Sample records for aav vector administration

  1. Preclinical safety evaluation of a recombinant AAV8 vector for X-linked retinoschisis after intravitreal administration in rabbits. (United States)

    Marangoni, Dario; Wu, Zhijian; Wiley, Henry E; Zeiss, Caroline J; Vijayasarathy, Camasamudram; Zeng, Yong; Hiriyanna, Suja; Bush, Ronald A; Wei, Lisa L; Colosi, Peter; Sieving, Paul A


    X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and one of the most common causes of macular degeneration in young men. Currently, no FDA-approved treatments are available for XLRS and a replacement gene therapy could provide a promising strategy. We have developed a novel gene therapy approach for XLRS, based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal route. On the basis of our prior study in an Rs1-KO mouse, this construct transduces efficiently all the retinal layers, resulting in an RS1 expression similar to that observed in the wild-type and improving retinal structure and function. In support of a clinical trial, we carried out a study to evaluate the ocular safety of intravitreal administration of AAV8-scRS/IRBPhRS into 39 New Zealand White rabbits. Two dose levels of vector, 2e(10) and 2e(11) vector genomes per eye (vg/eye), were tested and ocular inflammation was monitored over a 12-week period by serial ophthalmological and histopathological analysis. A mild ocular inflammatory reaction, consisting mainly of vitreous infiltrates, was observed within 4 weeks from injection, in both 2e(10) and 2e(11) vg/eye groups and was likely driven by the AAV8 capsid. At 12-week follow-up, ophthalmological examination revealed no clinical signs of vitreitis in either of the dose groups. However, while vitreous inflammatory infiltrate was significantly reduced in the 2e(10) vg/eye group at 12 weeks, some rabbits in the higher dose group still showed persistence of inflammatory cells, histologically. In conclusion, intravitreal administration of AAV8-scRS/IRBPhRS into the rabbit eye produces a mild and transient intraocular inflammation that resolves, at a 2e(10) vg/eye dose, within 3 months, and does not cause irreversible tissue damages. These data support the initiation of a clinical trial of intravitreal


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    Karen eNieto


    Full Text Available Since their discovery as a tool for gene transfer, vectors derived from the Adeno-Associated Virus (AAV have been used for gene therapy applications and attracted scientist to this field for their exceptional properties of efficiency of in vivo gene transfer and the level and duration of transgene expression. For many years, AAVs have been considered as low immunogenic vectors due to their ability to induce long term expression of non-self-proteins in contrast to what has been observed with other viral vectors, such as adenovirus (Ad, for which strong immune responses against the same transgene products were documented. The perceived low immunogenicity likely explains why the use of AAV vectors for vaccination was not seriously considered before the early 2000s. Indeed, while analyses conducted using a variety of transgenes and animal species slowly changed the vision of the immunological properties of AAVs, an increasing number of studies were also performed in the field of vaccination. Even if the comparison with other modes of vaccination was not systemically performed, the analyses conducted so far in the field of active immunotherapy strongly suggest that AAVs possess some interesting features to be used as tools to produce an efficient and sustained antibody (Ab response. In addition, recent studies also highlighted the potential of AAVs for passive immunotherapy. This review summarizes the main studies conducted to evaluate the potential of AAV vectors for vaccination against infectious agents and discusses their advantages and drawbacks. Altogether, the variety of studies conducted in this field contributes to the understanding of the immunological properties of this versatile virus and to the definition of its possible future applications.

  3. Development of rAAV2-CFTR: History of the First rAAV Vector Product to be Used in Humans. (United States)

    Loring, Heather S; ElMallah, Mai K; Flotte, Terence R


    The first human gene therapy trials using recombinant adeno-associated virus (rAAV) vectors were performed in cystic fibrosis (CF) patients. Over 100 CF patients were enrolled in 5 separate trials of rAAV2-CFTR administration via nasal, endobronchial, maxillary sinus, and aerosol delivery. Recombinant AAV vectors were designed to deliver the CF transmembrane regulator (CFTR) gene and correct the basic CFTR defect by restoring chloride transport and reverting the upregulation of proinflammatory cytokines. However, vector DNA expression was limited in duration because of the low incidence of integration and natural airway epithelium turnover. In addition, repeated administration of AAV-CFTR vector resulted in a humoral immune response that prevented effective gene transfer from subsequent doses of vector. AAV serotype 2 was used in human trials before the comparison with other serotypes and determination that serotypes 1 and 5 not only possess higher tropism for the airway epithelium, but also are capable of bypassing the binding and trafficking processes-both were important hindrances to the effectiveness of rAAV2. Although rAAV-CFTR gene therapy does not appear likely to supplant newer small-molecule CFTR modulators in the near future, early work with rAAV-CFTR provided an important foundation for later use of rAAV in humans.

  4. An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes. (United States)

    Kang, W; Wang, L; Harrell, H; Liu, J; Thomas, D L; Mayfield, T L; Scotti, M M; Ye, G J; Veres, G; Knop, D R


    Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human alpha-1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 x 10(11) DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 x 10(4) and 9.4 x 10(5) ng ml(-1) serum respectively, the latter being clinically relevant.

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

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    Ayşegül Akbay Yarpuzlu


    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

  6. Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

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    Chen Ling


    Full Text Available Although recombinant adeno-associated virus serotype 3 (AAV3 vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs from AAV3 (ITR3, as well as the trans-acting Rep proteins from AAV3 (Rep3 in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ∼10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492 were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of

  7. Small But Increasingly Mighty: Latest Advances in AAV Vector Research, Design, and Evolution. (United States)

    Grimm, Dirk; Büning, Hildegard


    Recombinant gene delivery vectors derived from naturally occurring or genetically engineered adeno-associated viruses (AAV) have taken center stage in human gene therapy, fueled by rapidly accumulating and highly encouraging clinical data. Nonetheless, it has also become evident that the current generation of AAV vectors will require improvements in transduction potency, antibody evasion, and cell specificity in order to realize their full potential and to widen applicability in larger patient cohorts. Fortunately, in the recent past, the field has seen a flurry of exciting new developments that enhance our understanding of AAV vector biology, including virus-host interactions, and/or that expand our arsenal of technologies for AAV capsid design and evolution. This review highlights a collection of latest advances in these areas, which, in the authors' opinion, hold particular promise to propel the AAV vector field forward in the near future, especially when applied in combination. These include fundamental novel insights into the AAV life cycle, from an unexpected role of autophagy and interactions with other viruses to the (re-)discovery of a universal AAV receptor and the function of AAV-AAP for capsid assembly. Concurrently, recent successes in the rational design of next-generation synthetic AAV capsids are pointed out, exemplified by the structure-guided derivation of AAV mutants displaying robust in vivo immune evasion. Finally, a variety of new and innovative strategies for high-throughput generation and screening of AAV capsid libraries are briefly reviewed, including Cre recombinase-based selection, ancestral AAV capsid reconstruction, and DNA barcoding of AAV genomes. All of these examples showcase the present momentum in the AAV field and, together with work by many other academic or industrial entities, raise substantial optimism that the remaining hurdles for human gene therapy with AAV vectors will (soon) be overcome.

  8. Mapping the AAV capsid host antibody response towards the development of second generation gene delivery vectors

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    Yu-Shan eTseng


    Full Text Available The recombinant Adeno-associated virus (rAAV gene delivery system is entering a crucial and exciting phase with the promise of more than 20 years of intense research now realized in a number of successful human clinical trials. However, as a natural host to AAV infection, anti-AAV antibodies are prevalent in the human population. For example, ~70% of human sera samples are positive for AAV serotype 2 (AAV2. Furthermore, low levels of pre-existing neutralizing antibodies in the circulation are detrimental to the efficacy of corrective therapeutic AAV gene delivery. A key component to overcoming this obstacle is the identification of regions of the AAV capsid that participate in interactions with host immunity, especially neutralizing antibodies, to be modified for neutralization escape. Three main approaches have been utilized to map antigenic epitopes on AAV capsids. The first is directed evolution in which AAV variants are selected in the presence of monoclonal antibodies or pooled human sera. This results in AAV variants with mutations on important neutralizing epitopes. The second is epitope searching, achieved by peptide scanning, peptide insertion or site-directed mutagenesis. The third, a structure biology-based approach, utilizes cryo-electron microscopy and image reconstruction of AAV capsids complexed to fragment antibodies, which are generated from monoclonal antibodies, to directly visualize the epitopes. In this review, the contribution of these three approaches to the current knowledge of AAV epitopes and success in their use to create second generation vectors will be discussed.

  9. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

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    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.


    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by ∼ 68% and ∼ 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy

  10. Dual reporter comparative indexing of rAAV pseudotyped vectors in chimpanzee airway. (United States)

    Flotte, Terence R; Fischer, Anne C; Goetzmann, Jason; Mueller, Christian; Cebotaru, Liudmila; Yan, Ziying; Wang, Lilli; Wilson, James M; Guggino, William B; Engelhardt, John F


    Selecting the most efficient recombinant adeno-associated virus (rAAV) serotype for airway gene therapy has been difficult due to cross-specific differences in tropism and immune response between humans and animal models. Chimpanzees--the closest surviving genetic relative of humans--provide a valuable opportunity to select the most effective serotypes for clinical trials in humans. However, designing informative experiments using this protected species is challenging due to limited availability and experimental regulations. We have developed a method using Renilla luciferase (RL) and firefly luciferase (FL) reporters to directly index the relative transduction and immune response of two promising rAAV serotypes following lung coinfection. Analysis of differential luciferase activity in chimpanzee airway brushings demonstrated a 20-fold higher efficiency for rAAV1 over rAAV5 at 90 days, a finding that was similar in polarized human airway epithelia. T-cell responses to AAV5 capsid were stronger than AAV1 capsid. This dual vector indexing approach may be useful in selecting lead vector serotypes for clinical gene therapy and suggests rAAV1 is preferred for cystic fibrosis.

  11. Synthetic AAV/CRISPR vectors for blocking HIV-1 expression in persistently infected astrocytes. (United States)

    Kunze, Christine; Börner, Kathleen; Kienle, Eike; Orschmann, Tanja; Rusha, Ejona; Schneider, Martha; Radivojkov-Blagojevic, Milena; Drukker, Micha; Desbordes, Sabrina; Grimm, Dirk; Brack-Werner, Ruth


    Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes. Analysis of AAV9P1 transduction efficiencies with single brain cell populations, including primary human brain cells, as well as human brain organoids demonstrated that AAV9P1 targeted terminally differentiated human astrocytes much more efficiently than neurons. We then investigated whether AAV9P1 can be used to deliver HIV-inhibitory genes to astrocytes. To this end we generated AAV9P1 vectors containing genes for HIV-1 proviral editing by CRISPR/Cas9. Latently HIV-1 infected astrocytes transduced with these vectors showed significantly diminished reactivation of proviruses, compared with untransduced cultures. Sequence analysis identified mutations/deletions in key HIV-1 transcriptional control regions. We conclude that AAV9P1 is a promising tool for gene delivery to astrocytes and may facilitate inactivation/destruction of persisting HIV-1 proviruses in astrocyte reservoirs. © 2017 Wiley Periodicals, Inc.

  12. Targeting Visceral Fat by Intraperitoneal Delivery of Novel AAV Serotype Vector Restricting Off-Target Transduction in Liver

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    Wei Huang


    Full Text Available It is challenging to genetically manipulate fat in adults. We demonstrate that intraperitoneal (i.p. injection of an engineered adeno-associated virus (AAV serotype Rec2 leads to high transduction of multiple visceral fat depots at a dose of 1 to 2 orders lower than commonly used doses for systemic gene delivery. To target adipose tissue, we develop a single AAV vector harboring two expression cassettes: one using the CBA promoter to drive transgene expression and one using the liver-specific albumin promoter to drive a microRNA-targeting WPRE sequence that only exists in this AAV vector. This dual-cassette vector achieves highly selective transduction of visceral fat while severely restricting off-target transduction of liver. As proof of efficacy, i.p. administration of an adipose-targeting Rec2 vector harboring the leptin gene corrects leptin deficiency, obesity, and metabolic syndromes of ob/ob mice. This study provides a powerful tool to genetically manipulate fat for basic research and gene therapies of genetic and acquired diseases.

  13. High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and Cap. (United States)

    Conway, J E; Rhys, C M; Zolotukhin, I; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J


    Recombinant adeno-associated virus type 2 (rAAV) vectors have recently been used to achieve long-term, high level transduction in vivo. Further development of rAAV vectors for clinical use requires significant technological improvements in large-scale vector production. In order to facilitate the production of rAAV vectors, a recombinant herpes simplex virus type I vector (rHSV-1) which does not produce ICP27, has been engineered to express the AAV-2 rep and cap genes. The optimal dose of this vector, d27.1-rc, for AAV production has been determined and results in a yield of 380 expression units (EU) of AAV-GFP produced from 293 cells following transfection with AAV-GFP plasmid DNA. In addition, d27.1-rc was also efficient at producing rAAV from cell lines that have an integrated AAV-GFP provirus. Up to 480 EU/cell of AAV-GFP could be produced from the cell line GFP-92, a proviral, 293 derived cell line. Effective amplification of rAAV vectors introduced into 293 cells by infection was also demonstrated. Passage of rAAV with d27. 1-rc results in up to 200-fold amplification of AAV-GFP with each passage after coinfection of the vectors. Efficient, large-scale production (>109 cells) of AAV-GFP from a proviral cell line was also achieved and these stocks were free of replication-competent AAV. The described rHSV-1 vector provides a novel, simple and flexible way to introduce the AAV-2 rep and cap genes and helper virus functions required to produce high-titer rAAV preparations from any rAAV proviral construct. The efficiency and potential for scalable delivery of d27.1-rc to producer cell cultures should facilitate the production of sufficient quantities of rAAV vectors for clinical application.

  14. Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment

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    J. Fraser Wright


    Full Text Available Adeno-associated virus (AAV-based vectors expressing therapeutic genes continue to demonstrate great promise for the treatment of a wide variety of diseases and together with other gene transfer vectors represent an emerging new therapeutic paradigm comparable in potential impact on human health to that achieved by recombinant proteins and vaccines. A challenge for the current pipeline of AAV-based investigational products as they advance through clinical development is the identification, characterization and lot-to-lot control of the process- and product-related impurities present in even highly purified preparations. Especially challenging are AAV vector product-related impurities that closely resemble the vector itself and are, in some cases, without clear precedent in established biotherapeutic products. The determination of acceptable levels of these impurities in vectors prepared for human clinical product development, with the goal of new product licensure, requires careful risk and feasibility assessment. This review focuses primarily on the AAV product-related impurities that have been described in vectors prepared for clinical development.

  15. AAV8 capsid variable regions at the two-fold symmetry axis contribute to high liver transduction by mediating nuclear entry and capsid uncoating

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    Tenney, Rebeca M.; Bell, Christie L.; Wilson, James M., E-mail:


    Adeno-associated virus serotype 8 (AAV8) is a promising vector for liver-directed gene therapy. Although efficient uncoating of viral capsids has been implicated in AAV8's robust liver transduction, much about the biology of AAV8 hepatotropism remains unclear. Our study investigated the structural basis of AAV8 liver transduction efficiency by constructing chimeric vector capsids containing sequences derived from AAV8 and AAV2 – a highly homologous yet poorly hepatotropic serotype. Engineered vectors containing capsid variable regions (VR) VII and IX from AAV8 in an AAV2 backbone mediated near AAV8-like transduction in mouse liver, with higher numbers of chimeric genomes detected in whole liver cells and isolated nuclei. Interestingly, chimeric capsids within liver nuclei also uncoated similarly to AAV8 by 6 weeks after administration, in contrast with AAV2, of which a significantly smaller proportion were uncoated. This study links specific AAV capsid regions to the transduction ability of a clinically relevant AAV serotype. - Highlights: • We construct chimeric vectors to identify determinants of AAV8 liver transduction. • An AAV2-based vector with 17 AAV8 residues exhibited high liver transduction in mice. • This vector also surpassed AAV2 in cell entry, nuclear entry and onset of expression. • Most chimeric vector particles were uncoated at 6 weeks, like AAV8 and unlike AAV2. • Chimera retained heparin binding and was antigenically distinct from AAV2 and AAV8.

  16. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

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    Susan D'Costa


    Full Text Available Clinical trials using recombinant adeno-associated virus (rAAV vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR is now the most common method to titer vector genomes (vg; however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  17. Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia. (United States)

    Ye, Guo-jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T Michael; Miller, Paul E; Sharma, Alok K; Ver Hoeve, James N; Smith, Leia M; Arndt, Tara; Calcedo, Roberto; Gaskin, Chantelle; Robinson, Paulette M; Knop, David R; Hauswirth, William W; Chulay, Jeffrey D


    Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated viral (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in cynomolgus macaques. Three groups of animals (n = 2 males and 2 females per group) received a subretinal injection in one eye of 300 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two concentrations (4 × 10(11) or 4 × 10(12) vector genomes/ml) and were evaluated over a 3-month period before being euthanized. Administration of rAAV2tYF-PR1.7-hCNGB3 was associated with a dose-related anterior and posterior segment inflammatory response that was greater than that observed in eyes injected with the vehicle control. Most manifestations of inflammation improved over time except that vitreous cells persisted in vector-treated eyes until the end of the study. One animal in the lower vector dose group was euthanized on study day 5, based on a clinical diagnosis of endophthalmitis. There were no test article-related effects on intraocular pressure, visual evoked potential responses, hematology or clinical chemistry parameters, or gross necropsy observations. Histopathological examination demonstrated minimal mononuclear infiltrates in all vector-injected eyes. Serum anti-AAV antibodies developed in all vector-injected animals. No animals developed antibodies to CNGB3. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

  18. Single Tyrosine Mutation in AAV8 Vector Capsid Enhances Gene Lung Delivery and Does Not Alter Lung Morphofunction in Mice

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    Sabrina V. Martini


    Full Text Available Background/Aims: Vectors derived from adeno-associated viruses (AAVs are important gene delivery tools for treating pulmonary diseases. Phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. We evaluated the pulmonary transduction efficiency of AAV8 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Male C57BL/6 mice (20-25 g, n=24 were randomly assigned into three groups: control group animals received intratracheal (i.t. instillation of saline (50 μl, wild-type AAV8 group, and capsid mutant Y733F AAV8 group, which received (i.t. AAV8 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics and morphometry, vector transduction (immunohistochemistry and mRNA expression of eGFP, and inflammatory cytokines and growth factor expression were analyzed. Results: Tyrosine-mutant AAV8 vectors displayed significantly increased transduction efficiency in the lung compared with their wild-type counterparts. No significant differences were observed in lung mechanics and morphometry between experimental groups. There was no evidence of inflammatory response in any group. Conclusion: AAV8 vectors may be useful for new therapeutic strategies for the treatment of pulmonary diseases.

  19. CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector

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    Giridhar Murlidharan


    Full Text Available Gene therapy using recombinant adeno-associated viral (AAV vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9, which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137 in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities.

  20. CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors

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    Rasmus O. Bak


    Full Text Available The CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno-associated viral (AAV vectors to serve as donor template DNA during homologous recombination (HR. However, the maximum AAV packaging capacity of ∼4.5 kb limits the donor size. Here, we overcome this constraint by showing that two co-transduced AAV vectors can serve as donors during consecutive HR events for the integration of large transgenes. Importantly, the method involves a single-step procedure applicable to primary cells with relevance to therapeutic genome editing. We use the methodology in primary human T cells and CD34+ hematopoietic stem and progenitor cells to site-specifically integrate an expression cassette that, as a single donor vector, would otherwise amount to a total of 6.5 kb. This approach now provides an efficient way to integrate large transgene cassettes into the genomes of primary human cells using HR-mediated genome editing with AAV vectors.

  1. Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors (United States)

    Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these ...

  2. Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia. (United States)

    Ye, Guo-jie; Budzynski, Ewa; Sonnentag, Peter; Nork, T Michael; Miller, Paul E; McPherson, Leslie; Ver Hoeve, James N; Smith, Leia M; Arndt, Tara; Mandapati, Savitri; Robinson, Paulette M; Calcedo, Roberto; Knop, David R; Hauswirth, William W; Chulay, Jeffrey D


    Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations.

  3. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S.; Odom, Guy L.; Hopkins, Stephanie; Case, Amanda; Wang, David B.; Chamberlain, Jeffrey S.; Garden, Gwenn A.


    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre-recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. PMID:25708596

  4. A Novel Retinal Ganglion Cell Promoter for Utility in AAV Vectors

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    Killian S. Hanlon


    Full Text Available Significant advances in gene therapy have enabled exploration of therapies for inherited retinal disorders, many of which are in preclinical development or clinical evaluation. Gene therapy for retinal conditions has led the way in this growing field. The loss of retinal ganglion cells (RGCs is a hallmark of a number of retinal disorders. As the field matures innovations that aid in refining therapies and optimizing efficacy are in demand. Gene therapies under development for RGC-related disorders, when delivered with recombinant adeno associated vectors (AAV, have typically been expressed from ubiquitous promoter sequences. Here we describe how a novel promoter from the murine Nefh gene was selected to drive transgene expression in RGCs. The Nefh promoter, in an AAV2/2 vector, was shown to drive preferential EGFP expression in murine RGCs in vivo following intravitreal injection. In contrast, EGFP expression from a CMV promoter was observed not only in RGCs, but throughout the inner nuclear layer and in amacrine cells located within the ganglion cell layer (GCL. Of note, the Nefh promoter sequence is sufficiently compact to be readily accommodated in AAV vectors, where transgene size represents a significant constraint. Moreover, this promoter should in principle provide a more targeted and potentially safer alternative for RGC-directed gene therapies.

  5. PTEN knockdown with the Y444F mutant AAV2 vector promotes axonal regeneration in the adult optic nerve

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    Zheng-ru Huang


    Full Text Available The lack of axonal regeneration is the major cause of vision loss after optic nerve injury in adult mammals. Activating the PI3K/AKT/mTOR signaling pathway has been shown to enhance the intrinsic growth capacity of neurons and to facilitate axonal regeneration in the central nervous system after injury. The deletion of the mTOR negative regulator phosphatase and tensin homolog (PTEN enhances regeneration of adult corticospinal neurons and ganglion cells. In the present study, we used a tyrosine-mutated (Y444F AAV2 vector to efficiently express a short hairpin RNA (shRNA for silencing PTEN expression in retinal ganglion cells. We evaluated cell survival and axonal regeneration in a rat model of optic nerve axotomy. The rats received an intravitreal injection of wildtype AAV2 or Y444F mutant AAV2 (both carrying shRNA to PTEN 4 weeks before optic nerve axotomy. Compared with the wildtype AAV2 vector, the Y444F mutant AAV2 vector enhanced retinal ganglia cell survival and stimulated axonal regeneration to a greater extent 6 weeks after axotomy. Moreover, post-axotomy injection of the Y444F AAV2 vector expressing the shRNA to PTEN rescued ~19% of retinal ganglion cells and induced axons to regenerate near to the optic chiasm. Taken together, our results demonstrate that PTEN knockdown with the Y444F AAV2 vector promotes retinal ganglion cell survival and stimulates long-distance axonal regeneration after optic nerve axotomy. Therefore, the Y444F AAV2 vector might be a promising gene therapy tool for treating optic nerve injury.

  6. Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult microglia. (United States)

    Su, Wei; Kang, John; Sopher, Bryce; Gillespie, James; Aloi, Macarena S; Odom, Guy L; Hopkins, Stephanie; Case, Amanda; Wang, David B; Chamberlain, Jeffrey S; Garden, Gwenn A


    Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia. © 2015 International Society for

  7. Elimination of contaminating cap genes in AAV vector virions reduces immune responses and improves transgene expression in a canine gene therapy model. (United States)

    Wang, Z; Halbert, C L; Lee, D; Butts, T; Tapscott, S J; Storb, R; Miller, A D


    Animal and human gene therapy studies utilizing AAV vectors have shown that immune responses to AAV capsid proteins can severely limit transgene expression. The main source of capsid antigen is that associated with the AAV vectors, which can be reduced by stringent vector purification. A second source of AAV capsid proteins is that expressed from cap genes aberrantly packaged into AAV virions during vector production. This antigen source can be eliminated by the use of a cap gene that is too large to be incorporated into an AAV capsid, such as a cap gene containing a large intron (captron gene). Here, we investigated the effects of elimination of cap gene transfer and of vector purification by CsCl gradient centrifugation on AAV vector immunogenicity and expression following intramuscular injection in dogs. We found that both approaches reduced vector immunogenicity and that combining the two produced the lowest immune responses and highest transgene expression. This combined approach enabled the use of a relatively mild immunosuppressive regimen to promote robust micro-dystrophin gene expression in Duchenne muscular dystrophy-affected dogs. Our study shows the importance of minimizing AAV cap gene impurities and indicates that this improvement in AAV vector production may benefit human applications.

  8. Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units

    International Nuclear Information System (INIS)

    Kuegler, S.; Lingor, P.; Schoell, U.; Zolotukhin, S.; Baehr, M.


    Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology

  9. Supramolecular polypseudorotaxane gels for controlled delivery of rAAV vectors in human mesenchymal stem cells for regenerative medicine. (United States)

    Rey-Rico, Ana; Babicz, Heiko; Madry, Henning; Concheiro, Angel; Alvarez-Lorenzo, Carmen; Cucchiarini, Magali


    The aim of this work was to investigate, for the first time, the possibility of using supramolecular polypseudorotaxane gels as scaffolds that can durably deliver rAAV vectors for applications in cartilage regeneration. Dispersions of Pluronic ® F68 (PF68) or Tetronic ® 908 (T908) containing either hyaluronic acid (HA) or chondroitin sulfate (CS) were prepared in PBS. Then, alpha-cyclodextrin (αCD) was added to some dispersions to form polypseudorotaxane gels. Polysaccharides and αCD reinforced the viscoelasticity of the gels, which could withstand autoclaving without changes. In vitro release of rAAV vectors and subsequent transduction of human mesenchymal stem cells (hMSCs) by rAAV vectors from the release medium and from gels in direct contact with the cells were investigated. Compared with free vectors, the gels provided higher levels of transgene expression. CS (or HA)/PF68/αCD gels rapidly released rAAV vectors while CS (or HA)/T908/αCD gels provided sustained release probably due to different interactions with the viral vectors. Incorporation of αCD into CS (or HA)/PF68 gels resulted on higher rAAV concentrations and sustained levels of transgene expression over time. HA increased the bioactivity and cytocompatibility of the gels, especially those based on T908. Overall, combining rAAV gene transfer with polypseudorotaxane gels may provide new, promising tools for human tissue engineering and regenerative medicine strategies. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Using AAV vectors expressing the β2-adrenoceptor or associated Gα proteins to modulate skeletal muscle mass and muscle fibre size (United States)

    Hagg, Adam; Colgan, Timothy D.; Thomson, Rachel E.; Qian, Hongwei; Lynch, Gordon S.; Gregorevic, Paul


    Anabolic β2-adrenoceptor (β2-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks’ treatment with the β2-AR agonist formoterol, and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration, and highlight novel methods for potentially modulating muscle mass in settings of disease. PMID:26972746

  11. Using AAV vectors expressing the β2-adrenoceptor or associated Gα proteins to modulate skeletal muscle mass and muscle fibre size. (United States)

    Hagg, Adam; Colgan, Timothy D; Thomson, Rachel E; Qian, Hongwei; Lynch, Gordon S; Gregorevic, Paul


    Anabolic β2-adrenoceptor (β2-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks' treatment with the β2-AR agonist formoterol, and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration, and highlight novel methods for potentially modulating muscle mass in settings of disease.

  12. Recombinant AAV9-TLK1B Administration Ameliorates Fractionated Radiation-Induced Xerostomia (United States)

    Shanmugam, Prakash Srinivasan Timiri; Dayton, Robert D.; Palaniyandi, Senthilnathan; Abreo, Fleurette; Caldito, Gloria; Klein, Ronald L.


    Abstract Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviral-mediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (1011 vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline- and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; pgland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection against radiation

  13. Impact of intravenous infusion time on AAV8 vector pharmacokinetics, safety, and liver transduction in cynomolgus macaques

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    Jenny A Greig


    Full Text Available Systemically delivered adeno-associated viral (AAV vectors are now in early-phase clinical trials for a variety of diseases. While there is a general consensus on inclusion and exclusion criteria for each of these trials, the conditions under which vectors are infused vary significantly. In this study, we evaluated the impact of intravenous infusion rate of AAV8 vector in cynomolgus macaques on transgene expression, vector clearance from the circulation, and potential activation of the innate immune system. The dose of AAV8 vector in terms of genome copies per kilogram body weight and its concentration were fixed, while the rate of infusion varied to deliver the entire dose over different time periods, including 1, 10, or 90 minutes. Analyses during the in-life phase of the experiment included sequential evaluation of whole blood for vector genomes and appearance of proinflammatory cytokines. Liver tissues were analyzed at the time of necropsy for enhanced green fluorescent protein (eGFP expression and vector genomes. The data were remarkable with a relative absence of any statistically significant effect of infusion time on vector transduction, safety, and clearance. However, some interesting and unexpected trends did emerge.

  14. Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector. (United States)

    Rincon, Melvin Y; de Vin, Filip; Duqué, Sandra I; Fripont, Shelly; Castaldo, Stephanie A; Bouhuijzen-Wenger, Jessica; Holt, Matthew G


    Until recently, adeno-associated virus 9 (AAV9) was considered the AAV serotype most effective in crossing the blood-brain barrier (BBB) and transducing cells of the central nervous system (CNS), following systemic injection. However, a newly engineered capsid, AAV-PHP.B, is reported to cross the BBB at even higher efficiency. We investigated how much we could boost CNS transgene expression by using AAV-PHP.B carrying a self-complementary (sc) genome. To allow comparison, 6 weeks old C57BL/6 mice received intravenous injections of scAAV2/9-GFP or scAAV2/PHP.B-GFP at equivalent doses. Three weeks postinjection, transgene expression was assessed in brain and spinal cord. We consistently observed more widespread CNS transduction and higher levels of transgene expression when using the scAAV2/PHP.B-GFP vector. In particular, we observed an unprecedented level of astrocyte transduction in the cortex, when using a ubiquitous CBA promoter. In comparison, neuronal transduction was much lower than previously reported. However, strong neuronal expression (including spinal motor neurons) was observed when the human synapsin promoter was used. These findings constitute the first reported use of an AAV-PHP.B capsid, encapsulating a scAAV genome, for gene transfer in adult mice. Our results underscore the potential of this AAV construct as a platform for safer and more efficacious gene therapy vectors for the CNS.

