gov (United States)

    Guenzel, Adam J; Collard, Renata; Kraus, Jan P; Matern, Dietrich; Barry, Michael A

    2015-03-01

    Propionic academia (PA) occurs because of mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase, a pivotal enzyme in the breakdown of certain amino acids and odd-chain fatty acids. There is no cure for PA, but dietary protein restriction and liver transplantation can attenuate its symptoms. We show here that a single intravenous injection of adeno-associated virus 2/8 (AAV8) or AAVrh10 expressing PCCA into PA hypomorphic mice decreased systemic propionylcarnitine and methyl citrate for up to 1.5 years. However, long-term phenotypic correction was always better in male mice. AAV-mediated PCCA expression was similar in most tissues in males and females at early time points and differed only in the liver. Over 1.5 years, luciferase and PCCA expression remained elevated in cardiac tissue for both sexes. In contrast, transgene expression in the liver and skeletal muscles of female, but not male, mice waned—suggesting that these tissues were major sinks for systemic phenotypic correction. These data indicate that single systemic intravenous therapy by AAV vectors can mediate long-term phenotype correction for PA. However, tissue-specific loss of expression in females reduces efficacy when compared with males. Whether similar sex-biased AAV effects occur in human gene therapy remains to be determined.

  15. HBV genes induce cytotoxic T-lymphocyte response upon adeno-associated virus (AAV) vector delivery into dendritic cells.

    Science.gov (United States)

    You, H; Liu, Y; Cong, M; Ping, W; You, C; Zhang, D; Mehta, J L; Hermonat, P L

    2006-09-01

    Hepatitis B virus (HBV) has been an increasing problem throughout the world and remains difficult to treat. But immunotherapeutic approaches offer new, effective treatments. Three recombinant adeno-associated virus (AAV) type 2 vectors, carrying one of the HBV S, C or X gene, were used to load (transduce) professional antigen-presenting dendritic cells (DC) for the purpose of stimulating cytotoxic T lymphocytes (CTL) in vitro. It was found that all three recombinant AAV/HBV antigen virus loaded DC at approximately 90% transduction efficiency. Most importantly, all three AAV-loaded DC stimulated rapid, antigen-specific and major histocompatibility complex (MHC)-restricted CTL. In vitro, these CTL killed (30-50%) synthetic antigen-positive autologous targets as well as HepG2 liver cell targets. In comparing the three antigens, it was found that AAV/HBV-C-derived CTL consistently had the highest killing efficiency. CTL derived from AAV/HBV-C-loaded DC also showed significantly higher killing of targets than that from bacterially generated C-protein-loaded DC. Further studies showed that AAV/HBV-C-derived CTL had higher interferon (IFN)-gamma. These data suggest that AAV/HBV antigen gene-loading of DC may be useful for immunotherapeutic protocols against HBV infection and that the HBV C antigen may be the most useful for this purpose.

  16. Transduction of nonhuman primate brain with adeno-associated virus serotype 1: vector trafficking and immune response.

    Science.gov (United States)

    Hadaczek, Piotr; Forsayeth, John; Mirek, Hanna; Munson, Keith; Bringas, John; Pivirotto, Phil; McBride, Jodi L; Davidson, Beverly L; Bankiewicz, Krystof S

    2009-03-01

    We used convection-enhanced delivery (CED) to characterize gene delivery mediated by adeno-associated virus type 1 (AAV1) by tracking expression of hrGFP (humanized green fluorescent protein from Renilla reniformis) into the striatum, basal forebrain, and corona radiata of monkey brain. Four cynomolgus monkeys received single infusions into corona radiata, putamen, and caudate. The other group (n = 4) received infusions into basal forebrain. Thirty days after infusion animals were killed and their brains were processed for immunohistochemical evaluation. Volumetric analysis of GFP-positive brain areas was performed. AAV1-hrGFP infusions resulted in approximately 550, 700, and 73 mm(3) coverage after infusion into corona radiata, striatum, and basal forebrain, respectively. Aside from targeted regions, other brain structures also showed GFP signal (internal and external globus pallidus, subthalamic nucleus), supporting the idea that AAV1 is actively trafficked to regions distal from the infusion site. In addition to neuronal transduction, a significant nonneuronal cell population was transduced by AAV1 vector; for example, oligodendrocytes in corona radiata and astrocytes in the striatum. We observed a strong humoral and cell-mediated response against AAV1-hrGFP in transduced monkeys irrespective of the anatomic location of the infusion, as evidenced by induction of circulating anti-AAV1 and anti-hrGFP antibodies, as well as infiltration of CD4(+) lymphocytes and upregulation of MHC-II in regions infused with vector. We conclude that transduction of antigen-presenting cells within the CNS is a likely cause of this response and that caution is warranted when foreign transgenes are used as reporters in gene therapy studies with vectors with broader tropism than AAV2.

  17. The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

    Science.gov (United States)

    Cao, Maohua; You, Hong; Hermonat, Paul L

    2014-01-01

    Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  18. Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

    Science.gov (United States)

    Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

    2017-10-15

    The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus

  19. Combined prophylactic and therapeutic intranasal vaccination against human papillomavirus type-16 using different adeno-associated virus serotype vectors.

    Science.gov (United States)

    Nieto, Karen; Kern, Andrea; Leuchs, Barbara; Gissmann, Lutz; Müller, Martin; Kleinschmidt, Jürgen A

    2009-01-01

    Cervical cancer is the second most frequent cancer among woman worldwide and is considered to be caused by infection with high-risk papilloma viruses. Genetic immunization using recombinant adeno-associated virus (rAAV) vectors has shown great promise for vaccination against human papillomavirus (HPV) infections. rAAV5, -8 and -9 vectors expressing an HPV16 L1/E7 fusion gene were generated and applied intranasally for combined prophylactic and therapeutic vaccination of mice. The rAAV5 and the rAAV9 vectors showed efficient induction of both humoral and cellular immune responses, whereas rAAV8 failed to immunize mice by the intranasal route. The L1-specific immune response evoked by expression of the L1/E7 fusion gene, however, was lower than that evoked by expression of the L1 antigen alone. This deficiency could be compensated by application of Escherichia coli heat-labile enterotoxin or monophsphoryl lipid as adjuvant upon vaccination with rAAV5-L1/E7. Coimmunization of rAAV9-L1/E7 with rAAV5-L1 or boosting of rAAV9-L1/E7 with rAAV5-L1 strongly increased L1-specific neutralizing antibody titres to levels above those achieved by vaccination with vectors expressing L1 alone. Both vectors elicited a vibrant cytotoxic T-lymphocyte response against L1 or E7. Nasal immunization with rAAV5 or rAAV9 was superior to vaccination with HPV16-L1 virus-like particles (VLPs) or HPV16-L1/E7 CVLPs with respect to humoral and cellular immune responses. Vaccination with the rAAV vectors led to a significant protection of animals against a challenge with different HPV tumour cell lines. Our results show that rAAV5 and rAAV9 vectors are promising candidates for a non-invasive nasal vaccination strategy.

  20. Avian adeno-associated parvovirus and Marek's disease virus: studies of viral interactions in chicken embryo fibroblasts. Brief report.

    Science.gov (United States)

    Bauer, H J; Monreal, G

    1988-01-01

    Using growth kinetics we demonstrate two effects based on interactions between the chicken herpesvirus, Marek's disease virus (MDV), and the dependovirus, avian adeno-associated virus (AAAV), in coinfected chicken embryo fibroblasts (CEF): (i) MDV provides helper activity for an efficient multiplication of AAAV; (ii) a high multiplicity of coinfecting AAAV inhibits completely the growth of MDV as well as AAAV.

  1. Recombinant adeno-associated virus vector carrying the thrombomodulin lectin-like domain for the treatment of abdominal aortic aneurysm.

    Science.gov (United States)

    Lai, Chao-Han; Wang, Kuan-Chieh; Kuo, Cheng-Hsiang; Lee, Fang-Tzu; Cheng, Tsung-Lin; Chang, Bi-Ing; Yang, Yu-Jen; Shi, Guey-Yueh; Wu, Hua-Lin

    2017-07-01

    Thrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl2-induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. The therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl2-induced AAA model and angiotensin II-infused AAA model, respectively. In the CaCl2-induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (10(11) genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. These findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Recombinant adeno-associated virus type 2, 4, and 5 vectors: Transduction of variant cell types and regions in the mammalian central nervous system

    OpenAIRE

    Davidson, Beverly L.; Stein, Colleen S.; Heth, Jason A.; Martins, Inês; Kotin, Robert M; Derksen, Todd A.; Zabner, Joseph; Ghodsi, Abdi; Chiorini, John A.

    2000-01-01

    Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in the central nervous system (CNS), but it is not known how other rAAV serotypes perform as CNS gene transfer vectors. Serotypes 4 and 5 are distinct from rAAV2 and from each other in their capsid regions, suggesting that they may direct binding and entry into different cell types. In this study, we examined the tropisms and transduction efficiencies of β-galactosidase-encoding vectors made...

  3. Self-complementary adeno-associated virus 2 (AAV)-T cell protein tyrosine phosphatase vectors as helper viruses to improve transduction efficiency of conventional single-stranded AAV vectors in vitro and in vivo.

    Science.gov (United States)

    Zhong, Li; Chen, Linyuan; Li, Yanjun; Qing, Keyun; Weigel-Kelley, Kirsten A; Chan, Rebecca J; Yoder, Mervin C; Srivastava, Arun

    2004-11-01

    Recombinant vectors based on adeno-associated virus type 2 (AAV) target the liver efficiently, but the transgene expression is limited to approximately 5% of hepatocytes. The lack of efficient transduction is due, in part, to the presence of a cellular protein, FKBP52, phosphorylated forms of which inhibit the viral second-strand DNA synthesis. We have documented that dephosphorylation of FKBP52 at tyrosine residues by the cellular T cell protein tyrosine phosphatase (TC-PTP) enhances AAV-mediated transduction in primary murine hematopoietic cells from TC-PTP-transgenic mice. We have also documented that AAV-mediated transduction is significantly enhanced in hepatocytes in TC-PTP-transgenic as well as in FKBP52-deficient mice because of efficient viral second-strand DNA synthesis. In this study, we evaluated whether co-infection of conventional single-stranded AAV vectors with self-complementary AAV-TC-PTP vectors leads to increased transduction efficiency of conventional AAV vectors in established human cell lines in vitro and in primary murine hepatocytes in vivo. We demonstrate here that scAAV-TC-PTP vectors serve as a helper virus in augmenting the transduction efficiency of conventional AAV vectors in vitro as well as in vivo which correlates directly with the extent of second-strand DNA synthesis of conventional single-stranded AAV vectors. Toxicological studies following tail-vein injections of scAAV-TC-PTP vectors in experimental mice show no evidence of any adverse effect in any of the organs in any of the mice for up to 13 weeks. Thus, this novel co-infection strategy should be useful in circumventing one of the major obstacles in the optimal use of recombinant AAV vectors in human gene therapy.

  4. Adeno-associated virus vector-mediated delivery of pigment epithelium-derived factor restricts neuroblastoma angiogenesis and growth.

    Science.gov (United States)

    Streck, Christian J; Zhang, Youbin; Zhou, Junfang; Ng, Catherine; Nathwani, Amit C; Davidoff, Andrew M

    2005-01-01

    The purpose of this study was to evaluate the ability of adeno-associated virus (AAV) vector-mediated delivery of pigment epithelium-derived factor (PEDF) to inhibit neuroblastoma (NB) xenograft growth. Pigment epithelium-derived factor was chosen for this study because, in addition to being a potent inhibitor of angiogenesis, it is capable of inducing neuronal differentiation. Cohorts of mice received either recombinant AAV encoding human PEDF (rAAV-hPEDF) at a range of doses or control vector via tail vein. Subsequent hPEDF expression was measured by enzyme-linked immunoassay. After 6 weeks, the mice were given human NB cells by retroperitoneal injection and then killed 5 weeks later. Tumor weight, microvessel density, tumor differentiation, apoptosis, and levels of intratumoral vascular endothelial growth factor (VEGF) expression were determined at that time. In subsequent cohorts of mice, AAV-mediated murine PEDF expression was tested against both human NB xenografts and murine tumors. In a series of in vitro studies, PEDF was shown to inhibit endothelial cell activation and to stimulate differentiation of NB cell lines. After tail vein injection of rAAV-hPEDF, stable transgene expression was generated and correlated with levels of vector administration. Human NB xenograft growth was restricted by hPEDF in a dose-dependent fashion. Intratumoral VEGF expression and microvessel density were decreased, and tumor cell apoptosis was increased in PEDF-treated mice. Treatment with PEDF had a significant impact on NB growth in mice when delivered continuously using a gene therapy-mediated approach. The activity of PEDF appears to be mediated in part by inhibition of tumor-induced angiogenesis through down-regulation of tumor-elaborated VEGF, with subsequent intratumoral apoptosis. Furthermore, hPEDF was able to induce NB differentiation in vitro and in vivo. In addition, antitumor efficacy was seen when mouse PEDF was used to treat syngeneic murine tumors. In our

  5. Adeno-Associated Virus Type 2 (AAV2) Capsid-Specific Cytotoxic T Lymphocytes Eliminate Only Vector-Transduced Cells Coexpressing the AAV2 Capsid In Vivo▿

    OpenAIRE

    Li, Chengwen; Hirsch, Matthew; Asokan, Aravind; Zeithaml, Brian; Ma, Hong; Kafri, Tal; Samulski, R. Jude

    2007-01-01

    A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kil...

  6. Adeno-associated virus for cystic fibrosis gene therapy

    Directory of Open Access Journals (Sweden)

    S.V. Martini

    2011-11-01

    Full Text Available Gene therapy is an alternative treatment for genetic lung disease, especially monogenic disorders such as cystic fibrosis. Cystic fibrosis is a severe autosomal recessive disease affecting one in 2500 live births in the white population, caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR. The disease is classically characterized by pancreatic enzyme insufficiency, an increased concentration of chloride in sweat, and varying severity of chronic obstructive lung disease. Currently, the greatest challenge for gene therapy is finding an ideal vector to deliver the transgene (CFTR to the affected organ (lung. Adeno-associated virus is the most promising viral vector system for the treatment of respiratory disease because it has natural tropism for airway epithelial cells and does not cause any human disease. This review focuses on the basic properties of adeno-associated virus and its use as a vector for cystic fibrosis gene therapy.

  7. Polyinosinic Acid Blocks Adeno-Associated Virus Macrophage Endocytosis In Vitro and Enhances Adeno-Associated Virus Liver-Directed Gene Therapy In Vivo

    NARCIS (Netherlands)

    van Dijk, Remco; Montenegro-Miranda, Paula S.; Riviere, Christel; Schilderink, Ronald; ten Bloemendaal, Lysbeth; van Gorp, Jacqueline; Duijst, Suzanne; de Waart, Dirk R.; Beuers, Ulrich; Haisma, Hidde J.; Bosma, Piter J.

    2013-01-01

    Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase

  8. Detection of adeno-associated virus in human semen: does viral infection play a role in the pathogenesis of male infertility?

    Science.gov (United States)

    Rohde, V; Erles, K; Sattler, H P; Derouet, H; Wullich, B; Schlehofer, J R

    1999-11-01

    To evaluate the occurrence of adeno-associated virus (AAV) DNA and/or human papillomavirus (HPV) DNA in the semen of infertile men as a possible factor in the pathogenesis of male infertility. Descriptive pilot study. University-based diagnostic and research laboratory. Semen specimens were collected from 30 men with diagnosed infertility and from 8 control subjects. Diagnostic spermiograms were made and the semen specimens were separated into seminal fluid, nonspermatozoal cells, and spermatozoa using a Ficoll gradient technique. The presence of AAV and HPV DNA in the different fractions of the ejaculates from the infertile men and the control subjects was detected by polymerase chain reaction. Semen quality was analyzed according to World Health Organization guidelines. Adeno-associated virus DNA was detected in 30% (9/30) of the ejaculates from the infertile men. No AAV DNA was found in the ejaculates from the 8 control subjects. In 8 of 9 samples, AAV DNA could be found only in the spermatozoal fraction of the specimen. Seven of 9 semen specimens that contained viral DNA also demonstrated oligoasthenozoospermia. Both AAV and HPV DNA was found in the spermatozoal fraction of 3 of 30 specimens. The data demonstrate for the first time the occurrence of AAV infection in human semen. Sperm motility seems to be affected by the presence of AAV.

  9. Engineering adeno-associated viruses for clinical gene therapy.

    Science.gov (United States)

    Kotterman, Melissa A; Schaffer, David V

    2014-07-01

    Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

  10. Prevention of dopaminergic neuron death by adeno-associated virus vector-mediated GDNF gene transfer in rat mesencephalic cells in vitro.

    Science.gov (United States)

    Fan, D; Ogawa, M; Ikeguchi, K; Fujimoto, K; Urabe, M; Kume, A; Nishizawa, M; Matsushita, N; Kiuchi, K; Ichinose, H; Nagatsu, T; Kurtzman, G J; Nakano, I; Ozawa, K

    1998-05-22

    Glial cell line-derived neurotrophic factor (GDNF) is known as a potent neurotrophic factor for dopaminergic neurons. Since adeno-associated virus (AAV) vector is a suitable vehicle for gene transfer into neurons, rat E14 mesencephalic cells were transduced with an AAV vector expressing GDNF. When compared with mock transduction, a larger number of dopaminergic neurons survived in AAV-GDNF-transduced cultures (234% and 325% of controls at 1 and 2 weeks, respectively; P neurons in the latter cultures grew more prominent neurites than those in the former. These findings suggest that AAV vector-mediated GDNF gene transfer may prevent dopaminergic neuron death, and is therefore a logical approach for the treatment of Parkinson's disease.

  11. Novel Vector Design and Hexosaminidase Variant Enabling Self-Complementary Adeno-Associated Virus for the Treatment of Tay-Sachs Disease.

    Science.gov (United States)

    Karumuthil-Melethil, Subha; Nagabhushan Kalburgi, Sahana; Thompson, Patrick; Tropak, Michael; Kaytor, Michael D; Keimel, John G; Mark, Brian L; Mahuran, Don; Walia, Jagdeep S; Gray, Steven J

    2016-07-01

    GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (α-β), "A" isoenzyme of lysosomal β-hexosaminidase (HexA). Mutations in the α-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA α-subunit, μ, incorporating critical sequences from the β-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP- GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter-intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the nervous system.

  12. Avian adeno-associated virus (AAAV) and fowl adenoviruses (FAV): studies of viral interactions in chicken cell cultures.

    Science.gov (United States)

    Bauer, H J; Hauschild, S; Logemann, K; Hehlein, K; Monreal, G

    1986-01-01

    As the growth kinetics of avian adeno-associated virus (AAAV) in chicken cells demonstrate, the three serotypes of fowl adenovirus (FAV), FAV -1, -5 and -8, provide complete helper activity for the production of infectious AAAV. Under one step conditions, the growth cycle of AAAV in primary chicken kidney cell (CKC) cultures is characterised by an eclipse phase of 8 hours and an exponential increase of the virus infectivity by 4 to 5 logs until 24 hours post-adsorption (p.a.). These growth characteristics do not depend on the serotype of FAV used as helper. In chicken embryo fibroblast (CEF) cultures the eclipse phase is prolonged to 12 hours p.a. and the virus infectivity increases only by 2 logs. In addition, the low efficiency of plating of FAV -1 in this cell system does not allow one step growth curves for AAAV. In CKC and CEF cultures coinfected with FAV and AAAV the multiplication of helper FAV is reduced. The degree of growth inhibition depends on the AAV multiplicity used. Sequential infection of CKC cultures with FAV -1 and AAAV modifies the AAAV growth cycle, i.e. there is a time reduction of the eclipse phase and a decrease of the virus yield. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious FAV by a plaque assay.

  13. Methods of treating Parkinson's disease using viral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Bankiewicz, Krystof; Cunningham, Janet

    2016-11-15

    Methods of delivering viral vectors, particularly recombinant adeno-associated virus (rAAV) virions, to the central nervous system (CNS) using convection enhanced delivery (CED) are provided. The rAAV virions include a nucleic acid sequence encoding a therapeutic polypeptide. The methods can be used for treating CNS disorders such as for treating Parkinson's Disease.

  14. Adeno-associated virus and lentivirus vectors mediate efficient and sustained transduction of cultured mouse and human dorsal root ganglia sensory neurons.

    Science.gov (United States)

    Fleming, J; Ginn, S L; Weinberger, R P; Trahair, T N; Smythe, J A; Alexander, I E

    2001-01-01

    Peripheral nervous system (PNS) sensory neurons are directly involved in the pathophysiology of numerous inherited and acquired neurological conditions. Therefore, efficient and stable gene delivery to these postmitotic cells has significant therapeutic potential. Among contemporary vector systems capable of neuronal transduction, only those based on herpes simplex virus have been extensively evaluated in PNS neurons. We therefore investigated the transduction performance of recombinant adeno-associated virus type 2 (AAV) and VSV-G-pseudotyped lentivirus vectors derived from human immunodeficiency virus (HIV-1) in newborn mouse and fetal human dorsal root ganglia (DRG) sensory neurons. In dissociated mouse DRG cultures both vectors achieved efficient transduction of sensory neurons at low multiplicities of infection (MOIs) and sustained transgene expression within a 28-day culture period. Interestingly, the lentivirus vector selectively transduced neurons in murine cultures, in contrast to human cultures, in which Schwann and fibroblast-like cells were also transduced. Recombinant AAV transduced all three cell types in both mouse and human cultures. After direct microinjection of murine DRG explants, maximal transduction efficiencies of 20 and 200 transducing units per neuronal transductant were achieved with AAV and lentivirus vectors, respectively. Most importantly, both vectors achieved efficient and sustained transduction of human sensory neurons in dissociated cultures, thereby directly demonstrating the exciting potential of these vectors for gene therapy applications in the PNS.

  15. Distinct transduction difference between adeno-associated virus type 1 and type 6 vectors in human polarized airway epithelia.

    Science.gov (United States)

    Yan, Z; Lei-Butters, D C M; Keiser, N W; Engelhardt, J F

    2013-03-01

    Of the many biologically isolated adeno-associated virus (AAV) serotypes, AAV1 and AAV6 share the highest degree of sequence homology, with only six different capsid residues. We compared the transduction efficiencies of rAAV1 and rAAV6 in primary polarized human airway epithelia and found significant differences in their abilities to transduce epithelia from the apical and basolateral membranes. rAAV1 transduction was ~10-fold higher than rAAV6 following apical infection, whereas rAAV6 transduction was ~10-fold higher than rAAV1 following basolateral infection. Furthermore, rAAV6 demonstrated significant polarity of transduction (100-fold; basolateral » apical), whereas rAAV1 transduced from both membranes with equal efficiency. To evaluate capsid residues responsible for the observed serotype differences, we mutated the six divergent amino acids either alone or in combination. Results from these studies demonstrated that capsid residues 418 and 531 most significantly controlled membrane polarity differences in transduction between serotypes, with the rAAV6-D418E/K531E mutant demonstrating decreased (~10-fold) basolateral transduction and the rAAV1-E418D/E531K mutant demonstrating a transduction polarity identical to rAAV6-WT (wild type). However, none of the rAAV6 mutants obtained apical transduction efficiencies of rAAV1-WT, suggesting that all six divergent capsid residues in AAV1 act in concert to improve apical transduction of HAE.

  16. Recombinant self-complementary adeno-associated virus serotype vector-mediated hematopoietic stem cell transduction and lineage-restricted, long-term transgene expression in a murine serial bone marrow transplantation model.

    Science.gov (United States)

    Maina, Njeri; Han, Zongchao; Li, Xiaomiao; Hu, Zhongbo; Zhong, Li; Bischof, Daniela; Weigel-Van Aken, Kirsten A; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

    2008-04-01

    Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.

  17. Long-term protection against human papillomavirus e7-positive tumor by a single vaccination of adeno-associated virus vectors encoding a fusion protein of inactivated e7 of human papillomavirus 16/18 and heat shock protein 70.

    Science.gov (United States)

    Zhou, Liqiao; Zhu, Tong; Ye, Xiaojing; Yang, Lin; Wang, Bing; Liang, Xiaoyan; Lu, Lina; Tsao, Yeou-Ping; Chen, Show-Li; Li, Juan; Xiao, Xiao

    2010-01-01

    We investigated a gene vaccine strategy against human papillomavirus (HPV)-induced cancer and premalignant diseases, using adeno-associated virus (AAV) vector encoding the viral E7 oncoproteins as the tumor antigens from HPV serotypes 16 (HPV16) and 18 (HPV18). Genetically inactivated E7 proteins were fused with a heat shock protein 70 (hsp70) to minimize the risk of cell transformation and enhance immune responses. The fusion protein gene was packaged in AAV serotype 1 or 2 (AAV1 or 2) for efficient in vivo gene expression. Our results showed that after a single intramuscular injection, the AAV1 vector elicited stronger HPV-specific cytotoxic T lymphocyte (CTL) responses and interferon-gamma secretion when compared with the AAV2 vector. Prophylactic immunization with AAV1 protected 100% of the mice from tumor growth for more than 1 year, whereas all the control mice immunized with either a LacZ vector or saline grew large tumors and died within 6 weeks after inoculation of E7-positive tumor cell line TC-1. In addition, this single-dose AAV1 vaccination completely protected the mice against second and third challenges with higher numbers of TC-1 cells. Despite lower CTL responses against the E7 antigens, AAV2 vector prophylactic immunization was also sufficient to protect 100% of the mice against the initial and second tumor challenges and 70% of the mice against the third challenge. In addition, therapeutic immunization with AAV1 after palpable tumor formation inhibited tumor growth and caused tumor regression in some mice. Thus, our studies support the potential of AAV vectors as a genetic vaccine for the prevention and treatment of HPV-induced malignancies.

  18. Rapid transgene expression in multiple precursor cell types of adult rat subventricular zone mediated by adeno-associated type 1 vectors.

    Science.gov (United States)

    Bockstael, Olivier; Melas, Catherine; Pythoud, Catherine; Levivier, Marc; McCarty, Douglas; Samulski, R Jude; De Witte, Olivier; Tenenbaum, Liliane

    2012-07-01

    The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.

  19. Insulated Foamy Viral Vectors

    Science.gov (United States)

    Browning, Diana L.; Collins, Casey P.; Hocum, Jonah D.; Leap, David J.; Rae, Dustin T.; Trobridge, Grant D.

    2016-01-01

    Retroviral vector-mediated gene therapy is promising, but genotoxicity has limited its use in the clinic. Genotoxicity is highly dependent on the retroviral vector used, and foamy viral (FV) vectors appear relatively safe. However, internal promoters may still potentially activate nearby genes. We developed insulated FV vectors, using four previously described insulators: a version of the well-studied chicken hypersensitivity site 4 insulator (650cHS4), two synthetic CCCTC-binding factor (CTCF)-based insulators, and an insulator based on the CCAAT box-binding transcription factor/nuclear factor I (7xCTF/NF1). We directly compared these insulators for enhancer-blocking activity, effect on FV vector titer, and fidelity of transfer to both proviral long terminal repeats. The synthetic CTCF-based insulators had the strongest insulating activity, but reduced titers significantly. The 7xCTF/NF1 insulator did not reduce titers but had weak insulating activity. The 650cHS4-insulated FV vector was identified as the overall most promising vector. Uninsulated and 650cHS4-insulated FV vectors were both significantly less genotoxic than gammaretroviral vectors. Integration sites were evaluated in cord blood CD34+ cells and the 650cHS4-insulated FV vector had fewer hotspots compared with an uninsulated FV vector. These data suggest that insulated FV vectors are promising for hematopoietic stem cell gene therapy. PMID:26715244

  20. Generation of recombinant adeno-associated virus (rAAV) from an adenoviral vector and functional reconstitution of the NADPH-oxidase.

    Science.gov (United States)

    Thrasher, A J; de Alwis, M; Casimir, C M; Kinnon, C; Page, K; Lebkowski, J; Segal, A W; Levinsky, R J

    1995-09-01

    The human parvovirus, adeno-associated virus-2 (AAV-2), has many attributes that recommend its use as a gene transfer vehicle, including a broad tissue tropism, the ability to integrate stably into the host genome, and efficient transduction of cells which proliferate slowly. However, application to human gene therapy is currently limited by existing methods for generation of recombinant AAV (rAAV), resulting in relatively low transducing titres. In an attempt to overcome some of these problems, we have developed a defective adenoviral vector which improves the efficiency of rAAV vector delivery to cells in which rAAV is propagated, and from which the rAAV genome can be efficiently rescued. A functional copy of the p47phox gene was successfully transferred to cell lines derived from patients with autosomal recessive chronic granulomatous disease (CGD) by rAAV recovered in this way, and function of the NADPH-oxidase was restored to levels which were stable for at least 8 weeks. This method for generation of rAAV, although still limited by the need for cotransfection of AAV Rep and Cap functions, may permit recovery of higher titre transducing stocks from cell lines in which these genes are stably incorporated, and significantly reduces the risk of contamination with wild-type adenovirus (wtAd).

  1. Enhanced survival of the LINCL mouse following CLN2 gene transfer using the rh.10 rhesus macaque-derived adeno-associated virus vector.

    Science.gov (United States)

    Sondhi, Dolan; Hackett, Neil R; Peterson, Daniel A; Stratton, Jamie; Baad, Michael; Travis, Kelly M; Wilson, James M; Crystal, Ronald G

    2007-03-01

    Late infantile neuronal ceroid lipofuscinosis (LINCL) is a lysosomal storage disorder caused by mutations in the CLN2 gene and a deficiency of tripeptidyl peptidase I (TPP-I). Prior studies with adeno-associated virus (AAV) serotype 2 or 5 mediated transfer of the CLN2 complementary DNA to the central nervous system (CNS) of CLN2(-/-) mice cleared CNS storage granules, but provided no improvement in the phenotype or survival of this model of LINCL. In this study, AAV serotypes (AAV2, AAV5, AAV8, and AAVrh.10) were compared for the delivery of the same CLN2 expression cassette. AAVrh.10, derived from rhesus macaque, provided the highest TPP-I level and maximum spread beyond the site of injection. The AAVrh.10-based vector functioned equally well in naive rats and in rats previously immunized against human serotypes of AAV. When administered to the CNS of CLN2(-/-) mice, the AAVrh.10CLN2 vector provided widespread TPP-I activity comparable to that in the wild-type mice. Importantly, the AAVrh.10CLN2-treated CLN2(-/-) mice had significant reduction in CNS storage granules and demonstrated improvement in gait, nest-making abilities, seizures, balance beam function, and grip strength, as well as having a survival advantage.

  2. A human parvovirus, adeno-associated virus, as a eucaryotic vector: transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase.

    Science.gov (United States)

    Tratschin, J D; West, M H; Sandbank, T; Carter, B J

    1984-01-01

    We have used the defective human parvovirus adeno-associated virus (AAV) as a novel eucaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p40 (pAVHiCAT) and p19 (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p19 is increased by E1A, whereas p40 yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus. Images PMID:6095038

  3. A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Tratschin, J.D.; West, M.H.P.; Sandbank, T.; Carter, B.J.

    1984-10-01

    The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/ (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.

  4. Biology of adeno-associated viral vectors in the central nervous system

    National Research Council Canada - National Science Library

    Murlidharan, Giridhar; Samulski, Richard J; Asokan, Aravind

    2014-01-01

    .... In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms, and transport mechanisms within the CNS...

  5. Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: results of a phase I trial.

    Science.gov (United States)

    Ghazi, Nicola G; Abboud, Emad B; Nowilaty, Sawsan R; Alkuraya, Hisham; Alhommadi, Abdulrahman; Cai, Huimin; Hou, Rui; Deng, Wen-Tao; Boye, Sanford L; Almaghamsi, Abdulrahman; Al Saikhan, Fahad; Al-Dhibi, Hassan; Birch, David; Chung, Christopher; Colak, Dilek; LaVail, Matthew M; Vollrath, Douglas; Erger, Kirsten; Wang, Wenqiu; Conlon, Thomas; Zhang, Kang; Hauswirth, William; Alkuraya, Fowzan S

    2016-03-01

    MERTK is an essential component of the signaling network that controls phagocytosis in retinal pigment epithelium (RPE), the loss of which results in photoreceptor degeneration. Previous proof-of-concept studies have demonstrated the efficacy of gene therapy using human MERTK (hMERTK) packaged into adeno-associated virus (AAV2) in treating RCS rats and mice with MERTK deficiency. The purpose of this study was to assess the safety of gene transfer via subretinal administration of rAAV2-VMD2-hMERTK in subjects with MERTK-associated retinitis pigmentosa (RP). After a preclinical phase confirming the safety of the study vector in monkeys, six patients (aged 14 to 54, mean 33.3 years) with MERTK-related RP and baseline visual acuity (VA) ranging from 20/50 to improved visual acuity in the treated eye following surgery, although the improvement was lost by 2 years in two of these patients. Gene therapy for MERTK-related RP using careful subretinal injection of rAAV2-VMD2-hMERTK is not associated with major side effects and may result in clinical improvement in a subset of patients.

  6. Neuroprotective effects of glial cell line-derived neurotrophic factor mediated by an adeno-associated virus vector in a transgenic animal model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Wang, Li-Jun; Lu, Yan-Yan; Muramatsu, Shin-ichi; Ikeguchi, Kunihiko; Fujimoto, Ken-ichi; Okada, Takashi; Mizukami, Hiroaki; Matsushita, Takashi; Hanazono, Yutaka; Kume, Akihiro; Nagatsu, Toshiharu; Ozawa, Keiya; Nakano, Imaharu

    2002-08-15

    Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive lethal disease that involves selective annihilation of motoneurons. Glial cell line-derived neurotrophic factor (GDNF) is proposed to be a promising therapeutic agent for ALS and other motor neuron diseases. Because adeno-associated virus (AAV) has been developed as an attractive gene delivery system with proven safety, we explored the therapeutic efficacy of intramuscular delivery of the GDNF gene mediated by an AAV vector (AAV-GDNF) in the G93A mouse model of ALS. We show here that AAV-GDNF leads to substantial and long-lasting expression of transgenic GDNF in a large number of myofibers with its accumulation at the sites of neuromuscular junctions. Detection of GDNF labeled with FLAG in the anterior horn neurons, but not beta-galactosidase expressed as a control, indicates that most of the transgenic GDNF observed there is retrogradely transported GDNF protein from the transduced muscles. This transgenic GDNF prevents motoneurons from their degeneration, preserves their axons innervating the muscle, and inhibits the treated-muscle atrophy. Furthermore, four-limb injection of AAV-GDNF postpones the disease onset, delays the progression of the motor dysfunction, and prolongs the life span in the treated ALS mice. Our finding thus indicates that AAV-mediated GDNF delivery to the muscle is a promising means of gene therapy for ALS.

  7. Introduction of tau mutation into cultured Rat1-R12 cells by gene targeting, using recombinant adeno-associated virus vector.

    Science.gov (United States)

    Shimada, Hiroko; Numazawa, Kahori; Sasaki, Tsukasa; Kato, Nobumasa; Ebisawa, Takashi

    2009-07-01

    We aim to develop a cultured cell model, to serve as a system with which the altered circadian phenotypes produced by the clock gene variations could be studied in vitro. Tau mutation, which shortens the circadian period of hamsters and mice, was introduced into the CK1epsilon locus of cultured Rat1-R12 cells by gene targeting mediated by a recombinant adeno-associated virus (rAAV) vector. After transduction of Rat1-R12 cells with rAAV, about 0.14% of the drug-resistant cells underwent gene targeting at CK1epsilon locus. Of the three clones isolated, only one carried the targeted allele of tau mutation and two carried the targeted wild-type allele. The clone with the targeted tau mutant allele exhibited a significantly shorter circadian period compared to the clone with targeted wild-type allele. rAAV-mediated gene targeting in cultured somatic cells is a convenient and powerful tool for analyzing the phenotypic outcome of clock gene variations, and for elucidating the pathogenesis of the disorders associated with abnormal circadian rhythmicity.

  8. A NEW RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV)-BASED RANDOM PEPTIDE DISPLAY LIBRARY SYSTEM: INFECTION-DEFECTIVE AAV1.9-3 AS A NOVEL DETARGETED PLATFORM FOR VECTOR EVOLUTION.

    Science.gov (United States)

    Adachi, Kei; Nakai, Hiroyuki

    2010-10-01

    Directed evolution through genetic engineering of viral capsids followed by selection has emerged as a powerful means to create novel recombinant adeno-associated virus (rAAV) vectors with desired tropism and enhanced properties. One of the most effective approaches uses rAAV-based random peptide display libraries. Here we report a novel system based on an infection-defective rAAV1.9-3 as a platform for random peptide display, and show that biopanning of the libraries in vitro effectively identifies the peptides that restore and enhance rAAV transduction. rAAV1.9-3 has a genetically engineered AAV1 capsid with amino acids 445-568 being replaced with those of AAV9, and has been identified as a variant exhibiting significantly impaired infectivity and delayed blood clearance when infused into mice. In this study, we generated rAAV1.9-3 variant libraries in which 7- or 12-mer random peptides were expressed at the capsid amino acid position 590. Three rounds of positive selection for primary human dermal fibroblasts successfully identified new rAAV-peptide variants that transduce them more efficiently than the prototype rAAV2. Thus our study demonstrates that an infection-defective rAAV variant serves as a novel detargeted platform for random peptide display libraries. We also describe a brief review of recent progress in rAAV-based random peptide display library approaches.

  9. A NEW RECOMBINANT ADENO-ASSOCIATED VIRUS (AAV)-BASED RANDOM PEPTIDE DISPLAY LIBRARY SYSTEM: INFECTION-DEFECTIVE AAV1.9-3 AS A NOVEL DETARGETED PLATFORM FOR VECTOR EVOLUTION*

    Science.gov (United States)

    Adachi, Kei; Nakai, Hiroyuki

    2011-01-01

    Directed evolution through genetic engineering of viral capsids followed by selection has emerged as a powerful means to create novel recombinant adeno-associated virus (rAAV) vectors with desired tropism and enhanced properties. One of the most effective approaches uses rAAV-based random peptide display libraries. Here we report a novel system based on an infection-defective rAAV1.9-3 as a platform for random peptide display, and show that biopanning of the libraries in vitro effectively identifies the peptides that restore and enhance rAAV transduction. rAAV1.9-3 has a genetically engineered AAV1 capsid with amino acids 445–568 being replaced with those of AAV9, and has been identified as a variant exhibiting significantly impaired infectivity and delayed blood clearance when infused into mice. In this study, we generated rAAV1.9-3 variant libraries in which 7- or 12-mer random peptides were expressed at the capsid amino acid position 590. Three rounds of positive selection for primary human dermal fibroblasts successfully identified new rAAV-peptide variants that transduce them more efficiently than the prototype rAAV2. Thus our study demonstrates that an infection-defective rAAV variant serves as a novel detargeted platform for random peptide display libraries. We also describe a brief review of recent progress in rAAV-based random peptide display library approaches. PMID:21603583

  10. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors

    Directory of Open Access Journals (Sweden)

    Sablitzky Fred

    2004-01-01

    Full Text Available Abstract Background Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV and lentiviral (LV vectors into discrete regions of the forebrain. Results Recombinant AAV-Cre, AAV-GFP (green fluorescent protein and LV-Cre-EGFP (enhanced GFP were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. Conclusion AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

  11. Efficient delivery of Cre-recombinase to neurons in vivo and stable transduction of neurons using adeno-associated and lentiviral vectors.

    Science.gov (United States)

    Ahmed, Bushra Y; Chakravarthy, Sridhara; Eggers, Ruben; Hermens, Wim T J M C; Zhang, Jing Ying; Niclou, Simone P; Levelt, Christiaan; Sablitzky, Fred; Anderson, Patrick N; Lieberman, A R; Verhaagen, Joost

    2004-01-30

    Inactivating genes in vivo is an important technique for establishing their function in the adult nervous system. Unfortunately, conventional knockout mice may suffer from several limitations including embryonic or perinatal lethality and the compensatory regulation of other genes. One approach to producing conditional activation or inactivation of genes involves the use of Cre recombinase to remove loxP-flanked segments of DNA. We have studied the effects of delivering Cre to the hippocampus and neocortex of adult mice by injecting replication-deficient adeno-associated virus (AAV) and lentiviral (LV) vectors into discrete regions of the forebrain. Recombinant AAV-Cre, AAV-GFP (green fluorescent protein) and LV-Cre-EGFP (enhanced GFP) were made with the transgene controlled by the cytomegalovirus promoter. Infecting 293T cells in vitro with AAV-Cre and LV-Cre-EGFP resulted in transduction of most cells as shown by GFP fluorescence and Cre immunoreactivity. Injections of submicrolitre quantities of LV-Cre-EGFP and mixtures of AAV-Cre with AAV-GFP into the neocortex and hippocampus of adult Rosa26 reporter mice resulted in strong Cre and GFP expression in the dentate gyrus and moderate to strong labelling in specific regions of the hippocampus and in the neocortex, mainly in neurons. The pattern of expression of Cre and GFP obtained with AAV and LV vectors was very similar. X-gal staining showed that Cre-mediated recombination had occurred in neurons in the same regions of the brain, starting at 3 days post-injection. No obvious toxic effects of Cre expression were detected even after four weeks post-injection. AAV and LV vectors are capable of delivering Cre to neurons in discrete regions of the adult mouse brain and producing recombination.

  12. Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene.

    Science.gov (United States)

    Ishiwata, Akira; Mimuro, Jun; Kashiwakura, Yuji; Niimura, Masanori; Takano, Katsuhiro; Ohmori, Tsukasa; Madoiwa, Seiji; Mizukami, Hiroaki; Okada, Takashi; Naka, Hiroyuki; Yoshioka, Akira; Ozawa, Keiya; Sakata, Yoichi

    2006-01-01

    Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9+/-1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1 x 10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.

  13. Major subsets of human dendritic cells are efficiently transduced by self-complementary adeno-associated virus vectors 1 and 2.

    Science.gov (United States)

    Veron, Philippe; Allo, Valérie; Rivière, Christel; Bernard, Jacky; Douar, Anne-Marie; Masurier, Carole

    2007-05-01

    Dendritic cells (DC) are antigen-presenting cells pivotal for inducing immunity or tolerance. Gene transfer into DC is an important strategy for developing immunotherapeutic approaches against infectious pathogens and cancers. One of the vectors previously described for the transduction of human monocytes or DC is the recombinant adeno-associated virus (rAAV), with a genome conventionally packaged as a single-stranded (ss) molecule. Nevertheless, its use is limited by the poor and variable transduction efficiency of DC. In this study, AAV type 1 (AAV1) and AAV2 vectors, which expressed the enhanced green fluorescent protein and were packaged as ss or self-complementary (sc) duplex strands, were used to transduce different DC subsets generated ex vivo and the immunophenotypes, states of differentiation, and functions of the subsets were carefully examined. We show here for the first time that a single exposure of monocytes (M(o)) or CD34(+) progenitors (CD34) to sc rAAV1 or sc rAAV2 leads to high transduction levels (5 to 59%) of differentiated M(o)-DC, M(o)-Langerhans cells (LC), CD34-LC, or CD34-plasmacytoid DC (pDC), with no impact on their phenotypes and functional maturation of these cells, compared to those of exposure to ss rAAV. Moreover, we show that all these DC subpopulations can also be efficiently transduced after commitment to their differentiation pathways. Furthermore, these DC subsets transduced with sc rAAV1 expressing a tumor antigen were potent activators of a CD8(+)-T-cell clone. Altogether, these results show the high potential of sc AAV1 and sc AAV2 vectors to transduce ex vivo conventional DC, LC, or pDC or to directly target them in vivo for the design of new DC-based immunotherapies.

  14. Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.

    Science.gov (United States)

    Hemphill, D D; McIlwraith, C W; Slayden, R A; Samulski, R J; Goodrich, L R

    2016-05-01

    IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I. Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes. Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis. This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response. Copyright © 2015. Published by Elsevier Ltd.

  15. Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain.

    Science.gov (United States)

    Broekman, M L D; Comer, L A; Hyman, B T; Sena-Esteves, M

    2006-01-01

    Adeno-associated virus (AAV) vectors have gained a preeminent position in the field of gene delivery to the normal brain through their ability to achieve extensive transduction of neurons and to mediate long-term gene expression with no apparent toxicity. In adult animals direct infusion of AAV vectors into the brain parenchyma results in highly efficient transduction of target structures. However AAV-mediated global delivery to the adult brain has been an elusive goal. In contrast, widespread global gene delivery has been obtained by i.c.v. injection of AAV1 or AAV2 in neonates. Among the novel AAV serotypes cloned and engineered for production of recombinant vectors, AAV8 has shown a tremendous potential for in vivo gene delivery with nearly complete transduction of many tissues in rodents after intravascular infusion. Here we compare the efficiency of an AAV8 serotyped vector with that of AAV1 and AAV2 serotyped vectors for the extent of gene delivery to the brain after neonatal injection into the lateral ventricles. The vectors all encoded green fluorescent protein (GFP) under control of a hybrid CMV enhancer/chicken beta-actin promoter with AAV2 inverted terminal repeats, but differed from each other with respect to the capsid type. A total of 6.8 x 10(10) genome copies were injected into the lateral ventricles of postnatal day 0 mice. Mice were killed at postnatal day 30 and brains analyzed for distribution of GFP-positive cells. AAV8 proved to be more efficient than AAV1 or AAV2 vectors for gene delivery to all of the structures analyzed, including the cerebral cortex, hippocampus, olfactory bulb, and cerebellum. Moreover the intensity of gene expression, assessed using a microarray reader, was considerably higher for AAV8 in all structures analyzed. In conclusion, the enhanced transduction achieved by AAV8 compared with AAV1 and AAV2 indicates that AAV8 is the superior serotype for gene delivery to the CNS.

  16. Different protein composition and functional properties of adeno-associated virus-6 vector manufactured from the culture medium and cell lysates

    Directory of Open Access Journals (Sweden)

    Jerome Denard

    2014-01-01

    Full Text Available Vectors based on recombinant adeno-associated viruses (rAAV attract a growing interest for human gene therapy. Recently, it was shown that many rAAV serotypes produced by transient transfection of human embryonic kidney 293 cell line (HEK293 are efficiently released into culture medium and functionally equivalent to those purified from cell lysates. Here, we report that HEK293 cells produce and secrete Galectin 3-binding protein (huG3BP, a protein that efficiently binds rAAV6 in vivo. Importantly, intracellular G3BP and secreted G3BP have different properties: while the secreted protein had the same electrophoretic mobility as serum huG3BP and interacted with rAAV6, intracellular protein migrated faster and did not bind rAAV6. Consequently, rAAV6 purified from culture medium (secreted, rAAV6-S was physically associated with huG3BP while rAAV6 harvested from cell lysates (cellular, rAAV6-C was huG3BP-free. After systemic injections, rAAV6-S bound to huG3BP was 3 times less efficient compared to rAAV6-C and induced an immune response against huG3BP protein. Our findings show that protein content of rAAVs purified from culture medium or from cell lysates can be different and these differences may impact vector efficacy and/or immune response.

  17. Adeno-associated virus (AAV) gene therapy for neurological disease.

    Science.gov (United States)

    Weinberg, Marc S; Samulski, R Jude; McCown, Thomas J

    2013-06-01

    Diseases of the central nervous system (CNS) have provided enormous opportunities for the therapeutic application of viral vector gene transfer. Adeno-associated virus (AAV) has been the vector of choice in recent clinical trials of neurological disease, including Parkinson's and Alzheimer's disease, due to the safety, efficacy, and stability of AAV gene transfer to the CNS. This review highlights the strategies employed for improving direct and peripheral targeting of therapeutic vectors to CNS tissue, and considers the significance of cellular and tissue transduction specificity, transgene regulation, and other variables that influence achievement of successful therapeutic goals. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Gangliosides Are Essential for Bovine Adeno-Associated Virus Entry

    OpenAIRE

    Schmidt, Michael; Chiorini, John A.

    2006-01-01

    Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. We have recently identified a bovine adeno-associated virus (BAAV) that demonstrates unique tropism and transduction activity compared to primate AAVs. To better understand the entry pathway and cell tropism of BAAV, we have characterized the initial cell surface interactions required for transduction with BAAV vectors. Like a number of AAVs, BAAV requires cell surface sialic acid groups for transduction and virus ...

  19. Mutants at the 2-Fold Interface of Adeno-associated Virus Type 2 (AAV2) Structural Proteins Suggest a Role in Viral Transcription for AAV Capsids.

    Science.gov (United States)

    Aydemir, Fikret; Salganik, Maxim; Resztak, Justyna; Singh, Jasbir; Bennett, Antonette; Agbandje-McKenna, Mavis; Muzyczka, Nicholas

    2016-08-15

    We previously reported that an amino acid substitution, Y704A, near the 2-fold interface of adeno-associated virus (AAV) was defective for transcription of the packaged genome (M. Salganik, F. Aydemir, H. J. Nam, R. McKenna, M. Agbandje-McKenna, and N. Muzyczka, J Virol 88:1071-1079, 2013, doi: http://dx.doi.org/10.1128/JVI.02093-13). In this report, we have characterized the defect in 6 additional capsid mutants located in a region ∼30 Å in diameter on the surface of the AAV type 2 (AAV2) capsid near the 2-fold interface. These mutants, which are highly conserved among primate serotypes, displayed a severe defect (3 to 6 logs) in infectivity. All of the mutants accumulated significant levels of uncoated DNA in the nucleus, but none of the mutants were able to accumulate significant amounts of genomic mRNA postinfection. In addition, wild-type (wt) capsids that were bound to the conformational antibody A20, which is known to bind the capsid surface in the region of the mutants, were also defective for transcription. In all cases, the mutant virus particles, as well as the antibody-bound wild-type capsids, were able to enter the cell, travel to the nucleus, uncoat, and synthesize a second strand but were unable to transcribe their genomes. Taken together, the phenotype of these mutants provides compelling evidence that the AAV capsid plays a role in the transcription of its genome, and the mutants map this functional region on the surface of the capsid near the 2-fold interface. This appears to be the first example of a viral structural protein that is also involved in the transcription of the viral genome that it delivers to the nucleus. Many viruses package enzymes within their capsids that assist in expressing their genomes postinfection, e.g., retroviruses. A number of nonenveloped viruses, including AAV, carry proteases that are needed for capsid maturation or for capsid modification during infection. We describe here what appears to be the first example of

  20. A High-Capacity, Capsid-Modified Hybrid Adenovirus/Adeno-Associated Virus Vector for Stable Transduction of Human Hematopoietic Cells

    OpenAIRE

    Dmitry M Shayakhmetov; Carlson, Cheryl A.; Stecher, Hartmut; Li, Qiliang; Stamatoyannopoulos, George; Lieber, André

    2002-01-01

    To achieve stable gene transfer into human hematopoietic cells, we constructed a new vector, ΔAd5/35.AAV. This vector has a chimeric capsid containing adenovirus type 35 fibers, which conferred efficient infection of human hematopoietic cells. The ΔAd5/35.AAV vector genome is deleted for all viral genes, allowing for infection without virus-associated toxicity. To generate high-capacity ΔAd5/35.AAV vectors, we employed a new technique based on recombination between two first-generation adenov...

  1. Glycated AAV vectors: chemical redirection of viral tissue tropism.

    Science.gov (United States)

    Horowitz, Eric D; Weinberg, Marc S; Asokan, Aravind

    2011-04-20

    A chemical approach for selective masking of arginine residues on viral capsids featuring an exogenous glycation reaction has been developed. Reaction of adeno-associated viral (AAV) capsids with the α-dicarbonyl compound, methylglyoxal, resulted in formation of arginine adducts. Specifically, surface-exposed guanidinium side chains were modified into charge neutral hydroimidazolones, thereby disrupting a continuous cluster of basic amino acid residues implicated in heparan sulfate binding. Consequent loss in heparin binding ability and decrease in infectivity were observed. Strikingly, glycated AAV retained the ability to infect neurons in the mouse brain and were redirected from liver to skeletal and cardiac muscle following systemic administration in mice. Further, glycated AAV displayed altered antigenicity demonstrating the potential for evading antibody neutralization. Generation of unnatural amino acid side chains through capsid glycation might serve as an orthogonal strategy to engineer AAV vectors displaying novel tissue tropisms for gene therapy applications.

  2. Comparative analysis of the transduction efficiency of five adeno associated virus serotypes and VSV-G pseudotype lentiviral vector in lung cancer cells.

    Science.gov (United States)

    Chen, Chiachen; Akerstrom, Victoria; Baus, James; Lan, Michael S; Breslin, Mary B

    2013-03-14

    Lung cancer is the leading cause of cancer-related deaths in the US. Recombinant vectors based on adeno-associated virus (AAV) and lentivirus are promising delivery tools for gene therapy due to low toxicity and long term expression. The efficiency of the gene delivery system is one of the most important factors directly related to the success of gene therapy. We infected SCLC cell lines, SHP-77, DMS 53, NCI-H82, NCI-H69, NCI-H727, NCI-H1155, and NSCLC cell lines, NCI-H23, NCI-H661, and NCI-H460 with VSV-G pseudo-typed lentivirus or 5 AAV serotypes, AAV2/1, AAV2/2, AAV2/4, AAV2/5, and AAV2/8 expressing the CMV promoter mCherry or green fluorescent protein transgene (EGFP). The transduction efficiency was analyzed by fluorescent microscopy and flow cytometry. Of all the serotypes of AAV examined, AAV2/1 was the optimal serotype in most of the lung cancer cell lines except for NCI-H69 and NCI-H82. The highest transduction rate achieved with AAV2/1 was between 30-50% at MOI 100. Compared to all AAV serotypes, lentivirus had the highest transduction efficiency of over 50% at MOI 1. Even in NCI-H69 cells resistant to all AAV serotypes, lentivirus had a 10-40% transduction rate. To date, AAV2 is the most widely-used serotype to deliver a transgene. Our results showed the transduction efficiency of AAVs tested was AAV2/1 > AA2/5 = AAV2/2> > AAV2/4 and AAV2/8. This study demonstrated that VSV-G pseudotyped lentivirus and AAV2/1 can mediate expression of a transgene for lung cancer gene therapy. Overall, our results showed that lentivirus is the best candidate to deliver a transgene into lung cancer cells for treatment.

  3. Cell-Based Measurement of Neutralizing Antibodies Against Adeno-Associated Virus (AAV).

    Science.gov (United States)

    Jungmann, Andreas; Müller, Oliver; Rapti, Kleopatra

    2017-01-01

    In recent years gene therapy using adeno-associated viral (AAV) vectors to treat cardiac disease has seen an unprecedented surge, owing to its safety, low immunogenicity relative to other vectors and high and long-term transduction efficiency. This field has also been hampered by the presence of preexisting neutralizing antibodies, not only in patients participating in clinical trials but also in preclinical large animal models. These conflicting circumstances have generated the need for a simple, efficient, and fast assay to screen subjects for the presence of neutralizing antibodies, or lack thereof, in order for them to be included in gene therapy trials.

  4. Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site

    OpenAIRE

    Huang, Lin-Ya; Patel, Ami; Ng, Robert; Miller, Edward Blake; Halder, Sujata; McKenna, Robert; Asokan, Aravind; Agbandje-McKenna, Mavis

    2016-01-01

    The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Densi...

  5. Evaluation of primitive murine hematopoietic stem and progenitor cell transduction in vitro and in vivo by recombinant adeno-associated virus vector serotypes 1 through 5.

    Science.gov (United States)

    Zhong, Li; Li, Weiming; Li, Yanjun; Zhao, Weihong; Wu, Jianqing; Li, Baozheng; Maina, Njeri; Bischof, Daniela; Qing, Keyun; Weigel-Kelley, Kirsten A; Zolotukhin, Irene; Warrington, Kenneth H; Li, Xiaomiao; Slayton, William B; Yoder, Mervin C; Srivastava, Arun

    2006-03-01

    Conflicting data exist on hematopoietic cell transduction by AAV serotype 2 (AAV2) vectors, and additional AAV serotype vectors have not been evaluated for their efficacy in hematopoietic stem/progenitor cell transduction. We evaluated the efficacy of conventional, single-stranded AAV serotype vectors 1 through 5 in primitive murine hematopoietic stem/progenitor cells in vitro as well as in vivo. In progenitor cell assays using Sca1+ c-kit+ Lin- hematopoietic cells, 9% of the colonies in cultures infected with AAV1 expressed the transgene. Coinfection of AAV1 with self-complementary AAV vectors carrying the gene for T cell protein tyrosine phosphatase (scAAV-TC-PTP) increased the transduction efficiency to 24%, indicating that viral secondstrand DNA synthesis is a rate-limiting step. This was further corroborated by the use of scAAV vectors, which bypass this requirement. In bone marrow transplantation studies involving lethally irradiated syngeneic mice, Sca1+ c-kit+ Lin- cells coinfected with AAV1 +/- scAAV-TC-PTP vectors led to transgene expression in 2 and 7.5% of peripheral blood (PB) cells, respectively, 6 months posttransplantation. In secondary transplantation experiments, 7% of PB cells and 3% of bone marrow (BM) cells expressed the transgene 6 months posttransplantation. Approximately 21% of BM-derived colonies harbored the proviral DNA sequences in integrated forms. These results document that AAV1 is thus far the most efficient vector in transducing primitive murine hematopoietic stem/progenitor cells. Further studies involving scAAV genomes and hematopoietic cell-specific promoters should further augment the transduction efficiency of AAV1 vectors, which should have implications in the optimal use of these vectors in hematopoietic stem cell gene therapy.

  6. Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains

    Science.gov (United States)

    Wu, Shi-Hao; Liao, Zhi-Xing; Rizak, Joshua D.; Zheng, Na; Zhang, Lin-Heng; Tang, Hen; He, Xiao-Bin; Wu, Yang; He, Xia-Ping; Yang, Mei-Feng; Li, Zheng-Hui; Qin, Dong-Dong; Hu, Xin-Tian

    2017-01-01

    Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non-human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin Ⅰ, or Ca2+/calmodulin-dependent protein kinase Ⅱ promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs). These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates. PMID:28409504

  7. Viral Vectors for Gene Therapy: Translational and Clinical Outlook.

    Science.gov (United States)

    Kotterman, Melissa A; Chalberg, Thomas W; Schaffer, David V

    2015-01-01

    In a range of human trials, viral vectors have emerged as safe and effective delivery vehicles for clinical gene therapy, particularly for monogenic recessive disorders, but there has also been early work on some idiopathic diseases. These successes have been enabled by research and development efforts focusing on vectors that combine low genotoxicity and immunogenicity with highly efficient delivery, including vehicles based on adeno-associated virus and lentivirus, which are increasingly enabling clinical success. However, numerous delivery challenges must be overcome to extend this success to many diseases; these challenges include developing techniques to evade preexisting immunity, to ensure more efficient transduction of therapeutically relevant cell types, to target delivery, and to ensure genomic maintenance. Fortunately, vector-engineering efforts are demonstrating promise in the development of next-generation gene therapy vectors that can overcome these barriers. This review highlights key historical trends in clinical gene therapy, the recent clinical successes of viral-based gene therapy, and current research that may enable future clinical application.

  8. Viral vector-based tools advance knowledge of basal ganglia anatomy and physiology.

    Science.gov (United States)

    Sizemore, Rachel J; Seeger-Armbruster, Sonja; Hughes, Stephanie M; Parr-Brownlie, Louise C

    2016-04-01

    Viral vectors were originally developed to deliver genes into host cells for therapeutic potential. However, viral vector use in neuroscience research has increased because they enhance interpretation of the anatomy and physiology of brain circuits compared with conventional tract tracing or electrical stimulation techniques. Viral vectors enable neuronal or glial subpopulations to be labeled or stimulated, which can be spatially restricted to a single target nucleus or pathway. Here we review the use of viral vectors to examine the structure and function of motor and limbic basal ganglia (BG) networks in normal and pathological states. We outline the use of viral vectors, particularly lentivirus and adeno-associated virus, in circuit tracing, optogenetic stimulation, and designer drug stimulation experiments. Key studies that have used viral vectors to trace and image pathways and connectivity at gross or ultrastructural levels are reviewed. We explain how optogenetic stimulation and designer drugs used to modulate a distinct pathway and neuronal subpopulation have enhanced our mechanistic understanding of BG function in health and pathophysiology in disease. Finally, we outline how viral vector technology may be applied to neurological and psychiatric conditions to offer new treatments with enhanced outcomes for patients. Copyright © 2016 the American Physiological Society.

  9. [Serotypes of adeno-associated viruses].

    Science.gov (United States)

    Lipiec, Agnieszka; Małecki, Maciej; Hajdukiewic, Karolina

    2009-01-01

    Cancer, monogenic and cardiovascular diseases are the main targets of clinical gene therapy. For the most part, the research is making use of virus gene carriers based on genome of well characterized adenoviruses and retroviruses. There is an intensive research being done on cloning vectors that would successfully deliver the therapeutic genes into the cells of interest without causing side effects. Experimental research and first clinical trials emphasize therapeutic significance of recombinant adeno-associated viruses (AAV). The presence of AAV serotypes and possibility of their cloning in vitro allow conducting clinical trials of gene therapy using gene products introducing therapeutic genes into targeted cells, tissues, organs. In the work twelve known serotypes of AAV are described. The cell tropism as well as biological activity were discussed. Research shows that AAV vectors mostly are able to infect skeletal muscles (AAV1, 8, 9), heart (AAV9), liver (AAV8) and sense organs (AAV3, 4, 5).

  10. Developmental stage determines efficiency of gene transfer to muscle satellite cells by in utero delivery of adeno-associated virus vector serotype 2/9

    Directory of Open Access Journals (Sweden)

    David H Stitelman

    2014-01-01

    Full Text Available Efficient gene transfer to muscle stem cells (satellite cells has not been achieved despite broad transduction of skeletal muscle by systemically administered adeno-associated virus serotype 2/9 (AAV-9 in mice. We hypothesized that cellular migration during fetal development would make satellite cells accessible for gene transfer following in utero intravascular injection. We injected AAV-9 encoding green fluorescent protein (GFP marker gene into the vascular space of mice ranging in ages from post-coital day 12 (E12 to postnatal day 1 (P1. Satellite cell transduction was examined using: immunohistochemistry and confocal microscopy, satellite cell migration assay, myofiber isolation and FACS analysis. GFP positive myofibers were detected in all mature skeletal muscle groups and up to 100% of the myofibers were transduced. We saw gestational variation in cardiac and skeletal muscle expression. E16 injection resulted in 27.7 ± 10.0% expression in satellite cells, which coincides with the timing of satellite cell migration, and poor satellite cell expression before and after satellite cell migration (E12 and P1. Our results demonstrate that efficient gene expression is achieved in differentiated myofibers and satellite cells after injection of AAV-9 in utero. These findings support the potential of prenatal gene transfer for muscle based treatment strategies.

  11. Gene therapy for haemophilia...yes, but...with non-viral vectors?

    Science.gov (United States)

    Liras, A; Olmedillas, S

    2009-05-01

    High-purity plasma-derived and recombinant factors are currently safe and efficient treatment for haemophilia. The mid-term future of haemophilia treatment will involve the use of modified recombinant factors to achieve advantages such as decreased immunogenicity in inhibitor formation and enhanced efficacy as a result of their longer half-life. In the long-term, gene therapy and cell therapy strategies will have to be considered. Achievements in cell therapy to date have been using embryonic stem cells and hepatic sinusoidal endothelial cells. Current gene therapy strategies for haemophilia are based on gene transfer using adeno-associated viruses and non-viral vectors. Gene therapy for haemophilia is justified because it is a chronic disease and because a very regular factor infusion is required that may involve fatal risks and because it is very expensive. Haemophilia is a very good candidate for use of gene therapy protocols because it is a monogenic disease, and even low expression is able to achieve reversion from a severe to a moderate phenotype. The current trends in haemophilia using adeno-associated viral vectors are safe but also involve immunogenicity problems. The other alternatives are non-viral vectors. There have been in recent years relevant advances in non-viral transfection that raise hope for considering this possibility. Several research groups are opting for this experimental alternative. An expression over 5%, representing a moderate phenotype, for a few months with a high safety, regarding vector, transfected cells, and implantation procedure, would already be a great success. This may represent an intermediate protocol in which the expression levels and times obtained are lower and shorter respectively as compared to viral vectors, but which provide a potential greater patient safety. This may more readily win acceptance among both patients and haematologists because fatal events in the past due to HIV/HCV infection may constrain the

  12. Co-vaccination with adeno-associated virus vectors encoding human papillomavirus 16 L1 proteins and adenovirus encoding murine GM-CSF can elicit strong and prolonged neutralizing antibody.

    Science.gov (United States)

    Liu, Dai-Wei; Chang, Junn-Liang; Tsao, Yeou-Ping; Huang, Chien-Wei; Kuo, Shu-Wen; Chen, Show-Li

    2005-01-01

    Non-infectious human papillomavirus-like particles (VLPs), encoded by the major capsid gene L1, have been shown to be effective as vaccines to prevent cervical cancer. We have developed the genetic immunization of the L1 gene to induce a neutralizing antibody. We constructed and generated a recombinant adeno-associated virus encoding human papillomavirus (HPV) 16 L1 protein that could form virus-like particles in transduced cells. Previous reports have demonstrated that the formation of VLP is necessary to induce high titers of neutralizing antibodies to protect an animal from viral challenge. Therefore, we carried out a single intramuscular (i.m.) injection with recombinant adeno-associated virus encoding HPV-16 L1 protein (rAAV-16L1) in BALB/c mice, which ultimately produced stronger and more prolonged neutralizing L1 antibodies, when compared to the DNA vaccine. Immunohistochemistry showed that the accumulation of antigen presenting cells, such as macrophages and dendritic cells, in rAAV-16L1 and L1 DNA-injected muscle fibers may be due to the L1 protein expression, but not to AAV infection. When compared to the L1 VLP vaccine, however, the titers of neutralizing L1 antibodies induced by VLP were higher than those induced by rAAV-16L1. Co-vaccinating with rAAV-16L1 and adenovirus encoding murine GM-CSF (rAAV-16L1/rAd-mGM-CSF) induced comparable higher levels of neutralizing L1 antibodies with those of VLP. This implies that a single i.m. co-injection with rAAV-16L1/rAd-mGM-CSF can achieve the same vaccine effect as a VLP vaccine requiring 3 booster injections.

  13. Exploiting high-throughput screens to optimize Adeno-Associated Viral Vectors for gene transfer into primary human keratinocytes

    OpenAIRE

    Sallach, Jessica

    2014-01-01

    Chronic non-healing wounds such as diabetic ulcers or burns represent a devastating health problem with significant clinical, physical and social implications. The healing can be frustrating and painful for patients. The difficult healing process requires advanced therapeutic strategies such as the use of primary human keratinocytes (HK) as autologous transplants, which may be considered for clinical use. To improve engraftment or to introduce therapeutic genes into primary HK, efficient and ...

  14. High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing.

    Science.gov (United States)

    Marsic, Damien; Méndez-Gómez, Héctor R; Zolotukhin, Sergei

    2015-01-01

    Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency.

  15. High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing

    Directory of Open Access Journals (Sweden)

    Damien Marsic

    2015-01-01

    Full Text Available Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency.

  16. Neutralizing Antibodies Against Adeno-Associated Virus (AAV): Measurement and Influence on Retinal Gene Delivery.

    Science.gov (United States)

    Desrosiers, Mélissa; Dalkara, Deniz

    2018-01-01

    Adeno-associated viral vectors have become widely used in the clinic for retinal gene therapy. Thanks to AAVs impeccable safety profile and positive functional outcomes in its clinical application, interest in retinal gene therapy has increased exponentially over the past decade. Although early clinical trials have shown there is little influence of neutralizing antibodies on the performance of AAV when vector is administered into the subretinal space, recent findings suggest neutralizing antibodies may play a role when AAV is delivered via the intravitreal route. These findings highlight the importance of microenvironment on gene delivery and stress the need for a versatile assay to screen subjects for the presence of AAV-neutralizing antibodies. Measuring NAb titers against AAV prior and after gene therapy will help us better understand the impact of preexisting immunity on gene transfer, especially when the vector is administered intravitreally.

  17. Adeno-associated viruses containing bFGF or BDNF are neuroprotective against excitotoxicity.

    Science.gov (United States)

    Schuettauf, Frank; Vorwerk, Christian; Naskar, Rita; Orlin, Anton; Quinto, Kristine; Zurakowski, David; Dejneka, Nadine S; Klein, Ronald L; Meyer, Edward M; Bennett, Jean

    2004-12-01

    Brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) hold much promise for the protection of retinal ganglion cells against excitotoxic cell death. We tested the possibility of delivering these growth factors to retinal ganglion cells via an adeno-associated viral (AAV) vector and tested their efficacy in two models of excitotoxicity. Rat retinas were infected with AAV vectors encoding bFGF or BDNF. A control vector containing green fluorescent protein (GFP) was injected in the contralateral eye. Eyes were subjected to either an intravitreal injection of N-methyl-D-aspartate (NMDA) or optic nerve crush, and ganglion cell survival was evaluated. AAV.CMV.bFGF and AAV.CBA.BDNF were neuroprotective against NMDA injection 1 month post-treatment. Additionally, AAV.CMV.bFGF was protective against optic nerve crush. AAV-mediated delivery of bFGF and BDNF can promote retinal cell survival following excitotoxic insult.

  18. Intramuscular administration of recombinant adeno-associated virus 2 alpha-1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model: safety and immunologic aspects.

    Science.gov (United States)

    Song, Sihong; Scott-Jorgensen, Marda; Wang, Jianming; Poirier, Amy; Crawford, James; Campbell-Thompson, Martha; Flotte, Terence R

    2002-09-01

    We performed a series of studies in baboons to evaluate the safety of intramuscular administration of rAAV vector expressing the alpha-1 antitrypsin (AAT) gene (SERPINA1) in a nonhuman primate model. Initial experiments performed with an rAAV vector expressing the human SERPINA1 gene (at doses of up to 5 x 10(12) vector genomes/kg) resulted in the generation of anti-human AAT antibodies, which correlated with a loss of detectable transgene expression. Subsequent studies made use of the baboon SERPINA1 gene tagged with a short (10-amino-acid) c-myc tag. When animals were sacrificed, 4 months after vector injection, transduced myofibers showed efficient transgene expression without detectable humoral immune responses. Mild inflammation was observed in and near the sites of injection in some vector- and saline-injected animals, but serum creatine kinase (CK) values were normal in nearly every case. Real-time PCR was also performed 4 months after injection on gonadal tissue to evaluate the risk of germline transmission. No vector sequences were detected in the gonadal tissues from these animals. These studies indicate that the risks of immune reaction and germline transmission after intramuscular injection of rAAV-SERPINA1 in nonhuman primates are relatively low within the range of vector doses studied.

  19. Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Daniel Barajas

    Full Text Available The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

  20. Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae

    Science.gov (United States)

    Aponte-Ubillus, Juan Jose; Akeefe, Hassibullah; Cinek, Tomas; Peltier, Joseph; Gold, Daniel

    2017-01-01

    The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors. PMID:28355224

  1. Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

    Directory of Open Access Journals (Sweden)

    Yarong Liu

    2014-01-01

    Full Text Available Adeno-associated virus type 2 (AAV2 is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs. We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.

  2. Molecular evolution of adeno-associated virus for enhanced glial gene delivery.

    Science.gov (United States)

    Koerber, James T; Klimczak, Ryan; Jang, Jae-Hyung; Dalkara, Deniz; Flannery, John G; Schaffer, David V

    2009-12-01

    The natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, leads to predominant transduction of neurons and epithelia within the central nervous system (CNS) and retina. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimer's and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of AAV vectors with valuable new gene delivery properties. We have engineered novel AAV variants capable of efficient glia transduction by employing directed evolution with a panel of four distinct AAV libraries, including a new semi-random peptide replacement strategy. These variants transduced both human and rat astrocytes in vitro up to 15-fold higher than their parent serotypes, and injection into the rat striatum yielded astrocyte transduction levels up to 16% of the total transduced cell population, despite the human astrocyte selection platform. Furthermore, one variant exhibited a substantial shift in tropism toward Müller glia within the retina, further highlighting the general utility of these variants for efficient glia transduction in multiple species within the CNS and retina.

  3. Recombinant adeno-associated virus-based vectors provide short-term rather than long-term transduction of primitive hematopoietic stem cells.