  15. Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism (United States)

    Wu, Pei; Xiao, Wu; Conlon, Thomas; Hughes, Jeffrey; Agbandje-McKenna, Mavis; Ferkol, Thomas; Flotte, Terence; Muzyczka, Nicholas


    Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be β-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag

  16. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice

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    Dario Marangoni


    Full Text Available X-linked retinoschisis (XLRS is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1 and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124 injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye, and a 6-month study in Rs1-KO mice (n = 162 dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

  17. Ocular and systemic safety of a recombinant AAV8 vector for X-linked retinoschisis gene therapy: GLP studies in rabbits and Rs1-KO mice. (United States)

    Marangoni, Dario; Bush, Ronald A; Zeng, Yong; Wei, Lisa L; Ziccardi, Lucia; Vijayasarathy, Camasamudram; Bartoe, Joshua T; Palyada, Kiran; Santos, Maria; Hiriyanna, Suja; Wu, Zhijian; Colosi, Peter; Sieving, Paul A


    X-linked retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding the protein retinoschisin (RS1) and is one of the most common causes of macular degeneration in young men. Our therapeutic approach for XLRS is based on the administration of AAV8-scRS/IRBPhRS, an adeno-associated viral vector coding the human RS1 protein, via the intravitreal (IVT) route. Two Good Laboratory Practice studies, a 9-month study in New Zealand White rabbits (n = 124) injected with AAV8-scRS/IRBPhRS at doses of 2E9, 2E10, 2E11, and 1.5E12 vector genomes/eye (vg/eye), and a 6-month study in Rs1-KO mice (n = 162) dosed with 2E9 and 2E10 vg/eye of the same vector were conducted to assess ocular and systemic safety. A self-resolving, dose-dependent vitreal inflammation was the main ocular finding, and except for a single rabbit dosed with 1.5E12 vg/eye, which showed a retinal detachment, no other ocular adverse event was reported. Systemic toxicity was not identified in either species. Biodistribution analysis in Rs1-KO mice detected spread of vector genome in extraocular tissues, but no evidence of organ or tissues damage was found. These studies indicate that IVT administration of AAV8-scRS/IRBPhRS is safe and well tolerated and support its advancement into a phase 1/2a clinical trial for XLRS.

  18. AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons: Implications for Gene Therapy and Disease Models

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    Katrina Albert


    Full Text Available Gene delivery using adeno-associated virus (AAV vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson′s disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson′s disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson′s disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson′s disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV-a-synuclein (a-syn to target substantia nigra dopamine neurons to produce an α-syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP, which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in

  19. Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy. (United States)

    Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R


    Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Intra-Arterial Delivery of AAV Vectors to the Mouse Brain After Mannitol Mediated Blood Brain Barrier Disruption (United States)

    Santillan, Alejandro; Sondhi, Dolan; Dyke, Jonathan P.; Crystal, Ronald G.; Gobin, Y. Pierre; Ballon, Douglas J.


    The delivery of therapeutics to neural tissue is greatly hindered by the blood brain barrier (BBB). Direct local delivery via diffusive release from degradable implants or direct intra-cerebral injection can bypass the BBB and obtain high concentrations of the therapeutic in the targeted tissue, however the total volume of tissue that can be treated using these techniques is limited. One treatment modality that can potentially access large volumes of neural tissue in a single treatment is intra-arterial (IA) injection after osmotic blood brain barrier disruption. In this technique, the therapeutic of interest is injected directly into the arteries that feed the target tissue after the blood brain barrier has been disrupted by exposure to a hyperosmolar mannitol solution, permitting the transluminal transport of the therapy. In this work we used contrast enhanced magnetic resonance imaging (MRI) studies of IA injections in mice to establish parameters that allow for extensive and reproducible BBB disruption. We found that the volume but not the flow rate of the mannitol injection has a significant effect on the degree of disruption. To determine whether the degree of disruption we observed with this method was sufficient for delivery of nanoscale therapeutics, we performed IA injections of an adeno-associated viral vector containing the CLN2 gene (AAVrh.10CLN2), which is mutated in the lysosomal storage disorder Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). We demonstrated that IA injection of AAVrh.10CLN2 after BBB disruption can achieve widespread transgene production in the mouse brain after a single administration. Further, we showed that there exists a minimum threshold of BBB disruption necessary to permit the AAV.rh10 vector to pass into the brain parenchyma from the vascular system. These results suggest that IA administration may be used to obtain widespread delivery of nanoscale therapeutics throughout the murine brain after a single

  1. Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering. (United States)

    Zhang, Yun; Liu, Xiaomei; Zhang, Jinju; Zhang, Chun


    To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins. AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed "CELiD" DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with "CELiD" DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %. The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.

  2. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side effects

    Directory of Open Access Journals (Sweden)

    Kai Gao


    Full Text Available Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots, and mixtures of empty and fully packaged virions with variable ratios of empty virions. The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP (enhanced green fluorescent protein or nuclear-targeted (n LacZ or secreted (human α1-antitrypsin (hA1AT reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT and 44% (nLacZ in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ALT levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side effects of rAAV8 EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.

  3. Long-term, efficient inhibition of microRNA function in mice using rAAV vectors. (United States)

    Xie, Jun; Ameres, Stefan L; Friedline, Randall; Hung, Jui-Hung; Zhang, Yu; Xie, Qing; Zhong, Li; Su, Qin; He, Ran; Li, Mengxin; Li, Huapeng; Mu, Xin; Zhang, Hongwei; Broderick, Jennifer A; Kim, Jason K; Weng, Zhiping; Flotte, Terence R; Zamore, Phillip D; Gao, Guangping


    Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA 'tough decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuDs depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122 TuD but not anti-let-7 TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High-throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuDs induced miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.

  4. Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung

    Directory of Open Access Journals (Sweden)

    Sabrina V. Martini


    Full Text Available Background/Aims: Adeno-associated virus (AAV vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Methods: Eighteen C57BL/6 mice were randomly assigned into three groups: (1 a control group (CTRL animals underwent intratracheal (i.t. instillation of saline, (2 the wild-type AAV9 group (WT-AAV9, 1010 vg, and (3 the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg, which received (i.t. self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP. Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. Results: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30% compared with their wild-type counterparts, without eliciting an inflammatory response. Conclusion: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

  5. Angiotensin 1-7 Overexpression Mediated by a Capsid-optimized AAV8 Vector Leads to Significant Growth Inhibition of Hepatocellular Carcinoma In vivo. (United States)

    Mao, Yingying; Pei, Nana; Chen, Xinglu; Chen, Huiying; Yan, Renhe; Bai, Na; Li, Andrew; Li, Jinlong; Zhang, Yanling; Du, Hongyan; Chen, Baihong; Sumners, Colin; Wang, Xuejun; Wang, Shengqi; Li, Hongwei


    Background: Angiotensin-(1-7) [Ang-(1-7)] has been identified to inhibit the growth of many types of tumor cells both in vitro and in vivo . However, the rapid degradation of Ang-(1-7) in vivo limits its clinical application. Adeno-associated virus (AAV) serotype-8 is a remarkable vector for long-term in vivo gene delivery. Method: This study was designed to investigate the effects of AAV-mediated Ang-(1-7) overexpression on hepatocellular carcinoma. We first generated three different tyrosine (Y) to phenylalanine (F) mutants of AAV8 (Y447F, Y703F, Y708F) and evaluated their in vivo transduction efficiencies. Results: The data indicated that the Y703F mutant elicited a significant enhancement of liver gene delivery when compared with wild-type AAV8 (wtAAV8). The anti-tumor effect of Ang-(1-7) mediated by this optimized vector was evaluated in H22 hepatoma-bearing mice. Our results demonstrated that AAV-Ang-(1-7) persistently inhibited the growth of hepatocellular carcinoma by significantly downregulating angiogenesis. This was confirmed by observed decreases in the levels of the proangiogenic factors VEGF and PIGF. Conclusion: Collectively, these data suggest that Ang-(1-7) overexpression mediated by the optimized vector may be an effective alternative for hepatocellular carcinoma therapy due to its long-term and significant anti-tumor activity.

  6. AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines. (United States)

    Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A


    Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

  7. Humoral immune response to AAV

    Directory of Open Access Journals (Sweden)

    Roberto eCalcedo


    Full Text Available Adeno-associated virus (AAV is a member of the family parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy.

  8. Biochemical and physiological improvement in a mouse model of Smith–Lemli–Opitz syndrome (SLOS following gene transfer with AAV vectors

    Directory of Open Access Journals (Sweden)

    Lee Ying


    Full Text Available Smith–Lemli–Opitz syndrome (SLOS is an inborn error of cholesterol synthesis resulting from a defect in 7-dehydrocholesterol reductase (DHCR7, the enzyme that produces cholesterol from its immediate precursor 7-dehydrocholesterol. Current therapy employing dietary cholesterol is inadequate. As SLOS is caused by a defect in a single gene, restoring enzyme functionality through gene therapy may be a direct approach for treating this debilitating disorder. In the present study, we first packaged a human DHCR7 construct into adeno-associated virus (AAV vectors having either type-2 (AAV2 or type-8 (AAV2/8 capsid, and administered treatment to juvenile mice. While a positive response (assessed by increases in serum and liver cholesterol was seen in both groups, the improvement was greater in the AAV2/8–DHCR7 treated mice. Newborn mice were then treated with AAV2/8–DHCR7 and these mice, compared to mice treated as juveniles, showed higher DHCR7 mRNA expression in liver and a greater improvement in serum and liver cholesterol levels. Systemic treatment did not affect brain cholesterol in any of the experimental groups. Both juvenile and newborn treatments with AAV2/8–DHCR7 resulted in increased rates of weight gain indicating that gene transfer had a positive physiological effect.

  9. Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

    Czech Academy of Sciences Publication Activity Database

    Tadokoro, T.; Miyanohara, A.; Navarro, M.; Kamizato, K.; Juhás, Štefan; Juhásová, Jana; Maršala, S.; Platoshyn, O.; Curtis, E.; Gabel, B.; Ciacci, J. D.; Lukáčová, N.; Bimbová, K.; Maršala, M.


    Roč. 125, č. 13 (2017), č. článku e55770. ISSN 1940-087X R&D Projects: GA MŠk(CZ) LO1609 Institutional support: RVO:67985904 Keywords : AAV9 * adult mouse Subject RIV: EI - Biotechnology ; Bionics OBOR OECD: Technologies involving the manipulation of cells, tissues, organs or the whole organism (assisted reproduction) Impact factor: 1.232, year: 2016

  10. Current strides in AAV-derived vectors and SIN channels further ...

    African Journals Online (AJOL)

    A.S. Odiba

    Vectors used in Gene Therapy Clinical Trials. Data Sourced from: The Journal of Gene. Medicine ( on 2nd June 2017. Vector. Gene Therapy. Clinical Trials. Number. %. Alphavirus (VEE) Replicon Vaccine. 1. 0. E. coli. 2. 0.1. Bifidobacterium longum. 1. 0. CRISPR-Cas9. 7. 0.3. Adenovirus + ...

  11. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial. (United States)

    Bennett, Jean; Wellman, Jennifer; Marshall, Kathleen A; McCague, Sarah; Ashtari, Manzar; DiStefano-Pappas, Julie; Elci, Okan U; Chung, Daniel C; Sun, Junwei; Wright, J Fraser; Cross, Dominique R; Aravand, Puya; Cyckowski, Laura L; Bennicelli, Jeannette L; Mingozzi, Federico; Auricchio, Alberto; Pierce, Eric A; Ruggiero, Jason; Leroy, Bart P; Simonelli, Francesca; High, Katherine A; Maguire, Albert M


    Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study. In this follow-on phase 1 trial, one dose of AAV2-hRPE65v2 (1.5 × 10(11) vector genomes) in a total volume of 300 μL was subretinally injected into the contralateral, previously uninjected, eyes of 11 children and adults (aged 11-46 years at second administration) with inherited retinal dystrophy caused by RPE65 mutations, 1.71-4.58 years after the initial subretinal injection. We assessed safety, immune response, retinal and visual function, functional vision, and activation of the visual cortex from baseline until 3 year follow-up, with observations ongoing. This study is registered with, number NCT01208389. No adverse events related to the AAV were reported, and those related to the procedure were mostly mild (dellen formation in three patients and cataracts in two). One patient developed bacterial endophthalmitis and was excluded from analyses. We noted improvements in efficacy outcomes in most patients without significant immunogenicity. Compared with baseline, pooled analysis of ten participants showed improvements in mean mobility and full-field light sensitivity in the injected eye by day 30 that persisted to year 3 (mobility p=0.0003, white light full-field sensitivity p0.49 for all time-points compared with baseline). To our knowledge, AAV2-hRPE65v2 is the first successful gene therapy administered to the contralateral eye. The results highlight the use of several outcome measures and help to delineate the variables that

  12. AAV9 supports wide-scale transduction of the CNS and TDP-43 disease modeling in adult rats

    Directory of Open Access Journals (Sweden)

    Kasey L Jackson

    Full Text Available AAV9 has emerged as an efficient adeno-associated virus (AAV serotype for gene transfer to the central nervous system. We have used this technique to study aspects of amyotrophic lateral sclerosis (ALS by administering AAV encoding the ALS-related gene transactive response DNA binding protein of 43 kDa (TDP-43 to neonatal rats. However, inducing the expression in adult subjects would be preferable to mimic the adult onset of symptoms in ALS. We expressed either green fluorescent protein (GFP or TDP-43 in adult rats after an intravenous (i.v. route of administration to attempt wide-scale transduction of the spinal cord for disease modeling. In order to optimize the gene transfer, we made comparisons of efficiency by age, gender, and across several AAV serotypes (AAV1, AAV8, AAV9, and AAV10. The data indicate more efficient neuronal transduction in neonates, with little evidence of glial transduction at either age, no gender-related differences in transduction, and that AAV9 was efficient in adults relative to the other serotypes tested. Based on these data, AAV9 TDP-43 was expressed at three vector doses in adult female rats yielding highly consistent, dose-dependent motor deficits. AAV9 can be delivered i.v. to adult rats to achieve consistent pathophysiological changes and a relevant adult-onset system for disease modeling.

  13. Repeated Delivery of Adeno-Associated Virus Vectors to the Rabbit Airway (United States)

    Beck, Suzanne E.; Jones, Lori A.; Chesnut, Kye; Walsh, Scott M.; Reynolds, Thomas C.; Carter, Barrie J.; Askin, Frederic B.; Flotte, Terence R.; Guggino, William B.


    Efficient local expression from recombinant adeno-associated virus (rAAV)-cystic fibrosis (CF) transmembrane conductance regulator (CFTR) vectors has been observed in the airways of rabbits and monkeys for up to 6 months following a single bronchoscopic delivery. However, it is likely that repeated administrations of rAAV vectors will be necessary for sustained correction of the CF defect in the airways. The current study was designed to test the feasibility of repeated airway delivery of rAAV vectors in the rabbit lung. After two doses of rAAV-CFTR to the airways, rabbits generated high titers of serum anti-AAV neutralizing antibodies. Rabbits then received a third dose of a rAAV vector containing the green fluorescent protein (GFP) reporter gene packaged in either AAV serotype 2 (AAV2) or serotype 3 (AAV3) capsids. Each dose consisted of 1 ml containing 5 × 109 DNase-resistant particles of rAAV vector, having no detectable replication-competent AAV or adenovirus. Three weeks later, GFP expression was observed in airway epithelial cells despite high anti-AAV neutralizing titers at the time of delivery. There was no significant difference in the efficiency of DNA transfer or expression between the rAAV3 and rAAV2 groups. No significant inflammatory responses to either repeated airway exposure to rAAV2-CFTR vectors or to GFP expression were observed. These experiments demonstrate that serum anti-AAV neutralizing antibody titers do not predict airway neutralization in vivo and that repeated airway delivery rAAV allows for safe and effective gene transfer. PMID:10516053

  14. Pre-clinical efficacy and dosing of an AAV8 vector expressing human methylmalonyl-CoA mutase in a murine model of methylmalonic acidemia (MMA). (United States)

    Chandler, Randy J; Venditti, Charles P


    We demonstrate that human methylmalonyl-CoA mutase (MUT), delivered using an AAV serotype 8 vector, rescues the lethal phenotype displayed by mice with MMA and provides long-term phenotypic correction. In addition to defining a lower limit of effective dosing, our studies establish that neither a species barrier to mitochondrial processing nor an apparent immune response to MUT limits the murine model as an experimental platform to test the efficacy of human gene therapy vectors for MMA. Published by Elsevier Inc.

  15. Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells. (United States)

    Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A


    Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Biochemical correction of short-chain acyl-coenzyme A dehydrogenase deficiency after portal vein injection of rAAV8-SCAD. (United States)

    Beattie, Stuart G; Goetzman, Eric; Conlon, Thomas; Germain, Sean; Walter, Glenn; Campbell-Thompson, Martha; Matern, Dietrich; Vockley, Jerry; Flotte, Terence R


    Recombinant adeno-associated viral vectors pseudotyped with serotype 5 and 8 capsids (AAV5 and AAV8) have been shown to be efficient gene transfer reagents for the liver. We have produced AAV5 and AAV8 vectors that express mouse short-chain acyl-CoA dehydrogenase (mSCAD) cDNA under the transcriptional control of the cytomegalovirus-chicken beta-actin hybrid promoter. We hypothesized that these vectors would produce sufficient hepatocyte transduction (after administration via the portal vein) and thus sufficient SCAD enzyme to correct the phenotype observed in the SCAD-deficient (BALB/cByJ) mouse, which includes elevated blood butyrylcarnitine and hepatic steatosis. Ten weeks after portal vein injection into 8-week-old mice, AAV8-treated livers contained acyl-CoA dehydrogenase activity (14.3 mU/mg) toward butyryl-CoA, compared with 7.6 mU/mg in mice that received phosphate-buffered saline. Immunohistochemistry showed expression of mSCAD within rAAV8-mSCAD-transduced hepatocytes, as seen by light microscopy. A significant reduction of circulating butyrylcarnitine was seen in AAV5-mSCAD- and AAV8-mSCAD-injected mice. Magnetic resonance spectroscopy of fasted mice demonstrated a significant reduction in relative lipid content within the livers of AAV8-mSCAD-treated mice. These results demonstrate biochemical correction of SCAD deficiency after AAV8-mediated SCAD gene delivery.

  17. Multi-parametric MRI at 14T for muscular dystrophy mice treated with AAV vector-mediated gene therapy.

    Directory of Open Access Journals (Sweden)

    Joshua Park

    Full Text Available The objective of this study was to investigate the efficacy of using quantitative magnetic resonance imaging (MRI as a non-invasive tool for the monitoring of gene therapy for muscular dystrophy. The clinical investigations for this family of diseases often involve surgical biopsy which limits the amount of information that can be obtained due to the invasive nature of the procedure. Thus, other non-invasive tools may provide more opportunities for disease assessment and treatment responses. In order to explore this, dystrophic mdx4cv mice were systemically treated with a recombinant adeno-associated viral (AAV vector containing a codon-optimized micro-dystrophin gene. Multi-parametric MRI of T2, magnetization transfer, and diffusion effects alongside 3-D volume measurements were then utilized to monitor disease/treatment progression. Mice were imaged at 10 weeks of age for pre-treatment, then again post-treatment at 8, 16, and 24 week time points. The efficacy of treatment was assessed by physiological assays for improvements in function and quantification of expression. Tissues from the hindlimbs were collected for histological analysis after the final time point for comparison with MRI results. We found that introduction of the micro-dystrophin gene restored some aspects of normal muscle histology and pathology such as decreased necrosis and resistance to contraction-induced injury. T2 relaxation values showed percentage decreases across all muscle types measured (tibialis anterior, gastrocnemius, and soleus when treated groups were compared to untreated groups. Additionally, the differences between groups were statistically significant for the tibialis anterior as well. The diffusion measurements showed a wider range of percentage changes and less statistical significance while the magnetization transfer effect measurements showed minimal change. MR images displayed hyper-intense regions of muscle that correlated with muscle pathology in

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

    LENUS (Irish Health Repository)

    Luo, Xiaoyan


    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.

  19. Systemically administered AAV9-sTRAIL combats invasive glioblastoma in a patient-derived orthotopic xenograft model

    Directory of Open Access Journals (Sweden)

    Matheus HW Crommentuijn


    Full Text Available Adeno-associated virus (AAV vectors expressing tumoricidal genes injected directly into brain tumors have shown some promise, however, invasive tumor cells are relatively unaffected. Systemic injection of AAV9 vectors provides widespread delivery to the brain and potentially the tumor/microenvironment. Here we assessed AAV9 for potential glioblastoma therapy using two different promoters driving the expression of the secreted anti-cancer agent sTRAIL as a transgene model; the ubiquitously active chicken β-actin (CBA promoter and the neuron-specific enolase (NSE promoter to restrict expression in brain. Intravenous injection of AAV9 vectors encoding a bioluminescent reporter showed similar distribution patterns, although the NSE promoter yielded 100-fold lower expression in the abdomen (liver, with the brain-to-liver expression ratio remaining the same. The main cell types targeted by the CBA promoter were astrocytes, neurons and endothelial cells, while expression by NSE promoter mostly occurred in neurons. Intravenous administration of either AAV9-CBA-sTRAIL or AAV9-NSE-sTRAIL vectors to mice bearing intracranial patient-derived glioblastoma xenografts led to a slower tumor growth and significantly increased survival, with the CBA promoter having higher efficacy. To our knowledge, this is the first report showing the potential of systemic injection of AAV9 vector encoding a therapeutic gene for the treatment of brain tumors.

  20. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

    Directory of Open Access Journals (Sweden)

    Daniel J Hui

    Full Text Available Adeno-associated virus (AAV has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC class I epitopes for common human leukocyte antigen (HLA types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

  1. A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro-Dystrophin Vector (United States)


    commonly used animal models for DMD.[18–22] Over the years, a number of different strategies have been developed to achieve effective AAV gene transfer in...and sarcoglycan gene therapies have signifi- cantly improved the cardiac outcome in animal models of DMD and LGMD, respectively. ● Targeting ...mediated local exon-skipping. [91,92] Compared to RNA editing with exon-skipping, targeted editing of themutated dystrophin gene has just entered an

  2. Intravitreal delivery of a novel AAV vector targets ON bipolar cells and restores visual function in a mouse model of complete congenital stationary night blindness. (United States)

    Scalabrino, Miranda L; Boye, Sanford L; Fransen, Kathryn M H; Noel, Jennifer M; Dyka, Frank M; Min, Seok Hong; Ruan, Qing; De Leeuw, Charles N; Simpson, Elizabeth M; Gregg, Ronald G; McCall, Maureen A; Peachey, Neal S; Boye, Shannon E


    Adeno-associated virus (AAV) effectively targets therapeutic genes to photoreceptors, pigment epithelia, Müller glia and ganglion cells of the retina. To date, no one has shown the ability to correct, with gene replacement, an inherent defect in bipolar cells (BCs), the excitatory interneurons of the retina. Targeting BCs with gene replacement has been difficult primarily due to the relative inaccessibility of BCs to standard AAV vectors. This approach would be useful for restoration of vision in patients with complete congenital stationary night blindness (CSNB1), where signaling through the ON BCs is eliminated due to mutations in their G-protein-coupled cascade genes. For example, the majority of CSNB1 patients carry a mutation in nyctalopin (NYX), which encodes a protein essential for proper localization of the TRPM1 cation channel required for ON BC light-evoked depolarization. As a group, CSNB1 patients have a normal electroretinogram (ERG) a-wave, indicative of photoreceptor function, but lack a b-wave due to defects in ON BC signaling. Despite retinal dysfunction, the retinas of CSNB1 patients do not degenerate. The Nyx(nob) mouse model of CSNB1 faithfully mimics this phenotype. Here, we show that intravitreally injected, rationally designed AAV2(quadY-F+T-V) containing a novel 'Ple155' promoter drives either GFP or YFP_Nyx in postnatal Nyx(nob) mice. In treated Nyx(nob) retina, robust and targeted Nyx transgene expression in ON BCs partially restored the ERG b-wave and, at the cellular level, signaling in ON BCs. Our results support the potential for gene delivery to BCs and gene replacement therapy in human CSNB1. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  3. AAV vectors expressing LDLR gain-of-function variants demonstrate increased efficacy in mouse models of familial hypercholesterolemia. (United States)

    Somanathan, Suryanarayan; Jacobs, Frank; Wang, Qiang; Hanlon, Alexandra L; Wilson, James M; Rader, Daniel J


    Familial hypercholesterolemia is a genetic disorder that arises because of loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous familial hypercholesterolemia is a candidate for gene therapy using adeno-associated viral vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen adeno-associated viral vector encoded LDLR expression. We sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 (hPCSK9) and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction. Amino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and human IDOL. A panel of mutant human LDLRs was initially screened in vitro for escape from PCSK9. The variant human LDLR-L318D was further evaluated using a mouse model of homozygous familial hypercholesterolemia lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme catalytic, APOBEC-1 (double knockout). Administration of wild-type human LDLR to double knockout mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9-mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\\C818A construct avoided human IDOL regulation and achieved stable reductions in serum cholesterol. An adeno-associated viral vector serotype 8.LDLR-L318D\\K809R\\C818A vector that carried all 3 amino acid substitutions conferred partial resistance to both hPCSK9- and human IDOL-mediated degradation. Amino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve on a potential gene therapeutic approach to treat homozygous familial hypercholesterolemia subjects. © 2014 American Heart Association, Inc.

  4. Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep (United States)

    Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.


    A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

  5. Transient and intensive pharmacological immunosuppression fails to improve AAV-based liver gene transfer in non-human primates

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    Unzu Carmen


    Full Text Available Abstract Background Adeno-associated vectors (rAAV have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD haploinsufficiency (acute intermittent porphyria benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this work, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression. Methods Three female Macaca fascicularis were intravenously injected with 1x1013 genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF, anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5x1012 genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression. Results Macaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As

  6. Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapy. (United States)

    Brantly, Mark L; Chulay, Jeffrey D; Wang, Lili; Mueller, Christian; Humphries, Margaret; Spencer, L Terry; Rouhani, Farshid; Conlon, Thomas J; Calcedo, Roberto; Betts, Michael R; Spencer, Carolyn; Byrne, Barry J; Wilson, James M; Flotte, Terence R


    Alpha-1 antitrypsin (AAT) deficiency is well-suited as a target for human gene transfer. We performed a phase 1, open-label, dose-escalation clinical trial of a recombinant adeno-associated virus (rAAV) vector expressing normal (M) AAT packaged into serotype 1 AAV capsids delivered by i.m. injection. Nine AAT-deficient subjects were enrolled sequentially in cohorts of 3 each at doses of 6.9 x 10(12), 2.2 x 10(13), and 6.0 x 10(13) vector genome particles per patient. Four subjects receiving AAT protein augmentation discontinued therapy 28 or 56 days before vector administration. Vector administration was well tolerated, with only mild local reactions and 1 unrelated serious adverse event (bacterial epididymitis). There were no changes in hematology or clinical chemistry parameters. M-specific AAT was expressed above background in all subjects in cohorts 2 and 3 and was sustained at levels 0.1% of normal for at least 1 year in the highest dosage level cohort, despite development of neutralizing antibody and IFN-gamma enzyme-linked immunospot responses to AAV1 capsid at day 14 in all subjects. These findings suggest that immune responses to AAV capsid that develop after i.m. injection of a serotype 1 rAAV vector expressing AAT do not completely eliminate transduced cells in this context.

  7. Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice. (United States)

    Osmon, Karlaina J L; Woodley, Evan; Thompson, Patrick; Ong, Katalina; Karumuthil-Melethil, Subha; Keimel, John G; Mark, Brian L; Mahuran, Don; Gray, Steven J; Walia, Jagdeep S


    GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 ganglioside. We investigated a self-complementary adeno-associated viral (scAAV) vector expressing HexM, through intravenous injections of the neonatal mice. We monitored one cohort for 8 weeks and another cohort long-term for survival benefit, behavioral, biochemical, and molecular analyses. Untreated Sandhoff disease (SD) control mice reached a humane endpoint at approximately 15 weeks, whereas treated mice had a median survival age of 40 weeks, an approximate 2.5-fold survival advantage. On behavioral tests, the treated mice outperformed their knockout age-matched controls and perform similarly to the heterozygous controls. Through the enzymatic and GM2 ganglioside analyses, we observed a significant decrease in the GM2 ganglioside level, even though the enzyme levels were not significantly increased. Molecular analyses revealed a global distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of a novel viral vector expressing the human HexM enzyme is effective in ameliorating the SD

  8. Safety and Efficacy Evaluation of rAAV2tYF-PR1.7-hCNGA3 Vector Delivered by Subretinal Injection in CNGA3 Mutant Achromatopsia Sheep. (United States)

    Gootwine, Elisha; Ofri, Ron; Banin, Eyal; Obolensky, Alexey; Averbukh, Edward; Ezra-Elia, Raaya; Ross, Maya; Honig, Hen; Rosov, Alexander; Yamin, Esther; Ye, Guo-Jie; Knop, David R; Robinson, Paulette M; Chulay, Jeffrey D; Shearman, Mark S


    Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector expressing the human CNGA3 gene designated AGTC-402 (rAAV2tYF-PR1.7-hCNGA3) for the treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. The results are herein reported of a study evaluating safety and efficacy of AGTC-402 in CNGA3-deficient sheep. Thirteen day-blind sheep divided into three groups of four or five animals each received a subretinal injection of an AAV vector expressing a CNGA3 gene in a volume of 500 μL in the right eye. Two groups (n = 9) received either a lower or higher dose of the AGTC-402 vector, and one efficacy control group (n = 4) received a vector similar in design to one previously shown to rescue cone photoreceptor responses in the day-blind sheep model (rAAV5-PR2.1-hCNGA3). The left eye of each animal received a subretinal injection of 500 μL of vehicle (n = 4) or was untreated (n = 9). Subretinal injections were generally well tolerated and not associated with systemic toxicity. Most animals had mild to moderate conjunctival hyperemia, chemosis, and subconjunctival hemorrhage immediately after surgery that generally resolved by postoperative day 7. Two animals treated with the higher dose of AGTC-402 and three of the efficacy control group animals had microscopic findings of outer retinal atrophy with or without inflammatory cells in the retina and choroid that were procedural and/or test-article related. All vector-treated eyes showed improved cone-mediated electroretinography responses with no change in rod-mediated electroretinography responses. Behavioral maze testing under photopic conditions showed significantly improved navigation times and reduced numbers of obstacle collisions in all vector-treated eyes compared to their contralateral control eyes or pre-dose results in the

  9. CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. (United States)

    Xu, L; Daly, T; Gao, C; Flotte, T R; Song, S; Byrne, B J; Sands, M S; Parker Ponder, K


    Although AAV vectors show promise for hepatic gene therapy, the optimal transcriptional regulatory elements have not yet been identified. In this study, we show that an AAV vector with the CMV enhancer/chicken beta-actin promoter results in 9.5-fold higher expression after portal vein injection than an AAV vector with the EF1 alpha promoter, and 137-fold higher expression than an AAV vector with the CMV promoter/enhancer. Although induction of the acute-phase response with the administration of lipopolysaccharide (LPS) activated the CMV promoter/enhancer from the context of an adenoviral vector in a previous study, LPS resulted in only a modest induction of this promoter from an AAV vector in vivo. An AAV vector with the CMV-beta-actin promoter upstream of the coagulation protein human factor X (hFX) was injected intravenously into neonatal mice. This resulted in expression of hFX at 548 ng/ml (6.8% of normal) for up to 1.2 years, and 0.6 copies of AAV vector per diploid genome in the liver at the time of sacrifice. Neonatal intramuscular injection resulted in expression of hFX at 248 ng/ml (3.1% of normal), which derived from both liver and muscle. We conclude that neonatal gene therapy with an AAV vector with the CMV-beta-actin promoter might correct hemophilia due to hFX deficiency.