    Science.gov (United States)

    van Os, R; Avraham, H; Banu, N; Mauch, P M; Whater, J; Yang, Y; Du, B

    1999-01-01

    Bone marrow stem cells collected from B6-Gpi-1a mice pretreated with 5-fluorouracil were incubated for 2 h at 37 degrees C in the presence of the recombinant adenovirus-associated virus-based vector (rAAV) SSV9. As measured in vitro immediately following transduction, SSV9 was found to be effective in transducing the primitive cobble-stone-area-forming cell (CAFC)-35 subset (60% transduction efficiency). However, this did not predict long-term expression as the presence of the transgene could not be detected six months after transplantation of 1-2 x 106 transduced bone marrow stem cells into lethally irradiated recipients. CAFC analysis of bone marrow cells and Southern blot analysis of bone marrow and spleen cells were negative, and polymerase chain reaction analysis showed less than 0.1% transduction in bone marrow cells. Therefore, based on our study we conclude that rAAV transiently transduces hematopoietic stem cells but fails to integrate into the genome, leading to the loss of the reporter gene within the first six months after transplantation in vivo.

  4. Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

    Science.gov (United States)

    Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

    2017-01-01

    Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192. © 2016 International Society for Neurochemistry.

  5. Gene therapy with recombinant adeno-associated vectors for neovascular age-related macular degeneration: 1 year follow-up of a phase 1 randomised clinical trial.

    Science.gov (United States)

    Rakoczy, Elizabeth P; Lai, Chooi-May; Magno, Aaron L; Wikstrom, Matthew E; French, Martyn A; Pierce, Cora M; Schwartz, Steven D; Blumenkranz, Mark S; Chalberg, Thomas W; Degli-Esposti, Mariapia A; Constable, Ian J

    2015-12-12

    Neovascular, or wet, age-related macular degeneration causes central vision loss and represents a major health problem in elderly people, and is currently treated with frequent intraocular injections of anti-VEGF protein. Gene therapy might enable long-term anti-VEGF therapy from a single treatment. We tested the safety of rAAV.sFLT-1 in treatment of wet age-related macular degeneration with a single subretinal injection. In this single-centre, phase 1, randomised controlled trial, we enrolled patients with wet age-related macular degeneration at the Lions Eye Institute and the Sir Charles Gairdner Hospital (Nedlands, WA, Australia). Eligible patients had to be aged 65 years or older, have age-related macular degeneration secondary to active subfoveal choroidal neovascularisation, with best corrected visual acuity (BCVA) of 3/60-6/24 and 6/60 or better in the other eye. Patients were randomly assigned (3:1) to receive either 1 × 10(10) vector genomes (vg; low-dose rAAV.sFLT-1 group) or 1 × 10(11) vg (high-dose rAAV.sFLT-1 group), or no gene-therapy treatment (control group). Randomisation was done by sequential group assignment. All patients and investigators were unmasked. Staff doing the assessments were masked to the study group at study visits. All patients received ranibizumab at baseline and week 4, and rescue treatment during follow-up based on prespecified criteria including BCVA measured on the Early Treatment Diabetic Retinopathy Study (EDTRS) scale, optical coherence tomography, and fluorescein angiography. The primary endpoint was ocular and systemic safety. This trial is registered with ClinicalTrials.gov, number NCT01494805. From Dec 16, 2011, to April 5, 2012, we enrolled nine patients of whom eight were randomly assigned to receive either intervention (three patients in the low-dose rAAV.sFLT-1 group and three patients in the high-dose rAAV.sFLT-1 group) or no treatment (two patients in the control group). Subretinal injection of r

  6. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype

    National Research Council Canada - National Science Library

    Ellis, Brian L; Hirsch, Matthew L; Barker, Jenny C; Connelly, Jon P; Steininger, 3rd, Robert J; Porteus, Matthew H

    2013-01-01

    .... Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity...

  7. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S. (Oregon HSU)

    2012-05-24

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  8. Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

    Energy Technology Data Exchange (ETDEWEB)

    Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

    2012-02-05

    Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites of AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.

  9. Study on the expression of human lysozyme in oviduct bioreactor mediated by recombinant avian adeno-associated virus.

    Science.gov (United States)

    Wang, A P; Wang, Y J; Wu, S; Zuo, W Y; Guo, C M; Hong, W M; Zhu, S Y

    2017-07-01

    Due to its antimicrobial properties and low toxicity, human lysozyme (hLYZ) has broad application in the medical field and as a preservative used by the food industry. However, limited availability hinders its widespread use. Hence, we constructed a recombinant avian adeno-associated virus (rAAAV) that would specifically express hLYZ in the chicken oviduct and harvested hLYZ from the egg whites of laying hens. The oviduct-specific human lysozyme expression cassette flanked by avian adeno-associated virus (AAAV) inverted terminal repeats (ITRs) was subcloned into the modified baculovirus transfer vector pFBX, and then the recombinant baculovirus rBac-ITRLYZ was generated. The recombinant avian adeno-associated virus was produced by co-infecting Sf9 cells with rBac-ITRLYZ and the other 2 baculoviruses containing AAAV functional genes and structural genes, respectively. Electron microscopy and real-time PCR revealed that the recombinant viral particles were generated successfully with a typical AAAV morphology and a high titer. After one intravenous injection of each laying hen with 2 × 1011 viral particles, oviduct-specific expression of recombinant human lysozyme (rhLYZ) was detected by reverse transcription-PCR. The expression level of rhLYZ in the first wk increased to 258 ± 11.5 μg/mL, reached a maximum of 683 ± 16.4 μg/mL at the fifth wk, and then progressively declined during the succeeding 7 wk of the study. Western blotting indicated that the oviduct-expressed rhLYZ had the same molecular weight as the natural enzyme. These results indicate that an efficient and convenient oviduct bioreactor mediated by rAAAV has been established, and it is useful for production of other recombinant proteins. © 2017 Poultry Science Association Inc.

  10. Gangliosides are essential for bovine adeno-associated virus entry.

    Science.gov (United States)

    Schmidt, Michael; Chiorini, John A

    2006-06-01

    Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. We have recently identified a bovine adeno-associated virus (BAAV) that demonstrates unique tropism and transduction activity compared to primate AAVs. To better understand the entry pathway and cell tropism of BAAV, we have characterized the initial cell surface interactions required for transduction with BAAV vectors. Like a number of AAVs, BAAV requires cell surface sialic acid groups for transduction and virus attachment. However, glycosphingolipids (GSLs), not cell surface proteins, were required for vector entry and transduction. Incorporation of gangliosides, ceramide-based glycolipids containing one or more sialic acid groups, into the cytoplasmic cell membranes of GSL-depleted COS cells partially reconstituted BAAV transduction. The dependency of BAAV on gangliosides for transduction was further confirmed by studies with C6 cells, a rat glioma cell line that is deficient in the synthesis of complex gangliosides. C6 cells were resistant to transduction by BAAV. Addition of gangliosides to C6 cells prior to transduction rendered the cells susceptible to transduction by BAAV. Therefore, gangliosides are a likely receptor for BAAV.

  11. Exploiting the Intron-splicing Mechanism of Insect Cells to Produce Viral Vectors Harboring Toxic Genes for Suicide Gene Therapy

    Directory of Open Access Journals (Sweden)

    Haifeng Chen

    2012-01-01

    Full Text Available Two mammalian introns, the human growth hormone intron and the Simian virus 40 large T antigen intron, were inserted into the coding sequences of diphtheria toxin fragment A (DT-A and barnase (Bar, respectively, to disrupt their open-reading frames (ORFs. Expression of these two toxic proteins were totally abolished, which enabled the production of normal levels of recombinant baculoviral and adeno-associated viral (AAV vectors in insect cells. When these viral vectors were introduced into mammalian cells, the introns were spliced out and the toxic proteins were expressed, which resulted in apoptosis in mammalian cells. This is the first report to show that viral vectors harboring toxin genes can be produced at normal levels by exploiting the intron-splicing mechanism of insect cells. Furthermore, viral vectors carrying the DT-A gene under control of tumor-specific promoters were able to exert tumor-specific cell killing. This novel method to produce viral vectors harboring toxic genes under control of tumor-specific promoter offers a powerful tool for further research, as well as for the development of toxin-based suicide gene therapy drugs.

  12. Viral vector-based influenza vaccines

    Science.gov (United States)

    de Vries, Rory D.; Rimmelzwaan, Guus F.

    2016-01-01

    ABSTRACT Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors. PMID:27455345

  13. Viral vector-based reversible neuronal inactivation and behavioral manipulation in the macaque monkey.

    Science.gov (United States)

    Nielsen, Kristina J; Callaway, Edward M; Krauzlis, Richard J

    2012-01-01

    Viral vectors are promising tools for the dissection of neural circuits. In principle, they can manipulate neurons at a level of specificity not otherwise achievable. While many studies have used viral vector-based approaches in the rodent brain, only a few have employed this technique in the non-human primate, despite the importance of this animal model for neuroscience research. Here, we report evidence that a viral vector-based approach can be used to manipulate a monkey's behavior in a task. For this purpose, we used the allatostatin receptor/allatostatin (AlstR/AL) system, which has previously been shown to allow inactivation of neurons in vivo. The AlstR was expressed in neurons in monkey V1 by injection of an adeno-associated virus 1 (AAV1) vector. Two monkeys were trained in a detection task, in which they had to make a saccade to a faint peripheral target. Injection of AL caused a retinotopic deficit in the detection task in one monkey. Specifically, the monkey showed marked impairment for detection targets placed at the visual field location represented at the virus injection site, but not for targets shown elsewhere. We confirmed that these deficits indeed were due to the interaction of AlstR and AL by injecting saline, or AL at a V1 location without AlstR expression. Post-mortem histology confirmed AlstR expression in this monkey. We failed to replicate the behavioral results in a second monkey, as AL injection did not impair the second monkey's performance in the detection task. However, post-mortem histology revealed a very low level of AlstR expression in this monkey. Our results demonstrate that viral vector-based approaches can produce effects strong enough to influence a monkey's performance in a behavioral task, supporting the further development of this approach for studying how neuronal circuits control complex behaviors in non-human primates.

  14. Characterization of the Adeno-Associated Virus 1 and 6 Sialic Acid Binding Site.

    Science.gov (United States)

    Huang, Lin-Ya; Patel, Ami; Ng, Robert; Miller, Edward Blake; Halder, Sujata; McKenna, Robert; Asokan, Aravind; Agbandje-McKenna, Mavis

    2016-06-01

    The adeno-associated viruses (AAVs), which are being developed as gene delivery vectors, display differential cell surface glycan binding and subsequent tissue tropisms. For AAV serotype 1 (AAV1), the first viral vector approved as a gene therapy treatment, and its closely related AAV6, sialic acid (SIA) serves as their primary cellular surface receptor. Toward characterizing the SIA binding site(s), the structure of the AAV1-SIA complex was determined by X-ray crystallography to 3.0 Å. Density consistent with SIA was observed in a pocket located at the base of capsid protrusions surrounding icosahedral 3-fold axes. Site-directed mutagenesis substitution of the amino acids forming this pocket with structurally equivalent residues from AAV2, a heparan sulfate binding serotype, followed by cell binding and transduction assays, further mapped the critical residues conferring SIA binding to AAV1 and AAV6. For both viruses five of the six binding pocket residues mutated (N447S, V473D, N500E, T502S, and W503A) abolished SIA binding, whereas S472R increased binding. All six mutations abolished or decreased transduction by at least 50% in AAV1. Surprisingly, the T502S substitution did not affect transduction efficiency of wild-type AAV6. Furthermore, three of the AAV1 SIA binding site mutants-S472R, V473D, and N500E-escaped recognition by the anti-AAV1 capsid antibody ADK1a. These observations demonstrate that common key capsid surface residues dictate both virus binding and entry processes, as well as antigenic reactivity. This study identifies an important functional capsid surface "hot spot" dictating receptor attachment, transduction efficiency, and antigenicity which could prove useful for vector engineering. The adeno-associated virus (AAV) vector gene delivery system has shown promise in several clinical trials and an AAV1-based vector has been approved as the first gene therapy treatment. However, limitations still exist with respect to transduction efficiency and

  15. Expression Profiles of Bovine Adeno-Associated Virus and Avian Adeno-Associated Virus Display Significant Similarity to That of Adeno-Associated Virus Type 5

    OpenAIRE

    Qiu, Jianming; Cheng, Fang; Pintel, David J.

    2006-01-01

    We present the first detailed expression profiles of nonprimate-derived adeno-associated viruses, namely, bovine adeno-associated virus (B-AAV) and avian adeno-associated virus (A-AAV), which were obtained after the infection of cell lines derived from their natural hosts. In general, the profiles of B-AAV and A-AAV were quite similar to that of AAV5; however, both exhibited features found for AAV2 as well. Like adeno-associated virus type 5 (AAV5), B-AAV and A-AAV utilized an internal polyad...

  16. Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy.

    Science.gov (United States)

    Li, Juan; Sun, Wunchang; Wang, Bing; Xiao, Xiao; Liu, Xiang-Qin

    2008-09-01

    Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Splitinteins catalyze precise ligation of two separate polypeptides through trans-splicing in a highly specific manner. Here we report a method of using protein trans-splicing to circumvent the packaging size limit of gene therapy vectors. To demonstrate this method, we chose a large dystrophin gene and an adeno-associated viral (AAV) vector, which has a small packaging size. A highly functional 6.3-kb Becker-form dystrophin cDNA was broken into two pieces and modified by adding appropriate split-intein coding sequences, resulting in splitgenes sufficiently small for packaging in AAV vectors. The two split-genes, after codelivery into target cells, produced two polypeptides that spontaneously trans-spliced to form the expected Becker-form dystrophin protein in cell culture in vitro. Delivering the split-genes by AAV1 vectors into the muscle of a mouse model of Duchenne muscular dystrophy rendered therapeutic gene expression and benefits.

  17. Neutralizing antibodies against adeno-associated viruses in inflammatory bowel disease patients: implications for gene therapy

    NARCIS (Netherlands)

    van der Marel, Sander; Comijn, Elisabeth M.; Verspaget, Hein W.; van Deventer, Sander; van den Brink, Gijs R.; Petry, Harald; Hommes, Daniel W.; Ferreira, Valerie

    2011-01-01

    Inflammatory bowel diseases (IBDs) are comprised of two major disorders: Crohn's disease (CD) and ulcerative colitis (UC). No curative treatment options are available, but gene therapy may offer an alternative therapeutic approach. For this a safe and reliable vector is needed. The adeno-associated

  18. Site-Specific PEGylated Adeno-Associated Viruses with Increased Serum Stability and Reduced Immunogenicity

    Directory of Open Access Journals (Sweden)

    Tianzhuo Yao

    2017-07-01

    Full Text Available Adeno-associated virus (AAV is one of the most extensively studied and utilized viral vectors in clinical gene transfer research. However, the serum instability and immunogenicity of AAV vectors significantly limit their application. Here, we endeavored to overcome these limitations by developing a straightforward approach for site-specific PEGylation of AAV via genetic code expansion. This technique includes incorporation of the azide moiety into the AAV capsid protein followed by orthogonal and stoichiometric conjugation of a variety of polyethylene glycols (PEGs through click chemistry. Using this approach, only the chosen site(s was consistently PEGylated under mild conditions, preventing nonselective conjugation. Upon a series of in vitro examinations, AAVs conjugated with 20-kD PEG at sites Q325+1, S452+1, and R585+1 showed a 1.7- to 2.4-fold stability improvement in pooled human serum and a nearly twofold reduction in antibody recognition. Subsequent animal research on Sprague Dawley rats displayed a promising 20% reduction in antibody inducement and a higher virus titer in the blood. Together, our data demonstrate successful protection of an AAV vector from antibody neutralization and blood clearance, thereby increasing the efficiency of therapeutic gene delivery.

  19. Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses.

    Science.gov (United States)

    Work, Lorraine M; Büning, Hildegard; Hunt, Ela; Nicklin, Stuart A; Denby, Laura; Britton, Nicola; Leike, Kristen; Odenthal, Margarete; Drebber, Uta; Hallek, Michael; Baker, Andrew H

    2006-04-01

    Virus-mediated gene delivery is restricted by the infectivity profile of the chosen vector. Targeting the vascular endothelium via systemic delivery has been attempted using peptides isolated in vitro (using either phage or vector display) and implicit reliance on target receptor expression in vivo. This has limited application since endothelial cells in vitro and in vivo differ vastly in receptor profiles and because of the existence of complex endothelial "zip codes" in vivo. We therefore tested whether in vivo phage display combined with adeno-associated virus (AAV) capsid modifications would allow in vivo homing to the endothelium residing in defined organs. Extensive in vivo biopanning in rats identified four consensus peptides homing to the lung or brain. Each was incorporated into the VP3 region of the AAV-2 capsid to display the peptide at the virion surface. Peptides that conferred heparan independence were shown to retarget virus to the expected vascular bed in vivo in a preferential manner, determined 28 days post-systemic injection by both virion DNA and transgene expression profiling. Our findings significantly impact the design of viral vectors for targeting individual vascular beds in vivo.

  20. Novel adeno-associated viruses derived from pig tissues transduce most major organs in mice.

    Science.gov (United States)

    Bello, Alexander; Chand, Allan; Aviles, Jenna; Soule, Geoff; Auricchio, Alberto; Kobinger, Gary P

    2014-10-22

    Recently, development of Adeno-associated virus (AAV) vectors has been focusing on expanding the genetic diversity of vectors from existing sequences via directed evolution or epitope remapping. Apart from intelligent design, AAV isolation from natural sources remains an important source of new AAVs with unique biological features. In this study, several new AAV sequences were isolated from porcine tissues (AAVpo2.1, -po4, -po5, and -po6), which aligned in divergent new clades. Viral particles generated from these sequences displayed tissue tropism and transduction efficiency profile specific to each porcine-derived AAV. When delivered systemically, AAVpo2.1 targeted the heart, kidney, and muscle, AAVpo5 performed poorly but was able to transduce muscle fibers when injected intramuscularly, whereas AAVpo4 and -po6 efficiently transduced all the major organs sampled, contending with 'gold-standard' AAVs. When delivered systemically, AAVpo4 and -po6 were detected by polymerase chain reaction (PCR) and histochemical staining of the transgene product in adult mouse brain, suggesting that these vectors can pass through the blood-brain barrier with efficiencies that may be useful for the development of therapeutic approaches. Porcine tissues are antigenically similar to human tissues and by inference, porcine AAVs may provide fresh tools to contribute to the development of gene therapy-based solutions to human diseases.

  1. Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

    Science.gov (United States)

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

  2. Viral vectors for cystic fibrosis gene therapy: What does the future hold?

    Directory of Open Access Journals (Sweden)

    Uta Griesenbach

    2010-12-01

    Full Text Available Uta Griesenbach1, Makoto Inoue2, Mamoru Hasegawa2, Eric WFW Alton11Department of Gene Therapy, Imperial College London, UK; The UK Cystic Fibrosis Gene Therapy Consortium; 2DNAVEC Corporation, Tsukuba, JapanAbstract: Gene transfer to the airway epithelium has been more difficult than originally anticipated, largely because of significant extra- and intracellular barriers in the lung. In general, viral vectors are more adapted to overcoming these barriers than nonviral gene transfer agents and are, therefore, more efficient in transferring genes into recipient cells. Viral vectors derived from adenovirus, adeno-associated virus, and Sendai virus, which all have a natural tropism for the airway epithelium, have been evaluated for cystic fibrosis (CF gene therapy. Although these vectors transduce airway epithelial cells efficiently, gene expression is transient and repeated administration is inefficient. They are, therefore, unlikely to be suitable for CF gene therapy. More recently, lentiviruses (LV have been assessed for lung gene transfer. In contrast to retroviruses, they transduce nondividing cells and randomly integrate into the genome. However, LVs do not have a natural tropism for the lung, and a significant amount of effort has been put into pseudotyping these vectors with proteins suitable for airway gene transfer. Several studies have shown that LV-mediated transduction leads to persistent gene expression (for the lifetime of the animal in the airways and, importantly, repeated administration is feasible. Thus, appropriately pseudotyped LV vectors are promising candidates for CF gene therapy. Here, we will review preclinical and clinical research related to viral CF gene therapy.Keywords: cystic fibrosis, gene therapy, adenovirus, AAV, lentivirus, Sendai virus

  3. Viral Vectors for in Vivo Gene Transfer

    Science.gov (United States)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  4. Recombinant avian adeno-associated virus-mediated oviduct-specific expression of recombinant human tissue kallikrein.

    Science.gov (United States)

    Wang, A P; Sun, H C; Wang, J Y; Wang, Y J; Yuan, W F

    2008-04-01

    Human tissue kallikrein (hK1) plays an important role in regulation of blood pressure, electrolyte and glucose transport, and renal function. To evaluate the feasibility of viral vector-mediated expression of recombinant human tissue kallikrein (rhK1) in the egg white of laying hens, human tissue kallikrein gene (hKLK1) cDNA-expression cassette was subcloned into avian adeno-associated virus (AAAV) transfer vector pAITR and transfected into AAV-293 cells with AAAV helper vector pcDNA-ARC and adenovirus helper vector pHelper. The recombinant viral particles with a typical AAAV morphology and relatively high titer were generated and identified by PCR and electron microscopy. After 1 intravenous injection of each laying hen with 2 x 10(10) viral particles, oviduct-specific expression of hKLK1 cDNA was demonstrated by reverse transcription-PCR. Secretion of rhK1 into the egg white was detected by enzymatic assay from d 2, reaching the highest level of 107 U/mL in wk 3, and lasted for more than 6 wk after injection. Western blotting showed that the oviduct-expressed rhK1 had the same molecular mass with the natural enzyme. These data suggest that rAAAV can mediate high level and long-lasting transgene expression in oviduct cells, and the established expression system is useful for production of other recombinant proteins.

  5. Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

    Science.gov (United States)

    Emmerling, Verena V; Pegel, Antje; Milian, Ernest G; Venereo-Sanchez, Alina; Kunz, Marion; Wegele, Jessica; Kamen, Amine A; Kochanek, Stefan; Hoerer, Markus

    2016-02-01

    Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Impact of capsid modifications by selected peptide ligands on recombinant adeno-associated virus serotype 2-mediated gene transduction.

    Science.gov (United States)

    Naumer, Matthias; Popa-Wagner, Ruth; Kleinschmidt, Jürgen A

    2012-10-01

    Vectors based on adeno-associated virus serotype 2 (AAV2) belong to today's most promising and most frequently used viral vectors in human gene therapy. Like in many other vector systems, the broad but non-specific tropism limits their use for certain cell types or tissues. One approach to screen for transduction-improved vectors is the selection of random peptide libraries displayed directly on the AAV2 capsid. Although the AAV2 library system has been widely applied for the successful selection of improved gene therapy vectors, it remains unknown which steps of the transduction process are most affected and therefore critical for the selection of targeting peptides. Attachment to the cell surface is the first essential step of AAV-mediated gene transduction; however, our experiments challenge the conventional belief that enhanced gene transfer is equivalent to more efficient cell binding of recombinant AAV2 vectors. A comparison of the various steps of gene transfer by vectors carrying a wild-type AAV2 capsid or displaying two exemplary peptide ligands selected from AAV2 random libraries on different human tumour cell lines demonstrated strong alterations in cell binding, cellular uptake, as well as intracellular processing of these vectors. Combined, our results suggest that entry and post-entry events are decisive for the selection of the peptides NDVRSAN and GPQGKNS rather than their cell binding efficiency.

  7. Adeno-associated virus-mediated gene transfer.

    Science.gov (United States)

    Srivastava, Arun

    2008-09-01

    Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. (c) 2008 Wiley-Liss, Inc.

  8. Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor

    Science.gov (United States)

    Pillay, Sirika; Zou, Wei; Cheng, Fang; Puschnik, Andreas S.; Meyer, Nancy L.; Ganaie, Safder S.; Deng, Xuefeng; Wosen, Jonathan E.; Davulcu, Omar; Yan, Ziying; Engelhardt, John F.; Brown, Kevin E.; Chapman, Michael S.

    2017-01-01

    ABSTRACT Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor. IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life

  9. High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing

    OpenAIRE

    Marsic, Damien; Méndez-Gómez, Héctor R; Zolotukhin, Sergei

    2015-01-01

    Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visua...

  10. Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

    OpenAIRE

    Li, Chengwen; DiPrimio, Nina; Bowles, Dawn E.; Hirsch, Matthew L.; Monahan, Paul E.; Asokan, Aravind; Rabinowitz, Joseph; Agbandje-McKenna, Mavis; Samulski, R. Jude

    2012-01-01

    Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize...

  11. Unique glycan signatures regulate adeno-associated virus tropism in the developing brain.

    Science.gov (United States)

    Murlidharan, Giridhar; Corriher, Travis; Ghashghaei, H Troy; Asokan, Aravind

    2015-04-01

    Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA(+) ependymal lining to 2,8-linked PSA(+) migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA- and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain. Viruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Viral Vector-Based Dissection of Marmoset GFAP Promoter in Mouse and Marmoset Brains.

    Directory of Open Access Journals (Sweden)

    Yoichiro Shinohara

    Full Text Available Adeno-associated virus (AAV vectors are small in diameter, diffuse easily in the brain, and represent a highly efficient means by which to transfer a transgene to the brain of a large animal. A major demerit of AAV vectors is their limited accommodation capacity for transgenes. Thus, a compact promoter is useful when delivering large transgenes via AAV vectors. In the present study, we aimed to identify the shortest astrocyte-specific GFAP promoter region that could be used for AAV-vector-mediated transgene expression in the marmoset brain. The 2.0-kb promoter region upstream of the GFAP gene was cloned from the marmoset genome, and short promoters (1.6 kb, 1.4 kb, 0.6 kb, 0.3 kb and 0.2 kb were obtained by progressively deleting the original 2.0-kb promoter from the 5' end. The short promoters were screened in the mouse cerebellum in terms of their strength and astrocyte specificity. We found that the 0.3-kb promoter maintained 40% of the strength of the original 2.0-kb promoter, and approximately 90% of its astrocyte specificity. These properties were superior to those of the 1.4-kb, 0.6-kb (20% promoter strength and 0.2-kb (70% astrocyte specificity promoters. Then, we verified whether the 0.3-kb GFAP promoter retained astrocyte specificity in the marmoset cerebral cortex. Injection of viral vectors carrying the 0.3-kb marmoset GFAP promoter specifically transduced astrocytes in both the cerebral cortex and cerebellar cortex of the marmoset. These results suggest that the compact 0.3-kb promoter region serves as an astrocyte-specific promoter in the marmoset brain, which permits us to express a large gene by AAV vectors that have a limited accommodation capacity.

  13. Advances in Non-Viral DNA Vectors for Gene Therapy

    Directory of Open Access Journals (Sweden)

    Cinnamon L. Hardee

    2017-02-01

    Full Text Available Uses of viral vectors have thus far eclipsed uses of non-viral vectors for gene therapy delivery in the clinic. Viral vectors, however, have certain issues involving genome integration, the inability to be delivered repeatedly, and possible host rejection. Fortunately, development of non-viral DNA vectors has progressed steadily, especially in plasmid vector length reduction, now allowing these tools to fill in specifically where viral or other non-viral vectors may not be the best options. In this review, we examine the improvements made to non-viral DNA gene therapy vectors, highlight opportunities for their further development, address therapeutic needs for which their use is the logical choice, and discuss their future expansion into the clinic

  14. Selective and rapid uptake of adeno-associated virus type 2 in brain.

    Science.gov (United States)

    Bartlett, J S; Samulski, R J; McCown, T J

    1998-05-20

    Recombinant adeno-associated virus (AAV) vectors effectively transfer and express foreign genes in the brain. The transferred genes, however, are selectively expressed in neurons, and the cause of this specificity is not understood. To address this question, wild-type AAV-2 capsids were covalently labeled with the fluorophore, Cy3, and infused into the inferior colliculus or the hippocampus. Using antibodies to identify neurons (NeuN), astrocytes (GFAP), or oligodendrocytes (OX-42), clear neuron-specific uptake of the virus was observed as early as 6 min after the start of the infusion. By 30 min postinfusion, AAV particles were present in the nucleus of neurons, yet in both the inferior colliculus and hippocampus, a subset of neurons did not take up the virus particles. No AAV particles were found in astrocytes 1.5 min or 24 hr after virus infusion. Interestingly, 1 hr postinfusion, no AAV particles were found in microglia, yet by 24 hr postinfusion, a punctate pattern of AAV particles was found in microglia. To test whether virus uptake correlated with vector-transduced cells, an rAAV-CMV-GFP virus was infused. By 3 days postinfusion, GFP was localized to neuronal populations with no expression in astrocytes or microglia, similar to that of fluorescent virus uptake. These findings demonstrate that in brain, AAV particles rapidly bind and enter primarily neurons with a pattern similar to that of in vivo vector transduction. In addition, these studies indicate that viral binding and uptake, independent of promoter tropism, can explain the specificity of AAV brain transduction. Thus, this first description of AAV kinetic disposition in vivo should facilitate targeted application of this vector for human brain gene therapy.

  15. Prevalence and pharmacological modulation of humoral immunity to AAV vectors in gene transfer to synovial tissue

    NARCIS (Netherlands)

    Mingozzi, F.; Chen, Y.; Edmonson, S. C.; Zhou, S.; Thurlings, R. M.; Tak, P. P.; High, K. A.; Vervoordeldonk, M. J.

    2013-01-01

    Antibodies against adeno-associated viral (AAV) vectors are highly prevalent in humans. Both preclinical and clinical studies showed that antibodies against AAV block transduction even at low titers, particularly when the vector is introduced into the bloodstream. Here we measured the neutralizing

  16. Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain

    OpenAIRE

    Murlidharan, Giridhar; Corriher, Travis; Ghashghaei, H. Troy; Asokan, Aravind

    2015-01-01

    Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and t...

  17. Viral vector-mediated selective and reversible blockade of the pathway for visual orienting in mice

    Directory of Open Access Journals (Sweden)

    Tadashi eIsa

    2013-10-01

    Full Text Available Recently, by using a combination of two viral vectors, we developed a technique for pathway-selective and reversible synaptic transmission blockade, and successfully induced a behavioral deficit of dexterous hand movements in macaque monkeys by affecting a population of spinal interneurons. To explore the capacity of this technique to work in other pathways and species, and to obtain fundamental methodological information, we tried to block the crossed tecto-reticular pathway, which is known to control orienting responses to visual targets, in mice. A neuron-specific retrograde gene transfer vector with the gene encoding enhanced tetanus neurotoxin (eTeNT tagged with enhanced green fluorescent protein (EGFP under the control of a tetracycline responsive element was injected into the left medial pontine reticular formation. 7–17 days later, an adeno-associated viral vector with a highly efficient Tet-ON sequence, rtTAV16, was injected into the right superior colliculus. 5–9 weeks later, the daily administration of doxycycline (Dox was initiated. Visual orienting responses toward the left side were impaired 1 - 4 days after Dox administration. Anti-GFP immunohistochemistry revealed that a number of neurons in the intermediate and deep layers of the right superior colliculus were positively stained, indicating eTeNT expression. After the termination of Dox administration, the anti-GFP staining returned to the baseline level within 28 days. A second round of Dox administration, starting from 28 days after the termination of the first Dox administration, resulted in the reappearance of the behavioral impairment. These findings showed that pathway-selective and reversible blockade of synaptic transmission causes behavioral effects also in rodents, and that the crossed tecto-reticular pathway surely controls visual orienting behaviors.

  18. Capsid Antibodies to Different Adeno-Associated Virus Serotypes Bind Common Regions

    Science.gov (United States)

    Gurda, Brittney L.; DiMattia, Michael A.; Miller, Edward B.; Bennett, Antonette; McKenna, Robert; Weichert, Wendy S.; Nelson, Christian D.; Chen, Wei-jun; Muzyczka, Nicholas; Olson, Norman H.; Sinkovits, Robert S.; Chiorini, John A.; Zolotutkhin, Sergei; Kozyreva, Olga G.; Samulski, R. Jude; Baker, Timothy S.; Parrish, Colin R.

    2013-01-01

    Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies. PMID:23760240

  19. Adeno-associated viral vector serotypes 1 and 5 targeted to the neonatal rat and pig striatum induce widespread transgene expression in the forebrain

    DEFF Research Database (Denmark)

    Kornum, Birgitte R; Stott, Simon R W; Mattsson, Bengt

    2010-01-01

    as striatal input and output areas, including large parts of the cortex. In both species, rAAV5 resulted in a more widespread transgene expression compared to rAAV1. In neonatal rats, rAAV5 also transduced several other areas such as the olfactory bulbs, hippocampus, and septum. Phenotypic analysis of the GFP...

  20. Effect of adeno-associated virus serotype and genomic structure on liver transduction and biodistribution in mice of both genders.

    Science.gov (United States)

    Pañeda, Astrid; Vanrell, Lucia; Mauleon, Itsaso; Crettaz, Julien S; Berraondo, Pedro; Timmermans, Eric J; Beattie, Stuart G; Twisk, Jaap; van Deventer, Sander; Prieto, Jesus; Fontanellas, Antonio; Rodriguez-Pena, Maria Sol; Gonzalez-Aseguinolaza, Gloria

    2009-08-01

    Recombinant adeno-associated viral (AAV) vectors have unique properties, which make them suitable vectors for gene transfer. Here we assess the liver transduction efficiency and biodistribution of AAV-pseudotyped capsids (serotypes) 1, 5, 6, and 8, combined with single-stranded and double-stranded genomic AAV2 structures carrying the luciferase reporter gene after systemic administration. The analysis was performed in vivo and ex vivo, in male and female mice. Gender-related differences in AAV-mediated transduction and biodistribution were shown for the four serotypes. Our data confirm the superiority of AAV8 over the rest of the serotypes, as well as a significant advantage of double-stranded genomes in terms of liver transduction efficiency, particularly in females. Regarding biodistribution, AAV5 displayed a narrower tropism than the other serotypes tested, transducing, almost exclusively, the liver. Interestingly, AAV1 and AAV8, in particular those having single-stranded genomes, showed high transduction efficiency of female gonads. However, no inadvertent germ line transmission of AAV genomes was observed after breeding single-stranded AAV8-injected female mice with untreated males. In conclusion, double-stranded AAV8 vectors led to the highest levels of liver transduction in mice, as demonstrated by luciferase expression. Nevertheless, the transduction of other organs with AAV8 vectors could favor the use of less efficient serotypes, such as AAV5, which display a narrow tropism.

  1. Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies.

    Science.gov (United States)

    Tseng, Yu-Shan; Vliet, Kim Van; Rao, Lavanya; McKenna, Robert; Byrne, Barry J; Asokan, Aravind; Agbandje-McKenna, Mavis

    2016-10-01

    Adeno-associated viruses (AAVs) are promising viral vectors for therapeutic gene delivery, and the approval of an AAV1 vector for the treatment of lipoprotein lipase deficiency has heralded a new and exciting era for this system. However, preclinical and clinical studies show that neutralization from pre-existing antibodies is detrimental for medical application and this hurdle must be overcome before full clinical realization can be achieved. Thus the binding sites for capsid antibodies must be identified and eliminated through capsid engineering. Towards this goal and to recapitulate patient polyclonal responses, a panel of six new mouse monoclonal antibodies (MAbs) has been generated against AAV8 and AAV9 capsids, two vectors being developed for therapeutic application. Native (capsid) dot blot assays confirmed the specificity of these antibodies for their parental serotypes, with the exception of one MAb, HL2372, selected to cross-react against both capsids. Furthermore, in vitro assays showed that these MAbs are capable of neutralizing virus infection. These MAbs will be utilized for structural mapping of antigenic footprints on their respective capsids to inform development of the next generation of rAAV vectors capable of evading antibody neutralization while retaining parental tropism. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Gene therapy of inherited retinopathies: a long and successful road from viral vectors to patients.

    Science.gov (United States)

    Colella, Pasqualina; Auricchio, Alberto

    2012-08-01

    Inherited retinopathies (IRs) are common and untreatable blinding conditions inherited mostly as monogenic due to mutations in genes expressed in retinal photoreceptors (PRs) and in retinal pigment epithelium (RPE). Over the last two decades, the retina has emerged as one of the most favorable target tissues for gene therapy given its small size and its enclosed and immune-privileged environment. Different types of viral vectors have been developed, especially those based on the adeno-associated virus (AAV), which efficiently deliver therapeutic genes to PRs or RPE upon subretinal injections. Dozens of successful proofs of concept of the efficacy of gene therapy for recessive and dominant IRs have been generated in small and large models that have paved the way to the first clinical trials using AAV in patients with Leber congenital amaurosis, a severe form of childhood blindness. The results from these initial trials suggest that retinal gene therapy with AAV is safe in humans, that vision can be improved in patients that have suffered from severe impairment of visual function, in some cases for decades, and that readministration of AAV to the subretinal space is feasible, effective, and safe. However, none of the trials could match the levels of efficacy of gene therapy observed in a dog model of the disease, suggesting that there is room for improvement. In conclusion, these results bode well for further testing of AAV-mediated retinal gene therapy in patients with other monogenic and complex forms of blindness.