  10. Intranasal Delivery of Recombinant NT4-NAP/AAV Exerts Potential Antidepressant Effect. (United States)

    Ma, Xian-Cang; Chu, Zheng; Zhang, Xiao-Ling; Jiang, Wen-Hui; Jia, Min; Dang, Yong-Hui; Gao, Cheng-Ge


    The present study was designed to construct a recombinant adeno-associated virus (rAAV) which can express NAP in the brain and examine whether this virus can produce antidepressant effects on C57 BL/6 mice that had been subjected to open field test and forced swimming test, via nose-to-brain pathway. When the recombinant plasmid pGEM-T Easy/NT4-NAP was digested by EcoRI, 297 bp fragments can be obtained and NT4-NAP sequence was consistent with the designed sequence confirmed by DNA sequencing. When the recombinant plasmid pSSCMV/NT4-NAP was digested by EcoRI, 297 bp fragments is visible. Immunohistochemical staining of fibroblasts revealed that expression of NAP was detected in NT4-NAP/AAV group. Intranasal delivery of NT4-NAP/AAV significantly reduced immobility time when the FST was performed after 1 day from the last administration. The effects observed in the FST could not be attributed to non-specific increases in activity since intranasal delivery of NT4-NAP/AAV did not alter the behavior of the mice during the open field test. The results indicated that a recombinant AAV vector which could express NAP in cells was successfully constructed and NAP may be a potential target for therapeutic action of antidepressant treatment.

  11. Intracisternal delivery of AAV9 results in oligodendrocyte and motor neuron transduction in the whole central nervous system of cats. (United States)

    Bucher, T; Dubreil, L; Colle, M-A; Maquigneau, M; Deniaud, J; Ledevin, M; Moullier, P; Joussemet, B


    Systemic and intracerebrospinal fluid delivery of adeno-associated virus serotype 9 (AAV9) has been shown to achieve widespread gene delivery to the central nervous system (CNS). However, after systemic injection, the neurotropism of the vector has been reported to vary according to age at injection, with greater neuronal transduction in newborns and preferential glial cell tropism in adults. This difference has not yet been reported after cerebrospinal fluid (CSF) delivery. The present study analyzed both neuronal and glial cell transduction in the CNS of cats according to age of AAV9 CSF injection. In both newborns and young cats, administration of AAV9-GFP in the cisterna magna resulted in high levels of motor neurons (MNs) transduction from the cervical (84±5%) to the lumbar (99±1%) spinal cord, demonstrating that the remarkable tropism of AAV9 for MNs is not affected by age at CSF delivery. Surprisingly, numerous oligodendrocytes were also transduced in the brain and in the spinal cord white matter of young cats, but not of neonates, indicating that (i) age of CSF delivery influences the tropism of AAV9 for glial cells and (ii) AAV9 intracisternal delivery could be relevant for both the treatment of MN and demyelinating disorders.

  12. In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease (United States)

    Lau, Cia-Hin; Suh, Yousin


    Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics. PMID:29333255

  13. 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.

  14. 75 FR 55808 - Prospective Grant of Exclusive License: Development of AAV5 Based Therapeutics To Treat Human... (United States)


    ... particles. The specific brain cells that are targeted by AAV5 belong to both non-neuronal/glial cells and... Thereof'' ; and U.S. Patent 6, 855, 314 entitled ``AAV5 Vector for Transducing Brain Cells and Lung Cells... of delivering nucleic acids to a cell by using the AAV5 vectors and particles. More specifically, the...

  15. B-cell depletion is protective against anti-AAV capsid immune response: a human subject case study

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    M Corti


    Full Text Available Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV vectors due to aspects of the clinical application, such as: (i administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; (ii progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and (iii a possibly faster depletion of pathogenic myofibers in this patient population. Immune response triggered by the first vector administration, and to subsequent doses, represents a major obstacle for successful gene transfer in young patients. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or enzyme replacement therapy (ERT for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. In this study, we evaluated the immune response of a Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate reactions against ERT. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

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

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    Harrison C Brown


    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.

  17. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice. (United States)

    Hordeaux, Juliette; Wang, Qiang; Katz, Nathan; Buza, Elizabeth L; Bell, Peter; Wilson, James M


    Improved delivery of adeno-associated virus (AAV) vectors to the CNS will greatly enhance their clinical utility. Selection of AAV9 variants in a mouse model led to the isolation of a capsid called PHP.B, which resulted in remarkable transduction of the CNS following intravenous infusion. However, we now show here that this enhanced CNS tropism is restricted to the model in which it was selected, i.e., a Cre transgenic mouse in a C57BL/6J background, and was not found in nonhuman primates or the other commonly used mouse strain BALB/cJ. We also report the potential for serious acute toxicity in NHP after systemic administration of high dose of AAV. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  18. Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery.

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    Zongchao Han

    Full Text Available Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV, makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs, critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.

  19. Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs. (United States)

    Jiang, Haiyan; Lillicrap, David; Patarroyo-White, Susannah; Liu, Tongyao; Qian, Xiaobing; Scallan, Ciaran D; Powell, Sandra; Keller, Tracey; McMurray, Morag; Labelle, Andrea; Nagy, Dea; Vargas, Joseph A; Zhou, Shangzhen; Couto, Linda B; Pierce, Glenn F


    Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

  20. In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Cia-Hin Lau


    Full Text Available Adeno-associated virus (AAV has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics.

  1. Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain. (United States)

    Matsuzaki, Yasunori; Konno, Ayumu; Mochizuki, Ryuta; Shinohara, Yoichiro; Nitta, Keisuke; Okada, Yukihiro; Hirai, Hirokazu


    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.

  2. Stable producer cell lines for adeno-associated virus (AAV) assembly. (United States)

    Chadeuf, Gilliane; Salvetti, Anna


    Stable producer cell lines containing both the rep and cap genes and recombinant adeno-associated virus (rAAV) vectors can be infected with a helper virus to provide reliable and efficient production of rAAV stocks. However, the development of these cell lines is time-consuming. The procedure described here is therefore recommended only for studies requiring the production of high amounts of rAAV, such as preclinical studies performed in large animals.

  3. Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results. (United States)

    Flotte, Terence R; Trapnell, Bruce C; Humphries, Margaret; Carey, Brenna; Calcedo, Roberto; Rouhani, Farshid; Campbell-Thompson, Martha; Yachnis, Anthony T; Sandhaus, Robert A; McElvaney, Noel G; Mueller, Christian; Messina, Louis M; Wilson, James M; Brantly, Mark; Knop, David R; Ye, Guo-jie; Chulay, Jeffrey D


    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.

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

    LENUS (Irish Health Repository)

    Flotte, Terence R


    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.

  5. Construction of adeno-associated virus packaging plasmids and cells that directly select for AAV helper functions. (United States)

    Whiteway, Alistair; Deru, Wale; Prentice, H Grant; Anderson, Robert


    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.

  6. Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse (United States)

    Park, TK; Wu, Z; Kjellstrom, S; Zeng, Y; Bush, RA; Sieving, PA; Colosi, P


    X-linked juvenile retinoschisis (XLRS) is a neurodevelopmental abnormality caused by retinoschisin gene mutations. XLRS is characterized by splitting through the retinal layers and impaired synaptic transmission of visual signals resulting in impaired acuity and a propensity to retinal detachment. Several groups have treated murine retinoschisis models successfully using adeno-associated virus (AAV) vectors. Owing to the fragile nature of XLRS retina, translating this therapy to the clinic may require an alternative to invasive subretinal vector administration. Here we show that all layers of the retinoschisin knockout (Rs1-KO) mouse retina can be transduced efficiently with AAV vectors administered by simple vitreous injection. Retinoschisin expression was restricted to the neuroretina using a new vector that uses a 3.5-kb human retinoschisin promoter and an AAV type 8 capsid. Intravitreal administration to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution similar to that observed in wild-type retina, including the expression by the photoreceptors lying deep in the retina. No off-target expression was observed. Rs1-KO mice treated with this vector showed a decrease in the schisis cavities and had improved retinal signaling evaluated by recording the electroretinogram 11–15 weeks after the application. PMID:19458650

  7. A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

    Directory of Open Access Journals (Sweden)

    Laura Adamson-Small


    Full Text Available Recombinant adeno-associated vectors based on serotype 9 (rAAV9 have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP and good manufacturing practice (GMP production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production.

  8. AAV-mediated gene therapy for liver diseases : the prime candidate for clinical application?

    NARCIS (Netherlands)

    van der Laan, Luc J. W.; Wang, Yigang; Tilanus, Hugo W.; Janssen, Harry L. A.; Pan, Qiuwei

    Areas covered: This review provides a summary of current literature on AAV-mediated gene therapies for both inherited and acquired liver diseases and outlines different strategies to overcome current clinical limitations. The unique properties of AAV over other viral vectors are highlighted as well

  9. Widespread gene transfer in the central nervous system of cynomolgus macaques following delivery of AAV9 into the cisterna magna

    Directory of Open Access Journals (Sweden)

    Christian Hinderer


    Full Text Available Adeno-associated virus serotype 9 (AAV9 vectors have recently been shown to transduce cells throughout the central nervous system of nonhuman primates when injected into the cerebrospinal fluid (CSF, a finding which could lead to a minimally invasive approach to treat genetic and acquired diseases affecting the entire CNS. We characterized the transduction efficiency of two routes of vector administration into the CSF of cynomolgus macaques—lumbar puncture, which is typically used in clinical practice, and suboccipital puncture, which is more commonly used in veterinary medicine. We found that delivery of vector into the cisterna magna via suboccipital puncture is up to 100-fold more efficient for achieving gene transfer to the brain. In addition, we evaluated the inflammatory response to AAV9-mediated GFP expression in the nonhuman primate CNS. We found that while CSF lymphocyte counts increased following gene transfer, there were no clinical or histological signs of immune toxicity. Together these data indicate that delivery of AAV9 into the cisterna magna is an effective method for achieving gene transfer in the CNS, and suggest that adapting this uncommon injection method for human trials could vastly increase the efficiency of gene delivery.

  10. AAV vector encoding human VEGF165–transduced pectineus muscular flaps increase the formation of new tissue through induction of angiogenesis in an in vivo chamber for tissue engineering: A technique to enhance tissue and vessels in microsurgically engineered tissue

    Directory of Open Access Journals (Sweden)

    Silvia Moimas


    Full Text Available In regenerative medicine, new approaches are required for the creation of tissue substitutes, and the interplay between different research areas, such as tissue engineering, microsurgery and gene therapy, is mandatory. In this article, we report a modification of a published model of tissue engineering, based on an arterio-venous loop enveloped in a cross-linked collagen–glycosaminoglycan template, which acts as an isolated chamber for angiogenesis and new tissue formation. In order to foster tissue formation within the chamber, which entails on the development of new vessels, we wondered whether we might combine tissue engineering with a gene therapy approach. Based on the well-described tropism of adeno-associated viral vectors for post-mitotic tissues, a muscular flap was harvested from the pectineus muscle, inserted into the chamber and transduced by either AAV vector encoding human VEGF165 or AAV vector expressing the reporter gene β-galactosidase, as a control. Histological analysis of the specimens showed that muscle transduction by AAV vector encoding human VEGF165 resulted in enhanced tissue formation, with a significant increase in the number of arterioles within the chamber in comparison with the previously published model. Pectineus muscular flap, transduced by adeno-associated viral vectors, acted as a source of the proangiogenic factor vascular endothelial growth factor, thus inducing a consistent enhancement of vessel growth into the newly formed tissue within the chamber. In conclusion, our present findings combine three different research fields such as microsurgery, tissue engineering and gene therapy, suggesting and showing the feasibility of a mixed approach for regenerative medicine.

  11. Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.

    Directory of Open Access Journals (Sweden)

    Peter Charbel Issa

    Full Text Available Adeno-associated viral vectors (AAV have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4(-/- mouse which is a model for Stargardt disease and in the Pde6b(rd1/rd1 mouse in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4(-/- mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.

  12. Recombinant AAV-mediated in vivo long-term expression and antitumour activity of an anti-ganglioside GM3(Neu5Gc) antibody. (United States)

    Piperno, G M; López-Requena, A; Predonzani, A; Dorvignit, D; Labrada, M; Zentilin, L; Burrone, O R; Cesco-Gaspere, M


    The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at ∼3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7.

  13. Apparently nonspecific enzyme elevations after portal vein delivery of recombinant adeno-associated virus serotype 2 vector in hepatitis C virus-infected chimpanzees. (United States)

    Flotte, Terence R; Goetzmann, Jason; Caridi, James; Paolillo, Joseph; Conlon, Thomas J; Potter, Mark; Mueller, Christian; Byrne, Barry J


    Hepatic gene transfer is envisioned as a substitute for protein replacement therapies, many of which are derived from blood products. Thus, the target populations may have a high prevalence of blood-borne pathogens, such as hepatitis C virus (HCV). We sought to determine whether the safety of recombinant adeno-associated virus serotype 2 (rAAV2) would be altered by preexisting HCV infection. Doses of approximately 1 x 10(13) vector genomes of an rAAV2-chimpanzee alpha(1)-antitrypsin (rAAV2-cAAT) vector were injected into the portal vein of each of three HCV genome-positive (HCV+) chimpanzees and three HCV-negative (HCV-) controls. Acute safety studies were performed up to 90 days after vector administration, along with analyses of the peripheral blood and liver tissue for rAAV2-cAAT genomes. Vector genome copy numbers in blood and liver tissue were similar in both groups. All animals demonstrated increases in liver and muscle enzyme levels after the pretreatment liver biopsy (5 days before vector injection) and after the vector injection. However, HCV+ animals demonstrated a substantially greater rise in aspartate aminotransferase, alanine aminotransferase, and creatinine phosphokinase values than HCV- animals. Histopathology demonstrated abnormal lipid accumulation (steatosis) in the hepatocytes of HCV+ animals, both before and after vector injection. These data indicate an increased susceptibility to subclinical liver toxicity from portal vein injection of rAAV2 in the presence of HCV infection.

  14. Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons. (United States)

    Caudal, D; Alvarsson, A; Björklund, A; Svenningsson, P


    Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague-Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8 weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8 weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3 weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic-pituitary-adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to

  15. Recent developments in recombinant AAV-mediated gene therapy for lung diseases. (United States)

    Flotte, Terence R


    Recent studies have shed light on a number of important obstacles to safe and effective gene transfer to the respiratory tract with recombinant AAV vectors. Among these are blocks at the level of receptor binding and internalizations, evasion of proteasomal degradation, inefficiency of nuclear entry, and nuclear factors that inhibit the conversion of rAAV genomes into active double-stranded DNA form. Other important issues have been the size constraints of the vector, the lack of retention of episomal forms of the vector genome, and immune responses which may limit the efficiency of repeated doses of rAAV. Each of these potential obstacles has been addressed with new vector designs. In addition, the availability of an abundance of novel rAAV serotypes, each with its own receptor tropism, has expanded the range of possibilities for long-term success of gene therapy in the respiratory tract.

  16. Rod Outer Segment Development Influences AAV-Mediated Photoreceptor Transduction After Subretinal Injection. (United States)

    Petit, Lolita; Ma, Shan; Cheng, Shun-Yun; Gao, Guangping; Punzo, Claudio


    Vectors based on the adeno-associated virus (AAV) are currently the preferred tools for delivering genes to photoreceptors (PR) in small and large animals. AAVs have been applied successfully in various models of PR dystrophies. However, unknown barriers still limit AAV's efficient application in several forms of severe PR degenerations due to insufficient transgene expression and/or treated cells at the time of injection. Optimizations of PR gene therapy strategies will likely benefit from the identification of the cellular factors that influence PR transduction. Interestingly, recent studies have shown that the AAV transduction profile of PRs differs significantly between neonatal and adult mouse retinas after subretinal injection. This phenomenon may provide clues to identify host factors that influence the efficiency of AAV-mediated PR transduction. This study demonstrates that rod outer segments are critical modulators of efficient AAV-mediated rod transduction. During retinal development, rod transduction correlated temporally and spatially with the differentiation order of PRs when vectors were introduced subretinally but not when introduced intravitreally. All subretinally injected vectors had an initial preference to transduce cones in the absence of formed rod outer segments and then displayed a preference for rods as the cells matured, independently of the expression cassette or AAV serotype. Consistent with this observation, altered development of rod outer segments was associated with a strong reduction of rod transduction and an increase in the percentage of transduced cones by 2- to 2.8-fold. A similar increase of cone transduction was observed in the adult retinal degeneration 1 (rd1) retina compared to wild-type mice. These results suggest that the loss of rod outer segments in diseased retinas could markedly affect gene transfer efficiency of AAV vectors by limiting the ability of AAVs to infect dying rods efficiently. This information could be

  17. Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting

    Directory of Open Access Journals (Sweden)

    Livia S. Carvalho


    Full Text Available Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for inherited retinal degeneration due to mutations in RPE65 have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies in vitro and in vivo using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both in vitro and in vivo compared to trans-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.

  18. Effective inhibition of specific gene by adenoassociated virus (AAV ...

    African Journals Online (AJOL)

    To perform functional tests on siRNA, which was expressed by the viral vector, recombinant AAVs, coding for siRNA against exogenous gene, EGFP, and endogenous gene, p53, were established and added into HEK293 cells, respectively. The results proved the expression of EGFP and p53 in cells were definitely ...

  19. Significant changes in endogenous retinal gene expression assessed 1 year after a single intraocular injection of AAV-CNTF or AAV-BDNF

    Directory of Open Access Journals (Sweden)

    Chrisna J LeVaillant


    Full Text Available Use of viral vectors to deliver therapeutic genes to the central nervous system holds promise for the treatment of neurodegenerative diseases and neurotrauma. Adeno-associated viral (AAV vectors encoding brain-derived neurotrophic factor (BDNF or ciliary derived neurotrophic factor (CNTF promote the viability and regeneration of injured adult rat retinal ganglion cells. However, these growth-inducing transgenes are driven by a constitutively active promoter, thus we examined whether long-term AAV-mediated secretion of BDNF or CNTF affected endogenous retinal gene expression. One year after the intravitreal injection of AAV-green fluorescent protein (GFP, bi-cistronic AAV-BDNF-GFP or AAV-CNTF-GFP, mRNA was extracted and analyzed using custom 96 well polymerase chain reaction arrays. Of 93 test genes, 56% showed significantly altered expression in AAV-BDNF-GFP and/or AAV-CNTF-GFP retinas compared with AAV-GFP controls. Of these genes, 73% showed differential expression in AAV-BDNF versus AAV-CNTF injected eyes. To focus on retinal ganglion cell changes, quantitative polymerase chain reaction was undertaken on mRNA (16 genes obtained from fixed retinal sections in which the ganglion cell layer was enriched. The sign and extent of fold changes in ganglion cell layer gene expression differed markedly from whole retinal samples. Sustained and global alteration in endogenous mRNA expression after gene therapy should be factored into any interpretation of experimental/clinical outcomes, particularly when introducing factors into the central nervous system that require secretion to evoke functionality.

  20. Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease

    Directory of Open Access Journals (Sweden)

    Piotr Hadaczek


    Full Text Available Huntington's disease (HD is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV, AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP, with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease.

  1. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

    Directory of Open Access Journals (Sweden)

    Atsushi Miyanohara


    Full Text Available Effective in vivo use of adeno-associated virus (AAV-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal. Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii potent retrograde transgene expression in brain motor centers (motor cortex and brain stem; and (iv the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.

  2. The structure of adeno-associated virus serotype 3B (AAV-3B): insights into receptor binding and immune evasion. (United States)

    Lerch, Thomas F; Xie, Qing; Chapman, Michael S


    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 2.6A 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. Copyright 2010 Elsevier Inc. All rights reserved.

  3. AAV2-mediated in vivo immune gene therapy of solid tumours

    LENUS (Irish Health Repository)

    Collins, Sara A


    Abstract Background Many strategies have been adopted to unleash the potential of gene therapy for cancer, involving a wide range of therapeutic genes delivered by various methods. Immune therapy has become one of the major strategies adopted for cancer gene therapy and seeks to stimulate the immune system to target tumour antigens. In this study, the feasibility of AAV2 mediated immunotherapy of growing tumours was examined, in isolation and combined with anti-angiogenic therapy. Methods Immune-competent Balb\\/C or C57 mice bearing subcutaneous JBS fibrosarcoma or Lewis Lung Carcinoma (LLC) tumour xenografts respectively were treated by intra-tumoural administration of AAV2 vector encoding the immune up-regulating cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) and the co-stimulatory molecule B7-1 to subcutaneous tumours, either alone or in combination with intra-muscular (IM) delivery of AAV2 vector encoding Nk4 14 days prior to tumour induction. Tumour growth and survival was monitored for all animals. Cured animals were re-challenged with tumourigenic doses of the original tumour type. In vivo cytotoxicity assays were used to investigate establishment of cell-mediated responses in treated animals. Results AAV2-mediated GM-CSF, B7-1 treatment resulted in a significant reduction in tumour growth and an increase in survival in both tumour models. Cured animals were resistant to re-challenge, and induction of T cell mediated anti-tumour responses were demonstrated. Adoptive transfer of splenocytes to naïve animals prevented tumour establishment. Systemic production of Nk4 induced by intra-muscular (IM) delivery of Nk4 significantly reduced subcutaneous tumour growth. However, combination of Nk4 treatment with GM-CSF, B7-1 therapy reduced the efficacy of the immune therapy. Conclusions Overall, this study demonstrates the potential for in vivo AAV2 mediated immune gene therapy, and provides data on the inter-relationship between tumour

  4. Systemic Correction of Murine Glycogen Storage Disease Type IV by an AAV-Mediated Gene Therapy. (United States)

    Yi, Haiqing; Zhang, Quan; Brooks, Elizabeth D; Yang, Chunyu; Thurberg, Beth L; Kishnani, Priya S; Sun, Baodong


    Deficiency of glycogen branching enzyme (GBE) causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a less branched, poorly soluble form of glycogen called polyglucosan (PG) in multiple tissues. This study evaluates the efficacy of gene therapy with an adeno-associated viral (AAV) vector in a mouse model of adult form of GSD IV (Gbe1 ys/ys ). An AAV serotype 9 (AAV9) vector containing a human GBE expression cassette (AAV-GBE) was intravenously injected into 14-day-old Gbe1 ys/ys mice at a dose of 5 × 10 11 vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at 3 months of age, GBE enzyme activity was highly elevated in heart, which is consistent with the high copy number of the viral vector genome detected. GBE activity also increased significantly in skeletal muscles and the brain, but not in the liver. The glycogen content was reduced to wild-type levels in muscles and significantly reduced in the liver and brain. At 9 months of age, though GBE activity was only significantly elevated in the heart, glycogen levels were significantly reduced in the liver, brain, and skeletal muscles of the AAV-treated mice. In addition, the AAV treatment resulted in an overall decrease in plasma activities of alanine transaminase, aspartate transaminase, and creatine kinase, and a significant increase in fasting plasma glucose concentration at 9 months of age. This suggests an alleviation of damage and improvement of function in the liver and muscles by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in Gbe1 ys/ys mice.

  5. Recombinant AAV serotype and capsid mutant comparison for pulmonary gene transfer of alpha-1-antitrypsin using invasive and noninvasive delivery. (United States)

    Liqun Wang, Rejean; McLaughlin, Thomas; Cossette, Travis; Tang, Qiushi; Foust, Kevin; Campbell-Thompson, Martha; Martino, Ashley; Cruz, Pedro; Loiler, Scott; Mueller, Christian; Flotte, Terence R


    Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing alpha-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 x 10(10) vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT ("long serpin") indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency-related emphysema.

  6. Recombinant AAV Serotype and Capsid Mutant Comparison for Pulmonary Gene Transfer of α-1-Antitrypsin Using Invasive and Noninvasive Delivery (United States)

    Liqun Wang, Rejean; McLaughlin, Thomas; Cossette, Travis; Tang, Qiushi; Foust, Kevin; Campbell-Thompson, Martha; Martino, Ashley; Cruz, Pedro; Loiler, Scott; Mueller, Christian; Flotte, Terence R


    Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing α-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 × 1010 vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT (“long serpin”) indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency–related emphysema. PMID:18941444

  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)


    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. Complexity of immune responses to AAV transgene products - Example of factor IX. (United States)

    Herzog, Roland W


    After two decades of research, in vivo gene transfer with adeno-associated viral (AAV) vectors has now resulted in successful treatments and even cures for several human diseases. However, the potential for immune responses against the therapeutic gene products remains one of the concerns as this approach is broadened to more patients, diverse diseases, and target organs. Immune responses following gene transfer of coagulation factor IX (FIX) for the treatment of the bleeding disorder hemophilia B has been extensively investigated in multiple animal models. Findings from these studies have not only influenced clinical trial design but have broader implications for other diseases. The impact of vector design and dose, as well as target organ/route of administration on humoral and cellular immune responses are reviewed. Furthermore, the potential for tolerance induction by hepatic gene transfer or combination with immune modulation is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro. (United States)

    Wu, Wenyi; Duan, Yajian; Ma, Gaoen; Zhou, Guohong; Park-Windhol, Cindy; D'Amore, Patricia A; Lei, Hetian


    Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.

  10. Sequential Adeno-Associated Viral Vector Serotype 9-Green Fluorescent Protein Gene Transfer Causes Massive Inflammation and Intense Immune Response in Rat Striatum. (United States)

    Yang, Chun; Hao, Fei; He, Jun; Lu, Tao; Klein, Ronald L; Zhao, Li-Ru; Duan, Wei-Ming


    Green fluorescent protein (GFP) is a broadly used live cell reporter for gene transduction although side effects associated with GFP in gene transfer are reported. The present study was designed to systematically examine host responses, including inflammatory and immune responses, induced by persistent overexpression of the GFP gene mediated by adeno-associated viral vector serotype 9 (AAV9), and their effects on GFP gene transduction in rat striatum. Our results show that host responses against AAV9-GFP transduction, and GFP transgene expression in the striatum exhibited a temporal and dose-dependent pattern. Both muscular and striatal delivery of AAV9-GFP increased levels of inflammation and immune reactions against sequential AAV9-GFP transduction in the striatum, leading to reduced levels of GFP expression. We also observed that rat sera from sequential administrations of AAV9-GFP group had significantly higher levels of neutralizing antibody against AAV9 vectors when compared with the age-matched rats. As excessive GFP can trigger vigorous inflammation and intense immune response after GFP gene transduction, the use of GFP as a live cell marker protein should be deliberated, especially in repeated administration studies.

  11. miRNA-mediated post-transcriptional silencing of transgenes leads to increased adeno-associated viral vector yield and targeting specificity. (United States)

    Reid, C A; Boye, S L; Hauswirth, W W; Lipinski, D M


    The production of high-titer recombinant adeno-associated virus (rAAV) vector is essential for treatment of genetic diseases affecting the retina and choroid, where anatomical constraints may limit injectable volumes. Problematically, cytotoxicity arising from overexpression of the transgene during vector production frequently leads to a reduction in vector yield. Herein, we evaluate the use of microRNA (miRNA)-mediated silencing to limit overexpression of cytotoxic transgenes during packaging as a method of increasing vector yield. We examined if post-transcriptional regulation of transgenes during packaging via miRNA technology would lead to increased rAAV yields. Our results demonstrate that silencing of cytotoxic transgenes during production resulted in up to a 22-fold increase in vector yield. The inclusion of organ-specific miRNA sequences improved biosafety by limiting off-target expression following systemic rAAV administration. The small size (22-23 bp) of the target site allows for the inclusion of multiple copies into the vector with minimal impact on coding capacity. Taken together, our results suggest that inclusion of miRNA target sites into the 3'-untranslated region of the AAV cassette allow for silencing of cytotoxic transgenes during vector production leading to improved vector yield, in addition to increasing targeting specificity without reliance on cell-specific promoters.

  12. Long-Term Efficacy Following Readministration of an Adeno-Associated Virus Vector in Dogs with Glycogen Storage Disease Type Ia (United States)

    Demaster, Amanda; Luo, Xiaoyan; Curtis, Sarah; Williams, Kyha D.; Landau, Dustin J.; Drake, Elizabeth J.; Kozink, Daniel M.; Bird, Andrew; Crane, Bayley; Sun, Francis; Pinto, Carlos R.; Brown, Talmage T.; Kemper, Alex R.


    Abstract Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV

  13. Better Targeting, Better Efficiency for Wide-scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B

    Directory of Open Access Journals (Sweden)

    Kasey L Jackson


    Full Text Available Widespread genetic modification of cells in the central nervous system (CNS with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin hybrid (CBA promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered AAV-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS-related protein TDP-43 with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

  14. Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B. (United States)

    Jackson, Kasey L; Dayton, Robert D; Deverman, Benjamin E; Klein, Ronald L


    Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

  15. Evaluation of Dose and Safety of AAV7m8 and AAV8BP2 in the Non-Human Primate Retina. (United States)

    Ramachandran, Pavitra S; Lee, Vivian; Wei, Zhangyong; Song, Ji Yun; Casal, Giulia; Cronin, Therese; Willett, Keirnan; Huckfeldt, Rachel; Morgan, Jessica I W; Aleman, Tomas S; Maguire, Albert M; Bennett, Jean


    Within the next decade, we will see many gene therapy clinical trials for eye diseases, which may lead to treatments for thousands of visually impaired people around the world. To target retinal diseases that affect specific cell types, several recombinant adeno-associated virus (AAV) serotypes have been generated and used successfully in preclinical mouse studies. Because there are numerous anatomic and physiologic differences between the eyes of mice and "men" and because surgical delivery approaches and immunologic responses also differ between these species, this study evaluated the transduction characteristics of two promising new serotypes, AAV7m8 and AAV8BP2, in the retinas of animals that are most similar to those of humans: non-human primates (NHPs). We report that while AAV7m8 efficiently targets a variety of cell types by subretinal injection in NHPs, transduction after intravitreal delivery was mostly restricted to the inner retina at lower doses that did not induce an immune response. AAV8BP2 targets the cone photoreceptors efficiently but bipolar cells inefficiently by subretinal injection. Additionally, transduction by both serotypes in the anterior chamber of the eye and the optic pathway of the brain was observed post-intravitreal delivery. Finally, we assessed immunogenicity, keeping in mind that these AAV capsids may be used in future clinical trials. We found that AAV8BP2 had a better safety profile compared with AAV7m8, even at the highest doses administered. These studies underscore the differences in AAV transduction between mice and primates, highlighting the importance of careful evaluation of therapeutic vectors in NHPs prior to moving to clinical trials.

  16. 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. (United States)

    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; Fraefel, Cornel


    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/G 2 -phase cells, while HSV-1 DNA replication is restricted to G 1 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/G 2 -phase cells, suggesting that the preference for S/G 2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G 2 -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

  17. Dual AAV Gene Therapy for Duchenne Muscular Dystrophy with a 7-kb Mini-Dystrophin Gene in the Canine Model. (United States)

    Kodippili, Kasun; Hakim, Chady H; Pan, Xiufang; Yang, Hsiao T; Yue, Yongping; Zhang, Yadong; Shin, Jin-Hong; Yang, N Nora; Duan, Dongsheng


    Dual adeno-associated virus (AAV) technology was developed in 2000 to double the packaging capacity of the AAV vector. The proof of principle has been demonstrated in various mouse models. Yet, pivotal evidence is lacking in large animal models of human diseases. Here we report expression of a 7-kb canine ΔH2-R15 mini-dystrophin gene using a pair of dual AAV vectors in the canine model of Duchenne muscular dystrophy (DMD). The ΔH2-R15 minigene is by far the most potent synthetic dystrophin gene engineered for DMD gene therapy. We packaged minigene dual vectors in Y731F tyrosine-modified AAV-9 and delivered to the extensor carpi ulnaris muscle of a 12-month-old affected dog at the dose of 2 × 10 13 viral genome particles/vector/muscle. Widespread mini-dystrophin expression was observed 2 months after gene transfer. The missing dystrophin-associated glycoprotein complex was restored. Treatment also reduced muscle degeneration and fibrosis and improved myofiber size distribution. Importantly, dual AAV therapy greatly protected the muscle from eccentric contraction-induced force loss. Our data provide the first clear evidence that dual AAV therapy can be translated to a diseased large mammal. Further development of dual AAV technology may lead to effective therapies for DMD and many other diseases in human patients.

  18. Novel strategy for generation and titration of recombinant adeno-associated virus vectors. (United States)

    Shiau, Ai-Li; Liu, Pu-Ste; Wu, Chao-Liang


    Recombinant adeno-associated virus (rAAV) vectors have many advantages for gene therapeutic applications compared with other vector systems. Several methods that use plasmids or helper viruses have been reported for the generation of rAAV vectors. Unfortunately, the preparation of large-scale rAAV stocks is labor-intensive. Moreover, the biological titration of rAAV is still difficult, which may limit its preclinical and clinical applications. For this study, we developed a novel strategy to generate and biologically titrate rAAV vectors. A recombinant pseudorabies virus (PrV) with defects in its gD, gE, and thymidine kinase genes was engineered to express the AAV rep and cap genes, yielding PS virus, which served as a packaging and helper virus for the generation of rAAV vectors. PS virus was useful not only for generating high-titer rAAV vectors by cotransfection with an rAAV vector plasmid, but also for amplifying rAAV stocks. Notably, the biological titration of rAAV vectors was also feasible when cells were coinfected with rAAV and PS virus. Based on this strategy, we produced an rAAV that expresses prothymosin alpha (ProT). Expression of the ProT protein in vitro and in vivo mediated by rAAV/ProT gene transfer was detected by immunohistochemistry and a bioassay. Taken together, our results demonstrate that the PrV vector-based system is useful for generating rAAV vectors carrying various transgenes.

  19. 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


    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.

  20. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo


    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... on the most salient vectors, and this works well, but many images contain a plethora of vectors, which makes their structure quite different from the linguistic transitivity structures with which Kress and van Leeuwen have compared ‘narrative’ images. It can also be asked whether facial expression vectors...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

  1. The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD. (United States)

    Wang, Jing-Zhang; Wu, Peng; Shi, Zhi-Min; Xu, Yan-Li; Liu, Zhi-Jun


    Mutations in the dystrophin gene (Dmd) result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which afflict many newborn boys. In 2016, Brain and Development published several interesting articles on DMD treatment with antisense oligonucleotide, kinase inhibitor, and prednisolone. Even more strikingly, three articles in the issue 6271 of Science in 2016 provide new insights into gene therapy of DMD and BMD via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). In brief, adeno-associated virus (AAV) vectors transport guided RNAs (gRNAs) and Cas9 into mdx mouse model, gRNAs recognize the mutated Dmd exon 23 (having a stop codon), and Cas9 cut the mutated exon 23 off the Dmd gene. These manipulations restored expression of truncated but partially functional dystrophin, improved skeletal and cardiac muscle function, and increased survival of mdx mice significantly. This review concisely summarized the related advancements and discussed their primary implications in the future gene therapy of DMD, including AAV-vector selection, gRNA designing, Cas9 optimization, dystrophin-restoration efficiency, administration routes, and systemic and long-term therapeutic efficacy. Future orientations, including off-target effects, safety concerns, immune responses, precision medicine, and Dmd-editing in the brain (potentially blocked by the blood-brain barrier) were also elucidated briefly. Collectively, the AAV-mediated and RNA-guided CRISPR/Cas9 system has major superiorities compared with traditional gene therapy, and might contribute to the treatment of DMD and BMD substantially in the near future. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  2. Full-length dystrophin reconstitution with adeno-associated viral vectors. (United States)

    Lostal, William; Kodippili, Kasun; Yue, Yongping; Duan, Dongsheng


    Duchenne muscular dystrophy (DMD) is the most common lethal muscle disorder in children. It is caused by mutations of the dystrophin gene. Adeno-associated virus (AAV)-mediated gene replacement therapy has been actively pursued to treat DMD. However, this promising therapeutic modality has been challenged by the small packaging capacity of the AAV vector. The size of the full-length dystrophin cDNA is >11 kb, while an AAV virus can carry only a 5 kb genome. Innovative high-capacity AAV vectors may offer an opportunity to express the full-length dystrophin coding sequence. Here we describe several sets of tri-AAV vectors for full-length human dystrophin delivery. In each set, the full-length human dystrophin cDNA was split into three fragments and independently packaged into separate recombinant AAV vectors. Each vector was engineered with unique recombination signals for directional recombination. Tri-AAV vectors were coinjected into the tibialis anterior muscle of dystrophin-deficient mdx4cv mice. Thirty-five days after injection, dystrophin expression was examined by immunofluorescence staining. Despite low reconstitution efficiency, full-length human dystrophin was successfully expressed from the tri-AAV vectors. Our results suggest that AAV can be engineered to express an extra-large (up to 15 kb) gene that is approximately three times the size of the wild-type AAV genome. Further optimization of the trivector strategy may expand the utility of AAV for human gene therapy.

  3. Targeting of breast metastases using a viral gene vector with tumour-selective transcription.

    LENUS (Irish Health Repository)

    Rajendran, Simon


    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.

  4. Vectors

    DEFF Research Database (Denmark)

    Boeriis, Morten; van Leeuwen, Theo


    This article revisits the concept of vectors, which, in Kress and van Leeuwen’s Reading Images (2006), plays a crucial role in distinguishing between ‘narrative’, action-oriented processes and ‘conceptual’, state-oriented processes. The use of this concept in image analysis has usually focused...... should be taken into account in discussing ‘reactions’, which Kress and van Leeuwen link only to eyeline vectors. Finally, the question can be raised as to whether actions are always realized by vectors. Drawing on a re-reading of Rudolf Arnheim’s account of vectors, these issues are outlined...

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

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


    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.

  6. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice

    Directory of Open Access Journals (Sweden)

    Vivian W Choi

    Full Text Available Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein. A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1 had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone and photopic (cone electroretinograms (ERGs. The effect was still present after 1 year.

  7. Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer. (United States)

    Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell'Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean


    We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

  8. beta-hexosaminidase lentiviral vectors: transfer into the CNS via systemic administration. (United States)

    Kyrkanides, Stephanos; Miller, Jennie H; Brouxhon, Sabine M; Olschowka, John A; Federoff, Howard J


    Brain inflammation in GM2 gangliosidosis has been recently realized as a key factor in disease development. The aim of this study was to investigate the effects of a FIV beta-hexosaminidase vector in the brain of HexB-deficient (Sandhoff disease) mice following intraperitoneal administration to pups of neonatal age. Since brain inflammation, lysosomal storage and neuromuscular dysfunction are characteristics of HexB deficiency, these parameters were employed as experimental outcomes in our study. The ability of the lentiviral vector FIV(HEX) to infect murine cells was initially demonstrated with success in normal mouse fibroblasts and human Tay-Sachs cells in vitro. Furthermore, systemic transfer of FIV(HEX) to P2 HexB-/- knockout pups lead to transduction of peripheral and central nervous system tissues. Specifically, beta-hexosaminidase expressing cells were immunolocalized in periventricular areas of the cerebrum as well as in the cerebellar cortex. FIV(HEX) neonatal treatment resulted in reduction of GM2 storage along with attenuation of the brain inflammation and amelioration of the attendant neuromuscular deterioration. In conclusion, these results demonstrate the effective transfer of a beta-hexosaminidase lentiviral vector to the brain of Sandhoff mice and resolution of the GM2 gangliosidosis after neonatal intraperitoneal administration.

  9. Tailored transgene expression to specific cell types in the central nervous system after peripheral injection with AAV9

    Directory of Open Access Journals (Sweden)

    Jonathan Dashkoff


    Full Text Available The capacity of certain adeno-associated virus (AAV vectors to cross the blood–brain barrier after intravenous delivery offers a unique opportunity for noninvasive brain delivery. However, without a well-tailored system, the use of a peripheral route injection may lead to undesirable transgene expression in nontarget cells or organs. To refine this approach, the present study characterizes the transduction profiles of new self-complementary AAV9 (scAAV9 expressing the green fluorescent protein (GFP either under an astrocyte (glial fibrillary acidic (GFA protein or neuronal (Synapsin (Syn promoter, after intravenous injection of adult mice (2 × 1013 vg/kg. ScAAV9-GFA-GFP and scAAV9-Syn-GFP robustly transduce astrocytes (11% and neurons (17%, respectively, without aberrant expression leakage. Interestingly, while the percentages of GFP-positive astrocytes with scAAV9-GFA-GFP are similar to the performances observed with scAAV9-CBA-GFP (broadly active promoter, significant higher percentages of neurons express GFP with scAAV9-Syn-GFP. GFP-positive excitatory as well as inhibitory neurons are observed, as well as motor neurons in the spinal cord. Additionally, both activated (GFAP-positive and resting astrocytes (GFAP-negative express the reporter gene after scAAV9-GFA-GFP injection. These data thoroughly characterize the gene expression specificity of AAVs fitted with neuronal and astrocyte-selective promoters after intravenous delivery, which will prove useful for central nervous system (CNS gene therapy approaches in which peripheral expression of transgene is a concern.

  10. Glial promoter selectivity following AAV-delivery to the immature brain.

    Directory of Open Access Journals (Sweden)

    Georg von Jonquieres

    Full Text Available Recombinant adeno-associated virus (AAV vectors are versatile tools for gene transfer to the central nervous system (CNS and proof-of-concept studies in adult rodents have shown that the use of cell type-specific promoters is sufficient to target AAV-mediated transgene expression to glia. However, neurological disorders caused by glial pathology usually have an early onset. Therefore, modelling and treatment of these conditions require expanding the concept of targeted glial transgene expression by promoter selectivity for gene delivery to the immature CNS. Here, we have investigated the AAV-mediated green fluorescent protein (GFP expression driven by the myelin basic protein (MBP or glial fibrillary acidic protein (GFAP promoters in the developing mouse brain. Generally, the extent of transgene expression after infusion at immature stages was widespread and higher than in adults. The GFAP promoter-driven GFP expression was found to be highly specific for astrocytes following vector infusion to the brain of neonates and adults. In contrast, the selectivity of the MBP promoter for oligodendrocytes was poor following neonatal AAV delivery, but excellent after vector injection at postnatal day 10. To extend these findings obtained in naïve mice to a disease model, we performed P10 infusions of AAV-MBP-GFP in aspartoacylase (ASPA-deficient mouse mutants presenting with early onset oligodendrocyte pathology. Spread of GFP expression and selectivity for oligodendrocytes in ASPA-mutants was comparable with our observations in normal animals. Our data suggest that direct AAV infusion to the developing postnatal brain, utilising cellular promoters, results in targeted and long-term transgene expression in glia. This approach will be relevant for disease modelling and gene therapy for the treatment of glial pathology.

  11. Therapeutic Efficacy of Vectored PGT121 Gene Delivery in HIV-1-Infected Humanized Mice. (United States)

    Badamchi-Zadeh, Alexander; Tartaglia, Lawrence J; Abbink, Peter; Bricault, Christine A; Liu, Po-Ting; Boyd, Michael; Kirilova, Marinela; Mercado, Noe B; Nanayakkara, Ovini S; Vrbanac, Vladimir D; Tager, Andrew M; Larocca, Rafael A; Seaman, Michael S; Barouch, Dan H


    Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required. IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Copyright © 2018 Badamchi-Zadeh et al.

  12. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    International Nuclear Information System (INIS)

    Zhao Weihong; Zhong Li; Wu Jianqing; Chen Linyuan; Qing Keyun; Weigel-Kelley, Kirsten A.; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H.; Srivastava, Arun


    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by ∼25-fold in WT MEFs, but only by ∼4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency ∼23-fold in WT MEFs, but only ∼4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, ∼59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only ∼28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant AAV vectors in human gene

  13. LIGHT induces distinct signals to clear an AAV-expressed persistent antigen in the mouse liver and to induce liver inflammation.

    Directory of Open Access Journals (Sweden)

    Michael L Washburn


    Full Text Available Infection with adeno-associated virus (AAV vector with liver tropism leads to persistent expression of foreign antigens in the mouse liver, with no significant liver inflammation or pathology. This provides a model to investigate antigen persistence in the liver and strategies to modulate host immunity to reduce or clear the foreign antigen expressed from AAV vector in the liver.We showed that expressing LIGHT with an adenovirus vector (Ad in mice with established AAV in the liver led to clearance of the AAV. Ad-LIGHT enhanced CD8 effector T cells in the liver, correlated with liver inflammation. LTbetaR-Ig proteins blocked Ad-LIGHT in clearing AAV. Interestingly, in LTbetaR-null mice, Ad-LIGHT still cleared AAV but caused no significant liver inflammation.Our data suggest that LIGHT interaction with the LTbetaR plays a critical role in liver inflammation but is not required for LIGHT-mediated AAV clearance. These findings will shed light on developing novel immuno-therapeutics in treating people chronically infected with hepato-tropic viruses.

  14. Convection-enhanced delivery of AAV2-PrPshRNA in prion-infected mice.

    Directory of Open Access Journals (Sweden)

    Misol Ahn

    Full Text Available Prion disease is caused by a single pathogenic protein (PrPSc, an abnormal conformer of the normal cellular prion protein PrPC. Depletion of PrPC in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA to suppress PrPC expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrPC levels were reduced by ∼ 70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrPSc formation in the thalamic infusion site by ∼ 75%, it did not suppress PrPSc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrPC in the brain is required for successful therapy of prion diseases.

  15. AAV2-mediated CLN2 gene transfer to rodent and non-human primate brain results in long-term TPP-I expression compatible with therapy for LINCL. (United States)

    Sondhi, D; Peterson, D A; Giannaris, E L; Sanders, C T; Mendez, B S; De, B; Rostkowski, A B; Blanchard, B; Bjugstad, K; Sladek, J R; Redmond, D E; Leopold, P L; Kaminsky, S M; Hackett, N R; Crystal, R G


    Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal, autosomal recessive disease resulting from mutations in the CLN2 gene with consequent deficiency in its product tripeptidyl peptidase I (TPP-I). In the central nervous system (CNS), the deficiency of TPP-I results in the accumulation of proteins in lysosomes leading to a loss of neurons causing progressive neurological decline, and death by ages 10-12 years. To establish the feasibility of treating the CNS manifestations of LINCL by gene transfer, an adeno-associated virus 2 (AAV2) vector encoding the human CLN2 cDNA (AAV2CUhCLN2) was assessed for its ability to establish therapeutic levels of TPP-I in the brain. In vitro studies demonstrated that AAV2CUhCLN2 expressed CLN2 and produced biologically active TPP-I protein of which a fraction was secreted as the pro-TPP-I precursor and was taken up by nontransduced cells (ie, cross-correction). Following AAV2-mediated CLN2 delivery to the rat striatum, enzymatically active TPP-I protein was detected. By immunohistochemistry TPP-I protein was detected in striatal neurons (encompassing nearly half of the target structure) for up to 18 months. At the longer time points following striatal administration, TPP-I-positive cell bodies were also observed in the substantia nigra, frontal cerebral cortex and thalamus of the injected hemisphere, and the frontal cerebral cortex of the noninjected hemisphere. These areas of the brain contain neurons that extend axons into the striatum, suggesting that CNS circuitry may aid the distribution of the gene product. To assess the feasibility of human CNS delivery, a total of 3.6 x 10(11) particle units of AAV2CUhCLN2 was administered to the CNS of African green monkeys in 12 distributed doses. Assessment at 5 and 13 weeks demonstrated widespread detection of TPP-I in neurons, but not glial cells, at all regions of injection. The distribution of TPP-I-positive cells was similar between the two time points at all injection

  16. Effect Of DNA-dependent protein kinase on the molecular fate of the rAAV2 genome in skeletal muscle (United States)

    Song, Sihong; Laipis, Philip J.; Berns, Kenneth I.; Flotte, Terence R.


    We report here that the DNA-dependent protein kinase (DNA-PK) affects the molecular fate of the recombinant adeno-associated virus (rAAV) genome in skeletal muscle. rAAV-human α1-antitrypsin (rAAV-hAAT) vectors were delivered by intramuscular injection to either C57BL/6 (DNA-PKcs+) or C57BL/6-SCID [severe combined immunodeficient (SCID), DNA-PKcs−] mice. In both strains, high levels of transgene expression were sustained for up to 1 year after a single injection. Southern blot analysis showed that rAAV genomes persisted as linear episomes for more than 1 year in SCID mice, whereas only circular episomal forms were observed in the C57BL/6 strain. These results indicate that DNA-PK is involved in the formation of circular rAAV episomes. PMID:11274433

  17. Sustained correction of FVII deficiency in dogs using AAV-mediated expression of zymogen FVII (United States)

    Marcos-Contreras, Oscar A.; Smith, Shannon M.; Bellinger, Dwight A.; Raymer, Robin A.; Merricks, Elizabeth; Faella, Armida; Pavani, Giulia; Zhou, Shangzhen; Nichols, Timothy C.; High, Katherine A.


    Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (>10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for >1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency. PMID:26702064

  18. Inflammation and Immune Response of Intra-Articular Serotype 2 Adeno-Associated Virus or Adenovirus Vectors in a Large Animal Model

    Directory of Open Access Journals (Sweden)

    Akikazu Ishihara


    Full Text Available Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad, serotype 2 adeno-associated virus vectors (rAAV2, or self-complementary (sc AAV2 vectors carrying green fluorescent protein (GFP. Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.

  19. Safety and biodistribution assessment of sc-rAAV2.5IL-1Ra administered via intra-articular injection in a mono-iodoacetate-induced osteoarthritis rat model

    Directory of Open Access Journals (Sweden)

    Gensheng Wang


    Full Text Available Interleukin-1 (IL-1 plays an important role in the pathophysiology of osteoarthritis (OA, and gene transfer of IL-1 receptor antagonist (IL-1Ra holds promise for OA treatment. A preclinical safety and biodistribution study evaluated a self-complementary adeno-associated viral vector carrying rat IL-1Ra transgene (sc-rAAV2.5rIL-1Ra at 5 × 108, 5 × 109, or 5 × 1010 vg/knee, or human IL-1Ra transgene (sc-rAAV2.5hIL-1Ra at 5 × 1010 vg/knee, in Wistar rats with mono-iodoacetate (MIA–induced OA at days 7, 26, 91, 180, and 364 following intra-articular injection. The MIA-induced OA lesions were consistent with the published data on this model. The vector genomes persisted in the injected knees for up to a year with only limited vector leakage to systemic circulation and uptake in tissues outside the knee. Low levels of IL-1Ra expression and mitigation of OA lesions were observed in the vector-injected knees, albeit inconsistently. Neutralizing antibodies against the vector capsid developed in a dose-dependent manner, but only the human vector induced a small splenic T-cell immune response to the vector capsid. No local or systemic toxicity attributable to vector administration was identified in the rats as indicated by clinical signs, body weight, feed consumption, clinical pathology, and gross and microscopic pathology through day 364. Taken together, the gene therapy vector demonstrated a favorable safety profile.

  20. The ANCA Vasculitis Questionnaire (AAV-PRO©) (United States)


    Eosinophilic Granulomatosis With Polyangiitis (Churg-Strauss) (EGPA); Churg-Strauss Syndrome (CSS); Granulomatosis With Polyangiitis (Wegener's) (GPA); Wegener Granulomatosis (WG); Microscopic Polyangiitis (MPA); ANCA-Associated Vasculitis (AAV); Vasculitis

  1. Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Anja K Gruenert

    Full Text Available Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2 vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss- DNA vector genome into double-stranded (ds- DNA. This step can be bypassed by using self-complementary (sc- AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV and 38.0±8.6% (ssAAV (p<0.001, respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.

  2. AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the Proteasome In Vivo (United States)

    Paulk, Nicole K.; Loza, Laura Marquez; Finegold, Milton J.


    Abstract Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah−/−Ku70−/− double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy. PMID:22486314

  3. Pharmacological modulation of humoral immunity in a nonhuman primate model of AAV gene transfer for hemophilia B. (United States)

    Mingozzi, Federico; Chen, Yifeng; Murphy, Samuel L; Edmonson, Shyrie C; Tai, Alex; Price, Sandra D; Metzger, Mark E; Zhou, Shangzhen; Wright, J Fraser; Donahue, Robert E; Dunbar, Cynthia E; High, Katherine A


    Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.

  4. AAV-mediated expression of CNTF promotes long-term survival and regeneration of adult rat retinal ganglion cells

    NARCIS (Netherlands)

    Leaver, Simone G; Cui, Qi; Plant, Giles W; Arulpragasam, A.; Hisheh, S; Verhaagen, J; Harvey, Alan R

    We compared the effects of intravitreal injection of bi-cistronic adeno-associated viral (AAV-2) vectors encoding enhanced green fluorescent protein (GFP) and either ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF) or growth-associated protein-43 (GAP43) on adult retinal

  5. Progressive neurodegenerative and behavioural changes induced by AAV-mediated overexpression of α-synuclein in midbrain dopamine neurons

    DEFF Research Database (Denmark)

    Decressac, M; Mattsson, Bente; Lundblad, M


    have failed to show a consistent behavioural phenotype and pronounced dopamine neurodegeneration. Using a more efficient adeno-associated viral (AAV) vector construct, which includes a WPRE enhancer element and uses the neuron-specific synapsin-1 promoter to drive the expression of human wild-type α...

  6. Development of a Novel AAV Gene Therapy Cassette with Improved Safety Features and Efficacy in a Mouse Model of Rett Syndrome

    Directory of Open Access Journals (Sweden)

    Kamal K.E. Gadalla


    Full Text Available Rett syndrome (RTT, caused by loss-of-function mutations in the MECP2 gene, is a neurological disorder characterized by severe impairment of motor and cognitive functions. The aim of this study was to investigate the impact of vector design, dosage, and delivery route on the efficacy and safety of gene augmentation therapy in mouse models of RTT. Our results show that AAV-mediated delivery of MECP2 to Mecp2 null mice by systemic administration, and utilizing a minimal endogenous promoter, was associated with a narrow therapeutic window and resulted in liver toxicity at higher doses. Lower doses of this vector significantly extended the survival of mice lacking MeCP2 or expressing a mutant T158M allele but had no impact on RTT-like neurological phenotypes. Modifying vector design by incorporating an extended Mecp2 promoter and additional regulatory 3′ UTR elements significantly reduced hepatic toxicity after systemic administration. Moreover, direct cerebroventricular injection of this vector into neonatal Mecp2-null mice resulted in high brain transduction efficiency, increased survival and body weight, and an amelioration of RTT-like phenotypes. Our results show that controlling levels of MeCP2 expression in the liver is achievable through modification of the expression cassette. However, it also highlights the importance of achieving high brain transduction to impact the RTT-like phenotypes.

  7. AAV-Nrf2 Promotes Protection and Recovery in Animal Models of Oxidative Stress. (United States)

    Liang, Katharine J; Woodard, Kenton T; Weaver, Mark A; Gaylor, John Paul; Weiss, Ellen R; Samulski, R Jude


    NRF2 is a transcription factor that drives antioxidant gene expression in multiple organ systems. We hypothesized that Nrf2 overexpression could be therapeutically applied toward diseases in which redox homeostasis is disrupted. In this study, adeno-associated virus (AAV)-Nrf2 was tested in a mouse model of acute acetaminophen-induced liver toxicity and successfully conferred protection from hepatotoxicity, validating the vector design and early onset of NRF2-mediated protection. Furthermore, therapeutic potential of AAV-Nrf2 in chronic disease also was tested in a light-induced mouse model of age-related macular degeneration. Adult BALB/c mice were intravitreally injected with AAV-Nrf2 and subject to light damage following injection. Retinal thickness and function were monitored following light damage using optical coherence tomography and electroretinography, respectively. By 3 months post-damage, injected eyes had greater retinal thickness compared to uninjected controls. At 1 month post-damage, AAV-Nrf2 injection facilitated full functional recovery from light damage. Our results suggest a therapeutic potential for Nrf2 overexpression in acute and long-term capacities in multiple organ systems, opening up doors for combination gene therapy where replacement gene therapy requires additional therapeutic support to prevent further degeneration. Published by Elsevier Inc.

  8. Inhalable delivery of AAV-based MRP4/ABCC4 silencing RNA prevents monocrotaline-induced pulmonary hypertension

    Directory of Open Access Journals (Sweden)

    Caroline Claude

    Full Text Available The ATP-binding cassette transporter MRP4 (encoded by ABCC4 regulates membrane cyclic nucleotides concentrations in arterial cells including smooth muscle cells. MRP4/ABCC4 deficient mice display a reduction in smooth muscle cells proliferation and a prevention of pulmonary hypertension in response to hypoxia. We aimed to study gene transfer of a MRP4/ABCC4 silencing RNA via intratracheal delivery of aerosolized adeno-associated virus 1 (AAV1.shMRP4 or AAV1.control in a monocrotaline-induced model of pulmonary hypertension in rats. Gene transfer was performed at the time of monocrotaline administration and the effect on the development of pulmonary vascular remodeling was assessed 35 days later. AAV1.shMRP4 dose-dependently reduced right ventricular systolic pressure and hypertrophy with a significant reduction with the higher doses (i.e., >1011 DRP/animal as compared to AAV1.control. The higher dose of AAV1.shMRP4 was also associated with a significant reduction in distal pulmonary arteries remodeling. AAV1.shMRP4 was finally associated with a reduction in the expression of ANF, a marker of cardiac hypertrophy. Collectively, these results support a therapeutic potential for downregulation of MRP4 for the treatment of pulmonary artery hypertension.

  9. 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

  10. Antidepressant effect of recombinant NT4-NAP/AAV on social isolated mice through intranasal route. (United States)

    Liu, Fei; Liu, You-Ping; Lei, Gang; Liu, Peng; Chu, Zheng; Gao, Cheng-Ge; Dang, Yong-Hui


    The purpose of the present study was to observe the depression-like behavior induced by social isolation; detect the antidepressant effect of a recombinant adeno-associated virus (AAV) expressing NAP on social isolation mice by intranasal delivery. After construction of NT4-NAP/AAV, expression of NAP was confirmed in vitro. 3-week-old C57/BL mice were bred individually in cages as social isolation-rearing. Six weeks later, the first subset of mice underwent behavioral tests and western blot; the second was for enzyme-linked immunosorbent assay. NT4-NAP/AAV was delivered quaque die by nasal administration for consecutive 10 days before behavioral test. Several depression-like behaviors were observed in social isolation mice, including decreased relative sucrose preference, longer immobility time in forced swimming test, lower plasma corticosterone and decreased brain-derived neurotrophic factor in hippocampus. Thus, social isolation procedure appears to be an animal model of depression with good face and construct validity. What's more, the antidepressant effect in social isolation-rearing mice was observed after intranasal administration of NT4-NAP/AAV, suggesting that this might be a promising therapeutic strategy for depressive disorder.

  11. CRISPR/Cas9-AAV Mediated Knock-in at NRL Locus in Human Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Xianglian Ge


    Full Text Available Clustered interspaced short palindromic repeats (CRISPR/CRISPR-associated protein 9 (Cas9-mediated genome engineering technologies are sparking a new revolution in biological research. This technology efficiently induces DNA double strand breaks at the targeted genomic sequence and results in indel mutations by the error-prone process of nonhomologous end joining DNA repair or homologous recombination with a DNA repair template. The efficiency of genome editing with CRISPR/Cas9 alone in human embryonic stem cells is still low. Gene targeting with adeno-associated virus (AAV vectors has been demonstrated in multiple human cell types with maximal targeting frequencies without engineered nucleases. However, whether CRISPR/Cas9-mediated double strand breaks and AAV based donor DNA mediated homologous recombination approaches could be combined to create a novel CRISPR/Cas9-AAV genetic tool for highly specific gene editing is not clear. Here we demonstrate that using CRISPR/Cas9-AAV, we could successfully knock-in a DsRed reporter gene at the basic motifleucine zipper transcription factor (NRL locus in human embryonic stem cells. For the first time, this study provides the proof of principle that these two technologies can be used together. CRISPR/Cas9-AAV, a new genome editing tool, offers a platform for the manipulation of human genome.

  12. Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy. (United States)

    Zhang, Yadong; Yue, Yongping; Li, Liang; Hakim, Chady H; Zhang, Keqing; Thomas, Gail D; Duan, Dongsheng


    Neuronal nitric oxide synthase (nNOS) membrane delocalization contributes to the pathogenesis of Duchenne muscular dystrophy (DMD) by promoting functional muscle ischemia and exacerbating muscle injury during exercise. We have previously shown that supra-physiological expression of nNOS-binding mini-dystrophin restores normal blood flow regulation and prevents functional ischemia in transgenic mdx mice, a DMD model. A critical next issue is whether systemic dual adeno-associated virus (AAV) gene therapy can restore nNOS-binding mini-dystrophin expression and mitigate muscle activity-related functional ischemia and injury. Here, we performed systemic gene transfer in mdx and mdx4cv mice using a pair of dual AAV vectors that expressed a 6 kb nNOS-binding mini-dystrophin gene. Vectors were packaged in tyrosine mutant AAV-9 and co-injected (5 × 10(12) viral genome particles/vector/mouse) via the tail vein to 1-month-old dystrophin-null mice. Four months later, we observed 30-50% mini-dystrophin positive myofibers in limb muscles. Treatment ameliorated histopathology, increased muscle force and protected against eccentric contraction-induced injury. Importantly, dual AAV therapy successfully prevented chronic exercise-induced muscle force drop. Doppler hemodynamic assay further showed that therapy attenuated adrenergic vasoconstriction in contracting muscle. Our results suggest that partial transduction can still ameliorate nNOS delocalization-associated functional deficiency. Further evaluation of nNOS binding mini-dystrophin dual AAV vectors is warranted in dystrophic dogs and eventually in human patients.

  13. Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.

    Directory of Open Access Journals (Sweden)

    Ashley T Martino


    Full Text Available Hepatic gene transfer, in particular using adeno-associated viral (AAV vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+ T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer.These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

  14. Recombinant Adeno-Associated Virus Vector Genomes Take the Form of Long-Lived, Transcriptionally Competent Episomes in Human Muscle (United States)

    Schnepp, Bruce C.; Chulay, Jeffrey D.; Ye, Guo-Jie; Flotte, Terence R.; Trapnell, Bruce C.; Johnson, Philip R.


    Gene augmentation therapy as a strategy to treat alpha-1 antitrypsin (AAT) deficiency has reached phase 2 clinical testing in humans. Sustained serum levels of AAT have been observed beyond one year after intramuscular administration of a recombinant adeno-associated virus (rAAV) vector expressing the AAT gene. In this study, sequential muscle biopsies obtained at 3 and 12 months after vector injection were examined for the presence of rAAV vector genomes. Each biopsy sample contained readily detectable vector DNA, the majority of which existed as double-stranded supercoiled and open circular episomes. Episomes persisted through 12 months, although at slightly lower levels than observed at 3 months. There was a clear dose response when comparing the low- and mid-vector-dose groups to the high-dose group. The highest absolute copy numbers were found in a high-dose subject, and serum AAT levels at 12 months confirmed that the high-dose group also had the highest sustained serum AAT levels. Sequence analysis revealed that the vast majority of episomes contained double-D inverted terminal repeats ranging from fully intact to severely deleted. Molecular clones of vector genomes derived directly from the biopsies were transcriptionally active, potentially identifying them as the source of serum AAT in the trial subjects. PMID:26650966

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

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    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.