  3. Recombinant Adeno-Associated Virus-Mediated Expression of Methamphetamine Antibody Attenuates Methamphetamine-Induced Hyperactivity in Mice.

    Science.gov (United States)

    Chen, Yun-Hsiang; Wu, Kuo-Jen; Wu, Kuang-Lun; Wu, Kun-Lieh; Tsai, Ho-Min; Chen, Mao-Liang; Chen, Yi-Wei; Hsieh, Wei; Lin, Chun-Ming; Wang, Yun

    2017-04-07

    Methamphetamine (Meth) is one of the most frequently abused drugs worldwide. Recent studies have indicated that antibodies with high affinity for Meth reduce its pharmacological effects. The purpose of this study was to develop a technique for virus-based passive immunization against Meth effects. We generated a recombinant adeno-associated virus serotype-8 vector (AAV-MethAb) carrying the gene for a Meth-specific monoclonal antibody (MethAb). Infection of 293 cells with AAV-MethAb resulted in the expression and secretion of antibodies which bind to Meth. The viral vector was then examined in adult ICR mice. Systemic administration of AAV-MethAb resulted in long-term expression of MethAb in the serum for up to 29 weeks. Serum collected from the animals receiving AAV-MethAb retained a high specificity for (+)-Meth. Animals were challenged with Meth five weeks after viral injection. Meth levels in the brain and serum were reduced while Meth-induced locomotor activity was significantly attenuated. In conclusion, AAV-MethAb administration effectively depletes Meth from brain and serum while reducing the behavioral response to Meth, and thus is a potential therapeutic approach for Meth abuse.

  4. Protection against infectious bursal disease virulent challenge conferred by a recombinant avian adeno-associated virus vaccine.

    Science.gov (United States)

    Perozo, F; Villegas, P; Estevez, C; Alvarado, I R; Purvis, L B; Williams, S

    2008-06-01

    The development and use of recombinant vaccine vectors for the expression of poultry pathogens proteins is an active research field. The adeno-associated virus (AAV) is a replication-defective virus member of the family Parvoviridae that has been successfully used for gene delivery in humans and other species. In this experiment, an avian adeno-associated virus (AAAV) expressing the infectious bursal disease virus (IBDV) VP2 protein (rAAAV-VP2) was evaluated for protection against IBDV-virulent challenge. Specific pathogen free (SPF) birds were inoculated with rAAAV-VP2 or with a commercial intermediate IBDV vaccine and then challenged with the Edgar strain. IBDV-specific antibody levels were observed in all vaccinated groups; titers were higher for the commercial vaccine group. The live, commercial vaccine induced adequate protection against morbidity and mortality; nevertheless, initial lymphoid depletion and follicular atrophy related to active viral replication was observed as early as day 14 and persisted up to day 28, when birds were challenged. No bursal tissue damage due to rAAAV-VP2 vaccination was observed. Eight-out-of-ten rAAAV-VP2-vaccinated birds survived the challenge and showed no clinical signs. The bursa:body weight ratio and bursa lesion scores in the rAAAV-VP2 group indicated protection against challenge. Therefore, transgenic expression of the VP2 protein after rAAAV-VP2 vaccination induced protective immunity against IBDV challenge in 80% of the birds, without compromising the bursa of Fabricius. The use of rAAAV virions for gene delivery represents a novel approach to poultry vaccination.

  5. How to Successfully Screen Random Adeno-Associated Virus Display Peptide Libraries In Vivo.

    Science.gov (United States)

    Körbelin, Jakob; Trepel, Martin

    2017-06-01

    Adeno-associated virus (AAV) has emerged as a very promising gene therapy vector. To enable tissue-directed gene expression, many artificially generated AAV variants have been established, often isolated from large pools of mutated capsids. Random peptide libraries displayed on AAV capsids have been used successfully to select vectors targeted to a given target cell or tissue in vitro and in vivo. However, the published methodology for screening of AAV libraries to isolate vectors with selective tissue tropism after intravenous administration in vivo has not been described in sufficient detail to address all critical steps. A step-by-step protocol is provided here.

  6. Neuropeptide Y Y1 receptor hippocampal overexpression via viral vectors is associated with modest anxiolytic-like and proconvulsant effects in mice

    DEFF Research Database (Denmark)

    Olesen, Mikkel V; Christiansen, Søren Hofman Oliveira; Gøtzsche, Casper René

    2012-01-01

    -like effect in rodents. The present study explored an alternative and more specific approach: overexpression of Y1 receptors. Using a recombinant adeno-associated viral vector (rAAV) encoding the Y1 gene (rAAV-Y1), we, for the first time, induced overexpression of functional transgene Y1 receptors...... in the hippocampus of adult mice and tested the animals in anxiety- and depression-like behavior. Hippocampal Y1 receptors have been suggested to mediate seizure-promoting effect, so the effects of rAAV-induced Y1 receptor overexpression were also tested in kainate-induced seizures. Y1 receptor transgene...... overexpression was found to be associated with modest anxiolytic-like effect in the open field and elevated plus maze tests, but no effect was seen on depression-like behavior using the tail suspension and forced swim tests. However, the rAAV-Y1 vector modestly aggravated kainate-induced seizures. These data...

  7. Safety of Liver Gene Transfer Following Peripheral Intravascular Delivery of Adeno-Associated Virus (AAV)-5 and AAV-6 in a Large Animal Model

    Science.gov (United States)

    Favaro, Patricia; Finn, Jonathan D.; Siner, Joshua I.; Wright, J. Fraser; High, Katherine A.

    2011-01-01

    Abstract Intravascular delivery of adeno-associated virus (AAV) vector is commonly used for liver-directed gene therapy. In humans, the high prevalence of neutralizing antibodies to AAV-2 capsid and the wide cross-reactivity with other serotypes hamper vector transduction efficacy. Moreover, the safety of gene-based approaches depends on vector biodistribution, vector dose, and route of administration. Here we sought to characterize the safety of AAV-5 and AAV-6 for liver-mediated human factor IX (hFIX) expression in rabbits at doses of 1 × 1012 or 1 × 1013 viral genomes/kg. Circulating therapeutic levels of FIX were observed in both cohorts of AAV-6-hFIX, whereas for AAV-5-hFIX only the high dose was effective. Long-lasting inhibitory antibodies to hFIX were detected in three of the 10 AAV-6-injected animals but were absent in the AAV-5 group. Overall, vector shedding in the semen was transient and vector dose-dependent. However, the kinetics of clearance were remarkably faster for AAV-5 (3–5 weeks) compared with AAV-6 (10–13 weeks). AAV-6 vector sequences outside the liver were minimal at 20–30 weeks post-injection. In contrast, AAV-5 exhibited relatively high amounts of vector DNA in tissues other than the liver. Together these data are useful to further define the safety and potential for clinical translation of these AAV vectors. PMID:21126217

  8. Adeno-associated virus-mediated L1 expression promotes functional recovery after spinal cord injury.

    Science.gov (United States)

    Chen, Jian; Wu, Junfang; Apostolova, Ivayla; Skup, Malgorzata; Irintchev, Andrey; Kügler, Sebastian; Schachner, Melitta

    2007-04-01

    Paucity of permissive molecules and abundance of inhibitory molecules in the injured spinal cord of adult mammals prevent axons from successful regeneration and, thus, contribute to the failure of functional recovery. Using an adeno-associated viral (AAV) vector, we expressed the regeneration-promoting cell adhesion molecule L1 in both neurons and glia in the lesioned spinal cord of adult mice. Exogenous L1, detectable already 1 week after thoracic spinal cord compression and immediate vector injection, was expressed at high levels up to 5 weeks, the longest time-period studied. Dissemination of L1-transduced cells throughout the spinal cord was wide, spanning over more than 10 mm rostral and 10 mm caudal to the lesion scar. L1 was not detectable in the fibronectin-positive lesion core. L1 overexpression led to improved stepping abilities and muscle coordination during ground locomotion over a 5-week observation period. Superior functional improvement was associated with enhanced reinnervation of the lumbar spinal cord by 5-HT axons. Corticospinal tract axons did not regrow beyond the lesion scar but extended distally into closer proximity to the injury site in AAV-L1-treated compared with control mice. The expression of the neurite outgrowth-inhibitory chondroitin sulphate proteoglycan NG2 was decreased in AAV-L1-treated spinal cords, along with reduction of the reactive astroglial marker GFAP. In vitro experiments confirmed that L1 inhibits astrocyte proliferation, migration, process extension and GFAP expression. Analyses of intracellular signalling indicated that exogenous L1 activates diverse cascades in neurons and glia. Thus, AAV-mediated L1 overexpression appears to be a potent means to favourably modify the local environment in the injured spinal cord and promote regeneration. Our study demonstrates a clinically feasible approach of promising potential.

  9. Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10

    Directory of Open Access Journals (Sweden)

    Flotte Terence R

    2005-02-01

    Full Text Available Abstract Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial activation or its downstream consequences. While several glial-selective drugs have been successful in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of the present studies was to explore a novel approach for controlling neuropathic pain. Here, an adeno-associated viral (serotype II; AAV2 vector was created that encodes the anti-inflammatory cytokine, interleukin-10 (IL-10. This anti-inflammatory cytokine is known to suppress the production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10 vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially infects meningeal cells surrounding the CSF space. Taken together, these data provide initial support that intrathecal gene therapy to drive the production of IL-10 may prove to be an efficacious treatment for neuropathic pain.

  10. Viral Vectors for Plant Genome Engineering.

    Science.gov (United States)

    Zaidi, Syed Shan-E-Ali; Mansoor, Shahid

    2017-01-01

    Recent advances in genome engineering (GE) has made it possible to precisely alter DNA sequences in plant cells, providing specifically engineered plants with traits of interest. Gene targeting efficiency depends on the delivery-method of both sequence-specific nucleases and repair templates, to plant cells. Typically, this is achieved using Agrobacterium mediated transformation or particle bombardment, both of which transform only a subset of cells in treated tissues. The alternate in planta approaches, stably integrating nuclease-encoding cassettes and repair templates into the plant genome, are time consuming, expensive and require extra regulations. More efficient GE reagents delivery methods are clearly needed if GE is to become routine, especially in economically important crops that are difficult to transform. Recently, autonomously replicating virus-based vectors have been demonstrated as efficient means of delivering GE reagents in plants. Both DNA viruses (Bean yellow dwarf virus, Wheat dwarf virus and Cabbage leaf curl virus) and RNA virus (Tobacco rattle virus) have demonstrated efficient gene targeting frequencies in model plants (Nicotiana benthamiana) and crops (potato, tomato, rice, and wheat). Here we discuss the recent advances using viral vectors for plant genome engineering, the current limitations and future directions.

  11. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses.

    Science.gov (United States)

    Calcedo, Roberto; Vandenberghe, Luk H; Gao, Guangping; Lin, Jianping; Wilson, James M

    2009-02-01

    Recombinant adeno-associated viruses (AAVs) have unique gene-transfer properties that speak to their potential as carriers for gene therapy or vaccine applications. However, the presence of neutralizing antibodies to AAV as a result of previous exposure can significantly limit effective gene transfer. In this study, we obtained 888 human serum samples from healthy volunteers in 10 countries around the world. Samples were assayed for neutralizing antibodies to AAV1, AAV2, AAV7, and AAV8, as well as to a novel, structurally distinct AAV vector, rh32.33, in an in vitro transduction inhibition assay. Our data revealed that neutralizing antibodies to AAV2 were the most prevalent antibodies in all regions, followed by antibodies to AAV1. The seroprevalences of antibodies to AAV7 and to AAV8 were lower than that for antibodies to AAV1, and neutralization of AAVrh32.33 was only rarely detected. Our data also indicate a strong linkage of seroreactivity between apparently distinct serotypes that has not been predicted previously in animal models.

  12. Adeno-associated virus rep protein synthesis during productive infection

    Energy Technology Data Exchange (ETDEWEB)

    Redemann, B.E.; Mendelson, E.; Carter, B.J.

    1989-02-01

    Adeno-associated virus (AAV) Rep proteins mediate viral DNA replication and can regulate expression from AAV genes. The authors studied the kinetics of synthesis of the four Rep proteins, Rep78, Rep68, Rep52, and Rep40, during infection of human 293 or KB cells with AAV and helper adenovirus by in vivo labeling with (/sup 35/S)methionine, immunoprecipitation, and immunoblotting analyses. Rep78 and Rep52 were readily detected concomitantly with detection of viral monomer duplex DNA replicating about 10 to 12 h after infection, and Rep68 and Rep40 were detected 2 h later. Rep78 and Rep52 were more abundant than Rep68 and Rep40 owing to a higher synthesis rate throughout the infectious cycle. In some experiments, very low levels of Rep78 could be detected as early as 4 h after infection. The synthesis rates of Rep proteins were maximal between 14 and 24 h and then decreased later after infection. Isotopic pulse-chase experiments showed that each of the Rep proteins was synthesized independently and was stable for at least 15 h. A slower-migrating, modified form of Rep78 was identified late after infection. AAV capsid protein synthesis was detected at 10 to 12 h after infection and also exhibited synthesis kinetics similar to those of the Rep proteins. AAV DNA replication showed at least two clearly defined stages. Bulk duplex replicating DNA accumulation began around 10 to 12 h and reached a maximum level at about 20 h when Rep and capsid protein synthesis was maximal. Progeny single-stranded DNA accumulation began about 12 to 13 h, but most of this DNA accumulated after 24 h when Rep and capsid protein synthesis had decreased.

  13. Adeno-Associated Virus (AAV) Type 5 Rep Protein Cleaves a Unique Terminal Resolution Site Compared with Other AAV Serotypes

    OpenAIRE

    Chiorini, John A.; Afione, Sandra; Kotin, Robert M

    1999-01-01

    Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome w...

  14. A molecular toolbox for rapid generation of viral vectors to up- or down-regulate in vivo neuronal gene expression

    Directory of Open Access Journals (Sweden)

    Melanie D. White

    2011-07-01

    Full Text Available We introduce a molecular toolbox for manipulation of neuronal gene expression in vivo. The toolbox includes promoters, ion channels, optogenetic tools, fluorescent proteins and intronic artificial microRNAs. The components are easily assembled into adeno-associated virus (AAV or lentivirus vectors using recombination cloning. We demonstrate assembly of toolbox components into lentivirus and AAV vectors and use these vectors for in vivo expression of inwardly rectifying potassium channels (Kir2.1, Kir3.1 and Kir3.2 and an artificial microRNA targeted against the ion channel HCN1 (HCN1 miR. We show that AAV assembled to express HCN1 miR produces efficacious and specific in vivo knockdown of HCN1 channels. Comparison of in vivo viral transduction using HCN1 miR with mice containing a germ line deletion of HCN1 reveals similar physiological phenotypes in cerebellar Purkinje cells. The easy assembly and re-usability of the toolbox components, together with the ability to up- or down-regulate neuronal gene expression in vivo, may be useful for applications in many areas of neuroscience.

  15. Characterization of Tissue Tropism Determinants of Adeno-Associated Virus Type 1

    OpenAIRE

    Hauck, Bernd; Xiao, Weidong

    2003-01-01

    Muscle is an attractive target for gene delivery because of its mass and because vectors can be delivered in a noninvasive fashion. Adeno-associated virus (AAV) has been shown to be effective for muscle-targeted gene transfer. Recent progress in characterization of AAV serotype 1 (AAV1) and AAV6 demonstrated that these two AAV serotypes are far more efficient in transducing muscle than is the traditionally used AAV2. Since all cis elements are identical in these vectors, the potential determi...

  16. Effects of Cellular Methylation on Transgene Expression and Site-Specific Integration of Adeno-Associated Virus.

    Science.gov (United States)

    Chanda, Diptiman; Hensel, Jonathan A; Higgs, Jerome T; Grover, Rajat; Kaza, Niroop; Ponnazhagan, Selvarangan

    2017-09-18

    DNA methylation is a major epigenetic event that affects not only cellular gene expression but that also has the potential to influence bacterial and viral DNA in their host-dependent functions. Adeno-associated virus (AAV) genome contains a high degree of CpG sequences capable of methylation in its terminal repeat sequences, which are the sole elements retained in AAV-based vectors used in gene therapy. The present study determined the influence of methylation status of the host cell on wild type (wt) AAV integration and recombinant (r) AAV transgene expression in HeLa cells. Results of the study indicated that hypo-methylation significantly enhanced both wtAAV chromosomal integration and transgene expression of rAAV. A direct influence of methylation on AAV integration was further confirmed by methylating the AAVS1 integration sites prior to viral infection with DNA trans-complementation assay. These results signify the importance of epigenetic status of target cells as one of the key factors in long-term transgene expression in AAV gene therapy.

  17. Effects of Cellular Methylation on Transgene Expression and Site-Specific Integration of Adeno-Associated Virus

    Directory of Open Access Journals (Sweden)

    Diptiman Chanda

    2017-09-01

    Full Text Available DNA methylation is a major epigenetic event that affects not only cellular gene expression but that also has the potential to influence bacterial and viral DNA in their host-dependent functions. Adeno-associated virus (AAV genome contains a high degree of CpG sequences capable of methylation in its terminal repeat sequences, which are the sole elements retained in AAV-based vectors used in gene therapy. The present study determined the influence of methylation status of the host cell on wild type (wt AAV integration and recombinant (r AAV transgene expression in HeLa cells. Results of the study indicated that hypo-methylation significantly enhanced both wtAAV chromosomal integration and transgene expression of rAAV. A direct influence of methylation on AAV integration was further confirmed by methylating the AAVS1 integration sites prior to viral infection with DNA trans-complementation assay. These results signify the importance of epigenetic status of target cells as one of the key factors in long-term transgene expression in AAV gene therapy.

  18. Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein.

    Science.gov (United States)

    Mori, Seiichiro; Wang, Lina; Takeuchi, Takamasa; Kanda, Tadahito

    2004-12-20

    We demonstrated the presence of two adeno-associated viruses (AAVs), designated AAV10 and AAV11, in cynomolgus monkeys by isolating and sequencing the entire viral coding regions from the monkey DNA. AAV10 and AAV11 capsid proteins shared 84% and 65%, respectively, of amino acids with AAV2. A phylogenetic analysis of AAV capsid proteins showed that AAV10 and AAV11 resembled most AAV8 and AAV4, respectively. To characterize the capsid protein, we pseudotyped an AAV2 vector with the monkey AAV capsid proteins and examined the resulting pseudotypes AAV2/10 and AAV2/11, in comparison with the AAV2 vector, for their host ranges in cell lines and tissue tropism in mice. AAV2/10 and AAV2/11 transduced primate cells less efficiently than AAV2. Whereas AAV2 transduced undifferentiated C2C12 mouse myoblasts more efficiently than differentiated ones, AAV2/10 and AAV2/11 transduced the undifferentiated myoblasts less efficiently than differentiated ones. Three weeks after injection to the muscle of the hind legs, AAV2/10 and AAV2 induced transgene expression similarly, but AAV2/11 did not transduce the skeletal muscle. Six weeks after systemic administration, transduced vector DNA was detected by PCR in the liver and spleen of mice inoculated with AAV2, in the liver, heart, muscle, lung, kidney, and uterus of mice with AAV2/10, and the muscle, kidney, spleen, lung, heart, and stomach of mice with AAV2/11. Mouse antisera against capsid protein VP2 of the three AAVs neutralized the respective vector particles in a type-specific manner. The results indicate that AAV10 and AAV11 capsid proteins, which are antigenically distinct from each other and AAV2, are likely to determine their host ranges and tissue tropism that are different from AAV2s, suggesting that cynomolgus AAVs could provide a broader choice of pseudotype AAV vectors for gene therapy.

  19. Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.

    Science.gov (United States)

    Wagner, Stefan; Thresher, Rosemary; Bland, Ross; Laible, Götz

    2015-10-14

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland.

  20. AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations

    NARCIS (Netherlands)

    Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, J.; Moloney, E.; Keynes, Roger J; Fawcett, James W; Rogers, John H

    2014-01-01

    As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression

  1. High density recombinant AAV particles are competent vectors for in vivo transduction

    Science.gov (United States)

    Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed heavier particles found in AAV preparations have traditionally been ignored due to its low in vitro infectivity. In this study, we systemically compared t...

  2. Inhalation of Nebulized Perfluorochemical Enhances Recombinant Adenovirus and Adeno-Associated Virus-Mediated Gene Expression in Lung Epithelium

    OpenAIRE

    Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J.; Wang, Lili; Gao, Guang Ping; Kolls, Jay K.; Bohm, Rudolf; Liggitt, Denny; Weiss, Daniel J.

    2012-01-01

    Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Reco...

  3. Adeno-Associated Virus Serotypes 1, 8, and 9 Share Conserved Mechanisms for Anterograde and Retrograde Axonal Transport

    OpenAIRE

    Castle, Michael J; Gershenson, Zachary T.; Giles, April R.; Holzbaur, Erika L. F.; Wolfe, John H

    2014-01-01

    Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector tr...

  4. Cloning and characterization of a bovine adeno-associated virus.

    Science.gov (United States)

    Schmidt, Michael; Katano, Hisako; Bossis, Ioannis; Chiorini, John A

    2004-06-01

    To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications.

  5. An essential receptor for adeno-associated virus infection.

    Science.gov (United States)

    Pillay, S; Meyer, N L; Puschnik, A S; Davulcu, O; Diep, J; Ishikawa, Y; Jae, L T; Wosen, J E; Nagamine, C M; Chapman, M S; Carette, J E

    2016-02-04

    Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr(-/-) (also known as Au040320(-/-) and Kiaa0319l(-/-)) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

  6. Recombinant adeno-associated virus-, polyethylenimine/plasmid- and lipofectamine/carboxyfluorescein-labeled small interfering RNA-based transfection in retinal pigment epithelial cells with ultrasound and/or SonoVue

    National Research Council Canada - National Science Library

    LI, HONGLI; WAN, CAIFENG; LI, FENGHUA

    2015-01-01

    ...)-mediated transfection of the type 2 recombinant adeno-associated virus (AAV) vectors encoding the enhanced green fluorescent protein (EGFP) gene (rAAV), polyethylenimine (PEI)/plasmid EGFP-N1 (pDNA) or lipofectamine (L...

  7. Hybrid viral vectors for vaccine and antibody production in plants.

    Science.gov (United States)

    Yusibov, Vidadi; Streatfield, Stephen J; Kushnir, Natasha; Roy, Gourgopal; Padmanaban, Annamalai

    2013-01-01

    Plants have a demonstrated potential for large-scale, rapid production of recombinant proteins for diverse product applications, including subunit vaccines and monoclonal antibodies. In this field, the accent has recently shifted from the engineering of "edible" vaccines based on stable expression of target protein in transgenic or transplastomic plants to the development of purified formulated vaccines that are delivered via injection. The injectable vaccines are commonly produced using transient expression of target gene delivered into genetically unmodified plant host via viral or bacterial vectors. Most viral vectors are based on plant RNA viruses, where nonessential sequences are replaced with the gene of interest. Utilization of viral hybrids that consist of genes and regulatory elements of different virus species, or transcomplementation systems (vector/transgene) had a substantial impact on the level of target protein expression. Development and introduction of agroviral hybrid vectors that combine genetic elements of bacterial binary plasmids and plant viral vectors, and agroinfiltration as a tool of the vector delivery have resulted in significant progress in large-scale production of recombinant vaccines and monoclonal antibodies in plants. This article presents an overview of plant hybrid viral vector expression systems developed so far.

  8. Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes.

    Science.gov (United States)

    Wu, Zhijian; Asokan, Aravind; Grieger, Joshua C; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Samulski, R Jude

    2006-11-01

    Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 cells in vitro and in mouse liver following portal vein administration. In corollary, the converse E531K mutation in AAV1 imparts heparin binding ability and increases transduction efficiency. Extraction of vector genomes from liver tissue suggests that the lysine 531 residue assists in preferential transduction of parenchymal cells by AAV6 vectors in comparison with AAV1. Lysine 531 is unique to AAV6 among other known AAV serotypes and is located in a basic cluster near the spikes that surround the icosahedral threefold axes of the AAV capsid. Similar to studies with autonomous parvoviruses, this study describes the first example of single amino acid changes that can explain differential phenotypes such as viral titer, receptor binding, and tissue tropism exhibited by closely related AAV serotypes. In particular, a single lysine residue appears to provide the critical minimum charged surface required for interacting with heparin through electrostatic interaction and simultaneously plays an unrelated yet critical role in the liver tropism of AAV6 vectors.

  9. Viral Vectors for Use in the Development of Biodefense Vaccines

    National Research Council Canada - National Science Library

    Lee, John S; Hadjipanayis, Angela G; Parker, Michael D

    2005-01-01

    ... agents of bioterrorism or biowarfare. The use of viruses, for example adenovirus, vaccinia virus, and Venezuelan equine encephalitis virus, as vaccine-vectors has enabled researchers to develop effective means for countering the threat of bioterrorism and biowarfare. An overview of the different viral vectors and the threats they counter will be discussed.

  10. Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer.

    Directory of Open Access Journals (Sweden)

    Lina Li

    Full Text Available Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or "CELiD", DNA. CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5 × 10(9 Sf9 cells, and 1-15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.

  11. Successful expansion but not complete restriction of tropism of adeno-associated virus by in vivo biopanning of random virus display peptide libraries.

    Directory of Open Access Journals (Sweden)

    Stefan Michelfelder

    Full Text Available Targeting viral vectors to certain tissues in vivo has been a major challenge in gene therapy. Cell type-directed vector capsids can be selected from random peptide libraries displayed on viral capsids in vitro but so far this system could not easily be translated to in vivo applications. Using a novel, PCR-based amplification protocol for peptide libraries displayed on adeno-associated virus (AAV, we selected vectors for optimized transduction of primary tumor cells in vitro. However, these vectors were not suitable for transduction of the same target cells under in vivo conditions. We therefore performed selections of AAV peptide libraries in vivo in living animals after intravenous administration using tumor and lung tissue as prototype targets. Analysis of peptide sequences of AAV clones after several rounds of selection yielded distinct sequence motifs for both tissues. The selected clones indeed conferred gene expression in the target tissue while gene expression was undetectable in animals injected with control vectors. However, all of the vectors selected for tumor transduction also transduced heart tissue and the vectors selected for lung transduction also transduced a number of other tissues, particularly and invariably the heart. This suggests that modification of the heparin binding motif by target-binding peptide insertion is necessary but not sufficient to achieve tissue-specific transgene expression. While the approach presented here does not yield vectors whose expression is confined to one target tissue, it is a useful tool for in vivo tissue transduction when expression in tissues other than the primary target is uncritical.

  12. Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 1

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Edward B.; Gurda-Whitaker, Brittney; Govindasamy, Lakshmanan; McKenna, Robert [Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610 (United States); Zolotukhin, Sergei [Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL 32610 (United States); Muzyczka, Nicholas [Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32610 (United States); Agbandje-McKenna, Mavis, E-mail: mckenna@ufl.edu [Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610 (United States)

    2006-12-01

    Crystals of baculovirus-expressed adeno-associated virus serotype 1 (AAV1) capsids have been grown in the rhombohedral space group R32 (unit-cell parameters a = 254.7 Å, α = 62.3°) and shown to diffract X-rays to at least 2.5 Å resolution. Crystals of baculovirus-expressed adeno-associated virus serotype 1 (AAV1) capsids have been grown in the rhombohedral space group R32 (unit-cell parameters a = 254.7 Å, α = 62.3°) and shown to diffract X-rays to at least 2.5 Å resolution. The diffraction data were subsequently processed and reduced with an overall R{sub sym} of 12.3% and a completeness of 89.0%. Based on the unit-cell volume, rotation-function and translation-function results and packing considerations, there is one virus capsid (60 viral proteins) per unit cell and there are ten viral proteins per crystallographic asymmetric unit. The AAV1 capsid shares both the twofold and threefold crystallographic symmetry operators. The AAV1 data have been initially phased using a polyalanine model (based on the crystal structure of AAV4) to 4.0 Å resolution and the structure determination and refinement is in progress using tenfold noncrystallographic symmetry electron-density averaging.

  13. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype

    OpenAIRE

    Ellis, Brian L.; Hirsch, Matthew L.; Barker, Jenny C.; Connelly, Jon P; Steininger, Robert J; Matthew H Porteus

    2013-01-01

    Background The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV ser...

  14. A viral vector expressing hypoxia-inducible factor 1 alpha inhibits hippocampal neuronal apoptosis

    OpenAIRE

    Chai, Xiqing; Kong, Weina; Liu, Lingyun; Yu, Wenguo; Zhang, Zhenqing; Sun, Yimin

    2014-01-01

    Hypoxia-inducible factor 1 (HIF-1) attenuates amyloid-beta protein neurotoxicity and decreases apoptosis induced by oxidative stress or hypoxia in cortical neurons. In this study, we constructed a recombinant adeno-associated virus (rAAV) vector expressing the human HIF-1α gene (rAAV-HIF-1α), and tested the assumption that rAAV-HIF-1α represses hippocampal neuronal apoptosis induced by amyloid-beta protein. Our results confirmed that rAAV-HIF-1α significantly reduces apoptosis induced by amyl...

  15. Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells

    Directory of Open Access Journals (Sweden)

    Jan Christoph Koch

    2014-09-01

    Full Text Available The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV to specifically downregulate RhoA via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC grown on the inhibitory substrate CSPG as well as neurite regeneration of primary midbrain neurons after scratch lesion. In the rat optic nerve crush model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGCs transduced with AAV expressing RhoA-shRNA was substantially increased at two weeks after optic nerve axotomy.Compared to previous data using pharmacological inhibitors to target RhoA, its upstream regulator Nogo or its main downstream target ROCK2, the specific effects of RhoA downregulation shown here were more pronounced in regard to promoting RGC survival while the stimulatory effects on neurite outgrowth were rather moderate. Taken together, we show here that specific knockdown of RhoA substantially increases neuronal survival after optic nerve axotomy and modestly increases neurite outgrowth in vitro and axonal regeneration after optic nerve crush.

  16. Viral vector-mediated downregulation of RhoA increases survival and axonal regeneration of retinal ganglion cells.

    Science.gov (United States)

    Koch, Jan Christoph; Tönges, Lars; Michel, Uwe; Bähr, Mathias; Lingor, Paul

    2014-01-01

    The Rho/ROCK pathway is a promising therapeutic target in neurodegenerative and neurotraumatic diseases. Pharmacological inhibition of various pathway members has been shown to promote neuronal regeneration and survival. However, because pharmacological inhibitors are inherently limited in their specificity, shRNA-mediated approaches can add more information on the function of each single kinase involved. Thus, we generated adeno-associated viral vectors (AAV) to specifically downregulate Ras homologous member A (RhoA) via shRNA. We found that specific knockdown of RhoA promoted neurite outgrowth of retinal ganglion cells (RGC) grown on the inhibitory substrate chondroitin sulfate proteoglycan (CSPG) as well as neurite regeneration of primary midbrain neurons (PMN) after scratch lesion. In the rat optic nerve crush (ONC) model in vivo, downregulation of RhoA significantly enhanced axonal regeneration compared to control. Moreover, survival of RGC transduced with AAV expressing RhoA-shRNA was substantially increased at 2 weeks after optic nerve axotomy. Compared to previous data using pharmacological inhibitors to target RhoA, its upstream regulator Nogo or its main downstream target ROCK, the specific effects of RhoA downregulation shown here were most pronounced in regard to promoting RGC survival but neurite outgrowth and axonal regeneration were also increased significantly. Taken together, we show here that specific knockdown of RhoA substantially increases neuronal survival after optic nerve axotomy and modestly increases neurite outgrowth in vitro and axonal regeneration after optic nerve crush.

  17. Production, Purification and Preliminary X-ray Crystallographic Studies of Adeno-Associated Virus Serotype 9

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, M.; Nam, H; Carter, A; McCall, A; Rence, C; Bennett, A; Gurda, B; McKenna, R; Porter, M; et. al.

    2009-01-01

    Adeno-associated virus (AAV) serotype 9, which is under development for gene-delivery applications, shows significantly enhanced capsid-associated transduction efficiency in muscle compared with other AAV serotypes. With the aim of characterizing the structural determinants of this property, the purification, crystallization and preliminary X-ray crystallographic analyses of the AAV9 viral capsid are reported. The crystals diffracted X-rays to 2.8 A resolution using synchrotron radiation and belonged to the trigonal space group P32, with unit-cell parameters a = b = 251.0, c = 640.0 A. There are three complete viral capsids in the crystal unit cell. The orientation and position of the asymmetric unit capsid have been determined by molecular-replacement methods and structure determination is in progress.

  18. Purification method for the avian adeno-associated virus.

    Science.gov (United States)

    Bauer, H J; Monreal, G

    1985-05-01

    Avian adeno-associated virus (AAAV) pre-purified by Uvasol extraction, one discontinuous and two CsCl equilibrium density gradients, was still considerably contaminated with avian adenovirus (CELO strain). Four different approaches were investigated in attempts to improve the elimination of the contaminating CELO virus. The contaminations were assayed by double immunodiffusion and indirect immunofluorescence. The immunoprecipitation of CELO virus with antiserum and protein A-Sepharose is the most effective method of obtaining purified AAAV free of CELO virus.

  19. Herpesviruses provide helper functions for avian adeno-associated parvovirus.

    Science.gov (United States)

    Bauer, H J; Monreal, G

    1986-01-01

    The avian herpesviruses infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as the mammalian herpesvirus pseudorabies virus (PRV) were able to provide complete helper activity for the production of infectious avian adeno-associated virus (AAAV) in chicken cells. The presence of AAAV in the infected chicken cell reduced the multiplication of HVT. ILTV or PRV, however, were not affected if used as helper viruses. Infectious AAAV was determined by an indirect immunofluorescence assay and infectious herpesvirus by plaque assays.

  20. Adeno-associated virus inverted terminal repeats stimulate gene editing

    OpenAIRE

    Hirsch, ML

    2014-01-01

    Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repea...

  1. Replicon RNA Viral Vectors as Vaccines

    Directory of Open Access Journals (Sweden)

    Kenneth Lundstrom

    2016-11-01

    Full Text Available Single-stranded RNA viruses of both positive and negative polarity have been used as vectors for vaccine development. In this context, alphaviruses, flaviviruses, measles virus and rhabdoviruses have been engineered for expression of surface protein genes and antigens. Administration of replicon RNA vectors has resulted in strong immune responses and generation of neutralizing antibodies in various animal models. Immunization of mice, chicken, pigs and primates with virus-like particles, naked RNA or layered DNA/RNA plasmids has provided protection against challenges with lethal doses of infectious agents and administered tumor cells. Both prophylactic and therapeutic efficacy has been achieved in cancer immunotherapy. Moreover, recombinant particles and replicon RNAs have been encapsulated by liposomes to improve delivery and targeting. Replicon RNA vectors have also been subjected to clinical trials. Overall, immunization with self-replicating RNA viruses provides high transient expression levels of antigens resulting in generation of neutralizing antibody responses and protection against lethal challenges under safe conditions.

  2. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

    Science.gov (United States)

    Ellis, Brian L; Hirsch, Matthew L; Barker, Jenny C; Connelly, Jon P; Steininger, Robert J; Porteus, Matthew H

    2013-03-06

    The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

  3. Avian adeno-associated virus-based expression of Newcastle disease virus hemagglutinin-neuraminidase protein for poultry vaccination.