  16. AAV5-Factor VIII Gene Transfer in Severe Hemophilia A. (United States)

    Rangarajan, Savita; Walsh, Liron; Lester, Will; Perry, David; Madan, Bella; Laffan, Michael; Yu, Hua; Vettermann, Christian; Pierce, Glenn F; Wong, Wing Y; Pasi, K John


    Patients with hemophilia A rely on exogenous factor VIII to prevent bleeding in joints, soft tissue, and the central nervous system. Although successful gene transfer has been reported in patients with hemophilia B, the large size of the factor VIII coding region has precluded improved outcomes with gene therapy in patients with hemophilia A. We infused a single intravenous dose of a codon-optimized adeno-associated virus serotype 5 (AAV5) vector encoding a B-domain-deleted human factor VIII (AAV5-hFVIII-SQ) in nine men with severe hemophilia A. Participants were enrolled sequentially into one of three dose cohorts (low dose [one participant], intermediate dose [one participant], and high dose [seven participants]) and were followed through 52 weeks. Factor VIII activity levels remained at 3 IU or less per deciliter in the recipients of the low or intermediate dose. In the high-dose cohort, the factor VIII activity level was more than 5 IU per deciliter between weeks 2 and 9 after gene transfer in all seven participants, and the level in six participants increased to a normal value (>50 IU per deciliter) that was maintained at 1 year after receipt of the dose. In the high-dose cohort, the median annualized bleeding rate among participants who had previously received prophylactic therapy decreased from 16 events before the study to 1 event after gene transfer, and factor VIII use for participant-reported bleeding ceased in all the participants in this cohort by week 22. The primary adverse event was an elevation in the serum alanine aminotransferase level to 1.5 times the upper limit of the normal range or less. Progression of preexisting chronic arthropathy in one participant was the only serious adverse event. No neutralizing antibodies to factor VIII were detected. The infusion of AAV5-hFVIII-SQ was associated with the sustained normalization of factor VIII activity level over a period of 1 year in six of seven participants who received a high dose, with

  17. In vivo expression of human ATP:cob(I)alamin adenosyltransferase (ATR) using recombinant adeno-associated virus (rAAV) serotypes 2 and 8. (United States)

    Erger, Kirsten E; Conlon, Thomas J; Leal, Nicole A; Zori, Robert; Bobik, Thomas A; Flotte, Terence R


    Methylmalonic aciduria (MMA) is an autosomal recessive disease with symptoms that include ketoacidosis, lethargy, recurrent vomiting, dehydration, respiratory distress, muscular hypotonia and death due to methylmalonic acid levels that are up to 1000-fold greater than normal. CblB MMA, a subset of the mutations leading to MMA, is caused by a deficiency in the enzyme cob(I)alamin adenosyltransferase (ATR). No animal model currently exists for this disease. ATR functions within the mitochondria matrix in the final conversion of cobalamin into coenzyme B(12), adenosylcobalamin (AdoCbl). AdoCbl is a required coenzyme for the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). The human ATR cDNA was cloned into a recombinant adeno-associated virus (rAAV) vector and packaged into AAV 2 or 8 capsids and delivered by portal vein injection to C57/Bl6 mice at a dose of 1 x 10(10) and 1 x 10(11) particles. Eight weeks post-injection RNA, genomic DNA and protein were then extracted and analyzed. Using primer pairs specific to the cytomegalovirus (CMV) enhancer/chicken beta-actin (CBAT) promoter within the rAAV vectors, genome copy numbers were found to be 0.03, 2.03 and 0.10 per cell in liver for the rAAV8 low dose, rAAV8 high dose and rAAV2 high dose, respectively. Western blotting performed on mitochondrial protein extracts demonstrated protein levels were comparable to control levels in the rAAV8 low dose and rAAV2 high dose animals and 3- to 5-fold higher than control levels were observed in high dose animals. Immunostaining demonstrated enhanced transduction efficiency of hepatocytes to over 40% in the rAAV8 high dose animals, compared to 9% and 5% transduction in rAAV2 high dose and rAAV8 low dose animals, respectively. These data demonstrate the feasibility of efficient ATR gene transfer to the liver as a prelude to future gene therapy experiments.

  18. Immunological Monitoring to Rationally Guide AAV Gene Therapy

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    Cedrik Michael Britten


    Full Text Available Recent successes with adeno-associated virus (AAV-based gene therapies fuel the hope for new treatments for hereditary diseases. Pre-existing as well as therapy-induced immune responses against both AAV and the encoded transgenes have been described and may impact on safety and efficacy of gene-therapy approaches. Consequently, monitoring of vector- and transgene-specific immunity is mandated and may rationally guide clinical development. Next to the humoral immune response, the cellular response is central in our understanding of the host reaction in gene therapy. But in contrast to the monitoring of antibodies, which has matured over many decades, sensitive and robust monitoring of T cells is a relatively new development. To make cellular immune assessments fit for purpose, investigators need to know, control and report the critical assay variables that influence the results. In addition, the quality of immune assays needs to be continuously adjusted to allow for exploratory hypothesis generation in early stages and confirmatory hypothesis validation in later stages of clinical development. The concept of immune assay harmonization which includes use of field-wide benchmarks, harmonization guidelines, and external quality control can support the context-specific evolution of immune assays. Multi-center studies pose particular challenges to sample logistics and quality control of sample specimens. Cooperative groups need to define if immune assessments should be performed in one central facility, in peripheral labs or including a combination of both. Finally, engineered reference samples that contain a defined number of antigen-specific T cells may become broadly applicable tools to control assay performance over time or across institutions.

  19. An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models

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    Catherine Gérard


    Full Text Available Friedreich ataxia (FRDA is a genetic disease due to increased repeats of the GAA trinucleotide in intron 1 of the frataxin gene. This mutation leads to a reduced expression of frataxin. We have produced an adeno-associated virus (AAV9 coding for human frataxin (AAV9-hFXN. This AAV was delivered by intraperitoneal (IP injection to young conditionally knockout mice in which the frataxin gene had been knocked-out in some tissues during embryogenesis by breeding them with mice expressing the Cre recombinase gene under the muscle creatine kinase (MCK or the neuron-specific enolase (NSE promoter. In the first part of the study, different doses of virus were tested from 6 × 1011 v.p. to 6 × 109 v.p. in NSE-cre mice and all leading to an increase in life spent of the mice. The higher and the lower dose were also tested in MCK-cre mice. A single administration of the AAV9-hFXN at 6 × 1011 v.p. more than doubled the life of these mice. In fact the MCK-cre mice treated with the AAV9-hFXN were sacrificed for further molecular investigations at the age of 29 weeks without apparent symptoms. Echography analysis of the heart function clearly indicated that the cardiac systolic function was better preserved in the mice that received 6 × 1011 v.p. of AAV9-hFXN. The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models. Thus, gene therapy with an AAV9-hFXN is a potential treatment of FRDA.

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

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    Bankiewicz, Krystof; Cunningham, Janet


    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.

  1. Intrathecal administration of IGF-I by AAVrh10 improves sensory and motor deficits in a mouse model of diabetic neuropathy

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    Judit Homs


    Full Text Available Different adeno-associated virus (AAV serotypes efficiently transduce neurons from central and peripheral nervous systems through various administration routes. Direct administration of the vectors to the cerebrospinal fluid (CSF could be an efficient and safe strategy. Here, we show that lumbar puncture of a nonhuman AAV leads to wide and stable distribution of the vector along the spinal cord in adult mice. AAVrh10 efficiently and specifically infects neurons, both in dorsal root ganglia (60% total sensory neurons and in the spinal cord (up to one-third of α-motor neurons. As a proof of concept, we demonstrate the efficacy of AAVrh10 in a mouse model of diabetic neuropathy, in which intrathecal delivery of the vector coding for insulin-like growth factor (IGF-I favored the release of the therapeutic protein into the CSF through its expression by sensory and motor neurons. IGF-I–treated diabetic animals showed increased vascular endothelial growth factor expression, activation of Akt/PI3K pathway, and stimulated nerve regeneration and myelination in injured limbs. Moreover, we achieved restoration of nerve conduction velocities in both sensory and motor nerves by AAVrh10, whereas we reached only sensory nerve improvement with AAV1. Our results indicate that intrathecal injection of AAVrh10 is a promising tool to design gene therapy approaches for sensorimotor diseases.

  2. Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals (United States)

    Gompf, Heinrich S.; Budygin, Evgeny A.; Fuller, Patrick M.; Bass, Caroline E.


    Techniques to genetically manipulate the activity of defined neuronal subpopulations have been useful in elucidating function, however applicability to translational research beyond transgenic mice is limited. Subtype targeted transgene expression can be achieved using specific promoters, but often currently available promoters are either too large to package into many vectors, in particular adeno-associated virus (AAV), or do not drive expression at levels sufficient to alter behavior. To permit neuron subtype specific gene expression in wildtype animals, we developed a combinatorial AAV targeting system that drives, in combination, subtype specific Cre-recombinase expression with a strong but non-specific Cre-conditional transgene. Using this system we demonstrate that the tyrosine hydroxylase promoter (TH-Cre-AAV) restricted expression of channelrhodopsin-2 (EF1α-DIO-ChR2-EYFP-AAV) to the rat ventral tegmental area (VTA), or an activating DREADD (hSyn-DIO-hM3Dq-mCherry-AAV) to  the  rat  locus  coeruleus  (LC). High expression levels were achieved in both regions. Immunohistochemistry (IHC) showed the majority of ChR2+ neurons (>93%) colocalized with TH in the VTA, and optical stimulation evoked striatal dopamine release. Activation of TH neurons in the LC produced sustained EEG and behavioral arousal. TH-specific hM3Dq expression in the LC was further compared with: (1) a Cre construct driven by a strong but non-specific promoter (non-targeting); and (2) a retrogradely-transported WGA-Cre delivery mechanism (targeting a specific projection). IHC revealed that the area of c-fos activation after CNO treatment in the LC and peri-LC neurons appeared proportional to the resulting increase in wakefulness (non-targeted > targeted > ACC to LC projection restricted). Our dual AAV targeting system effectively overcomes the large size and weak activity barrier prevalent with many subtype specific promoters by functionally separating subtype specificity from

  3. Heparan Sulfate Binding Promotes Accumulation of Intravitreally Delivered Adeno-associated Viral Vectors at the Retina for Enhanced Transduction but Weakly Influences Tropism. (United States)

    Woodard, Kenton T; Liang, Katharine J; Bennett, William C; Samulski, R Jude


    Many adeno-associated virus (AAV) serotypes efficiently transduce the retina when delivered to the subretinal space but show limited success when delivered to the vitreous due to the inner limiting membrane (ILM). Subretinal delivery of AAV serotype 2 (AAV2) and its heparan sulfate (HS)-binding-deficient capsid led to similar expression, indicating transduction of the outer retina occurred by HS-independent mechanisms. However, intravitreal delivery of HS-ablated recombinant AAV2 (rAAV2) led to a 300-fold decrease in transduction compared to AAV2. Fluorescence in situ hybridization of AAV transgenes was used to identify differences in retinal trafficking and revealed that HS binding was responsible for AAV2 accumulation at the ILM. This mechanism was tested on human ex vivo retinas and showed similar accumulation with HS-binding AAV2 capsid only. To evaluate if HS binding could be applied to other AAV serotypes to enhance their transduction, AAV1 and AAV8 were modified to bind HS with a single-amino-acid mutation and tested in mice. Both HS-binding mutants of AAV1 and AAV8 had higher intravitreal transduction than their non-HS-binding parent capsid due to increased retinal accumulation. To understand the influence that HS binding has on tropism, chimeric AAV2 capsids with dual-glycan usage were tested intravitreally in mice. Compared to HS binding alone, these chimeric capsids displayed enhanced transduction that was correlated with a change in tropism. Taken together, these data indicate that HS binding serves to sequester AAV capsids from the vitreous to the ILM but does not influence retinal tropism. The enhanced retinal transduction of HS-binding capsids provides a rational design strategy for engineering capsids for intravitreal delivery. Adeno-associated virus (AAV) has become the vector of choice for viral gene transfer and has shown great promise in clinical trials. The need for development of an easy, less invasive injection route for ocular gene therapy

  4. A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling.

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    Estanislao Nistal-Villan

    Full Text Available Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV. In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.

  5. Ab-externo AAV-mediated gene delivery to the suprachoroidal space using a 250 micron flexible microcatheter.

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    Marc C Peden


    Full Text Available The current method of delivering gene replacement to the posterior segment of the eye involves a three-port pars plana vitrectomy followed by injection of the agent through a 37-gauge cannula, which is potentially wrought with retinal complications. In this paper we investigate the safety and efficacy of delivering adeno-associated viral (AAV vector to the suprachoroidal space using an ab externo approach that utilizes an illuminated microcatheter.6 New Zealand White rabbits and 2 Dutch Belted rabbits were used to evaluate the ab externo delivery method. sc-AAV5-smCBA-hGFP vector was delivered into the suprachoroidal space using an illuminated iTrackTM 250A microcatheter. Six weeks after surgery, the rabbits were sacrificed and their eyes evaluated for AAV transfection using immunofluorescent antibody staining of GFP.Immunostaining of sectioned and whole-mounted eyes demonstrated robust transfection in all treated eyes, with no fluorescence in untreated control eyes. Transfection occurred diffusely and involved both the choroid and the retina. No apparent adverse effects caused by either the viral vector or the procedure itself could be seen either clinically or histologically.The ab externo method of delivery using a microcatheter was successful in safely and effectively delivering a gene therapy agent to the suprachoroidal space. This method presents a less invasive alternative to the current method of virally vectored gene delivery.

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

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    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: [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)


    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.

  7. Biomarkers for disease progression and AAV therapeutic efficacy in feline Sandhoff disease. (United States)

    Bradbury, Allison M; Gray-Edwards, Heather L; Shirley, Jamie L; McCurdy, Victoria J; Colaco, Alexandria N; Randle, Ashley N; Christopherson, Pete W; Bird, Allison C; Johnson, Aime K; Wilson, Diane U; Hudson, Judith A; De Pompa, Nicholas L; Sorjonen, Donald C; Brunson, Brandon L; Jeyakumar, Mylvaganam; Platt, Frances M; Baker, Henry J; Cox, Nancy R; Sena-Esteves, Miguel; Martin, Douglas R


    The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme β-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were largely normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated SD patients and those in future clinical trials. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Neoliberal and Neoconservative Vectors in the Administration of the Public School: policies and practices

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    Fernanda Martins


    Full Text Available With this article, we intend to approach the redefinition of the public school administration as a structural element in the mandates of the educational policies during the 80s and 90s in one central countrs, England. The selection of this context is due to the fact that this country have started shifts in the educational policies, namely regarding school administration. Moreover, this choice is also justified by the central position that this one country occupy. Thus, the policies regarding public schools administration in central countries become a point of reference to the analysis of the educational policies of countries that have a semi-periphery position, such as Portugal (cf.Afonso, 1998 and Sá, 2004. Besides this analysis to the policies, it is also intended to bring into the discussion the results of the already made investigations in this country which allow us to ponder on the impact of those new policies in the public schools/school actors. Underlying this option, is the supposed idea that in the educational policies analysis it is crucial to integrate the micro-political and the neopolitical dimensions, in order to break the “constant condescending look”, which doesn’t question the big political and legislative (central decisions, bearing in mind its rippling effect on the several management units (periphery (cf. Lima & Afonso, 1992:11.

  9. Treatment of experimental glioma by administration of adenoviral vectors expressing Fas ligand. (United States)

    Ambar, B B; Frei, K; Malipiero, U; Morelli, A E; Castro, M G; Lowenstein, P R; Fontana, A


    Fas ligand (FasL) is a cytokine, produced by activated T cells and NK cells, that triggers apoptosis of Fas-positive target cells including human glioma cells. As shown here, in vitro infection of rat F98 and human LN18 glioma cell lines with recombinant adenovirus (rAd) expressing FasL cDNA under control of the cytomegalovirus promoter (rAd-CMV-FasL) induced striking cytotoxicity in Fas-positive glioma cell lines but not in the Fas-negative F98 glioma subline F98/ZH. The extent of FasL-mediated cytotoxic effects outranged the expectations based on expression of beta-galactosidase (beta-Gal) by F98 cells infected with a control virus expressing the lacZ gene (rAd-CMV-lacZ). The detection of FasL bioactivity in supernatants of infected cells provides evidence of a bystander mechanism involving the cytotoxic action of FasL on uninfected cells. In F98 tumor-bearing rats, infection with rAd-CMV-FasL increased the mean survival time by 50% compared with infection with rAd-CMV-lacZ or untreated controls. These data suggest that viral vector transduction of the FasL gene could be part of a successful glioma gene therapy.

  10. A novel and highly efficient production system for recombinant adeno-associated virus vector. (United States)

    Wu, Zhijian; Wu, Xiaobing; Cao, Hui; Dong, Xiaoyan; Wang, Hong; Hou, Yunde


    Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/DeltaUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/DeltaUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28x10(4) particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.

  11. Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. (United States)

    Acland, Gregory M; Aguirre, Gustavo D; Bennett, Jean; Aleman, Tomas S; Cideciyan, Artur V; Bennicelli, Jeannette; Dejneka, Nadine S; Pearce-Kelling, Susan E; Maguire, Albert M; Palczewski, Krzysztof; Hauswirth, William W; Jacobson, Samuel G


    The short- and long-term effects of gene therapy using AAV-mediated RPE65 transfer to canine retinal pigment epithelium were investigated in dogs affected with disease caused by RPE65 deficiency. Results with AAV 2/2, 2/1, and 2/5 vector pseudotypes, human or canine RPE65 cDNA, and constitutive or tissue-specific promoters were similar. Subretinally administered vectors restored retinal function in 23 of 26 eyes, but intravitreal injections consistently did not. Photoreceptoral and postreceptoral function in both rod and cone systems improved with therapy. In dogs followed electroretinographically for 3 years, responses remained stable. Biochemical analysis of retinal retinoids indicates that mutant dogs have no detectable 11-cis-retinal, but markedly elevated retinyl esters. Subretinal AAV-RPE65 treatment resulted in detectable 11-cis-retinal expression, limited to treated areas. RPE65 protein expression was limited to retinal pigment epithelium of treated areas. Subretinal AAV-RPE65 vector is well tolerated and does not elicit high antibody levels to the vector or the protein in ocular fluids or serum. In long-term studies, wild-type cDNA is expressed only in target cells. Successful, stable restoration of rod and cone photoreceptor function in these dogs has important implications for treatment of human patients affected with Leber congenital amaurosis caused by RPE65 mutations.

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

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    Rajvinder eKarda


    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.

  13. AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model. (United States)

    Mätlik, Kert; Abo-Ramadan, Usama; Harvey, Brandon K; Arumäe, Urmas; Airavaara, Mikko


    For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Viral and Cellular Components of AAV2 Replication Compartments (United States)

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel


    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  15. Intracranial injection of AAV expressing NEP but not IDE reduces amyloid pathology in APP+PS1 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Nikisha Carty

    Full Text Available The accumulation of β-amyloid peptides in the brain has been recognized as an essential factor in Alzheimer's disease pathology. Several proteases, including Neprilysin (NEP, endothelin converting enzyme (ECE, and insulin degrading enzyme (IDE, have been shown to cleave β-amyloid peptides (Aβ. We have previously reported reductions in amyloid in APP+PS1 mice with increased expression of ECE. In this study we compared the vector-induced increased expression of NEP and IDE. We used recombinant adeno-associated viral vectors expressing either native forms of NEP (NEP-n or IDE (IDE-n, or engineered secreted forms of NEP (NEP-s or IDE (IDE-s. In a six-week study, immunohistochemistry staining for total Aβ was significantly decreased in animals receiving the NEP-n and NEP-s but not for IDE-n or IDE-s in either the hippocampus or cortex. Congo red staining followed a similar trend revealing significant decreases in the hippocampus and the cortex for NEP-n and NEP-s treatment groups. Our results indicate that while rAAV-IDE does not have the same therapeutic potential as rAAV-NEP, rAAV-NEP-s and NEP-n are effective at reducing amyloid loads, and both of these vectors continue to have significant effects nine months post-injection. As such, they may be considered reasonable candidates for gene therapy trials in AD.

  16. Sexually dimorphic patterns of episomal rAAV genome persistence in the adult mouse liver and correlation with hepatocellular proliferation. (United States)

    Dane, Allison P; Cunningham, Sharon C; Graf, Nicole S; Alexander, Ian E


    Recombinant adeno-associated virus vectors (rAAVs) show exceptional promise for liver-targeted gene therapy, with phenotype correction in small and large animal disease models being reported with increasing frequency. Success in humans, however, remains a considerable challenge that demands greater understanding of host-vector interactions, notably those governing the efficiency of initial gene transfer and subsequent long-term persistence of gene expression. In this study, we examined long-term enhanced green fluorescent protein (eGFP) expression and vector genome persistence in the mouse liver after rAAV2/8-mediated gene transfer in early adulthood. Two intriguing findings emerged of considerable scientific and clinical interest. First, adult female and male mice showed distinctly different patterns of persistence of eGFP expression across the hepatic lobule after exhibiting similar patterns initially. Female mice retained a predominantly perivenous pattern of expression, whereas male mice underwent inversion of this pattern with preferential loss of perivenous expression and relative retention of periportal expression. Second, these changing patterns of expression correlated with sexually dimorphic patterns of genome persistence that appear linked both spatially and temporally to underlying hepatocellular proliferation. Observation of the equivalent phenomenon in man could have significant implications for the long-term therapeutic efficacy of rAAV-mediated gene transfer, particularly in the context of correction of liver functions showing metabolic zonation.

  17. Preclinical Dose-Escalation Study of Intravitreal AAV-RS1 Gene Therapy in a Mouse Model of X-linked Retinoschisis: Dose-Dependent Expression and Improved Retinal Structure and Function. (United States)

    Bush, Ronald A; Zeng, Yong; Colosi, Peter; Kjellstrom, Sten; Hiriyanna, Suja; Vijayasarathy, Camasamudram; Santos, Maria; Li, Jinbo; Wu, Zhijian; Sieving, Paul A


    Gene therapy for inherited retinal diseases has been shown to ameliorate functional and structural defects in both animal models and in human clinical trials. X-linked retinoschisis (XLRS) is an early-age onset macular dystrophy resulting from loss of an extracellular matrix protein (RS1). In preparation for a human clinical gene therapy trial, we conducted a dose-range efficacy study of the clinical vector, a self-complementary AAV delivering a human retinoschisin (RS1) gene under control of the RS1 promoter and an interphotoreceptor binding protein enhancer (AAV8-scRS/IRBPhRS), in the retinoschisin knockout (Rs1-KO) mouse. The therapeutic vector at 1 × 10(6) to 2.5 × 10(9) (1E6-2.5E9) vector genomes (vg)/eye or vehicle was administered to one eye of 229 male Rs1-KO mice by intravitreal injection at 22 ± 3 days postnatal age (PN). Analysis of retinal function (dark-adapted electroretinogram, ERG), structure (cavities and outer nuclear layer thickness) by in vivo retinal imaging using optical coherence tomography, and retinal immunohistochemistry (IHC) for RS1 was done 3-4 months and/or 6-9 months postinjection (PI). RS1 IHC staining was dose dependent across doses ≥1E7 vg/eye, and the threshold for significant improvement in all measures of retinal structure and function was 1E8 vg/eye. Higher doses, however, did not produce additional improvement. At all doses showing efficacy, RS1 staining in Rs1-KO mouse was less than that in wild-type mice. Improvement in the ERG and RS1 staining was unchanged or greater at 6-9 months than at 3-4 months PI. This study demonstrates that vitreal administration of AAV8 scRS/IRBPhRS produces significant improvement in retinal structure and function in the mouse model of XLRS over a vector dose range that can be extended to a human trial. It indicates that a fully normal level of RS1 expression is not necessary for a therapeutic effect.

  18. Efficient Transduction of Vascular Endothelial Cells with Recombinant Adeno-Associated Virus Serotype 1 and 5 Vectors (United States)



    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 < 0.001) at 7 days posttransduction. Interestingly, expression was increased in cells transduced with rAAV5 to levels surpassing rAAV1 by day 14 and 21. 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. 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

  19. Chromosomal position effects on AAV-mediated gene targeting. (United States)

    Cornea, Anda M; Russell, David W


    The effects of chromosomal position and neighboring genomic elements on gene targeting in human cells remain largely unexplored. To study these, we used a shuttle vector system in which murine leukemia virus (MLV)-based proviral targets present at different chromosomal locations and containing mutations in the neomycin phosphotransferase (neo) gene were corrected by adeno-associated virus (AAV)-mediated gene targeting. Sixteen identical target loci present in HT-1080 human sarcoma cells were all successfully corrected by gene targeting. The gene targeting frequencies varied by as much as 10-fold, and there was a clear bias for correction of one of the targets in clones containing two target sites. The targeting frequency at each site was correlated to the proximity and density of various genomic elements, and we found a significant association of higher targeting frequencies at loci near a subset of dinucleotide microsatellite repeats (r = -0.55, P targeting frequencies at the target loci (r = 0.52, P targeting frequencies. Our results indicate that certain chromosomal positions are preferred sites for gene targeting in human cells.

  20. Systemic Errors in Quantitative Polymerase Chain Reaction Titration of Self-Complementary Adeno-Associated Viral Vectors and Improved Alternative Methods (United States)

    Fagone, Paolo; Wright, J. Fraser; Nathwani, Amit C.; Nienhuis, Arthur W.; Davidoff, Andrew M.


    Abstract Self-complementary AAV (scAAV) vector genomes contain a covalently closed hairpin derived from a mutated inverted terminal repeat that connects the two monomer single-stranded genomes into a head-to-head or tail-to-tail dimer. We found that during quantitative PCR (qPCR) this structure inhibits the amplification of proximal amplicons and causes the systemic underreporting of copy number by as much as 10-fold. We show that cleavage of scAAV vector genomes with restriction endonuclease to liberate amplicons from the covalently closed terminal hairpin restores quantitative amplification, and we implement this procedure in a simple, modified qPCR titration method for scAAV vectors. In addition, we developed and present an AAV genome titration procedure based on gel electrophoresis that requires minimal sample processing and has low interassay variability, and as such is well suited for the rigorous quality control demands of clinical vector production facilities. PMID:22428975

  1. Carbidopa-based modulation of the functional effect of the AAV2-hAADC gene therapy in 6-OHDA lesioned rats.

    Directory of Open Access Journals (Sweden)

    Agnieszka Ciesielska

    Full Text Available Progressively blunted response to L-DOPA in Parkinson's disease (PD is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC. We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the

  2. Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Aβ levels

    Directory of Open Access Journals (Sweden)

    Dicker Bridget L


    Full Text Available Abstract Background Alzheimer's disease (AD is characterized by a decline in cognitive function and accumulation of amyloid-β peptide (Aβ in extracellular plaques. Mutations in amyloid precursor protein (APP and presenilins alter APP metabolism resulting in accumulation of Aβ42, a peptide essential for the formation of amyloid deposits and proposed to initiate the cascade leading to AD. However, the role of Aβ40, the more prevalent Aβ peptide secreted by cells and a major component of cerebral Aβ deposits, is less clear. In this study, virally-mediated gene transfer was used to selectively increase hippocampal levels of human Aβ42 and Aβ40 in adult Wistar rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. Results Adeno-associated viral (AAV vectors encoding BRI-Aβ cDNAs were generated resulting in high-level hippocampal expression and secretion of the specific encoded Aβ peptide. As a comparison the effect of AAV-mediated overexpression of APPsw was also examined. Animals were tested for development of learning and memory deficits (open field, Morris water maze, passive avoidance, novel object recognition three months after infusion of AAV. A range of impairments was found, with the most pronounced deficits observed in animals co-injected with both AAV-BRI-Aβ40 and AAV-BRI-Aβ42. Brain tissue was analyzed by ELISA and immunohistochemistry to quantify levels of detergent soluble and insoluble Aβ peptides. BRI-Aβ42 and the combination of BRI-Aβ40+42 overexpression resulted in elevated levels of detergent-insoluble Aβ. No significant increase in detergent-insoluble Aβ was seen in the rats expressing APPsw or BRI-Aβ40. No pathological features were noted in any rats, except the AAV-BRI-Aβ42 rats which showed focal, amorphous, Thioflavin-negative Aβ42 deposits. Conclusion The results show that AAV-mediated gene transfer is a valuable tool to model aspects of AD pathology in

  3. E Pluribus Unum: 50 Years of Research, Millions of Viruses, and One Goal--Tailored Acceleration of AAV Evolution. (United States)

    Grimm, Dirk; Zolotukhin, Sergei


    Fifty years ago, a Science paper by Atchison et al. reported a newly discovered virus that would soon become known as adeno-associated virus (AAV) and that would subsequently emerge as one of the most versatile and most auspicious vectors for human gene therapy. A large part of its attraction stems from the ease with which the viral capsid can be engineered for particle retargeting to cell types of choice, evasion from neutralizing antibodies or other desirable properties. Particularly powerful and in the focus of the current review are high-throughput methods aimed at expanding the repertoire of AAV vectors by means of directed molecular evolution, such as random mutagenesis, DNA family shuffling, in silico reconstruction of ancestral capsids, or peptide display. Here, unlike the wealth of prior reviews on this topic, we especially emphasize and critically discuss the practical aspects of the different procedures that affect the ultimate outcome, including diversification protocols, combinatorial library complexity, and selection strategies. Our overall aim is to provide general guidance that should help users at any level, from novice to expert, to safely navigate through the rugged space of directed AAV evolution while avoiding the pitfalls that are associated with these challenging but promising technologies.

  4. Retro-Orbital Venous Sinus Delivery of rAAV9 Mediates High-Level Transduction of Brain and Retina Compared with Temporal Vein Delivery in Neonatal Mouse Pups. (United States)

    Gruntman, Alisha M; Su, Lin; Flotte, Terence R


    In order to pursue a clinical gene therapy for a human neurologic disease, it is often necessary to perform proof-of-concept trials in mouse models of that disease. In order to demonstrate a potential clinical efficacy, one must be able to select an appropriate vector and route of delivery for the appropriate age group in the disease model. Since many diseases require correction early in life, investigators often need to deliver recombinant adeno-associated viral (rAAV) vectors to neonatal mice. Herein, general central nervous system expression patterns of nuclear GFP following delivery of rAAV by three different routes are reported.

  5. 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


    -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...

  6. Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. (United States)

    Keeler, Allison M; Conlon, Thomas; Walter, Glenn; Zeng, Huadong; Shaffer, Scott A; Dungtao, Fu; Erger, Kirsten; Cossette, Travis; Tang, Qiushi; Mueller, Christian; Flotte, Terence R


    Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 10(12) vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD(-/-) mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD(-/-) mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD(-/-) mice maintained euglycemia, whereas untreated VLCAD(-/-) mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

  7. 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.


    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.

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


    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

  9. Administration

    DEFF Research Database (Denmark)

    Bogen handler om den praksis, vi kalder administration. Vi er i den offentlige sektor i Danmark hos kontorfolkene med deres sagsmapper, computere, telefoner,, lovsamlinger,, retningslinier og regneark. I bogen udfoldes en mangfoldighed af konkrete historier om det administrative arbejde fra...... forskellige områder i den offentlige sektor. Hensigten er at forstå den praksis og faglighed der knytter sig til det administrative arbejde...

  10. Comparison of the efficacy of four viral vectors for transducing hypothalamic magnocellular neurosecretory neurons in the rat supraoptic nucleus. (United States)

    Doherty, Faye C; Schaack, Jerome B; Sladek, Celia D


    Since transgenes were first cloned into recombinant adenoviruses almost 30 years ago, a variety of viral vectors have become important tools in genetic research. Viruses adeptly transport genetic material into eukaryotic cells, and replacing all or part of the viral genome with genes of interest or silencing sequences creates a method of gene expression modulation in which the timing and location of manipulations can be specific. The hypothalamo-neurohypophyseal system (HNS), consisting of the paraventricular (PVN) and supraoptic (SON) nuclei in the hypothalamus, regulates fluid balance homeostasis and is highly plastic, yet tightly regulated by extracellular fluid (ECF) osmolality and volume. Its reversible plasticity and physiological relevance make it a good system for studying interactions between gene expression and physiology. Here, four viral vectors were compared for their ability to transduce magnocellular neurosecretory neurons (MNCs) of the SON in adult rats. The vectors included an adenovirus, a lentivirus (HIV) and two serotypes of adeno-associated viruses (AAV5 and AAV2). Though adenovirus and AAV2 vectors have previously been used to transduce SON neurons, HIV and AAV5 have not. All four vectors transduced MNCs, but the AAV vectors were the most effective, transducing large numbers of MNCs, with minimal or no glial transduction. The AAV vectors were injected using a convection enhanced delivery protocol to maximize dispersal through the tissue, resulting in the transduction of neurons throughout the anterior to posterior length of the SON (∼1.5mm). AAV5, but not AAV2, showed some selectivity for SON neurons relative to those in the surrounding hypothalamus. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. DNA structure modulates the oligomerization properties of the AAV initiator protein Rep68.

    Directory of Open Access Journals (Sweden)

    Jorge Mansilla-Soto


    Full Text Available Rep68 is a multifunctional protein of the adeno-associated virus (AAV, a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3 helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag and bovine papillomavirus (BPV E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID and an AAA(+ motor domain. The AAA(+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA(+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration.

  12. AAV-mediated Sirt1 overexpression in skeletal muscle activates oxidative capacity but does not prevent insulin resistance

    Directory of Open Access Journals (Sweden)

    Laia Vil


    Full Text Available Type 2 diabetes is characterized by triglyceride accumulation and reduced lipid oxidation capacity in skeletal muscle. SIRT1 is a key protein in the regulation of lipid oxidation and its expression is reduced in the skeletal muscle of insulin resistant mice. In this tissue, Sirt1 up-regulates the expression of genes involved in oxidative metabolism and improves mitochondrial function mainly through PPARGC1 deacetylation. Here we examined whether Sirt1 overexpression mediated by adeno-associated viral vectors of serotype 1 (AAV1 specifically in skeletal muscle can counteract the development of insulin resistance induced by a high fat diet in mice. AAV1-Sirt1-treated mice showed up-regulated expression of key genes related to β-oxidation together with increased levels of phosphorylated AMP protein kinase. Moreover, SIRT1 overexpression in skeletal muscle also increased basal phosphorylated levels of AKT. However, AAV1-Sirt1 treatment was not enough to prevent high fat diet-induced obesity and insulin resistance. Although Sirt1 gene transfer to skeletal muscle induced changes at the muscular level related with lipid and glucose homeostasis, our data indicate that overexpression of SIRT1 in skeletal muscle is not enough to improve whole-body insulin resistance and that suggests that SIRT1 has to be increased in other metabolic tissues to prevent insulin resistance.

  13. Administration



    Cet imposant volume constitue un registre des cours magistraux tenus par l’auteur à l’École supérieure allemande des sciences administratives de Spire, enrichis des résultats de travaux scientifiques menés principalement à l'Institut Allemand de Recherche en Administration Publique (Deutsches Forschungsinstitut für öffentliche Verwaltung Speyer, FÖV). Il s’agit donc d’une entreprise au long cours, destinée à apporter un nouvel éclairage (quasi ?) exhaustif sur l’administration publique : son ...

  14. Adeno-associated viral vectors as agents for gene delivery : application in disorders and trauma of the central nervous system

    NARCIS (Netherlands)

    Ruitenberg, Marc J; Eggers, Ruben; Boer, Gerard J; Verhaagen, J.


    The use of viral vectors as agents for gene delivery provides a direct approach to manipulate gene expression in the mammalian central nervous system (CNS). The present article describes in detail the methodology for the injection of viral vectors, in particular adeno-associated virus (AAV) vectors,

  15. A gene therapy of experimental parkinsonism monkeys with intra-striatums implantation of AAV-TH with CT guiding

    International Nuclear Information System (INIS)

    Wang Wei; Mao Jun; Yu Xiaoping; Liu Sheng; Hu Weixin; Zeng Zhaojun; Cai Weijun; Wu Xiaobing


    Objective: To observe the transfection ability to neuron and the therapy effect of adeno-associated virus vector (AAV) mediated intracerebral expressing of human tyrosine hydroxylase (TH) in the Rhesus monkey of Parkinson disease with CT guiding. Methods: Six Rhesus monkeys were induced into hemi-parkinsonism models by infusion of MPTP into right internal carotid artery. Under the guidance of CT and stereotactic apparatus, the recombinant adeno-associated virus vectors-human tyrosine hydroxylase were injected into the right caudate nucleus of 5 PD monkeys. The motor ability of the PD model was evaluated for 6 months after implantation. The content of DA, DOPAC, and HVA in the caudate nucleus was assayed with HPLC, and the expression of the tyrosine hydroxylase gene was detected with immunohistochemistry and RT-PCR. Results: The method of stereotactic puncture guided with CT had the features of real-time operating, fine location, and mini-trauma. The motor ability of all five models was all ameliorated after stereotactical injection of AAV-hTH vectors from two weeks up to six months. One PD monkey did not show any twist in response to apomorphine test. The frequency of twirl in the other 4 PD monkeys in response to apomorphine test was decreased by 42%-70% compared with that preoperatively. The contents of DA and DA metabolites in the lesion caudate nucleus were increased. Many TH positive cells in the implantation sites of caudate nucleus were shown by immunohistochemistry, which was different from the contralateral caudate nucleus. TH-mRNA was found in the treated side of caudate nucleus with RT-PCR, but no positive detection in the untreated side of caudate nucleus or in other sites in the brain. The negative results of RT-PCR were also shown in samples of the heart, liver, kidney tissue, or right side caudate nucleus of the uninjected monkey. Conclusion: Under the guidance of CT and stereotactic apparatus, the AAV-hTH vector can be injected accurately into the

  16. 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


    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....... Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...... delivery to the neonatal rat and minipig striatum. The efficiency of GFP expression and the phenotype of GFP-positive cells were assessed within the forebrain at different time points up to 12 months after surgery. Both rAAV1-GFP and rAAV5-GFP delivery resulted in transduction of the striatum as well...

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


    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

  18. Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults. (United States)

    Brantly, Mark L; Spencer, L Terry; Humphries, Margaret; Conlon, Thomas J; Spencer, Carolyn T; Poirier, Amy; Garlington, Wendy; Baker, Dawn; Song, Sihong; Berns, Kenneth I; Muzyczka, Nicholas; Snyder, Richard O; Byrne, Barry J; Flotte, Terence R


    A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha1-antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging from 2.1 x 10(12) vector genomes (VG) to 6.9 x 10(13) VG, with three subjects per cohort. Subjects were injected sequentially in a dose-escalating fashion with a minimum of 14 days between patients. Subjects who had been receiving AAT protein replacement discontinued that therapy 28 days before vector administration. There were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were detected in the blood between 1 and 3 days after injection in nearly all patients receiving doses of 6.9 x 10(12) VG or higher. Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune responses were detected. One subject who had not been receiving protein replacement exhibited low-level expression of wild-type M-AAT in the serum (82 nM), which was detectable 30 days after receiving an injection of 2.1 x 10(13) VG. Unfortunately, residual but declining M-AAT levels from the washout of the protein replacement elevated background levels sufficiently to obscure any possible vector expression in that range in most of the other individuals in the higher dose cohorts.

  19. Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas. (United States)

    Nault, Jean-Charles; Datta, Shalini; Imbeaud, Sandrine; Franconi, Andrea; Mallet, Maxime; Couchy, Gabrielle; Letouzé, Eric; Pilati, Camilla; Verret, Benjamin; Blanc, Jean-Frédéric; Balabaud, Charles; Calderaro, Julien; Laurent, Alexis; Letexier, Mélanie; Bioulac-Sage, Paulette; Calvo, Fabien; Zucman-Rossi, Jessica


    Hepatocellular carcinomas (HCCs) are liver tumors related to various etiologies, including alcohol intake and infection with hepatitis B (HBV) or C (HCV) virus. Additional risk factors remain to be identified, particularly in patients who develop HCC without cirrhosis. We found clonal integration of adeno-associated virus type 2 (AAV2) in 11 of 193 HCCs. These AAV2 integrations occurred in known cancer driver genes, namely CCNA2 (cyclin A2; four cases), TERT (telomerase reverse transcriptase; one case), CCNE1 (cyclin E1; three cases), TNFSF10 (tumor necrosis factor superfamily member 10; two cases) and KMT2B (lysine-specific methyltransferase 2B; one case), leading to overexpression of the target genes. Tumors with viral integration mainly developed in non-cirrhotic liver (9 of 11 cases) and without known risk factors (6 of 11 cases), suggesting a pathogenic role for AAV2 in these patients. In conclusion, AAV2 is a DNA virus associated with oncogenic insertional mutagenesis in human HCC.

  20. Improved survival and reduced phenotypic severity following AAV9/MECP2 gene transfer to neonatal and juvenile male Mecp2 knockout mice. (United States)

    Gadalla, Kamal K E; Bailey, Mark E S; Spike, Rosemary C; Ross, Paul D; Woodard, Kenton T; Kalburgi, Sahana Nagabhushan; Bachaboina, Lavanya; Deng, Jie V; West, Anne E; Samulski, R Jude; Gray, Steven J; Cobb, Stuart R


    Typical Rett syndrome (RTT) is a pediatric disorder caused by loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. The demonstrated reversibility of RTT-like phenotypes in mice suggests that MECP2 gene replacement is a potential therapeutic option in patients. We report improvements in survival and phenotypic severity in Mecp2-null male mice after neonatal intracranial delivery of a single-stranded (ss) AAV9/chicken β-actin (CBA)-MECP2 vector. Median survival was 16.6 weeks for MECP2-treated versus 9.3 weeks for green fluorescent protein (GFP)-treated mice. ssAAV9/CBA-MECP2-treated mice also showed significant improvement in the phenotype severity score, in locomotor function, and in exploratory activity, as well as a normalization of neuronal nuclear volume in transduced cells. Wild-type (WT) mice receiving neonatal injections of the same ssAAV9/CBA-MECP2 vector did not show any significant deficits, suggesting a tolerance for modest MeCP2 overexpression. To test a MECP2 gene replacement approach in a manner more relevant for human translation, a self-complementary (sc) adeno-associated virus (AAV) vector designed to drive MeCP2 expression from a fragment of the Mecp2 promoter was injected intravenously (IV) into juvenile (4-5 weeks old) Mecp2-null mice. While the brain transduction efficiency in juvenile mice was low (~2-4% of neurons), modest improvements in survival were still observed. These results support the concept of MECP2 gene therapy for RTT.

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

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


    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

  2. Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes. (United States)

    Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A


    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

  3. Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes? (United States)

    Brown, Nolan; Song, Liujiang; Kollu, Nageswara R; Hirsch, Matthew L


    The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the

  4. Functional correction of neurological and somatic disorders at later stages of disease in MPS IIIA mice by systemic scAAV9-hSGSH gene delivery

    Directory of Open Access Journals (Sweden)

    Haiyan Fu


    Full Text Available The reversibility of neuropathic lysosomal storage diseases, including MPS IIIA, is a major goal in therapeutic development, due to typically late diagnoses and a large population of untreated patients. We used self-complementary adeno-associated virus (scAAV serotype 9 vector expressing human N-sulfoglucosamine sulfohydrolase (SGSH to test the efficacy of treatment at later stages of the disease. We treated MPS IIIA mice at 1, 2, 3, 6, and 9 months of age with an intravenous injection of scAAV9-U1a-hSGSH vector, leading to restoration of SGSH activity and reduction of glycosaminoglycans (GAG throughout the central nervous system (CNS and somatic tissues at a dose of 5E12 vg/kg. Treatment up to 3 months age improved learning ability in the Morris water maze at 7.5 months, and lifespan was normalized. In mice treated at 6 months age, behavioral performance was impaired at 7.5 months, but did not decline further when retested at 12 months, and lifespan was increased, but not normalized. Treatment at 9 months did not increase life-span, though the GAG storage pathology in the CNS was improved. The study suggests that there is potential for gene therapy intervention in MPS IIIA at intermediate stages of the disease, and extends the clinical relevance of our systemic scAAV9-hSGSH gene delivery approach.

  5. Vascular administration of adenoviral vector soaked in absorbable gelatin sponge particles (GSP) prolongs the transgene expression in hepatocytes. (United States)

    Park, Byeong-Ho; Lee, Jin-Hwa; Jeong, Jin-Sook; Rha, Seo-Hee; Kim, Seung-Eun; Kim, Jae-Seok; Kim, Jeong-Man; Hwang, Tae-Ho


    Transcatheter hepatic arterial chemoembolization using emulsions composed of anticancer agents and gelatin sponges (GS) has been an efficient and safe palliative treatment for inoperable hepatocellular carcinoma (HCC). We employed catheter-mediated left hepatic arterial embolization (CHAE) to increase transduction efficiency of adenoviral vector in canine hepatocytes. The emulsion was prepared by mixing pieces of GSP and adenoviral vectors expressing recombinant beta-galactosidase (Ad.LacZ) or human hepatocyte growth factor (Ad.hHGF). After the left hepatic artery was catheterized under angiography, CHAE with Ad.LacZ or Ad.hHGF was performed. Livers were removed and stained for LacZ activity on day 7. The expression pattern of LacZ staining was either scarce or patchy around the central hilum of the hepatic artery, or was homogeneously distributed in whole lobes, depending on whether large or small pieces of GSP were used. Hematological and serum biochemical changes during CHAE exhibited only a few effects. The chronological measurement of serum HGF concentration showed that the duration of transgene expression was greater after CHAE with Ad.hHGF. A similar pattern of transgene expression was observed in a rat model after hepatic arterial embolization with differential doses of Ad.hHGF soaked in GSP. These results suggest that hepatic arterial embolization by transcatheter mediated infusion with a mixture of adenovirus-GSP could be used for human HCC.

  6. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer (United States)

    Ou, Jingxing; Vijayasarathy, Camasamudram; Ziccardi, Lucia; Chen, Shan; Zeng, Yong; Marangoni, Dario; Pope, Jodie G.; Bush, Ronald A.; Wu, Zhijian; Li, Wei; Sieving, Paul A.


    Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor–depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1–signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology. PMID:26098217

  7. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-RS1 transfer. (United States)

    Ou, Jingxing; Vijayasarathy, Camasamudram; Ziccardi, Lucia; Chen, Shan; Zeng, Yong; Marangoni, Dario; Pope, Jodie G; Bush, Ronald A; Wu, Zhijian; Li, Wei; Sieving, Paul A


    Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor-depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1-signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology.

  8. Preclinical study design for rAAV. (United States)

    Flotte, Terence R; Conlon, Thomas J; Mueller, Christian


    The process of moving a novel drug such as an adeno-associated viral vector from the bench top to bedside is an arduous process requiring coordination and skill from multiple laboratories and regulatory agencies. Proceeding to a phase I safety trial in humans after most of the proof-of-concept data have been acquired may take several years. During this time, agencies including the FDA, NIH Office of Biotechnology Activities (OBA), and Recombinant DNA Advisory Committee (RAC) along with the investigator's team will develop a series of preclinical toxicology and biodistribution studies in order to develop a safety profile for the intended novel drug. In this chapter, key features of the pharm-tox study design and conduct will be discussed. Highlighted features include choosing a sufficient animal number and species to use in testing, dose determination, typical toxicological assays performed, the use of Standard Operating Procedures in respect to good laboratory practices compliancy, and role of the Quality Assurance Unit.

  9. Lipidomic Evaluation of Feline Neurologic Disease after AAV Gene Therapy

    Directory of Open Access Journals (Sweden)

    Heather L. Gray-Edwards


    Full Text Available GM1 gangliosidosis is a fatal lysosomal disorder, for which there is no effective treatment. Adeno-associated virus (AAV gene therapy in GM1 cats has resulted in a greater than 6-fold increase in lifespan, with many cats remaining alive at >5.7 years of age, with minimal clinical signs. Glycolipids are the principal storage product in GM1 gangliosidosis whose pathogenic mechanism is not completely understood. Targeted lipidomics analysis was performed to better define disease mechanisms and identify markers of disease progression for upcoming clinical trials in humans. 36 sphingolipids and subspecies associated with ganglioside biosynthesis were tested in the cerebrospinal fluid of untreated GM1 cats at a humane endpoint (∼8 months, AAV-treated GM1 cats (∼5 years old, and normal adult controls. In untreated GM1 cats, significant alterations were noted in 16 sphingolipid species, including gangliosides (GM1 and GM3, lactosylceramides, ceramides, sphingomyelins, monohexosylceramides, and sulfatides. Variable degrees of correction in many lipid metabolites reflected the efficacy of AAV gene therapy. Sphingolipid levels were highly predictive of neurologic disease progression, with 11 metabolites having a coefficient of determination (R2 > 0.75. Also, a specific detergent additive significantly increased the recovery of certain lipid species in cerebrospinal fluid samples. This report demonstrates the methodology and utility of targeted lipidomics to examine the pathophysiology of lipid storage disorders.

  10. 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


    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....... Our results show that striatal delivery of rAAV5 vectors in the neonatal brain represents a useful tool to express genes of interest both in the basal ganglia and the neocortex. Furthermore, we apply, for the first time, viral vector-mediated gene transfer to the pig brain providing the opportunity...

  11. Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

    Directory of Open Access Journals (Sweden)

    Raymond L Fields

    Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.

  12. Protective Role of rAAV-NDI1, Serotype 5, in an Acute MPTP Mouse Parkinson's Model

    Directory of Open Access Journals (Sweden)

    Jennifer Barber-Singh


    Full Text Available Defects in mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I have been implicated in a number of acquired and hereditary diseases including Leigh's syndrome and more recently Parkinson's disease. A limited number of strategies have been attempted to repair the damaged complex I with little or no success. We have recently shown that the non-proton-pumping, internal NADH-ubiquinone oxidoreductase (Ndi1 from Saccharomyces cerevisiae (baker's yeast can be successfully inserted into the mitochondria of mice and rats, and the enzyme was found to be fully active. Using recombinant adenoassociated virus vectors (serotype 5 carrying our NDI1 gene, we were able to express the Ndi1 protein in the substantia nigra (SN of C57BL/6 mice with an expression period of two months. The results show that the AAV serotype 5 was highly efficient in expressing Ndi1 in the SN, when compared to a previous model using serotype 2, which led to nearly 100% protection when using an acute MPTP model. It is conceivable that the AAV-serotype5 carrying the NDI1 gene is a powerful tool for proof-of-concept study to demonstrate complex I defects as the causable factor in diseases of the brain.

  13. Sustained secretion of human alpha-1-antitrypsin from murine muscle transduced with adeno-associated virus vectors (United States)

    Song, Sihong; Morgan, Michael; Ellis, Tamir; Poirier, Amy; Chesnut, Kye; Wang, Jianming; Brantly, Mark; Muzyczka, Nicholas; Byrne, Barry J.; Atkinson, Mark; Flotte, Terence R.


    Recombinant adeno-associated virus (AAV) vectors have been used to transduce murine skeletal muscle as a platform for secretion of therapeutic proteins. The utility of this approach for treating alpha-1-antitrypsin (AAT) deficiency was tested in murine myocytes in vitro and in vivo. AAV vectors expressing the human AAT gene from either the cytomegalovirus (CMV) promoter (AAV-C-AT) or the human elongation factor 1-α promoter (AAV-E-AT) were examined. In vitro in C2C12 murine myoblasts, the expression levels in transient transfections were similar between the two vectors. One month after transduction, however, the human elongation factor 1 promoter mediated 10-fold higher stable human AAT expression than the CMV promoter. In vivo transduction was performed by injecting doses of up to 1.4 × 1013 particles into skeletal muscles of several mouse strains (C57BL/6, BALB/c, and SCID). In vivo, the CMV vector mediated higher levels of expression, with sustained serum levels over 800 μg/ml in SCID and over 400 μg/ml in C57BL/6 mice. These serum concentrations are 100,000-fold higher than those previously observed with AAV vectors in muscle and are at levels which would be therapeutic if achieved in humans. High level expression was delayed for several weeks but was sustained for over 15 wk. Immune responses were dependent upon the mouse strain and the vector dosage. These data suggest that recombinant AAV vector transduction of skeletal muscle could provide a means for replacing AAT or other essential serum proteins but that immune responses may be elicited under certain conditions. PMID:9826709

  14. Adeno-associated virus vector transduction of vascular smooth muscle cells in vivo. (United States)

    Richter, M; Iwata, A; Nyhuis, J; Nitta, Y; Miller, A D; Halbert, C L; Allen, M D


    Adeno-associated virus (AAV) vectors might offer solutions for restenosis and angiogenesis by transducing nondividing cells and providing long-term gene expression. We investigated the feasibility of vascular cell transduction by AAV vectors in an in vivo rabbit carotid artery model. Time course of gene expression, inflammatory reaction to the vector, and effects of varying viral titer, exposure time, and intraluminal pressures on gene expression were examined. Recombinant AAV vectors with an Rous sarcoma virus promoter and alkaline phosphatase reporter gene were injected intraluminally into transiently isolated carotid segments. Following transduction, gene expression increased significantly over 14 days and then remained stable to 28 days, the last time point examined. Medial vascular smooth muscle cells were the main cell type transduced even with an intact endothelial layer. Increasing the viral titer and intraluminal pressure both enhanced transduction efficiency to achieve a mean of 34 +/- 7% of the subintimal layer of smooth muscle cells expressing gene product. A mild inflammatory reaction, composed of T cells with only rare macrophages, with minimal intimal thickening was demonstrated in 40% of transduced vessels; inflammatory cells were not detected in sham-operated control arteries. These findings demonstrate that AAV is a promising vector for intravascular applications in coronary and peripheral vascular diseases.

  15. Bioreactor production of recombinant herpes simplex virus vectors. (United States)

    Knop, David R; Harrell, Heather


    Serotypical application of herpes simplex virus (HSV) vectors to gene therapy (type 1) and prophylactic vaccines (types 1 and 2) has garnered substantial clinical interest recently. HSV vectors and amplicons have also been employed as helper virus constructs for manufacture of the dependovirus adeno-associated virus (AAV). Large quantities of infectious HSV stocks are requisite for these therapeutic applications, requiring a scalable vector manufacturing and processing platform comprised of unit operations which accommodate the fragility of HSV. In this study, production of a replication deficient rHSV-1 vector bearing the rep and cap genes of AAV-2 (denoted rHSV-rep2/cap2) was investigated. Adaptation of rHSV production from T225 flasks to a packed bed, fed-batch bioreactor permitted an 1100-fold increment in total vector production without a decrease in specific vector yield (pfu/cell). The fed-batch bioreactor system afforded a rHSV-rep2/cap2 vector recovery of 2.8 x 10(12) pfu. The recovered vector was concentrated by tangential flow filtration (TFF), permitting vector stocks to be formulated at greater than 1.5 x 10(9) pfu/mL.

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

    Directory of Open Access Journals (Sweden)

    Uta Griesenbach


    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

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


    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.

  18. Antigen fusion with C3d3 augments or inhibits humoral immunity to AAV genetic vaccines in a transgene-dependent manner. (United States)

    Logan, Grant J; Wang, Lina; Zheng, Maolin; Coppel, Ross L; Alexander, Ian E


    Genetic fusion of tandem repeats of the complement molecule C3d has been shown to considerably enhance immune responses to genetic vaccines. We have investigated the applicability of this approach to augment humoral immune responses toward vaccines delivered by recombinant adeno-associated virus (AAV) vectors. C3d(3)-fusion was found to markedly decrease antibody responses to merozoite surface protein 4/5 from Plasmodium yoelii and contrasted with greater than 50-fold enhancement in responses when this strategy was similarly applied to another AAV-encoded model antigen, hen egg lysozyme. These data indicate that the efficacy of the C3d(3) strategy operates in an antigen-dependent manner. Additional studies also showed that homologous recombination events between the C3d tandem repeats occurred during vector packaging and transduction resulting in expression of C3d(1)-, C3d(2)-, C3d(3)- and C3d(4)-fused antigen. This is the first report to apply the C3d approach to augment responses against a recombinant viral vector system and the consequences of these findings are discussed.

  19. 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.

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

    DEFF Research Database (Denmark)

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


    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...

  1. 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


    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.

  2. 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


    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...

  3. Smart and Controllable rAAV Gene Delivery Carriers in Progenitor Cells for Human Musculoskeletal Regenerative Medicine with a Focus on the Articular Cartilage. (United States)

    Rey-Rico, Ana; Cucchiarini, Magali


    Cell therapy using mesenchymal stem cells (MSCs) is a powerful tool for the treatment of various diseases and injuries. Still, important limitations including the large amounts of cells required for application in vivo and the age-related decline in lifespan, proliferation, and potency may hinder the use of MSCs in patients. In this regard, gene therapy may offer strong approaches to optimize the use of MSCs for regenerative medicine. Diverse nonviral and viral gene vehicles have been manipulated to genetically modify MSCs, among which the highly effective and relatively safe recombinant adeno-associated viral (rAAV) vectors that emerged as the preferred gene delivery system to treat human disorders. Yet, clinical adaptation of such gene vehicles may be limited by several hurdles, including the possibility of dissemination to nontarget sites and the presence of immune and toxic responses in the host organism that may impair their therapeutic actions. The use of smart biomaterials acting as interfaces to enhance the temporal and spatial presentation of therapeutic agents in the target place and/or acting as scaffolding for MSC growth is an innovative, valuable approach to overcome these shortcomings that else restrain the efficacy of such potent cell populations. Here, we provide an overview on the most recent tissue engineering approaches based on the use of biomaterials acting as vehicles for rAAV vectors to target MSCs directly in the recipient (in vivo strategy) or as supportive matrices for rAAV-modified MSCs for indirect cell reimplantation (ex vivo strategy) as means to activate the reparative processes in tissues of the musculoskeletal system. Copyright© Bentham Science Publishers; For any queries, please email at

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

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


    Background 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. Objective 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. Methods 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. Results 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

  5. Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis. (United States)

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


    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.

  6. AAV-mediated IL-10 gene transfer counteracts inflammation in the hypothalamic arcuate nucleus and obesity induced by high-fat diet. (United States)

    Nakata, Masanori; Yamamoto, Sawako; Okada, Takashi; Yada, Toshihiko


    Consumption of high-fat diet (HFD) induces energy imbalance and consequently obesity. In the pathogenesis of obesity, HFD triggers inflammation in the hypothalamus including arcuate nucleus (ARC). Interleukin-10 (IL-10) is a representative anti-inflammatory cytokine, known to ameliorate the adipose tissue inflammation and insulin resistance in obesity. However, the effect of IL-10 on the hypothalamic inflammation remains less defined. We here report the effect of over-expression of murine IL-10 using adeno-associated virus (AAV) vector on the inflammation in ARC and feeding behavior in HFD-induced obese (DIO) mice. DIO mice exhibited reduced POMC expression and elevated IKKs (IκB kinases) and SOCS3 expression in ARC. Overexpression of mIL-10 using AAV vector ameliorated obesity in parallel with restoration of ARC POMC expression in DIO mice. Moreover, IL-10 treatment suppressed IKKs activation and SOCS3 expression in ARC of DIO mice. These results suggest that IL-10 gene transfer provides an effective approach for counteracting HFD-induced inflammation and leptin resistance in ARC to prevent progression of obesity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial. (United States)

    Heier, Jeffrey S; Kherani, Saleema; Desai, Shilpa; Dugel, Pravin; Kaushal, Shalesh; Cheng, Seng H; Delacono, Cheryl; Purvis, Annie; Richards, Susan; Le-Halpere, Annaig; Connelly, John; Wadsworth, Samuel C; Varona, Rafael; Buggage, Ronald; Scaria, Abraham; Campochiaro, Peter A


    Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2 × 10 8 vector genomes (vg); cohort 2, 2 × 10 9 vg; cohort 3, 6 × 10 9 vg; and cohort 4, 2 × 10 10 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2 × 10 10 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with, number NCT01024998. 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 × 10 10 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2 × 10 10 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to

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


    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.

  9. A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro Dystrophin Vector (United States)


    pressure (MAP) at the baseline (Table 1). The baseline mean artery blood flow in the brachial artery (MABF) of DMD dogs only reached ~50% of that of... pressure ; BF, blood flow; NE, norepinephrine (NE). (C and D) MVC (mean vascular conductance) changes in normal (n = 11) and DMD dogs (n = 14). (E...published in peer -reviewed scientific journals or presented in academic conferences. Plan for future. With the support from the DoD MD130014 grant

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

    Directory of Open Access Journals (Sweden)

    Giridhar eMurlidharan


    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.

  11. Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice (United States)

    Goudy, Kevin; Song, Sihong; Wasserfall, Clive; Zhang, Y. Clare; Kapturczak, Matthias; Muir, Andrew; Powers, Matthew; Scott-Jorgensen, Marda; Campbell-Thompson, Martha; Crawford, James M.; Ellis, Tamir M.; Flotte, Terence R.; Atkinson, Mark A.


    The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders. PMID:11717448

  12. 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


    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...

  13. Preclinical evaluation of a recombinant adeno-associated virus vector expressing human alpha-1 antitrypsin made using a recombinant herpes simplex virus production method. (United States)

    Chulay, Jeffrey D; Ye, Guo-Jie; Thomas, Darby L; Knop, David R; Benson, Janet M; Hutt, Julie A; Wang, Gensheng; Humphries, Margaret; Flotte, Terence R


    Recombinant adeno-associated virus (rAAV) vectors offer promise for gene therapy of alpha-1 antitrypsin (AAT) deficiency. A toxicology study in mice evaluated intramuscular injection of an rAAV vector expressing human AAT (rAAV-CB-hAAT) produced using a herpes simplex virus (HSV) complementation system or a plasmid transfection (TFX) method at doses of 3 × 10(11) vg (1.2 × 10(13) vg/kg) for both vectors and 2 × 10(12) vg (8 × 10(13) vg/kg) for the HSV-produced vector. The HSV-produced vector had favorable in vitro characteristics in terms of purity, efficiency of transduction, and hAAT expression. There were no significant differences in clinical findings or hematology and clinical chemistry values between test article and control groups and no gross pathology findings. Histopathological examination demonstrated minimal to mild changes in skeletal muscle at the injection site, consisting of focal chronic interstitial inflammation and muscle degeneration, regeneration, and vacuolization, in vector-injected animals. At the 3 × 10(11) vg dose, serum hAAT levels were higher with the HSV-produced vector than with the TFX-produced vector. With the higher dose of HSV-produced vector, the increase in serum hAAT levels was dose-proportional in females and greater than dose-proportional in males. Vector copy numbers in blood were highest 24 hr after dosing and declined thereafter, with no detectable copies present 90 days after dosing. Antibodies to hAAT were detected in almost all vector-treated animals, and antibodies to HSV were detected in most animals that received the highest vector dose. These results support continued development of rAAV-CB-hAAT for treatment of AAT deficiency.