    Science.gov (United States)

    Perozo, F; Villegas, P; Estevez, C; Alvarado, I R; Purvis, L B; Saume, E

    2008-06-01

    The avian adeno-associated virus (AAAV) is a replication-defective nonpathogenic virus member of the family Parvoviridae that has been proved to be useful as a viral vector for gene delivery. The use of AAAV for transgenic expression of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein and its ability to induce immunity in chickens were assessed. Proposed advantages of this system include no interference with maternal antibodies, diminished immune response against the vector, and the ability to accommodate large fragments of genetic information. In this work the generation of recombinant AAAV virions expressing the HN protein (rAAAV-HN) was demonstrated by electron microscopy, immunocytochemistry, and western blot analysis. Serological evidence of HN protein expression after in ovo or intramuscular inoculation of the recombinant virus in specific-pathogen-free chickens was obtained. Serum from rAAAV-HN-vaccinated birds showed a systemic immune response evidenced by NDV-specific enzyme-linked immunosorbent assay and hemagglutination inhibition testing. Positive virus neutralization in embryonated chicken eggs and indirect immunofluorescence detection of NDV infected cells by serum from rAAAV-HN vaccinated birds is also reported. A vaccine-challenge experiment in commercial broiler chickens using a Venezuelan virulent viscerotropic strain of NDV was performed. All unvaccinated controls died within 5 days postchallenge. Protection up to 80% was observed in birds vaccinated in ovo and revaccinated at 7 days of age with the rAAAV-HN. The results demonstrate the feasibility of developing and using an AAAV-based gene delivery system for poultry vaccination.

  4. Gene delivery to the vasculature mediated by low-titre adeno-associated virus serotypes 1 and 5.

    Science.gov (United States)

    Sen, S; Conroy, S; Hynes, S O; McMahon, J; O'Doherty, A; Bartlett, J S; Akhtar, Y; Adegbola, T; Connolly, C E; Sultan, S; Barry, F; Katusic, Z S; O'Brien, T

    2008-02-01

    Vascular gene therapy requires safe and efficient gene transfer in vivo. Recombinant adeno-associated virus (AAV) is a promising viral vector but its use in the vasculature has produced conflicting results and serotypes other than AAV2 have not been intensively studied. We investigated the efficiency of alternative AAV serotypes for vascular gene delivery in vitro and in vivo. Vascular cell lines were transduced in vitro with AAV vectors. Rabbit carotid arteries were transduced with AAV1, 2 and 5 encoding enhanced green fluorescent protein (eGFP) ( approximately 1.4 x 10(9) DNAse-resistant particles (drp)). Gene transfer in vivo was assessed at 14 and 28 days. High-titre doses of AAV2 encoding beta-galactosidase in vivo were also studied. In vitro, transgene expression was not observed in endothelial cells using AAV2 whereas the use of serotypes 1 and 5 resulted in detectable levels of transgene expression. Coronary artery smooth muscle cells (CASMCs) transduced with AAV2 demonstrated higher levels of GFP expression than AAV1 or 5. Transgene expression in vivo was noted using low-titre AAV1 and AAV5 ( approximately 1.4 x 10(9) drp) in the media and adventitia. Only delivery of AAV1eGFP resulted in neointimal formation (3/7 vessels examined), with transgene expression noted in the neointima. Transgene expression with AAV2 was not detected in any layer of the blood vessel wall using low titre ( approximately 10(9) drp). However, high-titre ( approximately 10(11) drp) AAV2 resulted in transduction of cells in the media and adventitia but not the endothelium. AAV1 and AAV5 have advantages over AAV2 for vascular gene delivery at low titres.

  5. Efficient gene transfer into neurons in monkey brain by adeno-associated virus 8.

    Science.gov (United States)

    Masamizu, Yoshito; Okada, Takashi; Ishibashi, Hidetoshi; Takeda, Shin'ichi; Yuasa, Shigeki; Nakahara, Kiyoshi

    2010-04-21

    Although the adeno-associated virus (AAV) vector is a promising tool for gene transfer into neurons, especially for therapeutic purposes, neurotropism in primate brains is not fully elucidated for specific AAV serotypes. Here, we injected AAV serotype 8 (AAV8) vector carrying the enhanced green fluorescent protein (EGFP) gene under a ubiquitous promoter into the cerebral cortex, striatum and substantia nigra of common marmosets. Robust neuronal EGFP expression was observed at all injected sites. Cell typing with immunohistochemistry confirmed efficient AAV8-mediated gene transfer into the pyramidal neurons in the cortex, calbindin-positive medium spiny neurons in the striatum and dopaminergic neurons in the substantia nigra. The results indicate a preferential tropism of AAV8 for subsets of neurons, but not for glia, in monkey brains.

  6. Genome Engineering Using Adeno-associated Virus: Basic and Clinical Research Applications.

    Science.gov (United States)

    Gaj, Thomas; Epstein, Benjamin E; Schaffer, David V

    2016-03-01

    In addition to their broad potential for therapeutic gene delivery, adeno-associated virus (AAV) vectors possess the innate ability to stimulate homologous recombination in mammalian cells at high efficiencies. This process--referred to as AAV-mediated gene targeting--has enabled the introduction of a diverse array of genomic modifications both in vitro and in vivo. With the recent emergence of targeted nucleases, AAV-mediated genome engineering is poised for clinical translation. Here, we review key properties of AAV vectors that underscore its unique utility in genome editing. We highlight the broad range of genome engineering applications facilitated by this technology and discuss the strong potential for unifying AAV with targeted nucleases for next-generation gene therapy.

  7. Inhalation of nebulized perfluorochemical enhances recombinant adenovirus and adeno-associated virus-mediated gene expression in lung epithelium.

    Science.gov (United States)

    Beckett, Travis; Bonneau, Laura; Howard, Alan; Blanchard, James; Borda, Juan; Weiner, Daniel J; Wang, Lili; Gao, Guang Ping; Kolls, Jay K; Bohm, Rudolf; Liggitt, Denny; Weiss, Daniel J

    2012-04-01

    Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (pliquid perflubron, safely enhances lung gene expression.

  8. Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain

    OpenAIRE

    Sandro Alves; Julia Bode; Alexis-Pierre Bemelmans; Christof von Kalle; Nathalie Cartier; Björn Tews

    2016-01-01

    Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. ...

  9. Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease.

    Science.gov (United States)

    Gray-Edwards, Heather L; Randle, Ashley N; Maitland, Stacy A; Benatti, Hector R; Hubbard, Spencer M; Canning, Peter F; Vogel, Matthew B; Brunson, Brandon L; Hwang, Misako; Ellis, Lauren E; Bradbury, Allison M; Gentry, Atoska S; Taylor, Amanda R; Wooldridge, Anne A; Wilhite, Dewey R; Winter, Randolph L; Whitlock, Brian K; Johnson, Jacob A; Holland, Merilee; Salibi, Nouha; Beyers, Ronald J; Sartin, James L; Denney, Thomas S; Cox, Nancy R; Sena-Esteves, Miguel; Martin, Douglas R

    2017-09-18

    Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and β subunits separately (TSD α + β) injected at high (1.3 × 1013 vector genomes) or low (4.2 × 1012 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + β sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + β), and ganglioside clearance was most widespread in the TSD α + β high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

  10. Efficient and persistent transduction of exocrine and endocrine pancreas by adeno-associated virus type 8.

    Science.gov (United States)

    Cheng, Henrique; Wolfe, Stephanie H; Valencia, Valery; Qian, Keping; Shen, Leping; Phillips, M Ian; Chang, Lung-Ji; Zhang, Y Clare

    2007-09-01

    Efficient delivery of therapeutic proteins into the pancreas represents a major obstacle to gene therapy of pancreatic disorders. The current study compared the efficiency of recombinant lentivirus and adeno-associated virus (AAV) serotypes 1, 2, 5, 8 vectors delivered by intrapancreatic injection for gene transfer in vivo. Our results indicate that lentivirus and AAV 1, 2, 8 are capable of transducing pancreas with the order of efficiency AAV8 >AAV1 > AAV2 >/= lentivirus, whereas AAV5 was ineffective. AAV8 resulted in an efficient, persistent (150 days) and dose-dependent transduction in exocrine acinar cells and endocrine islet cells. Pancreatic ducts and blood vessels were also transduced. Extrapancreatic transduction was restricted to liver. Leukocyte infiltration was not observed in pancreas and blood glucose levels were not altered. Thus, AAV8 represents a safe and effective vehicle for therapeutic gene transfer to pancreas in vivo.

  11. Adeno-Associated Virus Serotype 8 Gene Transfer Rescues a Neonatal Lethal Murine Model of Propionic Acidemia

    Science.gov (United States)

    Chandler, Randy J.; Chandrasekaran, Suma; Carrillo-Carrasco, Nuria; Senac, Julien S.; Hofherr, Sean E.; Barry, Michael A.

    2011-01-01

    Abstract Propionic acidemia (PA) is an autosomal recessive disorder of metabolism caused by a deficiency of propionyl-coenzyme A carboxylase (PCC). Despite optimal dietary and cofactor therapy, PA patients still suffer from lethal metabolic instability and experience multisystemic complications. A murine model of PA (Pcca–/–) of animals that uniformly die within the first 48 hr of life was used to determine the efficacy of adeno-associated viral (AAV) gene transfer as a potential therapy for PA. An AAV serotype 8 (AAV8) vector was engineered to express the human PCCA cDNA and delivered to newborn mice via an intrahepatic injection. Greater than 64% of the Pcca–/– mice were rescued after AAV8-mediated gene transfer and survived until day of life 16 or beyond. Western analysis of liver extracts showed that PCC was completely absent from Pcca–/– mice but was restored to greater than wild-type levels after AAV gene therapy. The treated Pcca–/– mice also exhibited markedly reduced plasma levels of 2-methylcitrate compared with the untreated Pcca–/– mice, which indicates significant PCC enzymatic activity was provided by gene transfer. At the time of this report, the oldest treated Pcca–/– mice are over 6 months of age. In summary, AAV gene delivery of PCCA effectively rescues Pcca–/– mice from neonatal lethality and substantially ameliorates metabolic markers of the disease. These experiments demonstrate a gene transfer approach using AAV8 that might be used as a treatment for PA, a devastating and often lethal disorder desperately in need of new therapeutic options. PMID:20950151

  12. Trans-neuronal transduction of spinal neurons following cortical injection and anterograde axonal transport of a bicistronic AAV1 vector

    OpenAIRE

    Hutson, Thomas Haynes; Kathe, Claudia; Moon, Lawrence David Falcon

    2015-01-01

    Adeno-associated viral (AAV) vectors are one of the most promising gene delivery systems to the central nervous system. We now report, that AAV1 can be used to express transgenes trans-neuronally in neurons distant from the injection site. Specifically, intracortical injection of a bicistronic AAV1 vector trans-neuronally transduced spinal neurons as shown by fluorescence microscopy, the presence of AAV genome and AAV transcript in the contralateral spinal cord. Prior pyramidotomy abolished s...

  13. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    Energy Technology Data Exchange (ETDEWEB)

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.; Gurda-Whitaker, Brittney; Kalina, Amy [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Kohlbrenner, Erik [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Chiorini, John A. [GTTB, NIDCR, National Institutes of Health, Bethesda, MD 20892 (United States); McKenna, Robert [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Muzyczka, Nicholas [Department of Molecular Genetics and Microbiology and Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, FL 32610 (United States); Zolotukhin, Sergei [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Agbandje-McKenna, Mavis, E-mail: mckenna@ufl.edu [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States)

    2005-10-01

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  14. Methods of treating Parkinson's disease using viral vectors

    Energy Technology Data Exchange (ETDEWEB)

    Bankiewicz, Krys; Cunningham, Janet

    2012-11-13

    Methods of delivering viral vectors, particularly recombinant AAV virions, to the central nervous system (CNS) are provided for the treatment of CNS disorders, particularly those disorders which involve the neurotransmitter dopamine. The methods entail providing rAAV virions that comprise a transgene encoding aromatic amino acid decarboxylase (AADC) and administering the virions to the brain of a mammal using a non-manual pump.

  15. Tendon healing in vitro: adeno-associated virus-2 effectively transduces intrasynovial tenocytes with persistent expression of the transgene, but other serotypes do not.

    Science.gov (United States)

    Wang, Xiao Tian; Liu, Paul Y; Tang, Jin Bo; Mizukami, Hiroaki; Xin, Ke-Qin; Ozawa, Keiya; Ushijima, Hiroshi

    2007-01-01

    Transfer of exogenous growth factor genes to injured tendons offers a promising method for strengthening tendon repairs. Adeno-associated virus vectors have advantages of being both nonpathogenic and nontoxic. The authors explored the efficiency of transduction of intrasynovial tenocytes with different serotypes of adeno-associated virus (AAV) and the persistency of its expression of a growth factor transgene. Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed to 82 wells in eight culture plates and to 30 culture dishes. The tenocytes in the wells were treated with AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and AAV8 vectors containing the lacZ gene, and plasmid vectors (pCMVbeta-lacZ). The tenocytes were stained with in situ beta-galactosidase 5 days later. The basic fibroblast growth factor (bFGF) gene was cloned to the AAV2 vector to construct the AAV2-bFGF vector, which transduced tenocytes in culture dishes. Expression of the transgene was measured over 3 weeks and analyzed statistically. AAV2 effectively delivered exogenous genes to proliferating intrasynovial tenocytes. In contrast, other tested adeno-associated viruses transduced tenocytes minimally or not at all. The efficiency of gene transfer by AAV2, indicated by the percentage of cells with positive beta-galactosidase staining, was significantly greater than that by a plasmid vector (p = 0.001). Expression of the bFGF gene in tenocytes transduced with the AAV2-bFGF was significantly higher than that in the control over the 3-week period (p < 0.01). Gene transfer to tenocytes by AAV2 is more efficient than that by a plasmid vector. However, other adeno-associated virus serotypes cannot effectively transduce tenocytes. The bFGF gene can be delivered to intrasynovial tenocytes by the AAV2 vector effectively, and the gene transfer significantly increases expression of bFGF gene over 3 weeks.

  16. Identification of an adeno-associated virus binding epitope for AVB sepharose affinity resin

    Directory of Open Access Journals (Sweden)

    Qiang Wang

    Full Text Available Recent successes of adeno-associated virus (AAV–based gene therapy have created a demand for large-scale AAV vector manufacturing and purification techniques for use in clinical trials and beyond. During the development of purification protocols for rh.10, hu.37, AAV8, rh.64R1, AAV3B, and AAV9 vectors, based on a widely used affinity resin, AVB sepharose (GE, we found that, under the same conditions, different serotypes have different affinities to the resin, with AAV3B binding the best and AAV9 the poorest. Further analysis revealed a surface-exposed residue (amino acid number 665 in AAV8 VP1 numbering differs between the high-affinity AAV serotypes (serine in AAV3B, rh.10, and hu.37 and the low-affinity ones (asparagine in AAV8, rh.64R1, and AAV9. The residue locates within a surface-exposed, variable epitope flanked by highly conserved residues. The substitution of the epitope in AAV8, rh.64R1, and AAV9 with the corresponding epitope of AAV3B (SPAKFA resulted in greatly increased affinity to AVB sepharose with no reduction in the vectors’ in vitro potency. The presence of the newly identified AVB-binding epitope will be useful for affinity resin selection for the purification of novel AAV serotypes. It also suggests the possibility of vector engineering to yield a universal affinity chromatography purification method for multiple AAV serotypes.

  17. Recombinant Adeno-Associated Virus-Mediated Expression of Methamphetamine Antibody Attenuates Methamphetamine-Induced Hyperactivity in Mice

    OpenAIRE

    Yun-Hsiang Chen; Kuo-Jen Wu; Kuang-Lun Wu; Kun-Lieh Wu; Ho-Min Tsai; Mao-Liang Chen; Yi-Wei Chen; Wei Hsieh; Chun-Ming Lin; Yun Wang

    2017-01-01

    Methamphetamine (Meth) is one of the most frequently abused drugs worldwide. Recent studies have indicated that antibodies with high affinity for Meth reduce its pharmacological effects. The purpose of this study was to develop a technique for virus-based passive immunization against Meth effects. We generated a recombinant adeno-associated virus serotype-8 vector (AAV-MethAb) carrying the gene for a Meth-specific monoclonal antibody (MethAb). Infection of 293 cells with AAV-MethAb resulted i...

  18. Single Amino Acid Changes Can Influence Titer, Heparin Binding, and Tissue Tropism in Different Adeno-Associated Virus Serotypes▿

    OpenAIRE

    Wu, Zhijian; Asokan, Aravind; Joshua C Grieger; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Samulski, R. Jude

    2006-01-01

    Despite the high degree of sequence homology between adeno-associated virus (AAV) serotype 1 and 6 capsids (99.2%), these viruses have different liver transduction profiles when tested as vectors. Examination of the six amino acid residues that differ between AAV1 and AAV6 revealed that a lysine-to-glutamate change (K531E) suppresses the heparin binding ability of AAV6. In addition, the same mutation in AAV6 reduces transgene expression to levels similar to those achieved with AAV1 in HepG2 c...

  19. NLX-P101, an adeno-associated virus gene therapy encoding glutamic acid decarboxylase, for the potential treatment of Parkinson's disease.

    Science.gov (United States)

    Diaz-Nido, Javier

    2010-07-01

    Parkinson's disease (PD) is a neurodegenerative disease affecting nigrostriatal dopaminergic neurons. Dopamine depletion in the striatum leads to functional changes in several deep brain nuclei, including the subthalamic nucleus (STN), which becomes disinhibited and perturbs the control of body movement. Although there is no cure for PD, some pharmacological and surgical treatments can significantly improve the functional ability of patients, particularly in the early stages of the disease. Among neurodegenerative diseases, PD is a particularly suitable target for gene therapy because the neuropathology is largely confined to a relatively small region of the brain. Neurologix Inc is developing NLX-P101 (AAV2-GAD), an adeno-associated viral vector encoding glutamic acid decarboxylase (GAD), for the potential therapy of PD. As GAD potentiates inhibitory neurotransmission from the STN, sustained expression of GAD in the STN by direct delivery of NLX-P101 decreases STN overactivation. This procedure was demonstrated to be a safe and efficient method of reducing motor deficits in animal models of PD. A phase I clinical trial has demonstrated that NLX-P101 was safe and indicated the efficacy of this approach in patients with PD. Results from an ongoing phase II clinical trial of NLX-P101 are awaited to establish the clinical efficacy of this gene therapy.

  20. Viral paratransgenesis in the malaria vector Anopheles gambiae.

    Directory of Open Access Journals (Sweden)

    Xiaoxia Ren

    Full Text Available Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms, is being considered as a potential method to control vector-borne diseases such as malaria. The feasibility of paratransgenic malaria control has been hampered by the lack of candidate symbiotic microorganisms for the major vector Anopheles gambiae. In other systems, densonucleosis viruses (DNVs are attractive agents for viral paratransgenesis because they infect important vector insects, can be genetically manipulated and are transmitted to subsequent generations. However, An. gambiae has been shown to be refractory to DNV dissemination. We discovered, cloned and characterized the first known DNV (AgDNV capable of infection and dissemination in An. gambiae. We developed a flexible AgDNV-based expression vector to express any gene of interest in An. gambiae using a two-plasmid helper-transducer system. To demonstrate proof-of-concept of the viral paratransgenesis strategy, we used this system to transduce expression of an exogenous gene (enhanced green fluorescent protein; EGFP in An. gambiae mosquitoes. Wild-type and EGFP-transducing AgDNV virions were highly infectious to An. gambiae larvae, disseminated to and expressed EGFP in epidemiologically relevant adult tissues such as midgut, fat body and ovaries and were transmitted to subsequent mosquito generations. These proof-of-principle data suggest that AgDNV could be used as part of a paratransgenic malaria control strategy by transduction of anti-Plasmodium peptides or insect-specific toxins in Anopheles mosquitoes. AgDNV will also be extremely valuable as an effective and easy-to-use laboratory tool for transient gene expression or RNAi in An. gambiae.

  1. Characterization of tissue tropism determinants of adeno-associated virus type 1.

    Science.gov (United States)

    Hauck, Bernd; Xiao, Weidong

    2003-02-01

    Muscle is an attractive target for gene delivery because of its mass and because vectors can be delivered in a noninvasive fashion. Adeno-associated virus (AAV) has been shown to be effective for muscle-targeted gene transfer. Recent progress in characterization of AAV serotype 1 (AAV1) and AAV6 demonstrated that these two AAV serotypes are far more efficient in transducing muscle than is the traditionally used AAV2. Since all cis elements are identical in these vectors, the potential determinants for their differences in transducing muscle appear to be located within the AAV capsid proteins. In the present study, a series of AAV capsid mutants were generated to identify the major regions affecting AAV transduction efficiency in muscle. Replacement of amino acids 350 to 736 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector that behaved very similarly to AAV1 in vitro and in vivo in muscle. Characterization of additional mutants carrying smaller regions of the AAV1 VP1 amino acid sequence in the AAV2 capsid protein suggested that amino acids 350 to 430 of VP1 function as a major tissue tropism determinant. Further analysis showed that the heparin binding domain and the major antigenic determinants in the AAV capsid region were not necessary for the efficiency of AAV1 transduction of muscle.

  2. Regression of Schwannomas Induced by Adeno-Associated Virus-Mediated Delivery of Caspase-1

    Science.gov (United States)

    Prabhakar, Shilpa; Taherian, Mehran; Gianni, Davide; Conlon, Thomas J.; Fulci, Giulia; Brockmann, Jillian; Stemmer-Rachamimov, Anat; Sena-Esteves, Miguel; Breakefield, Xandra O.

    2013-01-01

    Abstract Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene therapy for schwannomas, using a model in which immortalized human NF2 schwannoma cells expressing a fluorescent protein and luciferase are implanted in the sciatic nerve of nude mice. Direct injection of an adeno-associated virus (AAV) serotype 1 vector encoding caspase-1 (ICE) under the Schwann-cell specific promoter, P0, leads to regression of these tumors with essentially no vector-mediated neuropathology, and no changes in sensory or motor function. In a related NF2 xenograft model designed to cause measurable pain behavior, the same gene therapy leads to tumor regression and concordant resolution of tumor-associated pain. This AAV1-P0-ICE vector holds promise for clinical treatment of schwannomas by direct intratumoral injection to achieve reduction in tumor size and normalization of neuronal function. PMID:23140466

  3. [Construction and identification of recombinant avian adeno-associated virus expressing GFP reporter gene].

    Science.gov (United States)

    Wang, An-ping; Sun, Huai-chang; Wang, Jian-ye; Wang, Yong-juan; Yuan, Wei-feng

    2007-07-01

    To generate recombinant avian adeno-associated virus (rAAAV) for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast (CEF) cells and CEL cell line were transduced with the recombinant virus, the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.

  4. Development of Viral Vectors for Gene Therapy for Chronic Pain

    Directory of Open Access Journals (Sweden)

    Yu Huang

    2011-01-01

    Full Text Available Chronic pain is a major health concern that affects millions of people. There are no adequate long-term therapies for chronic pain sufferers, leading to significant cost for both society and the individual. The most commonly used therapy for chronic pain is the application of opioid analgesics and nonsteroidal anti-inflammatory drugs, but these drugs can lead to addiction and may cause side effects. Further studies of the mechanisms of chronic pain have opened the way for development of new treatment strategies, one of which is gene therapy. The key to gene therapy is selecting safe and highly efficient gene delivery systems that can deliver therapeutic genes to overexpress or suppress relevant targets in specific cell types. Here we review several promising viral vectors that could be applied in gene transfer for the treatment of chronic pain and further discuss the possible mechanisms of genes of interest that could be delivered with viral vectors for the treatment of chronic pain.

  5. Induction of Robust Immune Responses against Human Immunodeficiency Virus Is Supported by the Inherent Tropism of Adeno-Associated Virus Type 5 for Dendritic Cells▿

    OpenAIRE

    Xin, Ke-Qin; MIZUKAMI, HIROAKI; Urabe, Masashi; Toda, Yoshihiko; Shinoda, Kaori; Yoshida, Atsushi; Oomura, Kenji; Kojima, Yoshitsugu; Ichino, Motohide; Klinman, Dennis; Ozawa, Keiya; Okuda, Kenji

    2006-01-01

    The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study o...

  6. Transcriptional activity of novel ALDH1L1 promoters in the rat brain following AAV vector-mediated gene transfer

    Directory of Open Access Journals (Sweden)

    Janitha M Mudannayake

    2016-01-01

    Full Text Available Aldehyde dehydrogenase family 1, member L1 (ALDH1L1 is a recently characterized pan-astrocytic marker that is more homogenously expressed throughout the brain than the classic astrocytic marker, glial fibrillary acidic protein. We generated putative promoter sequence variants of the rat ALDH1L1 gene for use in adeno-associated viral vector-mediated gene transfer, with an aim to achieve selective regulation of transgene expression in astrocytes in the rat brain. Unexpectedly, ALDH1L1 promoter variants mediated transcriptional activity exclusively in neurons in the substantia nigra pars compacta as assessed by luciferase reporter expression at 3 weeks postvector infusion. This selectivity for neurons in the substantia nigra pars compacta also persisted in the context of adeno-associated viral serotype 5, 8 or 9 vector-mediated gene delivery. An in vivo promoter comparison showed the highest performing ALDH1L1 promoter variant mediated higher transgene expression than the neuronal-specific synapsin 1 and tyrosine hydroxylase promoters. The ALDH1L1 promoter was also transcriptionally active in dentate granule neurons following intrahippocampal adeno-associated viral vector infusion, whereas transgene expression was detected in both striatal neurons and astrocytes following vector infusion into the striatum. Our results demonstrate the potential suitability of the ALDH1L1 promoter as a new tool in the development of gene therapy and disease modelling applications.

  7. Evolving lessons on nanomaterial-coated viral vectors for local and systemic gene therapy.

    Science.gov (United States)

    Kasala, Dayananda; Yoon, A-Rum; Hong, Jinwoo; Kim, Sung Wan; Yun, Chae-Ok

    2016-07-01

    Viral vectors are promising gene carriers for cancer therapy. However, virus-mediated gene therapies have demonstrated insufficient therapeutic efficacy in clinical trials due to rapid dissemination to nontarget tissues and to the immunogenicity of viral vectors, resulting in poor retention at the disease locus and induction of adverse inflammatory responses in patients. Further, the limited tropism of viral vectors prevents efficient gene delivery to target tissues. In this regard, modification of the viral surface with nanomaterials is a promising strategy to augment vector accumulation at the target tissue, circumvent the host immune response, and avoid nonspecific interactions with the reticuloendothelial system or serum complement. In the present review, we discuss various chemical modification strategies to enhance the therapeutic efficacy of viral vectors delivered either locally or systemically. We conclude by highlighting the salient features of various nanomaterial-coated viral vectors and their prospects and directions for future research.

  8. α2,3 and α2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

    OpenAIRE

    Wu, Zhijian; Miller, Edward; Agbandje-McKenna, Mavis; Samulski, Richard Jude

    2006-01-01

    Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotype...

  9. Chikungunya Virus Vaccines: Viral Vector-Based Approaches.

    Science.gov (United States)

    Ramsauer, Katrin; Tangy, Frédéric

    2016-12-15

    In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  10. Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success--a personal perspective.

    Science.gov (United States)

    Hastie, Eric; Samulski, R Jude

    2015-05-01

    Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications.

  11. High Prevalence of Infectious Adeno-associated Virus (AAV) in Human Peripheral Blood Mononuclear Cells Indicative of T Lymphocytes as Sites of AAV Persistence.

    Science.gov (United States)

    Hüser, Daniela; Khalid, Dina; Lutter, Timo; Hammer, Eva-Maria; Weger, Stefan; Heßler, Melanie; Kalus, Ulrich; Tauchmann, Yvonne; Hensel-Wiegel, Karin; Lassner, Dirk; Heilbronn, Regine

    2017-02-15

    Seroepidemiology shows that infections with adeno-associated virus (AAV) are widespread, but diverse AAV serotypes isolated from humans or nonhuman primates have so far not been proven to be causes of human disease. In view of the increasing success of AAV-derived vectors in human gene therapy, definition of the in vivo sites of wild-type AAV persistence and the clinical consequences of its reactivation is becoming increasingly urgent. Here, we identify the presumed cell type for AAV persistence in the human host by highly sensitive AAV PCRs developed for the full spectrum of human AAV serotypes. In genomic-DNA samples from leukocytes of 243 healthy blood donors, 34% were found to be AAV positive, predominantly AAV type 2 (AAV2) (77%), AAV5 (19%), and additional serotypes. Roughly 11% of the blood donors had mixed AAV infections. AAV prevalence was dramatically increased in immunosuppressed patients, 76% of whom were AAV positive. Of these, at least 45% displayed mixed infections. Follow-up of single blood donors over 2 years allowed repeated detection of the initial and/or additional AAV serotypes, suggestive of fluctuating, persistent infection. Leukocyte separation revealed that AAV resided in CD3+ T lymphocytes, perceived as the putative in vivo site of AAV persistence. Moreover, infectious AAVs of various serotypes could be rescued and propagated from numerous samples. The high prevalence and broad spectrum of human AAVs in leukocytes closely follow AAV seroepidemiology. Immunosuppression obviously enhances AAV replication in parallel with activation of human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6), reminiscent of herpesvirus-induced AAV activation. Adeno-associated virus is viewed as apathogenic and replication defective, requiring coinfection with adenovirus or herpesvirus for productive infection. In vivo persistence of a defective virus requires latency in specialized cell types to escape the host immune response until viral spread becomes

  12. Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

    Energy Technology Data Exchange (ETDEWEB)

    McCraw, Dustin M. [Department of Biochemistry and Molecular Biology, School of Medicine, Mail code L224, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098 (United States); O& #x27; Donnell, Jason K. [Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380 (United States); Taylor, Kenneth A. [Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295 (United States); Stagg, Scott M. [Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380 (United States); Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306 (United States); Chapman, Michael S., E-mail: chapmami@ohsu.edu [Department of Biochemistry and Molecular Biology, School of Medicine, Mail code L224, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098 (United States)

    2012-09-15

    The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.

  13. The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses.

    Directory of Open Access Journals (Sweden)

    Christina Hölscher

    2015-12-01

    Full Text Available Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including release from endosomes, nuclear transport and conversion of the single stranded DNA into a double stranded molecule. We hypothesized that the bottlenecks in AAV transduction are, in part, due to the presence of host cell restriction factors acting directly or indirectly on the AAV-mediated gene transduction. In order to identify such factors we performed a whole genome siRNA screen which identified a number of putative genes interfering with AAV gene transduction. A number of factors, yielding the highest scores, were identified as members of the SUMOylation pathway. We identified Ubc9, the E2 conjugating enzyme as well as Sae1 and Sae2, enzymes responsible for activating E1, as factors involved in restricting AAV. The restriction effect, mediated by these factors, was validated and reproduced independently. Our data indicate that SUMOylation targets entry of AAV capsids and not downstream processes of uncoating, including DNA single strand conversion or DNA damage signaling. We suggest that transiently targeting SUMOylation will enhance application of AAV in vitro and in vivo.

  14. Identification of the galactose binding domain of the adeno-associated virus serotype 9 capsid.

    Science.gov (United States)

    Bell, Christie L; Gurda, Brittney L; Van Vliet, Kim; Agbandje-McKenna, Mavis; Wilson, James M

    2012-07-01

    Adeno-associated virus serotype 9 (AAV9) vectors show promise for gene therapy of a variety of diseases due to their ability to transduce multiple tissues, including heart, skeletal muscle, and the alveolar epithelium of the lung. In addition, AAV9 is unique compared to other AAV serotypes in that it is capable of surpassing the blood-brain barrier and transducing neurons in the brain and spinal cord. It has recently been shown that AAV9 uses galactose as a receptor to transduce many different cell types in vitro, as well as cells of the mouse airway in vivo. In this study, we sought to identify the specific amino acids of the AAV9 capsid necessary for binding to galactose. By site-directed mutagenesis and cell binding assays, plus computational ligand docking studies, we discovered five amino acids, including N470, D271, N272, Y446, and W503, which are required for galactose binding that form a pocket at the base of the protrusions around the icosahedral 3-fold axes of symmetry. The importance of these amino acids for tissue tropism was also confirmed by in vivo studies in the mouse lung. Identifying the interactions necessary for AAV9 binding to galactose may lead to advances in vector engineering.

  15. Rapid, widespread transduction of the murine myocardium using self-complementary Adeno-associated virus.

    Science.gov (United States)

    Andino, Lourdes M; Conlon, Thomas J; Porvasnik, Stacy L; Boye, Sanford L; Hauswirth, William W; Lewin, Alfred S

    2007-12-10

    Adeno-associated virus (AAV) has shown great promise as a gene transfer vector. However, the incubation time needed to attain significant levels of gene expression is often too long for some clinical applications. Self-complementary AAV (scAAV) enters the cell as double stranded DNA, eliminating the step of second-strand synthesis, proven to be the rate-limiting step for gene expression of single-stranded AAV (ssAAV). The aim of this study was to compare the efficiency of these two types of AAV vectors in the murine myocardium. Four day old CD-1 mice were injected with either of the two AAV constructs, both expressing GFP and packaged into the AAV1 capsid. The animals were held for 4, 6, 11 or 21 days, after which they were euthanized and their hearts were excised. Serial sections of the myocardial tissue were used for real-time PCR quantification of AAV genome copies and for confocal microscopy. Although we observed similar numbers of AAV genomes at each of the different time points present in both the scAAV and the ssAAV infected hearts, microscopic analysis showed expression of GFP as early as 4 days in animals injected with the scAAV, while little or no expression was observed with the ssAAV constructs until day 11. AAV transduction of murine myocardium is therefore significantly enhanced using scAAV constructs.

  16. [Construction and expression of recombinant adeno-associated virus expressing brain-derived neurotrophic factor].

    Science.gov (United States)

    Li, Huiming; Qiu, Wei; Wang, Feng; Wei, Fang; Chen, Xiafang; Wu, Xiaobing; Huang, Qian

    2008-02-01

    A fusion gene called Ig-BDNF, in which brain-derived neurotrophic factor cDNA fused to the 3' end of signal peptide of Ig coding sequence, was constructed by PCR, digested and subcloned into shuttle plasmid pSNAV to obtain a recombinant plasmid pSNAV-Ig-BDNF. Then the plasmid encoding fusion protein was transfected into 293 cell lines and the stably transfected clones were selected with neomycin. AAV1 containing Ig-BDNF fusion gene vectors were obtained by super-infection by Herpes virus. The resultant adeno-associated virus vectors AAV-Ig-BDNF were confirmed by PCR, Western blotting and a sandwich enzyme-linked immunosorbent assay (ELISA) after infection of 293 cell lines. The results indicated that AAV-Ig-BDNF contained the target gene, and infected cells and produced the fusion protein into the supernatant. The content of BDNF in medium per 5x104 cells over a 24 h incubation period reached 1000 pg/mL. With the help of non-replicative adenovirus during AAV-Ig-BDNF infection, the expression of BDNF increased 7-8 fold, and the enhancement of BDNF gene expression was observed in a concentration-dependent manner. These results suggested that a functional AAV-Ig-BDNF was successfully constructed and it offers basis for further study for gene therapy of neural degeneration diseases.

  17. Adeno-associated virus inverted terminal repeats stimulate gene editing.

    Science.gov (United States)

    Hirsch, M L

    2015-02-01

    Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

  18. Directed evolution of adeno-associated virus for glioma cell transduction.

    Science.gov (United States)

    Maguire, Casey A; Gianni, Davide; Meijer, Dimphna H; Shaket, Lev A; Wakimoto, Hiroaki; Rabkin, Samuel D; Gao, Guangping; Sena-Esteves, Miguel

    2010-02-01

    Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10(4) genome copies/cell), and at low doses it was 1.45-1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.

  19. Targeting of breast metastases using a viral gene vector with tumour-selective transcription.

    LENUS (Irish Health Repository)

    Rajendran, Simon

    2012-01-31

    BACKGROUND: Adeno-associated virus (AAV) vectors have significant potential as gene delivery vectors for cancer gene therapy. However, broad AAV2 tissue tropism results in nonspecific gene expression. MATERIALS AND METHODS: We investigated use of the C-X-C chemokine receptor type 4 (CXCR4) promoter to restrict AAV expression to tumour cells, in subcutaneous MCF-7 xenograft mouse models of breast cancer and in patient samples, using bioluminescent imaging and flow cytometric analysis. RESULTS: Higher transgene expression levels were observed in subcutaneous MCF-7 tumours relative to normal tissue (muscle) using the CXCR4 promoter, unlike a ubiquitously expressing Cytomegalovirus promoter construct, with preferential AAVCXCR4 expression in epithelial tumour and CXCR4-positive cells. Transgene expression following intravenously administered AAVCXCR4 in a model of liver metastasis was detected specifically in livers of tumour bearing mice. Ex vivo analysis using patient samples also demonstrated higher AAVCXCR4 expression in tumour compared with normal liver tissue. CONCLUSION: This study demonstrates for the first time, the potential for systemic administration of AAV2 vector for tumour-selective gene therapy.

  20. Immune responses to rAAV6: The influence of canine parvovirus vaccination and neonatal administration of viral vector

    Directory of Open Access Journals (Sweden)

    Andrea L H Arnett

    2011-11-01

    Full Text Available Recombinant adeno-associated viral (rAAV vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV. rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, one month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice.

  1. Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

    Science.gov (United States)

    Chen, Yong; Chen, Qian; Li, Manman; Mao, Qianzhuo; Chen, Hongyan; Wu, Wei; Jia, Dongsheng; Wei, Taiyun

    2017-11-01

    Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.

  2. Pancreatic cell tracing, lineage tagging and targeted genetic manipulations in multiple cell types using pancreatic ductal infusion of adeno-associated viral vectors and/or cell-tagging dyes.

    Science.gov (United States)

    Xiao, Xiangwei; Guo, Ping; Prasadan, Krishna; Shiota, Chiyo; Peirish, Lauren; Fischbach, Shane; Song, Zewen; Gaffar, Iljana; Wiersch, John; El-Gohary, Yousef; Husain, Sohail Z; Gittes, George K

    2014-12-01

    Genetic manipulations, with or without lineage tracing for specific pancreatic cell types, are very powerful tools for studying diabetes, pancreatitis and pancreatic cancer. Nevertheless, the use of Cre/loxP systems to conditionally activate or inactivate the expression of genes in a cell type- and/or temporal-specific manner is not applicable to cell tracing and/or gene manipulations in more than one lineage at a time. Here we report a technique that allows efficient delivery of dyes for cell tagging into the mouse pancreas through the duct system, and that also delivers viruses carrying transgenes or siRNA under a specific promoter. When this technique is applied in genetically modified mice, it enables the investigator to perform either double lineage tracing or cell lineage tracing combined with gene manipulation in a second lineage. The technique requires <40 min.

  3. Adeno-associated virus-targeted disruption of the CFTR gene in cloned ferrets

    National Research Council Canada - National Science Library

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S; Chen, Juan; Zhang, Yulong; Welsh, Michael J; Leno, Gregory H; Engelhardt, John F

    2008-01-01

    .... In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning...

  4. Adeno-associated virus-mediated delivery of genes to mouse spermatogonial stem cells.

    Science.gov (United States)

    Watanabe, Satoshi; Kanatsu-Shinohara, Mito; Ogonuki, Narumi; Matoba, Shogo; Ogura, Atsuo; Shinohara, Takashi

    2017-01-01

    Spermatogenesis is a complicated process that originates from spermatogonial stem cells (SSCs), which have self-renewal activity. Because SSCs are the only stem cells in the body that transmit genetic information to the next generation, they are an attractive target for germline modification. Although several virus vectors have been successfully used to transduce SSCs, cell toxicity or insertional mutagenesis of the transgene has limited their usage. Adeno-associated virus (AAV) is unique among virus vectors because of its target specificity and low toxicity in somatic cells, and clinical trials have shown that it has promise for gene therapy. However, there are conflicting reports on the possibility of germline integration of AAV into the genome of male germ cells, including SSCs. Here, we examined the usefulness of AAV vectors for exploring germline gene modification in SSCs. AAV1 infected cultured SSCs without apparent toxicity. Moreover, SSCs that were infected in fresh testis cells generated normal appearing spermatogenic colonies after spermatogonial transplantation. A microinsemination experiment produced offspring that underwent excision of the floxed target gene by AAV1-mediated Cre expression. Analysis of the offspring DNA showed no evidence of AAV integration, suggesting a low risk of germline integration by AAV infection. Although more extensive experiments are required to assess the risk of germline integration, our results show that AAV1 is useful for genetic manipulation of SSCs, and gene transduction by AAV will provide a useful approach to overcome potential problems associated with previous virus vector-mediated gene transduction. © The Authors 2016. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Ultrasound Targeted Microbubble Destruction Stimulates Cellular Endocytosis in Facilitation of Adeno-Associated Virus Delivery

    Directory of Open Access Journals (Sweden)

    Lian-Fang Du

    2013-05-01

    Full Text Available The generally accepted mechanism for ultrasound targeted microbubble destruction (UTMD to enhance drug and gene delivery is through sonoporation. However, passive uptake of adeno-associated virus (AAV into cells following sonoporation does not adequately explain observations of enhanced transduction by UTMD. This study investigated alternative mechanisms of UTMD enhancement in AAV delivery. UTMD significantly enhanced transduction efficiency of AAV in a dose-dependent manner. UTMD stimulated a persistent uptake of AAV into the cytoplasm and nucleus. This phenomenon occurred over several hours, suggesting that some viral particles are endocytosed by cells rather than exclusively passing through pores created by sonoporation. Additionally, UTMD enhanced clathrin expression and accumulation at the plasma membrane suggesting greater clathrin-mediated endocytosis following UTMD. Transmission electron microscopy (TEM revealed that UTMD stimulated formation of clathrin-coated pits (CPs and uncoated pits (nCPs. Furthermore, inhibition of clathrin-mediated endocytosis partially blocked the enhancement of AAV uptake following UTMD. The results of this study implicate endocytosis as a mechanism that contributes to UTMD-enhanced AAV delivery.

  6. Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9

    Directory of Open Access Journals (Sweden)

    Jason L Picconi

    2014-01-01

    Full Text Available Gene therapy targeting of kidneys has been largely unsuccessful. Recently, a recombinant adeno-associated virus (rAAV vector was used to target adult mouse kidneys. Our hypothesis is that a pseudotyped rAAV 2/9 vector can produce fetal kidney-specific expression of the green fluorescent protein (GFP gene following maternal tail vein injection of pregnant mice. Pregnant mice were treated with rAAV2/9 vectors with either the ubiquitous cytomegalovirus promoter or the minimal NPHS1 promoter to drive kidney-specific expression of GFP. Kidneys from dams and pups were analyzed for vector DNA, gene expression, and protein. Vector DNA was identified in kidney tissue out to 12 weeks at low but stable levels, with levels higher in dams than that in pups. Robust GFP expression was identified in the kidneys of both dams and pups treated with the cytomegalovirus (CMV-enhanced green fluorescent protein (eGFP vector. When treated with the NPHS1-eGFP vector, dams and pups showed expression of GFP only in kidneys, localized to the glomeruli. An 80-fold increase in GFP mRNA expression in dams and a nearly 12-fold increase in pups was found out to 12 weeks of life. Selective targeting of the fetal kidney with a gene therapy vector was achieved by utilizing the pseudotyped rAAV 2/9 vector containing the NPHS1 promoter.

  7. Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain.

    Science.gov (United States)

    Matsuzaki, Yasunori; Konno, Ayumu; Mochizuki, Ryuta; Shinohara, Yoichiro; Nitta, Keisuke; Okada, Yukihiro; Hirai, Hirokazu

    2018-02-05

    Intravenous administration of adeno-associated virus (AAV)-PHP.B, a capsid variant of AAV9 containing seven amino acid insertions, results in a greater permeability of the blood brain barrier (BBB) than standard AAV9 in mice, leading to highly efficient and global transduction of the central nervous system (CNS). The present study aimed to examine whether the enhanced BBB penetrance of AAV-PHP.B observed in mice also occurs in non-human primates. Thus, a young adult (age, 1.6 years) and an old adult (age, 7.2 years) marmoset received an intravenous injection of AAV-PHP.B expressing enhanced green fluorescent protein (EGFP) under the control of the constitutive CBh promoter (a hybrid of cytomegalovirus early enhancer and chicken β-actin promoter). Age-matched control marmosets were treated with standard AAV9-capsid vectors. The animals were sacrificed 6 weeks after the viral injection. Based on the results, only limited transduction of neurons (0-2%) and astrocytes (0.1-2.5%) was observed in both AAV-PHP.B- and AAV9-treated marmosets. One noticeable difference between AAV-PHP.B and AAV9 was the marked transduction of the peripheral dorsal root ganglia neurons. Indeed, the soma and axons in the projection from the spinal cord to the nucleus cuneatus in the medulla oblongata were strongly labeled with EGFP by AAV-PHP.B. Thus, except for the peripheral dorsal root ganglia neurons, the AAV-PHP.B transduction efficiency in the CNS of marmosets was comparable to that of AAV9 vectors. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Molecular characterization and phylogenetic analysis of an avian adeno-associated virus originating from a chicken in China.

    Science.gov (United States)

    Wang, Jianye; Zhu, Liqian; Zhu, Jun; Sun, Huaichang; Zhu, Guoqiang

    2011-01-01

    The use of adeno-associated viruses (AAVs) as vectors for gene delivery is well established; however, genomic information about avian adeno-associated virus (AAAV) and its use are still limited. In this study, an AAAV strain, YZ-1, was isolated from healthy chickens in China, and the complete genome was sequenced. The genomic DNA of YZ-1 is 4,684 nucleotides long, including two ORFs encoding the nonstructural proteins (Rep) and the structural proteins (Cap), and an inverted terminal repeat (ITR) forming a typical T-shaped palindromic structure at each end. YZ-1 was 95.0 and 92.2% identical to the other two reported AAAV strains, DA-1 and VR-865, respectively, at the nucleotide sequence level. In comparison to VR-865, frameshift mutations or deletions in the N-terminal region of the Rep78 protein or VP2 protein were observed in YZ-1 and DA-1. Phylogenetic analysis revealed that YZ-1, DA-1 and VR-865 could be classified into the avian group of the AAV family. This group and other AAVs of mammalian origin displayed almost equal divergence from pathogenic waterfowl parvoviruses, revealing that AAAV has no direct evolutionary relationship to them. This study therefore provides new genomic information about AAAV.

  9. Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions.

    Science.gov (United States)

    Whiteway, Alistair; Deru, Wale; Prentice, H Grant; Anderson, Robert

    2003-12-01

    Recombinant adeno-associated virus type 2 (rAAV) has promise for use as a gene therapy vector. Potential problems in the production of rAAV stocks are both the limited amount of recombinant virus that is produced by traditional methods and the possibility of wild-type replication competent adeno-associated virus (wtAAV) contamination. The presence of these contaminants is largely dependent upon the helper plasmid used. Whilst wtAAV is not a pathogen, the presence of these contaminants is undesirable as they may affect experiments concerning the biology of rAAV. Additionally as protocols using rAAV with altered tropism are becoming more prevalent, it is important that no recombination be permitted that may cause the creation of a replication competent AAV with modified (targeting) capsids. Many experimental protocols require the generation of large amounts of high titre rAAV stocks. We describe the production of several AAV helper plasmids and cell lines designed to achieve this goal. These plasmids possess split AAV rep and cap genes to eliminate the production of wtAAV and they possess a selection mechanism which is operatively linked to expression from the AAV cap gene. This allows positive selection of those cells expressing the highest level of the structural capsid proteins and therefore those cells which yield the highest amount of rAAV.

  10. Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion.

    Science.gov (United States)

    Tse, Longping Victor; Klinc, Kelli A; Madigan, Victoria J; Castellanos Rivera, Ruth M; Wells, Lindsey F; Havlik, L Patrick; Smith, J Kennon; Agbandje-McKenna, Mavis; Asokan, Aravind

    2017-06-13

    Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.

  11. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

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    Dejia Li

    2009-08-01

    Full Text Available Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12 vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy and ameliorate muscle disease.

  12. Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey.

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    An, Heeyoung; Cho, Doo-Wan; Lee, Seung Eun; Yang, Young-Su; Han, Su-Cheol; Lee, C Justin

    2016-02-01

    Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys.

  13. Viral Hybrid Vectors for Somatic Integration - Are They the Better Solution?

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    Anja Ehrhardt

    2009-12-01

    Full Text Available The turbulent history of clinical trials in viral gene therapy has taught us important lessons about vector design and safety issues. Much effort was spent on analyzing genotoxicity after somatic integration of therapeutic DNA into the host genome. Based on these findings major improvements in vector design including the development of viral hybrid vectors for somatic integration have been achieved. This review provides a state-of-the-art overview of available hybrid vectors utilizing viruses for high transduction efficiencies in concert with various integration machineries for random and targeted integration patterns. It discusses advantages but also limitations of each vector system.

  14. Adeno-associated virus (AAV) serotypes 2, 4 and 5 display similar transduction profiles and penetrate solid tumor tissue in models of human glioma.

    Science.gov (United States)

    Thorsen, Frits; Afione, Sandra; Huszthy, Peter C; Tysnes, Berit B; Svendsen, Agnete; Bjerkvig, Rolf; Kotin, Robert M; Lønning, Per Eystein; Hoover, Frank

    2006-09-01

    Adeno-associated viral (AAV) vectors are potent delivery vehicles for gene transfer strategies directed at the central nervous system (CNS), muscle and liver. However, comparatively few studies have described AAV-mediated gene transfer to tumor tissues. We have previously demonstrated that while AAV2 and Adenoviral (Ad) 5 vectors have similar broad host ranges in tumor-derived cell lines, AAV2 was able to penetrate human glioblastoma biopsy spheroids and xenografts more efficiently than Ad 5 vectors. These results suggested that AAV vectors could be suitable for therapeutic gene delivery to solid tumor tissue. In the present work, the transduction efficacy of AAV serotypes 4 and 5 were compared to AAV2, both in vitro and in intracranial GBM xenografts derived from patient biopsies implanted into nude rats. AAV vector serotypes 2, 4, and 5 containing either the green fluorescent protein (GFP) or the bacterial beta-galactosidase (lacZ) reporter gene were added to five different human glioma cell lines, to multicellular spheroids generated from glioblastoma patient biopsies, and to spheroids xenografted intracranially in nude rats. Transduction efficiency was assessed by fluorescence imaging, histochemistry, immunohistochemistry and flow cytometry. While all three AAV serotypes were able to transduce the glioma cell lines when added individually or when they were administered in concert, AAV2 transduced the glioma cells most effectively compared to AAV4 or AAV5. Upon infecting glioblastoma spheroids in vitro, all three AAV serotypes efficiently transduced cells located at the surface as well as within deeper layers of the spheroids. In addition, similarly to what was observed for AAV2 16, both AAV4 and AAV5 were able to transduce human glioblastoma xenografts implanted intracranially. In addition to the widely used AAV2 serotype, AAV4 and AAV5 serotypes may also be used to transduce biologically diverse glioma cell lines. They also penetrate and transduce solid human

  15. Biodistribution of adeno-associated virus serotype 9 after intrathecal and intravenous delivery in mouse

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    Daniel J. Schuster

    2014-06-01

    Full Text Available Adeno-associated virus serotype 9 (AAV9-mediated gene transfer has been reported in central nervous system (CNS and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP across the mouse CNS and selected peripheral tissues after intrathecal (i.t. or intravenous (i.v. delivery of equivalent doses of single-stranded AAV9 vector. After i.t. delivery, GFP immunoreactivity (-ir was observed in spinal neurons, primary afferent fibers and corresponding primary sensory neurons at all spinal levels. Robust transduction was seen in small and large dorsal root ganglion (DRG neurons as well as trigeminal and vagal primary afferent neurons. Transduction efficiency in sensory ganglia was substantially lower in i.v. treated mice. In brain, i.v. delivery yielded GFP-immunoreactivity (-ir primarily in spinal trigeminal tract, pituitary, and scattered isolated neurons and astrocytes. In contrast, after i.t. delivery, GFP-ir was widespread throughout CNS, with greater intensity and more abundant neuropil-like staining at six weeks compared to three weeks. Brain regions with prominent GFP-ir included cranial nerve nuclei, ventral pons, cerebellar cortex, hippocampus, pituitary, choroid plexus, and selected nuclei of midbrain, thalamus and hypothalamus. In cortex, GFP-ir was associated with blood vessels, and was seen in both neurons and astrocytes. In the periphery, GFP-ir in colon and ileum was present in the enteric nervous system in both i.v. and i.t. treated mice. Liver and adrenal cortex, but not adrenal medulla, also showed abundant GFP-ir after both routes of delivery. In summary, i.t. delivery yielded higher transduction efficiency in sensory neurons and the CNS. The observation of comparable gene transfer to peripheral tissues using the two routes indicates that a component of i.t. delivered vector is redistributed from the subarachnoid space to the systemic circulation.

  16. Spinal nociceptive circuit analysis with recombinant adeno-associated viruses: the impact of serotypes and promoters.

    Science.gov (United States)

    Haenraets, Karen; Foster, Edmund; Johannssen, Helge; Kandra, Vinnie; Frezel, Noémie; Steffen, Timothy; Jaramillo, Valeria; Paterna, Jean-Charles; Zeilhofer, Hanns Ulrich; Wildner, Hendrik

    2017-09-01

    Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a > 20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.

  17. Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles.

    Science.gov (United States)

    Li, Chengwen; Diprimio, Nina; Bowles, Dawn E; Hirsch, Matthew L; Monahan, Paul E; Asokan, Aravind; Rabinowitz, Joseph; Agbandje-McKenna, Mavis; Samulski, R Jude

    2012-08-01

    Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration.

  18. Recombinant adeno-associated virus type 2-mediated gene transfer into human keratinocytes is influenced by both the ubiquitin/proteasome pathway and epidermal growth factor receptor tyrosine kinase.

    Science.gov (United States)

    Braun-Falco, Markus; Eisenried, Angelika; Büning, Hildegard; Ring, Johannes

    2005-05-01

    Efficient gene delivery into keratinocytes is a prerequisite for successful skin gene therapy. Vectors based on recombinant adeno-associated virus type 2 (rAAV-2) offer several promising features that make them attractive for cutaneous applications. However, highly efficient gene delivery may be hampered by different cellular factors, including lack of viral receptors, impairment of cytoplasmic trafficking or limitations in viral second-strand synthesis. This study was undertaken to find factors that influence rAAV-2-mediated in vitro gene transfer into human keratinocytes and, consequently, ways to optimize gene delivery. Transduction experiments using rAAV-2 vectors expressing green fluorescent protein (GFP) demonstrated that impaired cellular trafficking of vector particles and high levels of autophosphorylation at epidermal growth factor receptor tyrosine kinase (EGF-R TK) have a negative influence on gene transfer into keratinocytes. Treatment of keratinocytes with proteasome inhibitor MG132 resulted in a transient augmentation of GFP expression in up to 37% of cells. Treatment with EGF-R TK inhibitors (quinazoline type) enhanced transgene expression in 10-14.5% of the cells. Gene expression was stable for more than 10 weeks and persisted until proliferative senescence occurred. This stable gene expression allows speculation that keratinocyte stem cells have initially been transduced. These findings might have relevance for the use of rAAV-2 vectors in skin gene therapy: transient enhancement of rAAV-2 transduction with proteasome inhibitors might be useful for genetic promotion of wound healing or skin-directed vaccination. Treatment with quinazolines may increase rAAV-2 transduction of keratinocyte stem cells, which is important for gene therapy approaches to inherited diseases.

  19. Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

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    Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

    2017-01-01

    ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time

  20. Adeno-associated virus mediated delivery of a non-membrane targeted human soluble CD59 attenuates some aspects of diabetic retinopathy in mice.

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    Mehreen Adhi

    Full Text Available Diabetic retinopathy is the leading cause of visual dysfunction in working adults and is attributed to retinal vascular and neural cell damage. Recent studies have described elevated levels of membrane attack complex (MAC and reduced levels of membrane associated complement regulators including CD55 and CD59 in the retina of diabetic retinopathy patients as well as in animal models of this disease. We have previously described the development of a soluble membrane-independent form of CD59 (sCD59 that when delivered via a gene therapy approach using an adeno-associated virus vector (AAV2/8-sCD59 to the eyes of mice, can block MAC deposition and choroidal neovascularization. Here, we examine AAV2/8-sCD59 mediated attenuation of MAC deposition and ensuing complement mediated damage to the retina of mice following streptozotocin (STZ induced diabetes. We observed a 60% reduction in leakage of retinal blood vessels in diabetic eyes pre-injected with AAV2/8-sCD59 relative to negative control virus injected diabetic eyes. AAV2/8-sCD59 injected eyes also exhibited protection from non-perfusion of retinal blood vessels. In addition, a 200% reduction in retinal ganglion cell apoptosis and a 40% reduction in MAC deposition were documented in diabetic eyes pre-injected with AAV2/8-sCD59 relative to diabetic eyes pre-injected with the control virus. This is the first study characterizing a viral gene therapy intervention that targets MAC in a model of diabetic retinopathy. Use of AAV2/8-sCD59 warrants further exploration as a potential therapy for advanced stages of diabetic retinopathy.

  1. Synoviocyte infection with adeno-associated virus (AAV) is neutralized by human synovial fluid from arthritis patients and depends on AAV serotype.

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    Boissier, M-C; Lemeiter, D; Clavel, C; Valvason, C; Laroche, L; Begue, T; Bessis, N

    2007-06-01

    Intraarticular gene transfer with adeno-associated viral (AAV) vectors may allow efficient therapeutic transgene expression within the joint in diseases such as rheumatoid arthritis (RA), allowing high expression of the protein within the joint, preventing both systemic diffusion and side effects. However, humans demonstrate antibodies against AAV, which can influence gene transfer. To better understand critical obstacles to intraarticular gene therapy with AAV, we have previously shown that synovial fluid (SF) contains IgG to AAV that neutralizes chondrocyte infection in vitro. Our objective was therefore to compare neutralization exerted by SF from RA patients for four different AAV serotypes (AAV serotypes 1, 2, 5, and 8) on human primary synoviocytes. Serotype 2 infected synoviocytes most efficiently followed, in decreasing order, by serotypes 1, 5, and 8. SF from all patients partially inhibited infection of synoviocytes by at least one of the four serotypes. Infection with serotypes 1 and 2 was the most inhibited by SF, whereas inhibition was weak for serotypes 5 and 8. Last, we have shown that inhibition of AAV1/interleukin (IL)-4 infection of synoviocytes by SF could be reversed by increasing the number of AAV1/IL-4 particles, with a dose-dependent effect. We conclude that the most infectious AAV serotypes (1 and 2) in synoviocytes are also the serotypes most neutralized by SF. Thus, serotype 5 seems to demonstrate the best infection efficiency:immunogenicity ratio for local use in articular diseases. These data may be useful for tailoring intraarticular AAV-mediated gene therapy to individual patients.

  2. Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

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    DiMattia,M.; Govindasamy, L.; Levy, H.; Whitaker-Gurda, B.; Kohlbrenner, E.; Chiorini, J.; McKenna, R.; Muzyczka, N.; Zolotukhin, S.; Agbandje-McKenna, M.

    2005-01-01

    Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  3. Evidence for pH-Dependent Protease Activity in the Adeno-Associated Virus Capsid

    Science.gov (United States)

    Salganik, Maxim; Venkatakrishnan, Balasubramanian; Bennett, Antonette; Lins, Bridget; Yarbrough, Joseph; Agbandje-McKenna, Mavis

    2012-01-01

    Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection. PMID:22915820

  4. Structural insight into the unique properties of adeno-associated virus serotype 9.

    Science.gov (United States)

    DiMattia, Michael A; Nam, Hyun-Joo; Van Vliet, Kim; Mitchell, Matthew; Bennett, Antonette; Gurda, Brittney L; McKenna, Robert; Olson, Norman H; Sinkovits, Robert S; Potter, Mark; Byrne, Barry J; Aslanidi, George; Zolotukhin, Sergei; Muzyczka, Nicholas; Baker, Timothy S; Agbandje-McKenna, Mavis

    2012-06-01

    Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less "pointed" than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.

  5. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

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    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  6. Evidence for pH-dependent protease activity in the adeno-associated virus capsid.

    Science.gov (United States)

    Salganik, Maxim; Venkatakrishnan, Balasubramanian; Bennett, Antonette; Lins, Bridget; Yarbrough, Joseph; Muzyczka, Nicholas; Agbandje-McKenna, Mavis; McKenna, Robert

    2012-11-01

    Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.

  7. Pre-existing immunity to adeno-associated virus (AAV)2 limits transgene expression following intracerebral AAV2-based gene delivery in a 6-hydroxydopamine model of Parkinson's disease.

    Science.gov (United States)

    Janelidze, Shorena; Nordström, Ulrika; Kügler, Sebastian; Brundin, Patrik

    2014-01-01

    Adeno-associated virus (AAV) vectors are used to deliver potentially therapeutic genes in clinical trials in Parkinson's disease (PD). Pre-existing immunity to AAV and a local neuroinflammatory response might negatively affect the efficacy of such AAV-mediated gene delivery. We pre-immunized rats with wild-type AAV-2. Three months later, we created PD-like lesions by intrastriatal injections of 6-hydroxydopamine (6-OHDA) in 50% of the animals. One month later, we injected AAV2 vector expressing enhanced green fluorescent protein (eGFP) in the striatum. Using immunohistochemistry, we assessed eGFP expression, microglia activation and CD8 T cell infiltration. We also measured AAV-2 specific neutralizing antibody titers in the serum. The number of striatal cells transduced with AAV2 vector expressing eGFP was reduced by 71% in rats pre-immunized with wild-type AAV2 compared to non-immunized animals. We detected elevated numbers of OX6(+) activated microglia in the striatum and circulating AAV2-specific neutralizing antibodies in pre-immunized rats. We also observed that the intrastriatal 6-OHDA injection promoted CD8(+) T cell infiltration and enhanced microglia activation. Nevertheless, the 6-OHDA lesion did not alter AAV2-mediated expression of eGFP in either pre-immunized or non-immunized rats. Our findings indicate that intracerebral AAV2-based gene therapy is compromised in rats with pre-existing immunity to AAV2. By contrast, a local neuroinflammatory response, caused by intrastriatal a 6-OHDA injection, does not affect viral vector-mediated transgene expression. Our results emphasize the importance of monitoring circulating AAV-specific neutralizing antibodies in patients undergoing intracerebral gene therapy using AAV vectors. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5

    Directory of Open Access Journals (Sweden)

    Daniel J. Schuster

    2014-08-01

    Full Text Available We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5. Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated astrocytes and neurons. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space.

  9. Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain.

    Science.gov (United States)

    San Sebastian, W; Samaranch, L; Heller, G; Kells, A P; Bringas, J; Pivirotto, P; Forsayeth, J; Bankiewicz, K S

    2013-12-01

    We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.

  10. Agrobacterium-mediated viral vector-amplified transient gene expression in Nicotiana glutinosa plant tissue culture.

    Science.gov (United States)

    Collens, Jason I; Mason, Hugh S; Curtis, Wayne R

    2007-01-01

    A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector. All vectors were delivered to Nicotiana glutinosa plant cell suspension or hairy root cultures via auxotrophic Agrobacterium tumefaciens. Cell culture generated greater yield of reporter gene expression than did root culture, as a result of the limitation imposed on roots to express the protein only in surface tissue containing actively dividing cells. Reporter gene expression increased for cell culture when the reporter gene construct was co-delivered with the construct supplying both viral replication associated proteins (REP and REPA); gene expression decreased when the construct supplying only the viral REP protein was co-delivered. Reporter protein expression increased from 0.091% for the reporter construct alone to 0.22% total soluble protein (% TSP) when the viral Rep-supplying vector was co-delivered with the reporter gene construct. Reporter protein was generated 3 days after the initiation of bacterial co-culture, providing for rapid generation of heterologous protein in cell culture.

  11. Prospects for Foamy Viral Vector Anti-HIV Gene Therapy

    Directory of Open Access Journals (Sweden)

    Arun K. Nalla

    2016-03-01

    Full Text Available Stem cell gene therapy approaches for Human Immunodeficiency Virus (HIV infection have been explored in clinical trials and several anti-HIV genes delivered by retroviral vectors were shown to block HIV replication. However, gammaretroviral and lentiviral based retroviral vectors have limitations for delivery of anti-HIV genes into hematopoietic stem cells (HSC. Foamy virus vectors have several advantages including efficient delivery of transgenes into HSC in large animal models, and a potentially safer integration profile. This review focuses on novel anti-HIV transgenes and the potential of foamy virus vectors for HSC gene therapy of HIV.

  12. Viral gene therapy.

    Science.gov (United States)

    Mancheño-Corvo, P; Martín-Duque, P

    2006-12-01

    Cancer is a multigenic disorder involving mutations of both tumor suppressor genes and oncogenes. A large body of preclinical data, however, has suggested that cancer growth can be arrested or reversed by treatment with gene transfer vectors that carry a single growth inhibitory or pro-apoptotic gene or a gene that can recruit immune responses against the tumor. Many of these gene transfer vectors are modified viruses. The ability for the delivery of therapeutic genes, made them desirable for engineering virus vector systems. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses, adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Because these vector systems have unique advantages and limitations, each has applications for which it is best suited. Retroviral vectors can permanently integrate into the genome of the infected cell, but require mitotic cell division for transduction. Adenoviral vectors can efficiently deliver genes to a wide variety of dividing and nondividing cell types, but immune elimination of infected cells often limits gene expression in vivo. Herpes simplex virus can deliver large amounts of exogenous DNA; however, cytotoxicity and maintenance of transgene expression remain as obstacles. AAV also infects many non-dividing and dividing cell types, but has a limited DNA capacity. This review discusses current and emerging virusbased genetic engineering strategies for the delivery of therapeutic molecules or several approaches for cancer treatment.

  13. Filamentous Structures Induced by a Phytoreovirus Mediate Viral Release from Salivary Glands in Its Insect Vector.

    Science.gov (United States)

    Mao, Qianzhuo; Liao, Zhenfeng; Li, Jiajia; Liu, Yuyan; Wu, Wei; Chen, Hongyan; Chen, Qian; Jia, Dongsheng; Wei, Taiyun

    2017-06-15

    Numerous viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. These viruses circulate in the vector body, enter the salivary gland, and then are released into the apical plasmalemma-lined cavities, where saliva is stored. The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission. Here, we report a novel mechanism used by a persistent virus to overcome this essential barrier. We observed that the infection by rice gall dwarf virus (RGDV), a species of the genus Phytoreovirus in the family Reoviridae, induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector, Recilia dorsalis Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities, through a direct interaction of Pns11 of RGDV and actin of R. dorsalis Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary cavities, preventing viral transmission by R. dorsalis For the first time, we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities, thus overcoming the last membrane barrier for viral transmission by insect vectors.IMPORTANCE Understanding how persistent viruses overcome multiple tissue and membrane barriers within the insect vectors until final transmission is the key for viral disease control. The apical plasmalemma of the cavities where saliva is stored in the salivary glands is the last barrier for viral transmission by insect vectors; however, the mechanism is still poorly understood. Here we show that a virus has evolved to exploit virus-induced filaments to perform an exocytosis-like process that enables viral

  14. Improvement of Adeno-Associated Virus-Mediated Liver Transduction Efficacy by Regional Administration in Macaca fascicularis.

    Science.gov (United States)

    Zabaleta, Nerea; Salas, David; Paramo, Maria; Hommel, Mirja; Sier-Ferreira, Valerie; Hernandez-Alcoceba, Ruben; Prieto, Jesus; Bilbao, Jose I; Gonzalez-Aseguinolaza, Gloria

    2017-06-01

    The liver is a central organ in metabolism and can be affected by numerous inherited metabolic disorders. Recombinant adeno-associated virus (AAV)-based gene therapy represents a promising therapeutic approach for such diseases. AAVs have been demonstrated to be safe, and resulted in high and long-term expression in preclinical and clinical studies. However, there are still some concerns regarding the expression levels that can be achieved and the percentage of hepatocytes that can be transduced. Because of the cell-autonomous nature of most metabolic liver disorders, a high percentage of hepatocytes needs to be corrected in order to achieve a therapeutic effect. The goal of our work was to improve transduction efficacy of the liver by conveying the vector directly to hepatic tissue. Interventional radiology procedures were used to administer an AAV5 vector expressing a secreted form of human embryonic alkaline phosphatase (hSEAP) under the control of a liver-specific promoter to a clinically relevant animal model, Macaca fascicularis. Balloon occlusion of the regional hepatic venous flow was performed while injecting the vector either into the hepatic artery (HA) or, against flow, via the suprahepatic vein (SHV). In both cases the vector was injected into the right hepatic lobules, and the two routes were compared with conventional intravenous administration. Higher hSEAP levels were obtained when the vector was administered via SHV or HA than after intravenous injection. Furthermore, higher expression levels correlated with a higher number of vector genomes in the injected lobules. In conclusion, direct administration of AAV vectors via the hepatic blood flow with simultaneous balloon occlusion of the hepatic outflow increases liver transduction efficacy in comparison with systemic delivery and can be further improved in bigger animals or humans, where it would be technically feasible to inject the vector into the hepatic vasculature in the generality of lobules.

  15. Viral Vectors for Use in the Development of Biodefense Vaccines

    Science.gov (United States)

    2005-06-17

    development . . . . . . . . . . . . . . . . . . 1308 6. Anti-vector immune responses associated with virus-vectored vaccines...to influence the outcome of a local election. Another radical group in Japan, the Aum Shinrikyo, allegedly conducted research on BoNT, B. anthracis...aflotoxin and actively researched Clostridium perfringins, rotavirus , echovi- rus 71, and camelpox virus for use in biological warfare [2,3]. Their

  16. Viral vector-mediated transgenic cell therapy in regenerative medicine: safety of the process.

    Science.gov (United States)

    Liu, Yang; Wang, Dong-An

    2015-04-01

    There are safety concerns regarding viral vectors in regenerative medicine research because of adverse experiences in conventional gene therapy with systemic delivery of recombinant virus. Transgenic cell therapy emerges as an attractive strategy, in which the genes of interest are delivered in vitro into isolated cells first; instead of transgene vectors, these transgenic cells are then implanted back to the host. This ex vivo strategy enables the examination of cell viability and phenotype before subsequent transplantation and prevents to the most extent the potential delivery-related hazards caused by exposure of viral components to the host. The transgenic implants are often localized, thus traceable for safety monitoring except those cases involving systemic distribution of transgenic cells. The safety of ex vivo process used in viral vector-mediated transgenic cell therapy for regenerative medicine purpose. Safety concerns related to viral vector delivery can be dispelled in the majority of regenerative medicine applications by transgenic cell therapy. The ex vivo process executes in vitro transfection before subsequent transplantation of transgenic cells so that it avoids the exposure of viral components (particularly capsids or envelops) to the host, while this exposure is inevitable in conventional in vivo gene therapy. Besides, the practice of localized cell implantation and in vitro manipulation also reinforce the safety of transgenic cell therapy. Given the significantly reduced delivery-related hazard, viral vector-mediated transgenic cell therapy can be generally considered as a safe approach for most regenerative medicine applications.

  17. Detection and serological identification of adeno-associated virus in avian adenovirus stocks.

    Science.gov (United States)

    El Mishad, A M; McCormick, K J; Yates, V J; Trentin, J J

    1975-02-01

    Eleven avian adenovirus strains were tested for the presence of avian adeno-associated viruses (AAAV). Six strains contained AAAV. Electron microscopy using rabbit anti-AAAV serum was useful in detecting the satellite virus. The AAAV previously isolated from guail bronchitis virus was related to each of the six new isolates by immunoagglutination, complement fixation, immunodiffusion, and neutralization tests.

  18. Development of replication-competent viral vectors for HIV vaccine delivery.

    Science.gov (United States)

    Parks, Christopher L; Picker, Louis J; King, C Richter

    2013-09-01

    To briefly describe some of the replication-competent vectors being investigated for development of candidate HIV vaccines focusing primarily on technologies that have advanced to testing in macaques or have entered clinical trials. Replication-competent viral vectors have advanced to the stage at which decisions can be made regarding the future development of HIV vaccines. The viruses being used as replication-competent vector platforms vary considerably, and their unique attributes make it possible to test multiple vaccine design concepts and also mimic various aspects of an HIV infection. Replication-competent viral vectors encoding simian immunodeficiency virus or HIV proteins can be used to safely immunize macaques, and in some cases, there is evidence of significant vaccine efficacy in challenge protection studies. Several live HIV vaccine vectors are in clinical trials to evaluate immunogenicity, safety, the effect of mucosal delivery, and potential effects of preexisting immunity. A variety of DNA and RNA viruses are being used to develop replication-competent viral vectors for HIV vaccine delivery. Multiple viral vector platforms have proven to be well tolerated and immunogenic with evidence of efficacy in macaques. Some of the more advanced HIV vaccine prototypes based on vesicular stomatitis virus, vaccinia virus, measles virus, and Sendai virus are in clinical trials.