  14. Deletion of the B-B' and C-C' regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression. (United States)

    Zhou, Qingzhang; Tian, Wenhong; Liu, Chunguo; Lian, Zhonghui; Dong, Xiaoyan; Wu, Xiaobing


    Inverted terminal repeats (ITRs) of the adeno-associated virus (AAV) are essential for rescue, replication, packaging, and integration of the viral genome. While ITR mutations have been identified in previous reports, we designed a new truncated ITR lacking the B-B' and C-C' regions named as ITRΔBC and investigated its effects on viral genome replication, packaging, and expression of recombinant AAV (rAAV). The packaging ability was compared between ITRΔBC rAAV and wild-type (wt) ITR rAAV. Our results showed the productivity of ITRΔBC rAAV was reduced 4-fold, which is consistent with the 8-fold decrease in the replication of viral genomic DNA of ITRΔBC rAAV compared with wt ITR rAAV. Surprisingly, transgene expression was significantly higher for ITRΔBC rAAV. A preliminary exploration of the underlying mechanisms was carried out by inhibiting and degrading the ataxia telangiectasia mutated (ATM) protein and the Mre11 complex (MRN), respectively, since the rAAV expression was inhibited by the ATM and/or MRN through cis interaction or binding with wt ITRs. We demonstrated that the inhibitory effects were weakened on ITRΔBC rAAV expression. This study suggests deletion in ITR can affect the transgene expression of AAV, which provides a new way to improve the AAV expression through ITRs modification.

  15. Vector analysis

    CERN Document Server

    Newell, Homer E


    When employed with skill and understanding, vector analysis can be a practical and powerful tool. This text develops the algebra and calculus of vectors in a manner useful to physicists and engineers. Numerous exercises (with answers) not only provide practice in manipulation but also help establish students' physical and geometric intuition in regard to vectors and vector concepts.Part I, the basic portion of the text, consists of a thorough treatment of vector algebra and the vector calculus. Part II presents the illustrative matter, demonstrating applications to kinematics, mechanics, and e

  16. About vectors

    CERN Document Server

    Hoffmann, Banesh


    From his unusual beginning in ""Defining a vector"" to his final comments on ""What then is a vector?"" author Banesh Hoffmann has written a book that is provocative and unconventional. In his emphasis on the unresolved issue of defining a vector, Hoffmann mixes pure and applied mathematics without using calculus. The result is a treatment that can serve as a supplement and corrective to textbooks, as well as collateral reading in all courses that deal with vectors. Major topics include vectors and the parallelogram law; algebraic notation and basic ideas; vector algebra; scalars and scalar p

  17. Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

    Directory of Open Access Journals (Sweden)

    Victoria M. Velazquez


    Full Text Available Pre-existing antibodies (Abs to AAV pose a critical challenge for the translation of gene therapies. No effective approach is available to overcome pre-existing Abs. Given the complexity of Ab production, overcoming pre-existing Abs will require broad immune targeting. We generated a mouse model of pre-existing AAV9 Abs to test multiple immunosuppressants, including bortezomib, rapamycin, and prednisolone, individually or in combination. We identified an effective approach combining rapamycin and prednisolone, reducing serum AAV9 Abs by 70%–80% at 4 weeks and 85%–93% at 8 weeks of treatment. The rapamycin plus prednisolone treatment resulted in significant decreases in the frequency of B cells, plasma cells, and IgG-secreting and AAV9-specific Ab-producing plasma cells in bone marrow. The rapamycin plus prednisolone treatment also significantly reduced frequencies of IgD−IgG+ class-switched/FAS+CL7+ germinal center B cells, and of activated CD4+ T cells expressing PD1 and GL7, in spleen. These data suggest that rapamycin plus prednisolone has selective inhibitory effects on both T helper type 2 support of B cell activation in spleen and on bone marrow plasma cell survival, leading to effective AAV9 Abs depletion. This promising immunomodulation approach is highly translatable, and it poses minimal risk in the context of therapeutic benefits promised by gene therapy for severe monogenetic diseases, with a single or possibly a few treatments over a lifetime.

  18. Elementary vectors

    CERN Document Server

    Wolstenholme, E Œ


    Elementary Vectors, Third Edition serves as an introductory course in vector analysis and is intended to present the theoretical and application aspects of vectors. The book covers topics that rigorously explain and provide definitions, principles, equations, and methods in vector analysis. Applications of vector methods to simple kinematical and dynamical problems; central forces and orbits; and solutions to geometrical problems are discussed as well. This edition of the text also provides an appendix, intended for students, which the author hopes to bridge the gap between theory and appl

  19. Proof-of-concept: neonatal intravenous injection of adeno-associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine. (United States)

    Buckinx, R; Van Remoortel, S; Gijsbers, R; Waddington, S N; Timmermans, J-P


    Despite the success of viral vector technology in the transduction of the central nervous system in both preclinical research and gene therapy, its potential in neurogastroenterological research remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno-associated viral vectors (AAVs). Mice were intravenously injected at postnatal day one with AAV pseudotypes AAV8 or AAV9 carrying a cassette encoding enhanced green fluorescent protein (eGFP) as a reporter under the control of a cytomegalovirus promoter. At postnatal day 35, transduction of the myenteric and submucosal plexuses of the ileum and distal colon was evaluated in whole-mount preparations, using immunohistochemistry to neurochemically identify transduced enteric neurons. The pseudotypes AAV8 and AAV9 showed equal potential in transducing the enteric nervous system (ENS), with 25-30% of the neurons expressing eGFP. However, the percentage of eGFP-expressing colonic submucosal neurons was significantly lower. Neurochemical analysis showed that all enteric neuron subtypes, but not glia, expressed the reporter protein. Intrinsic sensory neurons were most efficiently transduced as nearly 80% of calcitonin gene-related peptide-positive neurons expressed the transgene. The pseudotypes AAV8 and AAV9 can be employed for gene delivery to both the myenteric and the submucosal plexus, although the transduction efficiency in the latter is region-dependent. These findings open perspectives for novel preclinical applications aimed at manipulating and imaging the ENS in the short term, and in gene therapy in the longer term. © 2015 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd.

  20. Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.

    Directory of Open Access Journals (Sweden)

    William E Grose

    Full Text Available The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9. Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes.

  1. Stimulation of IgY responses in gene gun immunized laying hens by combined administration of vector DNA coding for the target antigen Botulinum toxin A1 and for avian cytokine adjuvants. (United States)

    Niederstadt, Lars; Hohn, Oliver; Dorner, Brigitte G; Schade, Rüdiger; Bannert, Norbert


    DNA immunization is a convenient and effective way of inducing a specific antibody response. In mammals, co-administration of vectors encoding immunostimulatory cytokines can enhance the humoral response resulting in elevated antibody titers. We therefore set out to investigate the effect using avian interleukin 1β (IL-1β) and avian interleukin 6 (IL-6) as genetic adjuvants when immunizing laying hens. A BoNT A1 holotoxoid DNA immunogen carrying two inactivating mutations was evaluated for its ability to induce a specific and sustained IgY antibody response. Both the holotoxoid and the cytokine sequences were codon-optimized. In vitro, the proteins were efficiently expressed in transfected HEK 293T cells and the cytokines were secreted into the culture supernatants. Whereas eggs from hens immunized via gene gun using a prime boost strategy showed no differences in their total IgY content, the specific αBoNT A1 response was slightly elevated up to 1.4× by the IL-1β adjuvant vector and increased by 3.8× by the IL-6 vector. Finally, although hens receiving the IL-1β adjuvant had laying capacities above the average, hens receiving the IL-6 adjuvant experienced laying problems. Copyright © 2012 Elsevier B.V. All rights reserved.


    DEFF Research Database (Denmark)

    Glud, Andreas Nørgaard; Lillethorup, Thea Pinholt; Landeck, Natalie

    SYN) fibrils, overexpressing aSYN using Lentivirus (LV) and Adeno Assosiated Virus (AAV) vectors or proteasome inhibition in the nigrostriatal system, we hope to create a new porcine models for PD. Methods: Using conventional human-intended stereotaxic neurosurgery methods, we apply aSYN or preformed fibrils...

  3. Synergistic antitumor effect of AAV-mediated TRAIL expression combined with cisplatin on head and neck squamous cell carcinoma

    International Nuclear Information System (INIS)

    Jiang, Minghong; Liu, Zheng; Xiang, Yang; Ma, Hong; Liu, Shilian; Liu, Yanxin; Zheng, Dexian


    Adeno-associated virus-2 (AAV-2)-mediated gene therapy is quite suitable for local or regional application in head and neck cancer squamous cell carcinoma (HNSCC). However, its low transduction efficiency has limited its further development as a therapeutic agent. DNA damaging agents have been shown to enhance AAV-mediated transgene expression. Cisplatin, one of the most effective chemotherapeutic agents, has been recognized to cause cancer cell death by apoptosis with a severe toxicity. This study aims to evaluate the role of cisplatin in AAV-mediated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and the effect on HNSCC both in vitro and in vivo. Five human HNSCC cell lines were treated with recombinant soluble TRAIL (rsTRAIL) and infected with AAV/TRAIL to estimate the sensitivity of the cancer cells to TRAIL-induced cytotoxicity. KB cells were infected with AAV/EGFP with or without cisplatin pretreatment to evaluate the effect of cisplatin on AAV-mediated gene expression. TRAIL expression was detected by ELISA and Western blot. Cytotoxicity was measured by MTT assay and Western blot analysis for caspase-3 and -8 activations. Following the in vitro experiments, TRAIL expression and its tumoricidal activity were analyzed in nude mice with subcutaneous xenografts of HNSCC. HNSCC cell lines showed different sensitivities to rsTRAIL, and KB cells possessed both highest transduction efficacy of AAV and sensitivity to TRAIL among five cell lines. Preincubation of KB cells with subtherapeutic dosage of cisplatin significantly augmented AAV-mediated transgene expression in a heparin sulfate proteoglycan (HSPG)-dependent manner. Furthermore, cisplatin enhanced the killing efficacy of AAV/TRAIL by 3-fold on KB cell line. The AAV mediated TRAIL expression was observed in the xenografted tumors and significantly enhanced by cisplatin. AAV/TRAIL suppressed the tumors growth and cisplatin augmented the tumoricidal activity by two-fold. Furthermore

  4. Vector analysis

    CERN Document Server

    Brand, Louis


    The use of vectors not only simplifies treatments of differential geometry, mechanics, hydrodynamics, and electrodynamics, but also makes mathematical and physical concepts more tangible and easy to grasp. This text for undergraduates was designed as a short introductory course to give students the tools of vector algebra and calculus, as well as a brief glimpse into these subjects' manifold applications. The applications are developed to the extent that the uses of the potential function, both scalar and vector, are fully illustrated. Moreover, the basic postulates of vector analysis are brou

  5. associated virus (AAV)-mediated expression of small interfering RNA

    African Journals Online (AJOL)



    Apr 11, 2011 ... expressed from a DNA-based vector by the function of. *Corresponding .... cleotides were designed, which contained a sense strand of p53 or ..... Subthalamic GAD gene transfer in Parkinson disease patients who are candidates for deep brain stimulation. Hum. Gene Ther. 12(12): 1589-1591. Dufourny L ...

  6. AAV-Mediated Anterograde Transsynaptic Tagging: Mapping Corticocollicular Input-Defined Neural Pathways for Defense Behaviors. (United States)

    Zingg, Brian; Chou, Xiao-Lin; Zhang, Zheng-Gang; Mesik, Lukas; Liang, Feixue; Tao, Huizhong Whit; Zhang, Li I


    To decipher neural circuits underlying brain functions, viral tracers are widely applied to map input and output connectivity of neuronal populations. Despite the successful application of retrograde transsynaptic viruses for identifying presynaptic neurons of transduced neurons, analogous anterograde transsynaptic tools for tagging postsynaptically targeted neurons remain under development. Here, we discovered that adeno-associated viruses (AAV1 and AAV9) exhibit anterograde transsynaptic spread properties. AAV1-Cre from transduced presynaptic neurons effectively and specifically drives Cre-dependent transgene expression in selected postsynaptic neuronal targets, thus allowing axonal tracing and functional manipulations of the latter input-defined neuronal population. Its application in superior colliculus (SC) reveals that SC neuron subpopulations receiving corticocollicular projections from auditory and visual cortex specifically drive flight and freezing, two different types of defense behavior, respectively. Together with an intersectional approach, AAV-mediated anterograde transsynaptic tagging can categorize neurons by their inputs and molecular identity, and allow forward screening of distinct functional neural pathways embedded in complex brain circuits. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs

    Czech Academy of Sciences Publication Activity Database

    Miyanohara, A.; Kamizato, K.; Juhás, Štefan; Juhásová, Jana; Navarro, M.; Maršala, S.; Lukáčová, N.; Hruška-Plocháň, M.; Curtis, E.; Gabel, B.; Ciacci, J. D.; Ahrens, E. T.; Kaspar, B. K.; Cleveland, D.; Maršala, M.


    Roč. 3, č. 1 (2016), č. článku 16046. ISSN 2329-0501 R&D Projects: GA MŠk(CZ) LO1609 Institutional support: RVO:67985904 Keywords : AAV9 * rat * pig Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.610, year: 2016

  8. Biological effects of rAAV-caAlk2 coating on structural allograft healing

    DEFF Research Database (Denmark)

    Koefoed, Mette; Ito, Hiromu; Gromov, Kirill


    Structural bone allografts often fracture due to their lack of osteogenic and remodeling potential. To overcome these limitations, we utilized allografts coated with recombinant adeno-associated virus (rAAV) that mediate in vivo gene transfer. Using beta-galactosidase as a reporter gene, we show...

  9. Comparative impact of AAV and enzyme replacement therapy on respiratory and cardiac function in adult Pompe mice

    Directory of Open Access Journals (Sweden)

    Darin J Falk

    Full Text Available Pompe disease is an autosomal recessive genetic disorder characterized by a deficiency of the enzyme responsible for degradation of lysosomal glycogen (acid α-glucosidase (GAA. Cardiac dysfunction and respiratory muscle weakness are primary features of this disorder. To attenuate the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa−/− mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9-DES or bimonthly injections of recombinant human GAA (enzyme replacement therapy (ERT. Assessment of cardiac function and morphology was measured 1 and 3 months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at 3 months post-treatment in all groups. Gaa−/− animals receiving either AAV9-DES or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9-DES treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc at 3 months postinjection. Neither AAV nor ERT therapy altered minute ventilation during quiet breathing (eupnea. However, breathing frequency and expiratory time were significantly improved in AAV9-DES animals. These results indicate systemic delivery of either strategy improves cardiac function but AAV9-DES alone improves respiratory parameters at 3 months post-treatment in a murine model of Pompe disease.

  10. High Accuracy Vector Helium Magnetometer (United States)

    National Aeronautics and Space Administration — The proposed HAVHM instrument is a laser-pumped helium magnetometer with both triaxial vector and omnidirectional scalar measurement capabilities in a single...

  11. Vector velocimeter

    DEFF Research Database (Denmark)


    The present invention relates to a compact, reliable and low-cost vector velocimeter for example for determining velocities of particles suspended in a gas or fluid flow, or for determining velocity, displacement, rotation, or vibration of a solid surface, the vector velocimeter comprising a laser...

  12. Cloning vector (United States)

    Guilfoyle, R.A.; Smith, L.M.


    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site. 2 figures.

  13. Cloning vector (United States)

    Guilfoyle, Richard A.; Smith, Lloyd M.


    A vector comprising a filamentous phage sequence containing a first copy of filamentous phage gene X and other sequences necessary for the phage to propagate is disclosed. The vector also contains a second copy of filamentous phage gene X downstream from a promoter capable of promoting transcription in a bacterial host. In a preferred form of the present invention, the filamentous phage is M13 and the vector additionally includes a restriction endonuclease site located in such a manner as to substantially inactivate the second gene X when a DNA sequence is inserted into the restriction site.

  14. Delivery of AAV2/9-microdystrophin genes incorporating helix 1 of the coiled-coil motif in the C-terminal domain of dystrophin improves muscle pathology and restores the level of α1-syntrophin and α-dystrobrevin in skeletal muscles of mdx mice. (United States)

    Koo, Taeyoung; Malerba, Alberto; Athanasopoulos, Takis; Trollet, Capucine; Boldrin, Luisa; Ferry, Arnaud; Popplewell, Linda; Foster, Helen; Foster, Keith; Dickson, George


    Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

  15. AAV-mediated delivery of the transcription factor XBP1s into the striatum reduces mutant Huntingtin aggregation in a mouse model of Huntington's disease

    Energy Technology Data Exchange (ETDEWEB)

    Zuleta, Amparo [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago (Chile); Vidal, Rene L. [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Neurounion Biomedical Foundation, Santiago (Chile); Armentano, Donna; Parsons, Geoffrey [Department of Molecular Biology, Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Hetz, Claudio, E-mail: [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago (Chile); Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Av., Boston, MA 02446 (United States); Neurounion Biomedical Foundation, Santiago (Chile)


    Highlights: Black-Right-Pointing-Pointer The contribution of ER stress to HD has not been directly addressed. Black-Right-Pointing-Pointer Expression of XBP1s using AAVs decreases Huntingtin aggregation in vivo. Black-Right-Pointing-Pointer We describe a new in vivo model of HD based on the expression of a large fragment of mHtt-RFP. -- Abstract: Huntington's disease (HD) is caused by mutations that expand a polyglutamine region in the amino-terminal domain of Huntingtin (Htt), leading to the accumulation of intracellular inclusions and progressive neurodegeneration. Recent reports indicate the engagement of endoplasmic reticulum (ER) stress responses in human HD post mortem samples and animal models of the disease. Adaptation to ER stress is mediated by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that attenuates protein folding stress by controlling the expression of distinct transcription factors including X-Box binding protein 1 (XBP1). Here we targeted the expression of XBP1 on a novel viral-based model of HD. We delivered an active form of XBP1 locally into the striatum of adult mice using adeno-associated vectors (AAVs) and co-expressed this factor with a large fragment of mutant Htt as a fusion protein with RFP (Htt588{sup Q95}-mRFP) to directly visualize the accumulation of Htt inclusions in the brain. Using this approach, we observed a significant reduction in the accumulation of Htt588{sup Q95}-mRFP intracellular inclusion when XBP1 was co-expressed in the striatum. These results contrast with recent findings indicating a protective effect of XBP1 deficiency in neurodegeneration using knockout mice, and suggest a potential use of gene therapy strategies to manipulate the UPR in the context of HD.

  16. Transient Photoreceptor Deconstruction by CNTF Enhances rAAV-Mediated Cone Functional Rescue in Late Stage CNGB3-Achromatopsia (United States)

    Komáromy, András M; Rowlan, Jessica S; Corr, Amanda T Parton; Reinstein, Shelby L; Boye, Sanford L; Cooper, Ann E; Gonzalez, Amaliris; Levy, Britt; Wen, Rong; Hauswirth, William W; Beltran, William A; Aguirre, Gustavo D


    Achromatopsia is a genetic disorder of cones, and one of the most common forms is a channelopathy caused by mutations in the β-subunit, CNGB3, of the cone cyclic nucleotide-gated (CNG) channel. Recombinant adeno-associated virus of serotype 5 (rAAV5)-mediated gene transfer of human CNGB3 cDNA to mutant dog cones results in functional and structural rescue in dogs 1 year. We now test a new therapeutic concept by combining gene therapy with the administration of ciliary neurotrophic factor (CNTF). Intravitreal CNTF causes transient dedifferentiation of photoreceptors, a process called deconstruction, whereby visual cells become immature with short outer segments, and decreased retinal function and gene expression that subsequently return to normal. Cone function was successfully rescued in all mutant dogs treated between 14 and 42 months of age with this strategy. CNTF-mediated deconstruction and regeneration of the photoreceptor outer segments prepares the mutant cones optimally for gene augmentation therapy. PMID:23568263

  17. Vector geometry

    CERN Document Server

    Robinson, Gilbert de B


    This brief undergraduate-level text by a prominent Cambridge-educated mathematician explores the relationship between algebra and geometry. An elementary course in plane geometry is the sole requirement for Gilbert de B. Robinson's text, which is the result of several years of teaching and learning the most effective methods from discussions with students. Topics include lines and planes, determinants and linear equations, matrices, groups and linear transformations, and vectors and vector spaces. Additional subjects range from conics and quadrics to homogeneous coordinates and projective geom


    NARCIS (Netherlands)

    Thomas, E. G. F.


    This paper deals with the theory of integration of scalar functions with respect to a measure with values in a, not necessarily locally convex, topological vector space. It focuses on the extension of such integrals from bounded measurable functions to the class of integrable functions, proving

  19. Stereotaxic microinjection of viral vectors expressing Cre recombinase to study the role of target genes in cocaine conditioned place preference. (United States)

    Schierberl, Kathryn C; Rajadhyaksha, Anjali M


    Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

  20. Synthetic adeno-associated viral vector efficiently targets mouse and non-human primate retina in vivo. (United States)

    Carvalho, Livia S; Xiao, Ru; Wassmer, Sarah; Langsdorf, Aliete; Zinn, Eric; Pacouret, Simon; Shah, Samiksha; Comander, Jason I; Kim, Leo; Lim, Laurence; Vandenberghe, Luk H


    Gene therapy is a promising approach in the treatment of inherited and common complex disorders of the retina. Preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and efficient delivery vehicle for gene transfer. RPE and rods, and to a lesser extent, cone photoreceptors can be efficiently targeted with AAV. Other retinal cell types however are more challenging targets. The aim of this study was to characterize the transduction profile and efficiency of in silico designed, synthetic Anc80 AAVs for retinal gene transfer. Three Anc80 variants were evaluated for retinal targeting in mice and primates following subretinal delivery. In the murine retina Anc80L65 demonstrated high level of RPE and photoreceptor targeting with comparable cone photoreceptor affinity compared to other AAVs. Remarkably, Anc80L65 enhanced transduction kinetics with visible expression as early as day 1 and steady state mRNA levels at day 3. Inner retinal tropism of Anc80 variants demonstrated distinct transduction patterns of Müller glia, retinal ganglion cells and INL neurons. Finally, murine findings with Anc80L65 qualitatively translated to the Rhesus macaque in terms of cell targets, levels and onset of expression. Our findings support the use of Anc80L65 for therapeutic subretinal gene delivery.

  1. Long-term sex-biased correction of circulating propionic acidemia disease markers by adeno-associated virus vectors. (United States)

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


    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.

  2. 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


    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.

  3. Delivery and evaluation of recombinant adeno-associated viral vectors in the equine distal extremity for the treatment of laminitis. (United States)

    Mason, J B; Gurda, B L; Van Wettere, A; Engiles, J B; Wilson, J M; Richardson, D W


    Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. Randomised in vivo experiment. We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken β-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the

  4. An introduction to vectors, vector operators and vector analysis

    CERN Document Server

    Joag, Pramod S


    Ideal for undergraduate and graduate students of science and engineering, this book covers fundamental concepts of vectors and their applications in a single volume. The first unit deals with basic formulation, both conceptual and theoretical. It discusses applications of algebraic operations, Levi-Civita notation, and curvilinear coordinate systems like spherical polar and parabolic systems and structures, and analytical geometry of curves and surfaces. The second unit delves into the algebra of operators and their types and also explains the equivalence between the algebra of vector operators and the algebra of matrices. Formulation of eigen vectors and eigen values of a linear vector operator are elaborated using vector algebra. The third unit deals with vector analysis, discussing vector valued functions of a scalar variable and functions of vector argument (both scalar valued and vector valued), thus covering both the scalar vector fields and vector integration.

  5. AAVPG: A vigilant vector where transgene expression is induced by p53

    Energy Technology Data Exchange (ETDEWEB)

    Bajgelman, Marcio C.; Medrano, Ruan F.V.; Carvalho, Anna Carolina P.V.; Strauss, Bryan E., E-mail:


    Using p53 to drive transgene expression from viral vectors may provide on demand expression in response to physiologic stress, such as hypoxia or DNA damage. Here we introduce AAVPG, an adeno-associated viral (AAV) vector where a p53-responsive promoter, termed PG, is used to control transgene expression. In vitro assays show that expression from the AAVPG-luc vector was induced specifically in the presence of functional p53 (1038±202 fold increase, p<0.001). The AAVPG-luc vector was an effective biosensor of p53 activation in response to hypoxia (4.48±0.6 fold increase in the presence of 250 µM CoCl{sub 2}, p<0.001) and biomechanical stress (2.53±0.4 fold increase with stretching, p<0.05). In vivo, the vigilant nature of the AAVPG-luc vector was revealed after treatment of tumor-bearing mice with doxorubicin (pre-treatment, 3.4×10{sup 5}±0.43×10{sup 5} photons/s; post-treatment, 6.6×10{sup 5}±2.1×10{sup 5} photons/s, p<0.05). These results indicate that the AAVPG vector is an interesting option for detecting p53 activity both in vitro and in vivo. - Highlights: • AAV vector where transgene expression is controlled by the tumor suppressor p53. • The new vector, AAVPG, shown to function as a biosensor of p53 activity, in vitro and in vivo. • The p53 activity monitored by the AAVPG vector is relevant to cancer and other diseases. • AAVPG reporter gene expression was activated upon DNA damage, hypoxia and mechanical stress.

  6. Simultaneous subcutaneous and conjunctival administration of the influenza viral vector based Brucella abortus vaccine to pregnant heifers provides better protection against B. abortus 544 infection than the commercial B. abortus S19 vaccine. (United States)

    Tabynov, Kaissar; Orynbayev, Mukhit; Renukaradhya, Gourapura J; Sansyzbay, Abylai


    In this study, we explored possibility of increasing the protective efficacy of our novel influenza viral vector based B. abortus vaccine (Flu-BA) in pregnant heifers by adapting an innovative method of vaccine delivery. We administered the vaccine concurrently via the conjunctival and subcutaneous routes to pregnant heifers, and these routes were previously tested individually. The Flu-BA vaccination of pregnant heifers (n=9) against a challenge B. abortus 544 infection provided protection from abortion, infection of heifers and fetuses/calves by 88.8%, 100% and 100%, respectively (alpha=0.004-0.0007 vs. negative control; n=7). Our candidate vaccine using this delivery method provided slightly better protection than the commercial B. abortus S19 vaccine in pregnant heifers (n=8), which provided protection from abortion, infection of heifers and fetuses/calves by 87.5%, 75% and 87.5%, respectively. This improved method of the Flu-BA vaccine administration is highly recommended for the recovery of farms which has high prevalence of brucellosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. The Rep78 gene product of adeno-associated virus (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences. (United States)

    Smith, R H; Spano, A J; Kotin, R M


    The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences.

  8. Differential adenoassociated virus vector-driven expression of a neuropeptide Y gene in primary rat brain astroglial cultures after transfection with Sendai virosomes versus Lipofectin. (United States)

    de Fiebre, C M; Wu, P; Notabartolo, D; Millard, W J; Meyer, E M


    The ability of Sendai virosomes or Lipofectin to introduce an AAV vector into primary rat brain astroglial cultures was characterized. The pJDT95npy vector was constructed by inserting rat NPY cDNA downstream from the indigenous AAV p5, p19 and p40 promoters in pJDT95. Lipofectin-mediated transfection with pJDT95npy (10 micrograms) resulted in pronounced expression of several NPY mRNA species: p5-driven (3.3 kb), p19-driven (2.7 kb) and p40-driven (0.6, 0.8, 1.1, and 1.8 kb). Exposure to virosomally encapsulated pJDT95npy (50 or 100 ng) resulted in transient expression of some p40-driven mRNA species (0.8 and 1.8 kb). Neither method produced astroglia cells which synthesized mature NPY immunoreactivity. This demonstrates that an AAV-derived vector can drive gene expression in astroglia, that Sendai virosomes can infuse vectors into astroglia, but that the amount of DNA infused in this manner may limit long term expression.

  9. An Intrabody Drug (rAAV6-INT41 Reduces the Binding of N-Terminal Huntingtin Fragment(s to DNA to Basal Levels in PC12 Cells and Delays Cognitive Loss in the R6/2 Animal Model

    Directory of Open Access Journals (Sweden)

    I. Alexandra Amaro


    Full Text Available Huntington’s disease (HD is a fatal progressive disease linked to expansion of glutamine repeats in the huntingtin protein and characterized by the progressive loss of cognitive and motor function. We show that expression of a mutant human huntingtin exon-1-GFP fusion construct results in nonspecific gene dysregulation that is significantly reduced by 50% due to coexpression of INT41, an intrabody specific for the proline-rich region of the huntingtin protein. Using stable PC12 cell lines expressing either inducible human mutant huntingtin (mHtt, Q73 or normal huntingtin (nHtt, Q23, we investigated the effect of rAAV6-INT41, an adeno-associated virus vector with the INT41 coding sequence, on the subcellular distribution of Htt. Compartmental fractionation 8 days after induction of Htt showed a 6-fold increased association of a dominate N-terminal mHtt fragment with DNA compared to N-terminal nHtt. Transduction with rAAV6-INT41 reduced DNA binding of N-terminal mHtt 6.5-fold in the nucleus and reduced nuclear translocation of the detected fragments. Subsequently, when rAAV6-INT41 is delivered to the striatum in the R6/2 mouse model, treated female mice exhibited executive function statistically indistinguishable from wild type, accompanied by reductions in Htt aggregates in the striatum, suggesting that rAAV6-INT41 is promising as a gene therapy for Huntington’s disease.

  10. Inhibition of melanoma growth by subcutaneous administration of hTERTC27 viral cocktail in C57BL/6 mice.

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    Longfei Huo

    Full Text Available BACKGROUND: hTERTC27 is a 27 kDa C-terminal polypeptide of human telomerase reverse transcriptase that has previously been shown to reduce tumorigenicity of HeLa cells and suppress growth of xenografted glioblastoma in nude mice. Although ectopic expression of hTERTC27 upregulated genes that are involved in apoptosis, cell cycle, and immune response, the mechanism for hTERTC27-induced tumor suppression has not been completely elucidated. Since hTERT was identified as a universal tumor-associated antigen, we hypothesize that hTERTC27 inhibits tumor growth in vivo through activation of anti-tumor immune response. METHODOLOGY/PRINCIPAL FINDING: Immunocompetent C57BL/6 mice were used for mouse B16 melanoma model. Mice bearing B16 melanoma were administered rAAV-/rAdv viral cocktail expressing hTERTC27, and tumor growth was monitored after viral cocktail treatment. Blood and splenocytes were used to determine the level of cytokines and the activity of immune cells, respectively. B16 tumor growth was significantly inhibited by subcutaneous administration of a single dose of 1.5×10(11 vg rAAV-hTERTC27 and 2.5×10(9 pfu rAdv-hTERTC27 viral cocktail (rAAV-/rAdv-hTERTC27. The population and cytotoxicity of NK cells in the mice were significantly augmented by rAAV-/rAdv-hTERTC27 treatment, and selective depletion of the NK cell population in mice by intraperitoneal injection of anti-GM1 antibody abrogated the growth suppression of melanoma induced by rAAV-/rAdv-hTERTC27 administration. CONCLUSION: Activation of NK cells by administration of rAAV-/rAdv-hTERTC27 is critical for growth suppression of melanoma in mouse model.