  19. Vector serotype screening for use in ovine perinatal lung gene therapy.

    Science.gov (United States)

    McClain, Lauren E; Davey, Marcus G; Zoltick, Phillip W; Limberis, Maria P; Flake, Alan W; Peranteau, William H

    2016-06-01

    Successful in utero or perinatal gene therapy for congenital lung diseases, such as cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral (AAV) vector serotypes following intratracheal delivery. Six different AAV vector serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe of perinatal sheep via bronchoscopy. At 1 week, samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real-time polymerase chain reaction (qPCR). Fluorescent stereomicroscopy demonstrated GFP expression in the right upper lobe following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small- and medium-sized airways following intratracheal injection of AAV1, 6, 8, 9, and rh10. However, only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40 cycles, AAV1 did not show GFP gene amplification. Adeno-associated viral vector serotypes 6, 8, 9, and rh10 demonstrated efficient GFP transgene expression at early time points, and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Viral vectors: a look back and ahead on gene transfer technology.

    Science.gov (United States)

    Vannucci, Laura; Lai, Michele; Chiuppesi, Flavia; Ceccherini-Nelli, Luca; Pistello, Mauro

    2013-01-01

    No matter what their origin, strain and family, viruses have evolved exquisite strategies to reach and penetrate specific target cells where they hijack the cellular machinery to express viral genes and produce progeny particles. The ability to deliver and express genetic information to cells is the basis for exploiting viruses as "Trojan horses" to genetically modify the natural cell target or, upon manipulation of the viral receptor to retarget the virus, to genetically engineer different cell types. This process, known as transduction, is accomplished using viral vectors derived from parental wild type viruses whose viral genes, essential for replication and virulence, have been replaced with the heterologous gene(s) required for cell manipulation. Rearrangement of the viral genome to impede replication or generation of infectious virions but maintaining the ability to deliver nucleic acids has been the object of intense research since the early 1980s. Technological advances and the ever-growing knowledge of molecular virology and virus-host cell relationships have constantly improved the safety profile of viral vectors that are now used in vitro and in vivo to study cellular gene function, correct genetic defects (gene therapy), express therapeutic proteins, vaccinate against infectious agents and tumors, produce experimental animal models, and for other purposes. This review illustrates the strategies used to generate some of the most used viral vectors, and their advantages, limitations and principal applications.

  1. Generation of Helper Plasmids Encoding Mutant Adeno-associated Virus Type 2 Capsid Proteins with Increased Resistance against Proteasomal Degradation

    Directory of Open Access Journals (Sweden)

    Naghmeh Ahmadiankia

    2013-07-01

    Full Text Available   Objective(s: Adeno-associated virus type 2 (AAV2 vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y→F mutations.   Materials and Methods: A one-step, high-fidelity polymerase chain reaction (PCR cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant, subcloning (Y444730F and Y500730F mutants or a combination of both techniques (Y444500730F mutant. Results: Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping. Conclusion: PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

  2. Adenoviral and AAV-mediated gene transfer to the inner ear: role of serotype, promoter, and viral load on in vivo and in vitro infection efficiencies.

    Science.gov (United States)

    Luebke, Anne E; Rova, Cherokee; Von Doersten, Peter G; Poulsen, David J

    2009-01-01

    The lack of effective treatments for many forms of hearing and vestibular disorders has produced interest in virally mediated gene therapies. However, to develop a gene therapy strategy that would successfully treat inner ear disorders, appropriate viral vectors capable of transfecting cochlear and support cells must be identified. While virally mediated gene transfer into the inner ear has been accomplished using herpes simplex type I virus, vaccinia virus, retroviruses, adenovirus, and adeno-associated virus (AAV), we will restrict our discussion to AAV and adenoviral vectors. Issues such as vector toxicity and load, viral serotype and backbone, and promoter specificity are discussed and contrasted for both in vivo vs. in vitro inner ear gene transfer. Copyright (c) 2009 S. Karger AG, Basel.

  3. Adeno-Associated Virus Type 12 (AAV12): a Novel AAV Serotype with Sialic Acid- and Heparan Sulfate Proteoglycan-Independent Transduction Activity▿

    Science.gov (United States)

    Schmidt, Michael; Voutetakis, Antonis; Afione, Sandra; Zheng, Changyu; Mandikian, Danielle; Chiorini, John A.

    2008-01-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy. Recent isolations of novel AAV serotypes have led to significant advances by broadening the tropism and increasing the efficiency of gene transfer to the desired target cell. However, a major concern that remains is the strong preexisting immune responses to several vectors. In this paper, we describe the isolation and characterization of AAV12, an AAV serotype with unique biological and immunological properties. In contrast to those of all other reported AAVs, AAV12 cell attachment and transduction do not require cell surface sialic acids or heparan sulfate proteoglycans. Furthermore, rAAV12 is resistant to neutralization by circulating antibodies from human serum. The feasibility of rAAV12 as a vector was demonstrated in a mouse model in which muscle and salivary glands were transduced. These characteristics make rAAV12 an interesting candidate for gene transfer applications. PMID:18045941

  4. The structure of adeno-associated virus serotype 3B (AAV-3B): Insights into receptor binding and immune evasion

    Science.gov (United States)

    Lerch, Thomas F.; Xie, Qing; Chapman, Michael S.

    2010-01-01

    Adeno-associated viruses (AAVs) are leading candidate vectors for human gene therapy. AAV serotypes have broad cellular tropism and use a variety of cellular receptors. AAV serotype 3 binds to heparan sulfate proteoglycan prior to cell entry and is serologically distinct from other serotypes. The capsid features that distinguish AAV-3B from other serotypes are poorly understood. The structure of AAV-3B has been determined to 2.6Å resolution from twinned crystals of an infectious virus. The most distinctive structural features are located in regions implicated in receptor and antibody binding, providing insights into the cell entry mechanisms and antigenic nature of AAVs. We show that AAV-3B has a lower affinity for heparin than AAV-2, which can be rationalized by the distinct features of the AAV-3B capsid. The structure of AAV-3B provides an additional foundation for the future engineering of improved gene therapy vectors with modified receptor binding or antigenic characteristics. PMID:20444480

  5. Influence of HEK293 metabolism on the production of viral vectors and vaccine.

    Science.gov (United States)

    Petiot, Emma; Cuperlovic-Culf, Miroslava; Shen, Chun Fang; Kamen, Amine

    2015-11-04

    Mammalian cell cultures are increasingly used for the production of complex biopharmaceuticals including viral vectors and vaccines. HEK293 is the predominant cell line used for the transient expression of recombinant proteins and a well-established system for the production of viral vectors. Understanding metabolic requirements for high productivity in HEK293 cells remains an important area of investigation. Many authors have presented approaches for increased productivity through optimization of cellular metabolism from two distinct perspectives. One is a non-targeted approach, which is directed to improving feeding strategies by addition of exhausted or critical substrates and eventually removal of toxic metabolites. Alternatively, a targeted approach has attempted to identify specific targets for optimization through better understanding of the cellular metabolism under different operating conditions. This review will present both approaches and their successes with regards to improvement of viral production in HEK293 cells outlining the key relations between HEK293 cell metabolism and viral vector productivity. Also, we will summarize the current knowledge on HEK293 metabolism indicating remaining issues to address and problems to resolve to maximize the productivity of viral vectors in HEK293 cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency.

    Science.gov (United States)

    Hacker, Ulrich T; Wingenfeld, Lisa; Kofler, David M; Schuhmann, Natascha K; Lutz, Sandra; Herold, Tobias; King, Susan B S; Gerner, Franz M; Perabo, Luca; Rabinowitz, Joseph; McCarty, Douglas M; Samulski, Richard J; Hallek, Michael; Büning, Hildegard

    2005-11-01

    Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application. To exclude all influences other than those provided by the capsid, all serotypes contained the same transgene cassette flanked by the AAV2 inverted terminal repeats. We systematically compared these vectors for efficiency in human cancer cell directed gene transfer. In order to identify limiting steps, the influence of second-strand synthesis and proteasomal degradation of AAV in a poorly transducible cell line were examined. AAV2 was the most efficient serotype in all solid tumor cells and primary melanoma cells with transduction rates up to 98 +/- 0.3%. Transduction above 70% could be reached with serotypes 1 (in cervical and prostate carcinoma) and 3 (in cervical, breast, prostate and colon carcinoma) using 1000 genomic particles per cell. In the colon carcinoma cell line HT-29 proteasomal degradation limited AAV1-AAV4-mediated gene transfer. Moreover, inefficient second-strand synthesis prevents AAV2-mediated transgene expression in this cell line. Recent advances in AAV-vector technology suggest that AAV-based vectors can be used for cancer gene therapy. Our comparative analysis revealed that, although AAV2 is the most promising candidate for such an application, serotypes 1 and 3 are valid alternatives. Furthermore, the use of self-complementary AAV vectors and proteasome inhibitors significantly improves cancer cell transduction. Copyright (c) 2005 John Wiley & Sons, Ltd.

  7. A Narcissus mosaic viral vector system for protein expression and flavonoid production

    Science.gov (United States)

    2013-01-01

    Background With the explosive numbers of sequences generated by next generation sequencing, the demand for high throughput screening to understand gene function has grown. Plant viral vectors have been widely used as tools in down-regulating plant gene expression. However, plant viral vectors can also express proteins in a very efficient manner and, therefore, can also serve as a valuable tool for characterizing proteins and their functions in metabolic pathways in planta. Results In this study, we have developed a Gateway®-based high throughput viral vector cloning system from Narcissus Mosaic Virus (NMV). Using the reporter genes of GFP and GUS, and the plant genes PAP1 (an R2R3 MYB which activates the anthocyanin pathway) and selenium-binding protein 1 (SeBP), we show that NMV vectors and the model plant Nicotiana benthamiana can be used for efficient protein expression, protein subcellular localization and secondary metabolite production. Conclusions Our results suggest that not only can the plant viral vector system be employed for protein work but also can potentially be amenable to producing valuable secondary metabolites on a large scale, as the system does not require plant regeneration from seed or calli, which are stages where certain secondary metabolites can interfere with development. PMID:23849589

  8. Is avian adeno-associated virus an endogenous virus of chicken cells?

    Science.gov (United States)

    Dawson, G J; Yates, V J; Chang, P W; Oprandy, J J

    1982-08-05

    The adeno-associated viruses (AAV) are defective parvoviruses which produce infective progeny only in cells co-infected with a 'helper' adenovirus (Ad). Both human and simian AAV have been recovered from human and simian primary cell cultures following their inoculation with 'AAV-free' Ad. Whereas some studies have suggested that AAV exists in a latent state in these cells, others have indicated that the AAV genome is capable of establishing and maintaining a latent state in defined laboratory conditions which mimic the situation proposed for the 'latent' AAV recovered from human and simian tissues. Here, avian adeno-associated virus (AAAV) was consistently recovered from limiting dilutions of purified and unpurified avian Ad stocks propagated in embryonating chicken eggs derived from two independently raised flocks of White Leghorn (WL) chickens but not when these Ad stocks were propagated in duck cells. These observations suggest that AAAV is a latent endogenous virus of at least some flocks of WL chickens.

  9. Viral vector-based prime-boost immunization regimens : a possible involvement of T-cell competition

    NARCIS (Netherlands)

    de Mare, A.; Lambeck, A. J. A.; Regts, J.; van Dam, G. M.; Nijman, H. W.; Snippe, H.; Wilschut, J.; Daemen, T.

    Vaccination with recombinant viral vectors may be impeded by preexisting vector-specific immunity or by vector-specific immunity induced during the priming immunization. It is assumed that virus-neutralizing antibodies represent the principal effector mechanism of vector-specific immunity, while

  10. Cloning of an Avian Adeno-Associated Virus (AAAV) and Generation of Recombinant AAAV Particles

    OpenAIRE

    Bossis, Ioannis; Chiorini, John A.

    2003-01-01

    Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The ent...

  11. Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

    OpenAIRE

    An, Heeyoung; Cho, Doo-Wan; Lee, Seung Eun; Yang, Young-Su; Han, Su-Cheol; Lee, C. Justin

    2016-01-01

    Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey br...

  12. Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5

    OpenAIRE

    Schuster, Daniel J.; Belur, Lalitha R.; Riedl, Maureen S.; Schnell, Stephen A.; Podetz-Pedersen, Kelly M; Kitto, Kelley F.; R. Scott McIvor; Lucy eVulchanova; Fairbanks, Carolyn A.

    2014-01-01

    We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5). Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir) in thalamus was most prominent in medial geniculate nu...

  13. Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma

    OpenAIRE

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Zucman-Rossi, Jessica

    2016-01-01

    Adeno-associated virus type 2 (AAV2) is a defective DNA virus that was previously considered to be non-pathogenic. We identified somatic AAV2 integration in a subset of 11 hepatocellular carcinomas (HCC) that mainly developed in normal liver without known etiology through recurrent insertional mutagenesis in cancer driver genes such as telomerase reverse transcriptase (TERT), cyclin A2 (CCNA2), cyclin E1 (CCNE1), tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10), and lysine (K)-...

  14. Adeno-Associated Virus-Mediated Correction of a Canine Model of Glycogen Storage Disease Type Ia

    Science.gov (United States)

    Weinstein, David A.; Correia, Catherine E.; Conlon, Thomas; Specht, Andrew; Verstegen, John; Onclin-Verstegen, Karine; Campbell-Thompson, Martha; Dhaliwal, Gurmeet; Mirian, Layla; Cossette, Holly; Falk, Darin J.; Germain, Sean; Clement, Nathalie; Porvasnik, Stacy; Fiske, Laurie; Struck, Maggie; Ramirez, Harvey E.; Jordan, Juan; Andrutis, Karl; Chou, Janice Y.; Byrne, Barry J.

    2010-01-01

    Abstract Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-α. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver. PMID:20163245

  15. Alpha2,3 and alpha2,6 N-linked sialic acids facilitate efficient binding and transduction by adeno-associated virus types 1 and 6.

    Science.gov (United States)

    Wu, Zhijian; Miller, Edward; Agbandje-McKenna, Mavis; Samulski, Richard Jude

    2006-09-01

    Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either alpha2,3-linked or alpha2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcalpha2-3GalNAcbeta1-4GlcNAc, as well as two glycoproteins with alpha2,3 and alpha2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support alpha2,3 and alpha2,6 sialic acids that

  16. α2,3 and α2,6 N-Linked Sialic Acids Facilitate Efficient Binding and Transduction by Adeno-Associated Virus Types 1 and 6

    Science.gov (United States)

    Wu, Zhijian; Miller, Edward; Agbandje-McKenna, Mavis; Samulski, Richard Jude

    2006-01-01

    Recombinant adeno-associated viruses (AAVs) are promising vectors in the field of gene therapy. Different AAV serotypes display distinct tissue tropism, believed to be related to the distribution of their receptors on target cells. Of the 11 well-characterized AAV serotypes, heparan sulfate proteoglycan and sialic acid have been suggested to be the attachment receptors for AAV type 2 and types 4 and 5, respectively. In this report, we identify the receptor for the two closely related serotypes, AAV1 and AAV6. First, we demonstrate using coinfection experiments and luciferase reporter analysis that AAV1 and AAV6 compete for similar receptors. Unlike heparin sulfate, enzymatic or genetic removal of sialic acid markedly reduced AAV1 and AAV6 binding and transduction. Further analysis using lectin staining and lectin competition assays identified that AAV1 and AAV6 use either α2,3-linked or α2,6-linked sialic acid when transducing numerous cell types (HepG2, Pro-5, and Cos-7). Treatment of cells with proteinase K but not glycolipid inhibitor reduced AAV1 and AAV6 infection, supporting the hypothesis that the sialic acid that facilitates infection is associated with glycoproteins rather than glycolipids. In addition, we determined by inhibitor (N-benzyl GalNAc)- and cell line-specific (Lec-1) studies that AAV1 and AAV6 require N-linked and not O-linked sialic acid. Furthermore, a resialylation experiment on a deficient Lec-2 cell line confirmed a 2,3 and 2,6 N-linked sialic acid requirement, while studies of mucin with O-linked sialic acid showed no inhibition effect for AAV1 and AAV6 transduction on Cos-7 cells. Finally, using a glycan array binding assay we determined that AAV1 efficiently binds to NeuAcα2-3GalNAcβ1-4GlcNAc, as well as two glycoproteins with α2,3 and α2,6 N-linked sialic acids. Taken together, competition, genetic, inhibitor, enzymatic reconstitution, and glycan array experiments support α2,3 and α2,6 sialic acids that are present on N

  17. Potentiation of anthrax vaccines using protective antigen-expressing viral replicon vectors.

    Science.gov (United States)

    Wang, Hai-Chao; An, Huai-Jie; Yu, Yun-Zhou; Xu, Qing

    2015-02-01

    DNA vaccines require improvement for human use because they are generally weak stimulators of the immune system in humans. The efficacy of DNA vaccines can be improved using a viral replicon as vector to administer antigen of pathogen. In this study, we comprehensively evaluated the conventional non-viral DNA, viral replicon DNA or viral replicon particles (VRP) vaccines encoding different forms of anthrax protective antigen (PA) for specific immunity and protective potency against anthrax. Our current results clearly suggested that these viral replicon DNA or VRP vaccines derived from Semliki Forest virus (SFV) induced stronger PA-specific immune responses than the conventional non-viral DNA vaccines when encoding the same antigen forms, which resulted in potent protection against challenge with the Bacillus anthracis strain A16R. Additionally, the naked PA-expressing SFV replicon DNA or VRP vaccines without the need for high doses or demanding particular delivery regimens elicited robust immune responses and afforded completely protective potencies, which indicated the potential of the SFV replicon as vector of anthrax vaccines for use in clinical application. Therefore, our results suggest that these PA-expressing SFV replicon DNA or VRP vaccines may be suitable as candidate vaccines against anthrax. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. VSV-GP: a potent viral vaccine vector that boosts the immune response upon repeated applications.

    Science.gov (United States)

    Tober, Reinhard; Banki, Zoltan; Egerer, Lisa; Muik, Alexander; Behmüller, Sandra; Kreppel, Florian; Greczmiel, Ute; Oxenius, Annette; von Laer, Dorothee; Kimpel, Janine

    2014-05-01

    Antivector immunity limits the response to homologous boosting for viral vector vaccines. Here, we describe a new, potent vaccine vector based on replication-competent vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP), which we previously showed to be safe in mice. In mice, VSV and VSV-GP encoding ovalbumin (OVA) as a model antigen (VSV-OVA and VSV-GP-OVA) induced equal levels of OVA-specific humoral and cellular immune responses upon a single immunization. However, boosting with the same vector was possible only for VSV-GP-OVA as neutralizing antibodies to VSV limited the immunogenicity of the VSV-OVA boost. OVA-specific cytotoxic T-lymphocyte (CTL) responses induced by VSV-GP-OVA were at least as potent as those induced by an adenoviral state-of-the-art vaccine vector and completely protected mice in a Listeria monocytogenes challenge model. VSV-GP is so far the only replication-competent vaccine vector that does not lose efficacy upon repeated application. Although there has been great progress in treatment and prevention of infectious diseases in the past several years, effective vaccines against some of the most serious infections, e.g., AIDS, malaria, hepatitis C, or tuberculosis, are urgently needed. Here, several approaches based on viral vector vaccines are under development. However, for all viral vaccine vectors currently in clinical testing, repeated application is limited by neutralizing antibodies to the vector itself. Here, we have exploited the potential of vesicular stomatitis virus pseudotyped with the glycoprotein of the lymphocytic choriomeningitis virus (VSV-GP) as a vaccine platform. VSV-GP is the first replication-competent viral vector vaccine that does not induce vector-specific humoral immunity, i.e., neutralizing antibodies, and therefore can boost immune responses against a foreign antigen by repeated applications. The vector allows introduction of various antigens and

  19. Updates in inducible transgene expression using viral vectors: from transient to stable expression.

    Science.gov (United States)

    Mortimer, Cara L; Dugdale, Benjamin; Dale, James L

    2015-04-01

    The prospect of economically producing useful biologics in plants has greatly increased with the advent of viral vectors. The ability of viral vectors to amplify transgene expression has seen them develop into robust transient platforms for the high-level, rapid production of recombinant proteins. To adapt these systems to stably transformed plants, new ways of deconstructing the virus machinery and linking its expression and replication to chemically controlled promoters have been developed. The more advanced of these stable, inducible hyper-expression vectors provide both activated and amplified heterologous transgene expression. Such systems could be deployed in broad acre crops and provide a pathway to fully exploit the advantages of plants as a platform for the manufacture of a wide spectrum of products. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

    Directory of Open Access Journals (Sweden)

    Daniel C. Chung

    2011-11-01

    Full Text Available Background/Aims: Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods: Adeno-associated virus (AAV offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber’s congenital amaurosis, a disease of childhood blindness. Results: Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions: We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.

  1. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    Energy Technology Data Exchange (ETDEWEB)

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  2. Humoral and cellular capsid-specific immune responses to adeno-associated virus type 1 in randomized healthy donors.

    Science.gov (United States)

    Veron, Philippe; Leborgne, Christian; Monteilhet, Virginie; Boutin, Sylvie; Martin, Samia; Moullier, Philippe; Masurier, Carole

    2012-06-15

    A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8(+) cells. IFN-γ-producing cells were also observed among effector memory CD4(+) cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.

  3. Adeno-associated virus serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport.

    Science.gov (United States)

    Castle, Michael J; Gershenson, Zachary T; Giles, April R; Holzbaur, Erika L F; Wolfe, John H

    2014-08-01

    Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane, rather

  4. Viral vector-based reversible neuronal inactivation and behavioral manipulation in the macaque monkey

    Directory of Open Access Journals (Sweden)

    Kristina Juliane Nielsen

    2012-06-01

    Full Text Available Viral vectors are promising tools for the dissection of neural circuits. In principle, they can manipulate neurons at a level of specificity not otherwise achievable. While many studies have used viral vector-based approaches in the rodent brain, only a few have employed this technique in the non-human primate, despite the importance of this animal model for neuroscience research. Here, we report for the first time that a viral vector-based approach can be used to manipulate a monkey’s behavior in a task. For this purpose, we used the allatostatin receptor/allatostatin (AlstR/AL system, which has previously been shown to allow inactivation of neurons in vivo. The AlstR was expressed in neurons in monkey V1 by injection of an AAV1 vector. Two monkeys were trained in a detection task, in which they had to make a saccade to a faint peripheral target. Injection of AL caused a retinotopic deficit in the detection task in one monkey. Specifically, the monkey showed marked impairment for detection targets placed at the visual field location represented at the virus injection site, but not for targets shown elsewhere. We confirmed that these deficits indeed were due to the interaction of AlstR and AL by injecting saline, or AL at a V1 location without AlstR expression. Post-mortem histology confirmed AlstR expression in this monkey. We failed to replicate the behavioral results in a second monkey, as AL injection did not impair the second monkey’s performance in the detection task. However, post-mortem histology revealed a very low level of AlstR expression in this monkey. Our results demonstrate that viral vector-based approaches can produce effects strong enough to influence a monkey’s performance in a behavioral task, supporting the further development of this approach for studying how neuronal circuits control complex behaviors in non-human primates.

  5. Role of T cell competition in the induction of cytotoxic T lymphocyte activity during viral vector-based immunization regimens.

    NARCIS (Netherlands)

    Lambeck, A.J.A.; Nijman, H.W.; Hoogeboom, B.N.; Regts, J.; Mare, A. de; Wilschut, J.; Daemen, T.

    2010-01-01

    T cell competition between antigen- and vector-specific T cells may determine the outcome of viral vector-based immunization regimens, as we previously proposed. Here, we unravelled the interplay between antigen- and vector-specific immunity, using recombinant Semliki Forest virus (rSFV). Priming of

  6. The AAV vector toolkit: poised at the clinical crossroads

    National Research Council Canada - National Science Library

    Asokan, Aravind; Schaffer, David V; Samulski, R Jude

    2012-01-01

    The discovery of naturally occurring adeno-associated virus (AAV) isolates in different animal species and the generation of engineered AAV strains using molecular genetics tools have yielded a versatile AAV vector toolkit...

  7. Infectious bursal disease virus as a replication-incompetent viral vector expressing green fluorescent protein.

    Science.gov (United States)

    Mosley, Yung-Yi C; Wu, Ching Ching; Lin, Tsang Long

    2017-01-01

    Infectious bursal disease virus (IBDV) has been established as a replication-competent viral vector capable of carrying an epitope at multiple loci in the genome. To enhance the safety and increase the insertion capacity of IBDV as a vector, a replication-incompetent IBDV vector was developed in the present study. The feasibility of replacing one of the viral gene loci, including pvp2, vp3, vp1, or the polyprotein vp243, with the sequence of green fluorescent protein (GFP) was explored. A method combining TCID50 and immunoperoxidase monolayer assay (IPMA) determined the most feasible locus for gene replacement to be pvp2. The genomic segment containing gfp at the pvp2 locus was able to be encapsidated into IBDV particles. Furthermore, the expression of GFP in GFP-IBDV infected cells was confirmed by Western blotting and GFP-IBDV particles showed similar morphology and size to that of wildtype IBDV by electron microscopy. By providing the deleted protein in trans in a packaging cell line (pVP2-DF1), replication-incompetent GFP-IBDV particles were successfully plaque-quantified. The gfp sequence from the plaque-forming GFP-IBDV in pVP2-DF1 was confirmed by RT-PCR and sequencing. To our knowledge, GFP-IBDV developed in the present study is the first replication-incompetent IBDV vector which expresses a foreign protein in infected cells without the capability to produce viral progeny. Additionally, such replication-incompetent IBDV vectors could serve as bivalent vaccine vectors for conferring protection against infections with IBDV and other economically important, or zoonotic, avian pathogens.

  8. Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9

    OpenAIRE

    DiMattia, Michael A.; Nam, Hyun-Joo; Van Vliet, Kim; Mitchell, Matthew; Bennett, Antonette; Gurda, Brittney L.; McKenna, Robert; Olson, Norman H.; Sinkovits, Robert S.; Potter, Mark; Byrne, Barry J.; Aslanidi, George; Zolotukhin, Sergei; Muzyczka, Nicholas; Baker, Timothy S.

    2012-01-01

    Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsi...

  9. Identification and characterization of novel adeno-associated virus isolates in ATCC virus stocks.

    Science.gov (United States)

    Schmidt, Michael; Grot, Emmanuelle; Cervenka, Peter; Wainer, Sandra; Buck, Charles; Chiorini, John A

    2006-05-01

    Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for cell binding and transduction, differences were observed in lectin competition, resulting in distinct tropisms in human cancer cell lines.

  10. Identification and Characterization of Novel Adeno-Associated Virus Isolates in ATCC Virus Stocks

    OpenAIRE

    Schmidt, Michael; Grot, Emmanuelle; Cervenka, Peter; Wainer, Sandra; Buck, Charles; Chiorini, John A.

    2006-01-01

    Adeno-associated viruses (AAVs) depend on a helper virus for efficient replication. To identify novel AAV isolates, we screened a diverse set of virus isolates for the presence of AAV DNA. AAVs found in 10 simian adenovirus isolates showed greater than 96% homology to AAV1 and AAV6 but had distinct biological properties. Two representatives of this group, AAV(VR-195) and AAV(VR-355), were studied in more detail. While the novel AAVs had high sequence homologies and required sialic acid for ce...

  11. Structural Characterization of the Dual Glycan Binding Adeno-Associated Virus Serotype 6▿ †

    OpenAIRE

    Ng, Robert; Govindasamy, Lakshmanan; Gurda, Brittney L.; McKenna, Robert; Kozyreva, Olga G.; Samulski, R. Jude; Parent, Kristin N.; Baker, Timothy S.; Agbandje-McKenna, Mavis

    2010-01-01

    The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consiste...

  12. Adeno-associated virus type 2 as an oncogenic virus in human hepatocellular carcinoma.

    Science.gov (United States)

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Zucman-Rossi, Jessica

    2016-03-01

    Adeno-associated virus type 2 (AAV2) is a defective DNA virus that was previously considered to be non-pathogenic. We identified somatic AAV2 integration in a subset of 11 hepatocellular carcinomas (HCC) that mainly developed in normal liver without known etiology through recurrent insertional mutagenesis in cancer driver genes such as telomerase reverse transcriptase (TERT), cyclin A2 (CCNA2), cyclin E1 (CCNE1), tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10), and lysine (K)-specific methyltransferase 2B (KMT2B).

  13. Selective optical control of synaptic transmission in the subcortical visual pathway by activation of viral vector-expressed halorhodopsin.

    Directory of Open Access Journals (Sweden)

    Katsuyuki Kaneda

    Full Text Available The superficial layer of the superior colliculus (sSC receives visual inputs via two different pathways: from the retina and the primary visual cortex. However, the functional significance of each input for the operation of the sSC circuit remains to be identified. As a first step toward understanding the functional role of each of these inputs, we developed an optogenetic method to specifically suppress the synaptic transmission in the retino-tectal pathway. We introduced enhanced halorhodopsin (eNpHR, a yellow light-sensitive, membrane-targeting chloride pump, into mouse retinal ganglion cells (RGCs by intravitreously injecting an adeno-associated virus serotype-2 vector carrying the CMV-eNpHR-EYFP construct. Several weeks after the injection, whole-cell recordings made from sSC neurons in slice preparations revealed that yellow laser illumination of the eNpHR-expressing retino-tectal axons, putatively synapsing onto the recorded cells, effectively inhibited EPSCs evoked by electrical stimulation of the optic nerve layer. We also showed that sSC spike activities elicited by visual stimulation were significantly reduced by laser illumination of the sSC in anesthetized mice. These results indicate that photo-activation of eNpHR expressed in RGC axons enables selective blockade of retino-tectal synaptic transmission. The method established here can most likely be applied to a variety of brain regions for studying the function of individual inputs to these regions.

  14. The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization.

    Science.gov (United States)

    Maggin, Jenna E; James, Jeffrey A; Chappie, Joshua S; Dyda, Fred; Hickman, Alison Burgess

    2012-03-01

    The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies. However, Rep40 and Rep52 have not been observed to form stable oligomers on their own or with DNA, suggesting that important determinants of helicase multimerization lie outside the helicase domain. Here, we report that when the 23-residue linker that connects the endonuclease and helicase domains is appended to the adeno-associated virus type 5 (AAV5) helicase domain, the resulting protein forms discrete complexes on DNA consistent with single or double hexamers. The formation of these complexes does not require the Rep binding site sequence, nor is it nucleotide dependent. These complexes have stimulated ATPase and helicase activities relative to the helicase domain alone, indicating that they are catalytically relevant, a result supported by negative-stain electron microscopy images of hexameric rings. Similarly, the addition of the linker region to the AAV5 Rep endonuclease domain also confers on it the ability to bind and multimerize on nonspecific double-stranded DNA. We conclude that the linker is likely a key contributor to Rep68/78 DNA-dependent oligomerization and may play an important role in mediating Rep68/78's conversion from site-specific DNA binding to nonspecific DNA unwinding.

  15. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

    Directory of Open Access Journals (Sweden)

    Zheng Liu

    2012-04-01

    Full Text Available Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Methods Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. Results The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. Conclusion These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy.

  16. Effects of adeno-associated virus serotype and tissue-specific expression on circulating biomarkers of propionic acidemia.

    Science.gov (United States)

    Guenzel, Adam J; Hillestad, Matthew L; Matern, Dietrich; Barry, Michael A

    2014-09-01

    Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca(-/-)(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease.

  17. Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome.

    Directory of Open Access Journals (Sweden)

    Jennifer L Daily

    Full Text Available Angelman syndrome (AS, a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286 and Thr(305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.

  18. Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress.

    Science.gov (United States)

    Kyostio-Moore, S; Bangari, D S; Ewing, P; Nambiar, B; Berthelette, P; Sookdeo, C; Hutto, E; Moran, N; Sullivan, J; Matthews, G L; Scaria, A; Armentano, D

    2013-02-01

    To evaluate the role of synovial oxidative stress on joint pathology in a spontaneous mouse model of osteoarthritis (OA) by intra-articular (IA) delivery of recombinant adeno-associated virus (rAAV) expressing anti-oxidant protein heme oxygenase-1 (HO-1). Joint transduction by rAAV vectors was evaluated with serotype 1, 2, 5 and 8 capsids carrying LacZ gene administered by IA injections into STR/ort mice. Transduced cell types were identified by β-galactosidase staining in sectioned joints. Effect of oxidative stress on AAV transduction of primary synoviocytes in vitro was quantitated by fluorescence-activated cell sorting (FACS) analysis. In vivo, the efficacy of rAAV1/HO-1 was tested by IA administration into STR/ort mice followed by histopathological scoring of cartilage. Levels of 3-nitrotyrosine (3-NT) and HO-1 were assessed by immunohistochemistry (IHC) of joint sections. Administration of a rAAV1 based vector into OA mouse joints resulted in transduction of the synovium, joint capsule, adipocytes and skeletal muscle while none of the serotypes showed significant cartilage transduction. All OA joints exhibited significantly elevated levels of oxidative stress marker, 3-NT, in the synovium compared to OA-resistant CBA-strain of mice. In vitro studies demonstrated that AAV transgene expression in primary synoviocytes was augmented by oxidative stress induced by H(2)O(2) and that a rAAV expressing HO-1 reduced the levels of oxidative stress. In vivo, HO-1 was increased in the synovium of STR/ort mice. However, delivery of rAAV1/HO-1 into OA joints did not reduce cartilage degradation. AAV-mediated HO-1 delivery into OA joints during active disease was not sufficient to improve cartilage pathology in this model. Copyright © 2012 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

  19. Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver

    National Research Council Canada - National Science Library

    Loiler, S A; Conlon, T J; Song, S; Tang, Q; Warrington, K H; Agarwal, A; Kapturczak, M; Li, C; Ricordi, C; Atkinson, M A; Muzyczka, N; Flotte, T R

    2003-01-01

    .... Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting...

  20. Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver.

    Science.gov (United States)

    Loiler, S A; Conlon, T J; Song, S; Tang, Q; Warrington, K H; Agarwal, A; Kapturczak, M; Li, C; Ricordi, C; Atkinson, M A; Muzyczka, N; Flotte, T R

    2003-09-01

    Human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type I diabetes. However, limits to the efficiency of rAAV serotype 2 (rAAV2)-mediated gene transfer have been reported for both of these cell targets. Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting. In order to test this, we generated rAAV particles that display a ligand (ApoE) that targets the low-density lipoprotein receptor, which is present on both of these cell types. The rAAV/ApoE viruses greatly enhanced the efficiency of transduction of both islet cells ex vivo and murine hepatocytes in vivo when compared to native rAAV2 serotype (220- and four-fold, respectively). The use of receptor-targeted rAAV particles may circumvent the lower abundance of receptors on certain nonpermissive cell types.

  1. Genetic manipulation of specific neural circuits by use of a viral vector system.

    Science.gov (United States)

    Kobayashi, Kenta; Kato, Shigeki; Kobayashi, Kazuto

    2017-01-05

    To understand the mechanisms underlying higher brain functions, we need to analyze the roles of specific neuronal pathways or cell types forming the complex neural networks. In the neuroscience field, the transgenic approach has provided a useful gene engineering tool for experimental studies of neural functions. The conventional transgenic technique requires the appropriate promoter regions that drive a neuronal type-specific gene expression, but the promoter sequences specifically functioning in each neuronal type are limited. Previously, we developed novel types of lentiviral vectors showing high efficiency of retrograde gene transfer in the central nervous system, termed highly efficient retrograde gene transfer (HiRet) vector and neuron-specific retrograde gene transfer (NeuRet) vector. The HiRet and NeuRet vectors enable genetical manipulation of specific neural pathways in diverse model animals in combination with conditional cell targeting, synaptic transmission silencing, and gene expression systems. These newly developed vectors provide powerful experimental strategies to investigate, more precisely, the machineries exerting various neural functions. In this review, we give an outline of the HiRet and NeuRet vectors and describe recent representative applications of these viral vectors for studies on neural circuits.

  2. Induction of Robust Immune Responses against Human Immunodeficiency Virus Is Supported by the Inherent Tropism of Adeno-Associated Virus Type 5 for Dendritic Cells▿

    Science.gov (United States)

    Xin, Ke-Qin; Mizukami, Hiroaki; Urabe, Masashi; Toda, Yoshihiko; Shinoda, Kaori; Yoshida, Atsushi; Oomura, Kenji; Kojima, Yoshitsugu; Ichino, Motohide; Klinman, Dennis; Ozawa, Keiya; Okuda, Kenji

    2006-01-01

    The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy. PMID:17005662

  3. Induction of robust immune responses against human immunodeficiency virus is supported by the inherent tropism of adeno-associated virus type 5 for dendritic cells.

    Science.gov (United States)

    Xin, Ke-Qin; Mizukami, Hiroaki; Urabe, Masashi; Toda, Yoshihiko; Shinoda, Kaori; Yoshida, Atsushi; Oomura, Kenji; Kojima, Yoshitsugu; Ichino, Motohide; Klinman, Dennis; Ozawa, Keiya; Okuda, Kenji

    2006-12-01

    The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the human immunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells (DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1, AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity study of AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 gene induced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had been transduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cell-mediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells) and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of AAV-based vaccine and gene therapy.

  4. Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.

    OpenAIRE

    Krasnykh, V N; Mikheeva, G V; Douglas, J T; Curiel, D.T.

    1996-01-01

    To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has bee...

  5. Viral vectors for cystic fibrosis gene therapy: What does the future hold?

    OpenAIRE

    Uta Griesenbach; Makoto Inoue; Mamoru Hasegawa; et al

    2010-01-01

    Uta Griesenbach1, Makoto Inoue2, Mamoru Hasegawa2, Eric WFW Alton11Department of Gene Therapy, Imperial College London, UK; The UK Cystic Fibrosis Gene Therapy Consortium; 2DNAVEC Corporation, Tsukuba, JapanAbstract: Gene transfer to the airway epithelium has been more difficult than originally anticipated, largely because of significant extra- and intracellular barriers in the lung. In general, viral vectors are more adapted to overcoming these barriers than nonviral gene transfer agents and...

  6. Carbohydrate-Based Ice Recrystallization Inhibitors Increase Infectivity and Thermostability of Viral Vectors

    Science.gov (United States)

    Ghobadloo, Shahrokh M.; Balcerzak, Anna K.; Gargaun, Ana; Muharemagic, Darija; Mironov, Gleb G.; Capicciotti, Chantelle J.; Briard, Jennie G.; Ben, Robert N.; Berezovski, Maxim V.

    2014-07-01

    The inability of vaccines to retain sufficient thermostability has been an obstacle to global vaccination programs. To address this major limitation, we utilized carbohydrate-based ice recrystallization inhibitors (IRIs) to eliminate the cold chain and stabilize the potency of Vaccinia virus (VV), Vesicular Stomatitis virus (VSV) and Herpes virus-1 (HSV-1). The impact of these IRIs was tested on the potency of the viral vectors using a plaque forming unit assay following room temperature storage, cryopreservation with successive freeze-thaw cycles and lyophilization. Viral potency after storage with all three conditions demonstrated that N-octyl-gluconamide (NOGlc) recovered the infectivity of shelf stored VV, 5.6 Log10 PFU mL-1 during 40 days, and HSV-1, 2.7 Log10 PFU mL-1 during 9 days. Carbon-linked antifreeze glycoprotein analogue ornithine-glycine-glycine-galactose (OGG-Gal) increases the recovery of VV and VSV more than 1 Log10 PFU mL-1 after 10 freeze-thaw cycles. In VSV, cryostorage with OGG-Gal maintains high infectivity and reduces temperature-induced aggregation of viral particles by 2 times that of the control. In total, OGG-Gal and NOGlc preserve virus potency during cryostorage. Remarkably, NOGlc has potential to eliminate the cold chain and permit room temperature storage of viral vectors.

  7. Defective interfering viruses and their impact on vaccines and viral vectors.

    Science.gov (United States)

    Frensing, Timo

    2015-05-01

    Defective interfering particles (DIPs) have been found for many important viral pathogens and it is believed that most viruses generate DIPs. This article reviews the current knowledge of the generation and amplification of DIPs, which possess deletions in the viral genome but retain the ability to replicate in the presence of a complete helper virus. In addition, mechanisms are discussed by which DIPs interfere with the replication of their helper virus leading to the production of mainly progeny DIPs by coinfected cells. Even though DIPs cannot replicate on their own, they are biologically active and it is well known that they have a huge impact on virus replication, evolution, and pathogenesis. Moreover, defective genomes are potent inducers of the innate immune response. Yet, little attention has been paid to DIPs in recent years and their impact on biotechnological products such as vaccines and viral vectors remains elusive in most cases. With a focus on influenza virus, this review demonstrates that DIPs are important for basic research on viruses and for the production of viral vaccines and vectors. Reducing the generation and/or amplification of DIPs ensures reproducible results as well as high yields and consistent product quality in virus production. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Disruption of vector transmission by a plant-expressed viral glycoprotein.

    Science.gov (United States)

    Montero-Astúa, Mauricio; Rotenberg, Dorith; Leach-Kieffaber, Alexandria; Schneweis, Brandi A; Park, Sunghun; Park, Jungeun K; German, Thomas L; Whitfield, Anna E

    2014-03-01

    Vector-borne viruses are a threat to human, animal, and plant health worldwide, requiring the development of novel strategies for their control. Tomato spotted wilt virus (TSWV) is one of the 10 most economically significant plant viruses and, together with other tospoviruses, is a threat to global food security. TSWV is transmitted by thrips, including the western flower thrips, Frankliniella occidentalis. Previously, we demonstrated that the TSWV glycoprotein GN binds to thrips vector midguts. We report here the development of transgenic plants that interfere with TSWV acquisition and transmission by the insect vector. Tomato plants expressing GN-S protein supported virus accumulation and symptom expression comparable with nontransgenic plants. However, virus titers in larval insects exposed to the infected transgenic plants were three-log lower than insects exposed to infected nontransgenic control plants. The negative effect of the GN-S transgenics on insect virus titers persisted to adulthood, as shown by four-log lower virus titers in adults and an average reduction of 87% in transmission efficiencies. These results demonstrate that an initial reduction in virus infection of the insect can result in a significant decrease in virus titer and transmission over the lifespan of the vector, supportive of a dose-dependent relationship in the virus-vector interaction. These findings demonstrate that plant expression of a viral protein can be an effective way to block virus transmission by insect vectors.

  9. Mutagenic Analysis of an Adeno-Associated Virus Variant Capable of Simultaneously Promoting Immune Resistance and Robust Gene Delivery.

    Science.gov (United States)

    Kim, Yoojin; Kim, Eunmi; Oh, Seokmin; Yoon, Ye-Eun; Jang, Jae-Hyung

    2018-01-01

    In addition to the ability to boost gene delivery efficiency in many therapeutically relevant cells, the capability of circumventing neutralizing antibody (NAb) inactivation is a key prerequisite that gene carriers must fulfill for their extensive applications as therapeutic agents in many gene therapy trials, especially for cancer treatments. This study revealed that a genetically engineered adeno-associated virus (AAV) variant, AAVr3.45, inherently possesses dual beneficial properties as a gene carrier: (i) efficiently delivering therapeutic genes to many clinically valuable cells (e.g., stem or cancer cells) and (ii) effectively bypassing immunoglobulin (IgG) neutralization. Detailed interpretation of the structural features of AAVr3.45, which was previously engineered from AAV2, demonstrated that the LATQVGQKTA peptide at the heparan sulfate proteoglycan binding domain, especially the presence of cationic lysine on the peptide, served as a key motif for dramatically enhancing its gene delivery capabilities, ultimately broadening its tropisms for many cancer cell lines. Furthermore, the substitution of valine on the AAV2 capsid at the amino acid 719 site to methionine functioned as a coordinator for promoting viral resistance against IgG inactivation. The NAb-resistant characteristics of AAVr3.45 were possibly associated with the LATQVGQKTA sequence itself, indicating that its synergistic cooperation with the point mutation (V719M) is required for maximizing its ability to evade NAb inactivation. The potential of AAVr3.45 as a cancer gene therapy agent was confirmed by provoking apoptosis in breast adenocarcinoma by efficiently delivering a pro-apoptotic gene, BIM (Bcl-2-like protein 11), under high titers of human IgG. Thus, the superior aspects of the NAb-resistant AAVr3.45 as a potential therapeutic agent for systemic injection approaches, especially for cancer gene therapy, were highlighted in this study.

  10. Replicating viral vectors as HIV vaccines: Summary report from IAVI Sponsored Satellite Symposium, International AIDS Society Conference, July 22, 2007

    NARCIS (Netherlands)

    Koff, W. C.; Parks, C. L.; Berkhout, B.; Ackland, J.; Noble, S.; Gust, I. D.

    2008-01-01

    At the International AIDS Society Conference oil Pathogenesis, Treatment and Prevention held in Sydney, Australia, in July 2007, the International AIDS Vaccine Initiative (IAVI) convened a satellite symposium entitled 'Accelerating the Development of Replicating Viral Vectors for AIDS Vaccines.' Its

  11. Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice.

    Science.gov (United States)

    Petry, H; Brooks, A; Orme, A; Wang, P; Liu, P; Xie, J; Kretschmer, P; Qian, H S; Hermiston, T W; Harkins, R N

    2008-01-01

    Neutralizing antibodies (nAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a clinical DNA delivery system. The present study was designed to investigate if AAV re-administration in muscle tissue is dependent on the nAB titer. Recombinant (r)AAV serotype 1, as a promising candidate for targeting skeletal muscle, was used for gene delivery. C57Bl/6 mice were infected intramuscularly with doses between 1 x 10(9) and 5 x 10(10) virus particles (vp) of AAV1-expressing luciferase (AAV1-luc) or human interferon-beta (AAV1-hIFNbeta). Increasing transgene expression was observed over the first 2 months and anti-AAV1 nAB titers peaked between weeks 4 and 8. Six months after the first administration, 5 x 10(10) vp of AAV1-IFNbeta were re-administered. Following re-administration, nAB titers increased but did not significantly affect transgene expression from the AAV vector that had been administered first. In contrast, hIFNbeta expression originating from the second vector administration was significantly diminished and reflected the nAB titer present at the day of re-administration. The present study extends earlier observations that preexisting nAB affects AAV1 re-administration. The level of nAB is proportional to the virus dose used for the first injection and transgene expression following re-administration is dependent on preexisting nAB titer.

  12. Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

    Directory of Open Access Journals (Sweden)

    Busch Melanie

    2009-12-01

    Full Text Available Abstract Background Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC on complete Freund's adjuvant-induced inflammatory pain in rats. Results POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin. Conclusion Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.

  13. Viral and vector zoonotic exploitation of a homo-sociome memetic complex.

    Science.gov (United States)

    Rupprecht, C E; Burgess, G W

    2015-05-01

    As most newly characterized emerging infectious diseases are considered to be zoonotic, a modern pre-eminence ascribed within this classification lies clearly within the viral taxonomic realm. In particular, RNA viruses deserve special concern given their documented impact on conservation biology, veterinary medicine and public health, with an unprecedented ability to promote an evolutionary host-pathogen arms race from the ultimate infection and immunity perspective. However, besides the requisite molecular/gross anatomical and physiological bases for infectious diseases to transmit from one host to another, both viral pathogens and their reservoirs/vectors exploit a complex anthropological, cultural, historical, psychological and social suite that specifically defines the phylodynamics within Homo sapiens, unlike any other species. Some of these variables include the ecological benefits of living in groups, decisions on hunting and foraging behaviours and dietary preferences, myths and religious doctrines, health economics, travel destinations, population planning, political decisions on agricultural product bans and many others, in a homo-sociome memetic complex. Taken to an extreme, such complexities elucidate the underpinnings of explanations as to why certain viral zoonoses reside in neglected people, places and things, whereas others are chosen selectively and prioritized for active mitigation. Canine-transmitted rabies serves as one prime example of how a neglected viral zoonosis may transition to greater attention on the basis of renewed advocacy, social media, local champions and vested international community engagement. In contrast, certain bat-associated and arboviral diseases suffer from basic ignorance and perpetuated misunderstanding of fundamental reservoir and vector ecology tenets, translated into failed control policies that only exacerbate the underlying environmental conditions of concern. Beyond applied biomedical knowledge, epidemiological

  14. pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

    Directory of Open Access Journals (Sweden)

    Ogris Manfred

    2010-03-01

    Full Text Available Abstract Background The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP and directed into a 2000 bp long matrix attachment region sequence (MARS derived from the human β-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression. Results Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P element that is known to be less affected by epigenetic silencing events. Conclusions The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

  15. CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

    Science.gov (United States)

    Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

    2017-12-07

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

  16. Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors.

    Science.gov (United States)

    Monjezi, R; Miskey, C; Gogishvili, T; Schleef, M; Schmeer, M; Einsele, H; Ivics, Z; Hudecek, M

    2017-01-01

    Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

  17. Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity

    Directory of Open Access Journals (Sweden)

    Justin Judd

    2012-01-01

    Full Text Available Adeno-associated virus (AAV-derived vectors are promising gene delivery systems, and a number of design strategies have been pursued to improve their performance. For example, genetic insertion of proteins into the capsid may be used to achieve vector retargeting, reduced immunogenicity, or to track vector transport. Unfortunately, rational approaches to genetic insertion have experienced limited success due to the unpredictable context-dependent nature of protein folding and the complexity of the capsid's macroassembly. We report the construction and use of a frame-enriched DNase-based random insertion library based on AAV2 cap, called pAAV2_RaPID (Random Peptide Insertion by DNase. The fluorescent mCherry protein was inserted randomly throughout the AAV2 capsid and the library was selected for fluorescent and infectious variants. A capsid site was identified in VP3 that can tolerate the large protein insertion. In contrast to previous efforts to incorporate fluorescent proteins into the AAV2 capsid, the isolated mCherry mutant maintains native infectivity while displaying robust fluorescence. Collectively, these results demonstrate that the pAAV2_RaPID platform library can be used to create fully infectious AAV vectors carrying large functional protein domains on the capsid.

  18. Lenti-viral vector- mediated genetic modification of the neural scar: predominant transduction of astrocytes but not meningeal cells

    NARCIS (Netherlands)

    Hendriks, W.T.J.; Eggers, R.; Verhaagen, J.; Boer, G.J.

    2007-01-01

    Viral vector-mediated overexpression of neurotrophins in cells constituting the neural scar may represent a powerful approach to rendering scar tissue of a central nervous system (CNS) lesion permissive for neuronal regrowth. In this study a lentiviral vector encoding green fluorescent protein

  19. Systemic gene delivery to the central nervous system using Adeno-associated virus

    Directory of Open Access Journals (Sweden)

    Mathieu eBOURDENX

    2014-06-01

    Full Text Available Adeno-associated virus (AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain-barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  20. Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment.

    Science.gov (United States)

    Ausubel, L J; Meseck, M; Derecho, I; Lopez, P; Knoblauch, C; McMahon, R; Anderson, J; Dunphy, N; Quezada, V; Khan, R; Huang, P; Dang, W; Luo, M; Hsu, D; Woo, S L C; Couture, L

    2011-04-01

    Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

  1. Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

    Science.gov (United States)

    Wu, Hong-Xia; Wang, Hua-Lei; Guo, Xiao-Feng; Yang, Yu-Jiao; Ma, Jin-Zhu; Wang, Tie-Cheng; Gao, Yu-Wei; Zhao, Yong-Kun; Yang, Song-Tao; Xia, Xian-Zhu

    2013-10-01

    To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

  2. Insulin Therapy Improves Adeno-Associated Virus Transduction of Liver and Skeletal Muscle in Mice and Cultured Cells.

    Science.gov (United States)

    Carrig, Sean; Bijjiga, Enoch; Wopat, Mitchell J; Martino, Ashley T

    2016-11-01

    Adeno-associated virus (AAV) gene transfer is a promising treatment for genetic abnormalities. Optimal AAV vectors are showing success in clinical trials. Gene transfer to skeletal muscle and liver is being explored as a potential therapy for some conditions, that is, α1-antitrypsin (AAT) disorder and hemophilia B. Exploring approaches that enhance transduction of liver and skeletal muscle, using these vectors, is beneficial for gene therapy. Regulating hormones as an approach to improve AAV transduction is largely unexplored. In this study we tested whether insulin therapy improves liver and skeletal muscle gene transfer. In vitro studies demonstrated that the temporary coadministration (2, 8, and 24 hr) of insulin significantly improves AAV2-CMV-LacZ transduction of cultured liver cells and differentiated myofibers, but not of lung cells. In addition, there was a dose response related to this improved transduction. Interestingly, when insulin was not coadministered with the virus but given 24 hr afterward, there was no increase in the transgene product. Insulin receptor gene (INSR) expression levels were increased 5- to 13-fold in cultured liver cells and differentiated myofibers when compared with lung cells. Similar INSR gene expression profiles occurred in mouse tissues. Insulin therapy was performed in mice, using a subcutaneously implanted insulin pellet or a high-carbohydrate diet. Insulin treatment began just before intramuscular delivery of AAV1-CMV-schFIX or liver-directed delivery of AAV8-CMV-schFIX and continued for 28 days. Both insulin augmentation therapies improved skeletal muscle- and liver-directed gene transduction in mice as seen by a 3.0- to 4.5-fold increase in human factor IX (hFIX) levels. The improvement was observed even after the insulin therapy ended. Monitoring insulin showed that insulin levels increased during the brief period of rAAV delivery and during the entire insulin augmentation period (28 days). This study demonstrates

  3. Sindbis viral vector induced apoptosis requires translational inhibition and signaling through Mcl-1 and Bak

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    Meruelo Daniel

    2010-02-01

    Full Text Available Abstract Background Sindbis viral vectors are able to efficiently target and kill tumor cells in vivo, as shown using pancreatic and ovarian cancer models. Infection results in apoptosis both in vitro and in vivo. Sindbis vector uptake is mediated by the LAMR, which is upregulated on a number of different tumor types, thus conferring specificity of the vector to a wide range of cancers. In this study we elucidate the mechanism of apoptosis in two tumor cell lines, MOSEC, derived from the ovarian epithelium and Pan02, derived from a pancreatic adenocarcinoma. A comprehensive understanding of the mechanism of apoptosis would facilitate the design of more effective vectors for cancer therapy. Results The initial phase of Sindbis vector induced apoptosis in MOSEC and Pan02 models reconfirms that viral infection is sensed by PKR due to double-stranded RNA intermediates associated with genomic replication. PKR activation results in translation inhibition through eIF2α phosphorylation and initiation of the stress response. Our studies indicate that the roles of two proteins, Mcl-1 and JNK, intimately link Sindbis induced translational arrest and cellular stress. Translational arrest inhibits the synthesis of anti-apoptotic Bcl-2 protein, Mcl-1. JNK activation triggers the release of Bad from 14-3-3, which ultimately results in apoptosis. These signals from translational arrest and cellular stress are propagated to the mitochondria where Bad and Bik bind to Bcl-xl and Mcl-1 respectively. Formation of these heterodimers displaces Bak, which results in caspase 9 cleavage and signaling through the mitochondrial pathway of apoptosis. Conclusion The host cell response to Sindbis is triggered through PKR activation. Our studies demonstrate that PKR activation and subsequent translational arrest is linked to both cellular stress and apoptosis. We have also found the linkage point between translational arrest and apoptosis to be Mcl-1, a protein whose constant

  4. Adeno-associated virus (AAV) capsid genes isolated from rat and mouse liver genomic DNA define two new AAV species distantly related to AAV-5.

    Science.gov (United States)

    Lochrie, Michael A; Tatsuno, Gwen P; Arbetman, Alejandra E; Jones, Kris; Pater, Cheryl; Smith, Peter H; McDonnell, Jennifer W; Zhou, Shang-Zhen; Kachi, Shu; Kachi, Michiko; Campochiaro, Peter A; Pierce, Glenn F; Colosi, Peter

    2006-09-15

    Using polymerase chain reactions and genome walking strategies, adeno-associated virus (AAV)-like capsid genes were isolated from rat and mouse liver genomic DNA, where they are present at AAVs since their amino acid sequences are AAV capsid. They are most similar to the AAV-5 and goat AAV capsids. A recombinant vector with the mouse AAV capsid and a lacZ transgene (rAAV-mo.1 lacZ) was able to transduce rodent cell lines in vitro. However, it was not able to transduce eight human cell lines or primary human fibroblasts in vitro. It did not bind heparin and its ability to transduce cells in vitro was not inhibited by heparin, mucin, or sialic acid suggesting it uses a novel entry receptor. rAAV-mo.1 lacZ was 29 times more resistant to in vitro neutralization by pooled, purified human IgG than AAV-2. In vivo, rAAV-mo.1 lacZ efficiently transduced murine ocular cells after a subretinal injection. Intramuscular injection of a rAAV-mo.1 human factor IX (hFIX) vector into mice resulted in no detectable hFIX in plasma, but intravenous injection resulted in high plasma levels of hFIX, equivalent to that obtained from a rAAV-8 hFIX vector. Biodistribution analysis showed that rAAV-mo.1 primarily transduced liver after an intravenous injection. These AAV capsids may be useful for gene transfer in rodents.

  5. Adeno-associated virus liver transduction efficiency measured by in vivo [18F]FHBG positron emission tomography imaging in rodents and nonhuman primates.

    Science.gov (United States)

    Pañeda, Astrid; Collantes, Maria; Beattie, Stuart G; Otano, Itzia; Snapper, Jolanda; Timmermans, Eric; Guembe, Laura; Petry, Harald; Lanciego, Jose Luis; Benito, Alberto; Prieto, Jesus; Rodriguez-Pena, Maria Sol; Peñuelas, Iván; Gonzalez-Aseguinolaza, Gloria

    2011-08-01

    Recombinant adeno-associated virus 5 (rAAV5) represents a candidate vector with unique advantages for the treatment of hepatic disorders because of its narrow hepatic tropism. Noninvasive in vivo imaging of transgene expression provides an important tool with which to quantify the transduction efficiency, and duration and location, of transgene expression. In this study, we used positron emission tomography (PET) and positron emission tomography-computed tomography (PET-CT) imaging to monitor liver transduction efficacy in rodents and nonhuman primates that received rAAV5 vector encoding herpes simplex virus thymidine kinase (HSV-TK). HSV-TK expression in liver was also measured by immunohistochemistry. Notable differences in liver transduction efficiency were found, dependent on the animal species and sex. Male rodents were better transduced than females, as previously described. Moreover, male nonhuman primates also displayed increased hepatic expression of the rAAV5-delivered transgene, indicating that differences in rAAV-mediated liver transduction can be anticipated in humans. Our results demonstrate the high sensitivity and reproducibility of PET, using HSV-TK and [(18)F]FHBG, to detect gene expression after rAAV vector administration into living animals, confirming the utility of this technology in the quantification of transgene expression, even at low expression levels. However, we also describe how an immune response against HSV-TK hampered analysis of long-term expression in nonhuman primates.

  6. Host and vector-dependent effects on the risk of germline transmission of AAV vectors.

    Science.gov (United States)

    Favaro, Patricia; Downey, Harre D; Zhou, J Shangzhen; Wright, J Fraser; Hauck, Bernd; Mingozzi, Federico; High, Katherine A; Arruda, Valder R

    2009-06-01

    The assessment of the risk of germline transmission of vector-coded sequences is critical for clinical translation of gene transfer strategies. We used rabbit models to analyze the risk of germline transmission of adeno-associated viral (AAV) vectors. Intravenous injection of AAV-2 or AAV-8 resulted in liver-mediated, long-term expression of therapeutic levels of human factor IX (hFIX) in a dose-dependent manner. In high-dose cohorts, AAV-8 resulted in twofold higher levels of circulating hFIX and of vector DNA in liver compared to AAV-2. Vector sequences were found in the semen of all rabbits. The kinetics of vector clearance from semen was dose- and time-dependent but serotype-independent. No late recurrence of AAV-8 sequences was found in the semen over several consecutive cycles of spermatogenesis. In a novel rabbit model, AAV-2 or AAV-8 sequences were detected in the semen of vasectomized animals that lack germ cells. Therefore, structures of the genitourinary (GU) tract, as well as the testis, contribute significantly to vector shedding in the semen. Collectively, data from these two models suggest that the risk of inadvertent germline transmission in males by AAV-8 vectors is low, similar to that of AAV-2, and that AAV dissemination to the semen is in part modulated by host-dependent factors.

  7. [Vesicular stomatitis virus (VSV) as a vaccine vector for immunization against viral infections].

    Science.gov (United States)

    Tomczyk, Tomasz; Orzechowska, Beata

    2013-01-11

    Vesicular stomatitis virus (VSV), a member of the Rhabdoviridae family, is a promising candidate for potential use in construction of antiviral vaccines. In the natural environment VSV is a pathogen of wild ungulates and livestock. Some of the features that make VSV an excellent platform for the development of a range of viral therapeutics includes its immunogenicity and ability to grow to high titers in cell lines approved for vaccine use. Infection in humans is rare and usually asymptomatic, with mild flu-like symptoms. Moreover, due to affinity of VSV envelope glycoprotein to the LDL (low-density lipoprotein) receptor, VSV is effective at targeting a variety of tissues in vivo. A series of research results confirm the possibility of developing VSV-based vaccines against human papilloma viruses (HPV), human immunodeficiency virus (HIV), hepatitis B virus (HBV) and filoviruses (MARV, ZEBOV and SEBOV), as well as the potential use of a successfully developed vaccine against hepatitis C virus (HCV). VSV is neurotropic and infection can cause a viral encephalitis in experimental animals. Therefore, intensive studies are being undertaken to achieve satisfactory expression of the viral antigens while maintaining the safety of the constructed vectors.

  8. Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

    Directory of Open Access Journals (Sweden)

    Claudia Merkl

    Full Text Available Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4 into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

  9. Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.

    Science.gov (United States)

    Li, Hua; Zhang, Feng-Lan; Shi, Wen-Jie; Bai, Xue-Jia; Jia, Shu-Qin; Zhang, Chen-Guang; Ding, Wei

    2015-01-01

    The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

  10. Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants.

    Directory of Open Access Journals (Sweden)

    Hua Li

    Full Text Available The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol propionate (SPDP. Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.

  11. Examining the cross-reactivity and neutralization mechanisms of a panel of mAbs against adeno-associated virus serotypes 1 and 5.

    Science.gov (United States)

    Harbison, Carole E; Weichert, Wendy S; Gurda, Brittney L; Chiorini, John A; Agbandje-McKenna, Mavis; Parrish, Colin R

    2012-02-01

    Neutralizing antibodies play a central role in the prevention and clearance of viral infections, but can be detrimental to the use of viral capsids for gene delivery. Antibodies present a major hurdle for ongoing clinical trials using adeno-associated viruses (AAVs); however, relatively little is known about the antigenic epitopes of most AAV serotypes or the mechanism(s) of antibody-mediated neutralization. We developed panels of AAV mAbs by repeatedly immunizing mice with AAV serotype 1 (AAV1) capsids, or by sequentially immunizing with AAV1 followed by AAV5 capsids, in order to examine the efficiency and mechanisms of antibody-mediated neutralization. The antibodies were not cross-reactive between heterologous AAV serotypes except for a low level of recognition of AAV1 capsids by the AAV5 antibodies, probably due to the initial immunization with AAV1. The neutralization efficiency of different IgGs varied and Fab fragments derived from these antibodies were generally poorly neutralizing. The antibodies appeared to display various alternative mechanisms of neutralization, which included inhibition of receptor-binding and interference with a post-attachment step.

  12. Cooler temperatures destabilize RNA interference and increase susceptibility of disease vector mosquitoes to viral infection.

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    Zach N Adelman

    Full Text Available The impact of global climate change on the transmission dynamics of infectious diseases is the subject of extensive debate. The transmission of mosquito-borne viral diseases is particularly complex, with climatic variables directly affecting many parameters associated with the prevalence of disease vectors. While evidence shows that warmer temperatures often decrease the extrinsic incubation period of an arthropod-borne virus (arbovirus, exposure to cooler temperatures often predisposes disease vector mosquitoes to higher infection rates. RNA interference (RNAi pathways are essential to antiviral immunity in the mosquito; however, few experiments have explored the effects of temperature on the RNAi machinery.We utilized transgenic "sensor" strains of Aedes aegypti to examine the role of temperature on RNA silencing. These "sensor" strains express EGFP only when RNAi is inhibited; for example, after knockdown of the effector proteins Dicer-2 (DCR-2 or Argonaute-2 (AGO-2. We observed an increase in EGFP expression in transgenic sensor mosquitoes reared at 18°C as compared with 28°C. Changes in expression were dependent on the presence of an inverted repeat with homology to a portion of the EGFP sequence, as transgenic strains lacking this sequence, the double stranded RNA (dsRNA trigger for RNAi, showed no change in EGFP expression when reared at 18°C. Sequencing small RNAs in sensor mosquitoes reared at low temperature revealed normal processing of dsRNA substrates, suggesting the observed deficiency in RNAi occurs downstream of DCR-2. Rearing at cooler temperatures also predisposed mosquitoes to higher levels of infection with both chikungunya and yellow fever viruses.This data suggest that microclimates, such as those present in mosquito breeding sites, as well as more general climactic variables may influence the dynamics of mosquito-borne viral diseases by affecting the antiviral immunity of disease vectors.

  13. Safety of the novel influenza viral vector Brucella abortus vaccine in pregnant heifers

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    Kaissar Tabynov

    2016-01-01

    Full Text Available ABSTRACT: The present study provides the first information about the safety of a new influenza viral vector vaccine expressing the Brucella ribosomal protein L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10 or subcutaneous (n=10 route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with B. abortus S19 (n=10 or B. abortus RB51 (n=10 and a negative (PBS+Montanide Gel01; n=10 control group. Clinical studies, thermometry, assessment of local reactogenicity and observation of abortion showed that the vector vaccine via the conjunctival or subcutaneous route was completely safe for pregnant heifers compared to the commercial vaccines B. abortus S19 or B. abortus RB51. The only single adverse event was the formation of infiltration at the site of subcutaneous injection; this reaction was not observed for the conjunctival route.

  14. Chikungunya viral fitness measures within the vector and subsequent transmission potential.

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    Rebecca C Christofferson

    Full Text Available Given the recent emergence of chikungunya in the Americas, the accuracy of forecasting and prediction of chikungunya transmission potential in the U.S. requires urgent assessment. The La Reunion-associated sub-lineage of chikungunya (with a valine substitution in the envelope protein was shown to increase viral fitness in the secondary vector, Ae. albopictus. Subsequently, a majority of experimental and modeling efforts focused on this combination of a sub-lineage of the East-Central-South African genotype (ECSA-V-Ae. albopictus, despite the Asian genotype being the etiologic agent of recent chikungunya outbreaks world-wide. We explore a collection of data to investigate relative transmission efficiencies of the three major genotypes/sub-lineages of chikungunya and found difference in the extrinsic incubation periods to be largely overstated. However, there is strong evidence supporting the role of Ae. albopictus in the expansion of chikungunya that our R0 calculations cannot attribute to fitness increases in one vector over another. This suggests other ecological factors associated with the Ae. albopictus-ECSA-V cycle may drive transmission intensity differences. With the apparent bias in literature, however, we are less prepared to evaluate transmission where Ae. aegypti plays a significant role. Holistic investigations of CHIKV transmission cycle(s will allow for more complete assessment of transmission risk in areas affected by either or both competent vectors.

  15. Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat.

    Science.gov (United States)

    Bish, Lawrence T; Morine, Kevin; Sleeper, Meg M; Sanmiguel, Julio; Wu, Di; Gao, Guangping; Wilson, James M; Sweeney, H Lee

    2008-12-01

    Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9. Neonatal mice were injected with 2.5 x 10(11) genome copies (GC) of AAV serotype 1, 6, 7, 8, or 9 expressing LacZ under the control of the constitutive chicken beta-actin promoter with cytomegalovirus enhancer promoter via intrapericardial injection and monitored for up to 1 year. Adult rats were injected with 5 x 10(11) GC of the AAV vectors via direct cardiac injection and monitored for 1 month. Cardiac distribution of LacZ expression was assessed by X-Gal histochemistry, and beta-galactosidase activity was quantified in a chemiluminescence assay. Cardiac functional data and biodistribution data were also collected in the rat. AAV9 provided global cardiac gene transfer stable for up to 1 year that was superior to other serotypes. LacZ expression was relatively cardiac specific, and cardiac function was unaffected by gene transfer. AAV9 provides high-level, stable expression in the mouse and rat heart and may provide a simple alternative to the creation of cardiac-specific transgenic mice. AAV9 should be used in rodent cardiac studies and may be the vector of choice for clinical trials of cardiac gene transfer.

  16. A directed evolution approach to select for novel Adeno-associated virus capsids on an HIV-1 producer T cell line.

    Science.gov (United States)

    Wooley, Dawn P; Sharma, Priyanka; Weinstein, John R; Kotha Lakshmi Narayan, Poornima; Schaffer, David V; Excoffon, Katherine J D A

    2017-12-01

    A directed evolution approach was used to select for Adeno-associated virus (AAV) capsids that would exhibit more tropism toward an HIV-1 producer T cell line with the long-term goal of developing improved gene transfer vectors. A library of AAV variants was used to infect H9 T cells previously infected or uninfected by HIV-1 followed by AAV amplification with wild-type adenovirus. Six rounds of biological selection were performed, including negative selection and diversification after round three. The H9 T cells were successfully infected with all three wild-type viruses (AAV, adenovirus, and HIV-1). Four AAV cap mutants best representing the small number of variants emerging after six rounds of selection were chosen for further study. These mutant capsids were used to package an AAV vector and subsequently used to infect H9 cells that were previously infected or uninfected by HIV-1. A quantitative polymerase chain reaction assay was performed to measure cell-associated AAV genomes. Two of the four cap mutants showed a significant increase in the amount of cell-associated genomes as compared to wild-type AAV2. This study shows that directed evolution can be performed successfully to select for mutants with improved tropism for a T cell line in the presence of HIV-1. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Adeno-associated virus-mediated expression of myostatin propeptide improves the growth of skeletal muscle and attenuates hyperglycemia in db/db mice.

    Science.gov (United States)

    Jiang, J G; Shen, G F; Li, J; Qiao, C; Xiao, B; Yan, H; Wang, D W; Xiao, X

    2017-03-01

    Inhibition of myostatin, a negative growth modulator for muscle, can functionally enhance muscle mass and improve glucose and fat metabolism in myostatin propeptide (MPRO) transgenic mice. This study was to investigate whether myostatin inhibition by adeno-associated virus (AAV)-mediated gene delivery of MPRO could improve muscle mass and achieve therapeutic effects on glucose regulation and lipid metabolism in the db/db mice and the mechanisms involved in that process. Eight-week-old male db/db mice were administered saline, AAV-GFP and AAV-MPRO/Fc vectors and monitored random blood glucose levels and body weight for 36 weeks. Body weight gain was not different during follow-up among the groups, but AAV-MPRO/Fc vectors resulted high level of MPRO in the blood companied by an increase in skeletal muscle mass and muscle hypertrophy. In addition, AAV-MPRO/Fc-treated db/db mice showed significantly lower blood glucose and insulin levels and significantly increased glucose tolerance and insulin sensitivity compared with the control groups (Pmuscle, although no difference was observed in fat pad weights and serum TG and FFA levels. Finally, AAV-MPRO/Fc-treated mice had enhanced insulin signaling in the skeletal muscle. These data suggest that AAV-mediated MPRO therapy may provide an important clue for potential clinical applications to prevent type II diabetes, and these studies confirm that MPRO is a therapeutic target for type II diabetes.

  18. Molecular characterization of two strains of the avian adeno-associated virus (AAAV).

    Science.gov (United States)

    Hess, M; Paul, G; Kling, S; Monreal, G

    1995-01-01

    An avian adeno-associated virus (AAAV) was isolated after propagating a field isolate of the CELO virus (fowl adenovirus serotype 1 (FAV1)) in embryonated eggs. The isolated dependovirus was compared with the known AAAV obtained from the American Type Culture Collection (ATCC VR-865). The genomes were analysed by digestion with several restriction endonucleases. Although both DNAs have the same size, most restriction enzymes produced different restriction patterns. Double digests were used to construct for the first time restriction maps for avian dependoviruses. The two DNAs rendered different restriction maps in which the different restriction sites were mainly located in the middle and right part of the genomes. The effect of these differences on the structure proteins was shown by western blot analysis. In the immunoblot, the immunofluorescence and immunodiffusion test the two dependoviruses were serologically indistinguishable and therefore can be regarded as two different strains of the same virus. To differentiate between both strains we named the original one as AAAV VR-865 compared with the isolated AAAV DA-1.

  19. Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

    Science.gov (United States)

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

    2008-01-01

    Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model in the domestic ferret using recombinant adeno-associated virus–mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338

  20. The effect of DNA-dependent protein kinase on adeno-associated virus replication.

    Directory of Open Access Journals (Sweden)

    Young-Kook Choi

    Full Text Available BACKGROUND: DNA-dependent protein kinase (DNA-PK is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, w