  11. Inner ear gene transfection in neonatal mice using adeno-associated viral vector: a comparison of two approaches.

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    Li Xia

    Full Text Available Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs and 17% of outer hair cells (OHCs were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively was slightly higher, but the difference was not significant.

  12. Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites. (United States)

    Flotte, Terence R; Conlon, Thomas J; Poirier, Amy; Campbell-Thompson, Martha; Byrne, Barry J


    To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg. A mouse biodistribution study was performed in 5 cohorts of 10 mice, receiving intramuscular injections at the same doses; as well as in a lower dose cohort (3 x 10(8) VG; equivalent to 1.2 x 10(10)VG/kg); and in 4 other cohorts (excluding the vehicle control) injected with identical doses intravenously. Finally, biodistribution was examined in rabbits, with serial collection of blood and semen, as well as terminal tissue collection. Two significant findings were present, both of which were dose dependent. First, inflammatory cell infiltrates were detected at the site of injection 21, 60, or 90 days after intramuscular injection of 1 x 10(13)VG. This was not associated with loss of transgene expression. Second, vector DNA sequences were detected in most animals, levels being highest with the highest doses and earliest time points. Vector DNA was also present in liver, spleen, kidneys, and a number of other organs, including the gonads of animals receiving the highest dose. Likewise, vector DNA was present in the semen of male rabbits at higher doses. The copy number of vector DNA in the blood and semen declined over time throughout the study. These two dose-dependent findings have served to guide to the design of a phase 1 human trial of rAAV1-AAT.

  13. All optical vector magnetometer, Phase I (United States)

    National Aeronautics and Space Administration — This Phase I research project will investigate a novel method of operating an atomic magnetometer to simultaneously measure total magnetic fields and vector magnetic...


    National Aeronautics and Space Administration — This data set contains the Magellan Surface Characteristics Vector Data Record (SCVDR) which is an orbit-by-orbit reduction of Magellan scattering and emission...

  15. A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

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    Liu Yi


    Full Text Available Abstract Background Gene targeting is a powerful method that can be used for examining the functions of genes. Traditionally, the construction of knockout (KO vectors requires an amplification step to obtain two homologous, large fragments of genomic DNA. Restriction enzymes that cut at unique recognitions sites and numerous cloning steps are then carried out; this is often a time-consuming and frustrating process. Results We have developed a one-step cloning method for the insertion of two arms into a KO vector using exonuclease III. We modified an adeno-associated virus KO shuttle vector (pTK-LoxP-NEO-AAV to yield pAAV-LIC, which contained two cassettes at the two multiple-cloning sites. The vector was digested with EcoRV to give two fragments. The two homologous arms, which had an overlap of 16 bases with the ends of the vector fragments, were amplified by polymerase chain reaction. After purification, the four fragments were mixed and treated with exonuclease III, then transformed into Escherichia coli to obtain the desired clones. Using this method, we constructed SirT1 and HDAC2 KO vectors, which were used to establish SirT1 KO cells from the colorectal cancer cell line (HCT116 and HDAC2 KO cells from the colorectal cancer cell line (DLD1. Conclusions Our method is a fast, simple, and efficient technique for cloning, and has great potential for high-throughput construction of KO vectors.

  16. An AAV promoter-driven neuropeptide Y gene delivery system using Sendai virosomes for neurons and rat brain. (United States)

    Wu, P; de Fiebre, C M; Millard, W J; King, M A; Wang, S; Bryant, S O; Gao, Y P; Martin, E J; Meyer, E M


    An adeno-associated virus (AAV)-derived construct (pJDT95npy) containing rat neuropeptide Y (NPY) cDNA inserted downstream of endogenous AAV promoters was used to investigate AAV-driven NPY expression in postmitotic neurons in vitro and in the brain. NPY mRNA was expressed in NT2/N and rat brain primary neuronal cultures after transfection. There was a corresponding increase in the number of neurons staining for NPY-like immunoreactivity and an increase in NPY release during depolarization in the primary cultures. Injections of Sendai-virosome encapsulated pJDT95npy into neocortex increased NPY-like immunoreactivity in neurons but not glia indicating that the latter cell type did not have the translational, post-translational or storage capacity to accumulate the peptide. Injections into the rat hypothalamic para-ventricular nucleus increased body weight and food intake for 21 days, though NPY-like immunoreactivity remained elevated for at least 50 days. These studies demonstrate that AAV-derived constructs may be useful for delivering genes into post-mitotic neurons, and that Sendai virosomes are effective for delivering these constructs in vivo.

  17. rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study. (United States)

    Rocca, C J; Ur, S N; Harrison, F; Cherqui, S


    Effective gene therapy strategies for the treatment of kidney disorders remain elusive. We report an optimized kidney-targeted gene delivery strategy using recombinant adeno-associated virus (rAAV) administered via retrograde renal vein injection in mice. Renal vein injection of rAAV consistently resulted in superior kidney transduction compared with tail vein injection using as little as half the tail vein dose. We compared rAAV5, 6, 8 and 9, containing either green fluorescent protein (GFP) or luciferase reporter genes driven by the Cytomegalovirus promoter. We demonstrated that although rAAV6 and 8 injected via renal vein transduced the kidney, transgene expression was mainly restricted to the medulla. Transgene expression was systematically low after rAAV5 injection, attributed to T-cell immune response, which could be overcome by transient immunosuppression. However, rAAV9 was the only serotype that permitted high-transduction efficiency of both the cortex and medulla. Moreover, both the glomeruli and tubules were targeted, with a higher efficiency within the glomeruli. To improve the specificity of kidney-targeted gene delivery with rAAV9, we used the parathyroid hormone receptor 'kidney-specific' promoter. We obtained a more efficient transgene expression within the kidney, and a significant reduction in other tissues. Our work represents the first comprehensive and clinically relevant study for kidney gene delivery.

  18. Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors


    Capasso, Cristian; Hirvinen, Mari; Cerullo, Vincenzo


    Viral vectors have been extensively studied due to their great transduction efficiency compared to non-viral vectors. These vectors have been used extensively in gene therapy, enabling the comprehension of, not only the advantages of these vectors, but also the limitations, such as the activation of the immune system after vector administration. Moreover, the need to control the target of the vector has led to the development of chemical and non-chemical modifications of the vector surface, a...

  19. AAV-mediated overexpression of the CB1 receptor in the mPFC of adult rats alters cognitive flexibility, social behavior and emotional reactivity

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    Matthias eKlugmann


    Full Text Available The endocannabinoid (ECB system is strongly involved in the regulation of cognitive processing and emotional behavior and evidence indicates that ECB signaling might affect these behavioral abilities by modulations of prefrontal cortical functions. The aim of the present study was to examine the role of the CB1 receptor in the medial prefrontal cortex (mPFC on cognitive flexibility and emotional behavior. Therefore, the CB1 receptor was overexpressed by adeno-associated virus (AAV vector-mediated gene transfer specifically in the mPFC of adult Wistar rats. Animals were then tested in different anxiety-related paradigms for emotional reactivity (e.g. elevated plus maze (EPM, light/dark emergence test (EMT, social interaction and the attentional set shift task (ASST - an adaptation of the human Wisconsin card sorting test - for cognitive abilities and behavioral flexibility. A subtle increase in exploratory behavior was found in CB1 receptor overexpressing animals (CB1-R compared to empty vector injected controls (Empty in the EMT and EPM, although general locomotor activity did not differ between the groups. During social interaction testing, social contact behavior towards the unknown conspecific was found to be decreased, whereas social withdrawal was increased in CB1-R animals and they showed an inadequate increase in exploratory behavior compared to control animals. In the ASST, impaired reversal learning abilities were detected in CB1-R animals compared to controls, indicating reduced behavioral flexibility. In conclusion, upregulation of the CB1 receptor specifically in the rat mPFC induces alterations in emotional reactivity, leads to inadequate social behavior and impairs cognitive flexibility. These findings might be relevant for neuropsychiatric disorders, since higher cortical CB1 receptor expression levels as well as similar behavioral impairments as observed in the present study have been described in schizophrenic patients.

  20. Towards a Non-Human Primate Model of Alpha-Synucleinopathy for Development of Therapeutics for Parkinson's Disease: Optimization of AAV1/2 Delivery Parameters to Drive Sustained Expression of Alpha Synuclein and Dopaminergic Degeneration in Macaque.

    Directory of Open Access Journals (Sweden)

    James B Koprich

    Full Text Available Recent failures in clinical trials for disease modification in Parkinson's disease have highlighted the need for a non-human primate model of the synucleinopathy underpinning dopaminergic neuron degeneration. The present study was defined to begin the development of such a model in cynomolgus macaque. We have validated surgical and vector parameters to define a means to provide a robust over-expression of alpha-synuclein which is associated with Lewy-like pathology and robust degeneration of the nigrostriatal pathway. Thus, an AAV1/2 vector incorporating strong transcription and transduction regulatory elements was used to deliver the gene for the human A53T mutation of alpha-synuclein. When injected into 4 sites within each substantia nigra (7 μl per site, 1.7 x 1012 gp/ml, this vector provided expression lasting at least 4 months, and a 50% loss of nigral dopaminergic neurons and a 60% reduction in striatal dopamine. Further studies will be required to develop this methodology into a validated model of value as a drug development platform.

  1. Onchocerca volvulus infection and serological prevalence, ocular onchocerciasis and parasite transmission in northern and central Togo after decades of Simulium damnosum s.l. vector control and mass drug administration of ivermectin. (United States)

    Komlan, Kossi; Vossberg, Patrick S; Gantin, Richard G; Solim, Tchalim; Korbmacher, Francois; Banla, Méba; Padjoudoum, Koffi; Karabou, Potchoziou; Köhler, Carsten; Soboslay, Peter T


    Mass drug administration (MDA) of ivermectin has become the main intervention to control onchocerciasis or "river blindness". In Togo, after many years of MDA, Onchocerca volvulus infection has declined dramatically, and elimination appears achievable, but in certain river basins the current situation remains unknown. We have conducted parasitological, serological, ophthalmological, and entomological assessments in northern and central Togo within the river basins of Ôti, Kéran and Mô. Examinations were completed in 1,455 participants from 11 onchocerciasis sentinel villages, and O. volvulus transmission by Simulium damnosum sensu lato (s.l.) was evaluated. In children (aged 1-10 years), the prevalence of microfilariae (Mf) was 2.3% and in adults it ranged from 5.1 to 13.3%. Positive IgG4 responses to O. volvulus adult (crude) worm antigen (OvAg) and the recombinant Ov16 antigen were in all-ages 48.7% and 34.4%, and 29.1% and 14.9% in children, respectively. In the river basin villages of Kéran, Mô and Ôti, the IgG4 seroprevalences to OvAg in children were 51.7%, 23.5% and 12.7%, respectively, and to the Ov16 antigen 33.3% (Kéran) and 5.2% (Ôti). Onchocerciasis ocular lesions (punctate keratitis, evolving iridocyclitis and chorioretinitis) were observed in children and young adults. O. volvulus-specific DNA (Ov150) was detected by poolscreen in vector samples collected from Tchitchira/Kéran(22.8%), Bouzalo/Mô(11.3%), Baghan/Mô(2.9%) and Pancerys/Ôti(4.9%); prevalences of O. volvulus infection in S. damnosum s.l. were, respectively, 1%, 0.5%, 0.1% and 0.2%. In the northern and central river basins in Togo, interruption of O. volvulus transmission has not yet been attained. Patent O. volvulus infections, positive antibody responses, progressive ocular onchocerciasis were diagnosed, and parasite transmission by S. damnosum s.l. occurred close to the survey locations. Future interventions may require approaches selectively targeted to non-complying endemic

  2. rAAV-compatible MiniPromoters for restricted expression in the brain and eye. (United States)

    de Leeuw, Charles N; Korecki, Andrea J; Berry, Garrett E; Hickmott, Jack W; Lam, Siu Ling; Lengyell, Tess C; Bonaguro, Russell J; Borretta, Lisa J; Chopra, Vikramjit; Chou, Alice Y; D'Souza, Cletus A; Kaspieva, Olga; Laprise, Stéphanie; McInerny, Simone C; Portales-Casamar, Elodie; Swanson-Newman, Magdalena I; Wong, Kaelan; Yang, George S; Zhou, Michelle; Jones, Steven J M; Holt, Robert A; Asokan, Aravind; Goldowitz, Daniel; Wasserman, Wyeth W; Simpson, Elizabeth M


    Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

  3. Test of Understanding of Vectors: A Reliable Multiple-Choice Vector Concept Test (United States)

    Barniol, Pablo; Zavala, Genaro


    In this article we discuss the findings of our research on students' understanding of vector concepts in problems without physical context. First, we develop a complete taxonomy of the most frequent errors made by university students when learning vector concepts. This study is based on the results of several test administrations of open-ended…

  4. Viral Vector Mediated Over-Expression of Estrogen Receptor–α in Striatum Enhances the Estradiol-induced Motor Activity in Female Rats and Estradiol Modulated GABA Release (United States)

    Schultz, Kristin N.; von Esenwein, Silke A.; Hu, Ming; Bennett, Amy L.; Kennedy, Robert T.; Musatov, Sergei; Toran-Allerand, C. Dominique; Kaplitt, Michael G.; Young, Larry J.; Becker, Jill B.


    Classical estrogen receptor signaling mechanisms involve estradiol binding to intracellular nuclear receptors (estrogen receptor-α (ERα) and estrogen receptor-β (ERβ)) to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K+- evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERα located on the membrane of medium spiny GABAergic neurons. This experiment examined whether over-expression of ERα in the striatum would enhance the effect of estradiol on rotational behavior and the K+- evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERα cDNA (AAV.ERα) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERα in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared to controls and exhibited behavioral sensitization of contralateral rotations induced by a low dose of amphetamine. ERα over-expression also enhanced the inhibitory effect of estradiol on K+- evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior. PMID:19211896

  5. Viral vector-mediated overexpression of estrogen receptor-alpha in striatum enhances the estradiol-induced motor activity in female rats and estradiol-modulated GABA release. (United States)

    Schultz, Kristin N; von Esenwein, Silke A; Hu, Ming; Bennett, Amy L; Kennedy, Robert T; Musatov, Sergei; Toran-Allerand, C Dominique; Kaplitt, Michael G; Young, Larry J; Becker, Jill B


    Classical estrogen receptor-signaling mechanisms involve estradiol binding to intracellular nuclear receptors [estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta)] to promote changes in protein expression. Estradiol can also exert effects within seconds to minutes, however, a timescale incongruent with genomic signaling. In the brain, estradiol rapidly potentiates stimulated dopamine release in the striatum of female rats and enhances spontaneous rotational behavior. Furthermore, estradiol rapidly attenuates the K(+)-evoked increase of GABA in dialysate. We hypothesize that these rapid effects of estradiol in the striatum are mediated by ERalpha located on the membrane of medium spiny GABAergic neurons. This experiment examined whether overexpression of ERalpha in the striatum would enhance the effect of estradiol on rotational behavior and the K(+)-evoked increase in GABA in dialysate. Ovariectomized female rats were tested for rotational behavior or underwent microdialysis experiments after unilateral intrastriatal injections of a recombinant adeno-associated virus (AAV) containing the human ERalpha cDNA (AAV.ERalpha) into the striatum; controls received either the same vector into areas outside the striatum or an AAV containing the human alkaline phosphatase gene into the striatum (AAV.ALP). Animals that received AAV.ERalpha in the striatum exhibited significantly greater estradiol-induced contralateral rotations compared with controls and exhibited behavioral sensitization of contralateral rotations induced by a low-dose of amphetamine. ERalpha overexpression also enhanced the inhibitory effect of estradiol on K(+)-evoked GABA release suggesting that disinhibition of dopamine release from terminals in the striatum resulted in the enhanced rotational behavior.

  6. How to Successfully Screen Random Adeno-Associated Virus Display Peptide Libraries In Vivo. (United States)

    Körbelin, Jakob; Trepel, Martin


    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.

  7. Raster images vectorization system


    Genytė, Jurgita


    The problem of raster images vectorization was analyzed and researched in this work. Existing vectorization systems are quite expensive, the results are inaccurate, and the manual vectorization of a large number of drafts is impossible. That‘s why our goal was to design and develop a new raster images vectorization system using our suggested automatic vectorization algorithm and the way to record results in a new universal vectorial file format. The work consists of these main parts: analysis...

  8. Systemic injection of AAV9-GDNF provides modest functional improvements in the SOD1G93A ALS rat but has adverse side effects. (United States)

    Thomsen, G M; Alkaslasi, M; Vit, J-P; Lawless, G; Godoy, M; Gowing, G; Shelest, O; Svendsen, C N


    Injecting proteins into the central nervous system that stimulate neuronal growth can lead to beneficial effects in animal models of disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has shown promise in animal and cell models of Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS). Here, systemic AAV9-GDNF was delivered via tail vein injections to young rats to determine whether this could be a safe and functional strategy to treat the SOD1 G93A rat model of ALS and, therefore, translated to a therapy for ALS patients. We found that GDNF administration in this manner resulted in modest functional improvement, whereby grip strength was maintained for longer and the onset of forelimb paralysis was delayed compared to non-treated rats. This did not, however, translate into an extension in survival. In addition, ALS rats receiving GDNF exhibited slower weight gain, reduced activity levels and decreased working memory. Collectively, these results confirm that caution should be applied when applying growth factors such as GDNF systemically to multiple tissues.

  9. Kochen-Specker vectors

    International Nuclear Information System (INIS)

    Pavicic, Mladen; Merlet, Jean-Pierre; McKay, Brendan; Megill, Norman D


    We give a constructive and exhaustive definition of Kochen-Specker (KS) vectors in a Hilbert space of any dimension as well as of all the remaining vectors of the space. KS vectors are elements of any set of orthonormal states, i.e., vectors in an n-dimensional Hilbert space, H n , n≥3, to which it is impossible to assign 1s and 0s in such a way that no two mutually orthogonal vectors from the set are both assigned 1 and that not all mutually orthogonal vectors are assigned 0. Our constructive definition of such KS vectors is based on algorithms that generate MMP diagrams corresponding to blocks of orthogonal vectors in R n , on algorithms that single out those diagrams on which algebraic (0)-(1) states cannot be defined, and on algorithms that solve nonlinear equations describing the orthogonalities of the vectors by means of statistically polynomially complex interval analysis and self-teaching programs. The algorithms are limited neither by the number of dimensions nor by the number of vectors. To demonstrate the power of the algorithms, all four-dimensional KS vector systems containing up to 24 vectors were generated and described, all three-dimensional vector systems containing up to 30 vectors were scanned, and several general properties of KS vectors were found

  10. Pre-clinical evaluation of AAV5-miHTT gene therapy of Huntington´s disease

    Czech Academy of Sciences Publication Activity Database

    Konstantinová, P.; Miniarikova, J.; Blits, B.; Zimmer, V.; Spoerl, A.; Southwell, A.; Hayden, M.; van Deventer, S.; Deglon, N.; Motlík, Jan; Juhás, Štefan; Juhásová, Jana; Richard, Ch.; Petry, H.


    Roč. 78, Supl 2 (2015), s. 8-8 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. 08.11.2015-10.11.2015, Liblice] R&D Projects: GA MŠk ED2.1.00/03.0124 Institutional support: RVO:67985904 Keywords : Huntington´s disease * gene therapy * AAV5-miHTT Subject RIV: EB - Gene tics ; Molecular Biology

  11. AAV-based shRNA silencing of NF-κB ameliorates muscle pathologies in mdx mice. (United States)

    Yang, Q; Tang, Y; Imbrogno, K; Lu, A; Proto, J D; Chen, A; Guo, F; Fu, F H; Huard, J; Wang, B


    Chronic inflammation, promoted by an upregulated NF-kappa B (NF-κB) pathway, has a key role in Duchenne muscular dystrophy (DMD) patients' pathogenesis. Blocking the NF-κB pathway has been shown to be a viable approach to diminish chronic inflammation and necrosis in the dystrophin-defective mdx mouse, a murine DMD model. In this study, we used the recombinant adeno-associated virus serotype 9 (AAV9) carrying an short hairpin RNA (shRNA) specifically targeting the messenger RNA of NF-κB/p65 (p65-shRNA), the major subunit of NF-κB associated with chronic inflammation in mdx mice. We examined whether i.m. AAV9-mediated delivery of p65-shRNA could decrease NF-κB activation, allowing for amelioration of muscle pathologies in 1- and 4-month-old mdx mice. At 1 month after treatment, NF-κB/p65 levels were significantly decreased by AAV gene transfer of p65-shRNA in the two ages of treatment groups, with necrosis significantly decreased compared with controls. Quantitative analysis revealed that central nucleation (CN) of the myofibers of p65-shRNA-treated 1-month-old mdx muscles was reduced from 67 to 34%, but the level of CN was not significantly decreased in treated 4-month-old mdx mice. Moreover, delivery of the p65-shRNA enhanced the capacity of myofiber regeneration in old mdx mice treated at 4 months of age when the dystrophic myofibers were most exhausted; however, such p65 silencing diminished the myofiber regeneration in young mdx mice treated at 1 month of age. Taken together, these findings demonstrate that the AAV-mediated delivery of p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by selectively reducing NF-κB/p65 activity.

  12. Healthy and diseased corticospinal motor neurons are selectively transduced upon direct AAV2-2 injection into the motor cortex. (United States)

    Jara, J H; Stanford, M J; Zhu, Y; Tu, M; Hauswirth, W W; Bohn, M C; DeVries, S H; Özdinler, P H


    Direct gene delivery to the neurons of interest, without affecting other neuron populations in the cerebral cortex, represent a challenge owing to the heterogeneity and cellular complexity of the brain. Genetic modulation of corticospinal motor neurons (CSMN) is required for developing effective and long-term treatment strategies for motor neuron diseases, in which voluntary movement is impaired. Adeno-associated viruses (AAV) have been widely used for neuronal transduction studies owing to long-term and stable gene expression as well as low immunoreactivity in humans. Here we report that AAV2-2 transduces CSMN with high efficiency upon direct cortex injection and that transduction efficiencies are similar during presymptomatic and symptomatic stages in hSOD1(G93A) transgenic amyotrophic lateral sclerosis (ALS) mice. Our findings reveal that choice of promoter improves selectivity as AAV2-2 chicken β-actin promoter injection results in about 70% CSMN transduction, the highest percentage reported to date. CSMN transduction in both wild-type and transgenic ALS mice allows detailed analysis of single axon fibers within the corticospinal tract in both cervical and lumbar spinal cord and reveals circuitry defects, which mainly occur between CSMN and spinal motor neurons in hSOD1(G93A) transgenic ALS mice. Our findings set the stage for CSMN gene therapy in ALS and related motor neuron diseases.

  13. Peripheral blood aspirates overexpressing IGF-I via rAAV gene transfer undergo enhanced chondrogenic differentiation processes. (United States)

    Frisch, Janina; Orth, Patrick; Rey-Rico, Ana; Venkatesan, Jagadeesh Kumar; Schmitt, Gertrud; Madry, Henning; Kohn, Dieter; Cucchiarini, Magali


    Implantation of peripheral blood aspirates induced towards chondrogenic differentiation upon genetic modification in sites of articular cartilage injury may represent a powerful strategy to enhance cartilage repair. Such a single-step approach may be less invasive than procedures based on the use of isolated or concentrated MSCs, simplifying translational protocols in patients. In this study, we provide evidence showing the feasibility of overexpressing the mitogenic and pro-anabolic insulin-like growth factor I (IGF-I) in human peripheral blood aspirates via rAAV-mediated gene transfer, leading to enhanced proliferative and chondrogenic differentiation (proteoglycans, type-II collagen, SOX9) activities in the samples relative to control (reporter rAAV-lacZ) treatment over extended periods of time (at least 21 days, the longest time-point evaluated). Interestingly, IGF-I gene transfer also triggered hypertrophic, osteo- and adipogenic differentiation processes in the aspirates, suggesting that careful regulation of IGF-I expression may be necessary to contain these events in vivo. Still, the current results demonstrate the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage injuries. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  14. Sustained Inhibition of HBV Replication In Vivo after Systemic Injection of AAVs Encoding Artificial Antiviral Primary MicroRNAs. (United States)

    Maepa, Mohube Betty; Ely, Abdullah; Grayson, Wayne; Arbuthnot, Patrick


    Chronic infection with hepatitis B virus (HBV) remains a problem of global significance and improving available treatment is important to prevent life-threatening complications arising in persistently infected individuals. HBV is susceptible to silencing by exogenous artificial intermediates of the RNA interference (RNAi) pathway. However, toxicity of Pol III cassettes and short duration of silencing by effectors of the RNAi pathway may limit anti-HBV therapeutic utility. To advance RNAi-based HBV gene silencing, mono- and trimeric artificial primary microRNAs (pri-miRs) derived from pri-miR-31 were placed under control of the liver-specific modified murine transthyretin promoter. The sequences, which target the X sequence of HBV, were incorporated into recombinant hepatotropic self-complementary adeno-associated viruses (scAAVs). Systemic intravenous injection of the vectors into HBV transgenic mice at a dose of 1 × 10 11 per animal effected significant suppression of markers of HBV replication for at least 32 weeks. The pri-miRs were processed according to the intended design, and intrahepatic antiviral guide sequences were detectable for 40 weeks after the injection. There was no evidence of toxicity, and innate immunostimulation was not detectable following the injections. This efficacy is an improvement on previously reported RNAi-based inhibition of HBV replication and is important to clinical translation of the technology. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Vector regression introduced

    Directory of Open Access Journals (Sweden)

    Mok Tik


    Full Text Available This study formulates regression of vector data that will enable statistical analysis of various geodetic phenomena such as, polar motion, ocean currents, typhoon/hurricane tracking, crustal deformations, and precursory earthquake signals. The observed vector variable of an event (dependent vector variable is expressed as a function of a number of hypothesized phenomena realized also as vector variables (independent vector variables and/or scalar variables that are likely to impact the dependent vector variable. The proposed representation has the unique property of solving the coefficients of independent vector variables (explanatory variables also as vectors, hence it supersedes multivariate multiple regression models, in which the unknown coefficients are scalar quantities. For the solution, complex numbers are used to rep- resent vector information, and the method of least squares is deployed to estimate the vector model parameters after transforming the complex vector regression model into a real vector regression model through isomorphism. Various operational statistics for testing the predictive significance of the estimated vector parameter coefficients are also derived. A simple numerical example demonstrates the use of the proposed vector regression analysis in modeling typhoon paths.

  16. [Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT]. (United States)

    Wang, Jian-Sheng; Zhang, Ming-Xin; Liu, De-Chun; Duan, Xiao-Yi; Zhou, Su-Na; Zhang, Guang-Jian; Yang, Guang-Xiao; Wang, Quan-Ying


    To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Apoptin-HA2-TAT, laying a foundation for gene therapy research of malignant tumors. The Apoptin and HA2-TAT gene were inserted in pUC19/NT4 vector after digested with restriction enzyme. The fusion gene of NT4-Apoptin-HA2-TAT was sub-cloned into the shuttle plasmid of adeno-associated virus; the products were co-transferred into HEK-293 cell line with helper plasmid pAAV/Ad and adeno-plasmid pFG140.The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was measured by quantitative dot blot hybridization. The effect of AAV-NT4-Apoptin -HA2-TAT on HepG2 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. The NT4-Apoptin-HA2-TAT was confirmed by restriction enzyme digestion and DNA sequencing. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cells (3.14 x 10(15) pfu/L). AAV-NT4-Apoptin-HA2-TAT had strong deduce apoptosis effect on HepG2 cells. Compared with Adeno-associated mock virus and in normal cell line NIH3T3, Aden-associated virus NT4-Apoptin-HA2-TAT significantly decreased the survival rate of HepG2 cells. The recombinant adeno-associated virus vector encoding gene NT4-Apoptin-HA2-TAT has been successfully constructed in this experiment by molecular cloning and in vitro recombination techniques, laying a foundation for further research of gene therapy of cancer.

  17. Light axial vector mesons (United States)

    Chen, Kan; Pang, Cheng-Qun; Liu, Xiang; Matsuki, Takayuki


    Inspired by the abundant experimental observation of axial-vector states, we study whether the observed axial-vector states can be categorized into the conventional axial-vector meson family. In this paper we carry out an analysis based on the mass spectra and two-body Okubo-Zweig-Iizuka-allowed decays. Besides testing the possible axial-vector meson assignments, we also predict abundant information for their decays and the properties of some missing axial-vector mesons, which are valuable for further experimental exploration of the observed and predicted axial-vector mesons.

  18. In vivo post-transcriptional gene silencing of alpha-1 antitrypsin by adeno-associated virus vectors expressing siRNA. (United States)

    Cruz, Pedro E; Mueller, Christian; Cossette, Travis L; Golant, Alexandra; Tang, Qiushi; Beattie, Stuart G; Brantly, Mark; Campbell-Thompson, Martha; Blomenkamp, Keith S; Teckman, Jeffrey H; Flotte, Terence R


    alpha-1 Antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population. Homozygosity for the most common mutation (Glu342Lys, PI(*)Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted. This lack of secretion causes severe serum deficiency predisposing to chronic lung disease. Twelve to fifteen percent of patients with PI(*)ZZ also develop liver disease, which can be severe, even in infancy. This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte. Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes. In this report, we describe studies of small-interfering RNAs (siRNAs) designed to downregulate endogenous AAT within hepatocytes. Three different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone, either singly or as a trifunctional (3X) construct. Each had activity independently, but the levels of AAT expression in cell culture models showed the greatest decrease with the 3X construct, resulting in levels that were five-fold lower than controls. The rAAV-3X-siRNA was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT overexpressing transgenic mice. Those studies showed a decrease in total human AAT, a clearing of Z-AAT accumulation by immunohistochemistry, and a decrease in monomer Z-AAT within the liver within 3 weeks after vector injection. The rAAV8-3X-siRNA vector may hold promise as a potential therapy for patients with AAT liver disease.

  19. VectorBase (United States)

    U.S. Department of Health & Human Services — VectorBase is a Bioinformatics Resource Center for invertebrate vectors. It is one of four Bioinformatics Resource Centers funded by NIAID to provide web-based...

  20. Custodial vector model

    DEFF Research Database (Denmark)

    Becciolini, Diego; Franzosi, Diogo Buarque; Foadi, Roshan


    We analyze the Large Hadron Collider (LHC) phenomenology of heavy vector resonances with a $SU(2)_L\\times SU(2)_R$ spectral global symmetry. This symmetry partially protects the electroweak S-parameter from large contributions of the vector resonances. The resulting custodial vector model spectrum...

  1. [Construction and identification of recombinant adeno-associated virus vector harboring fusion gene NT4-Ant-Shepherdin[79-87 (United States)

    Tang, Xiao-Jiang; Ping, Bao-Hua; Pan, Cheng-En; Yang, Guang-Xiao; Wang, Quan-Ying


    To construct a recombinant adeno-associated virus vector harboring fusion gene NT4-Ant-Shepherdin[79-87] and investigate Survivin as a anticancer therapeutic target by use of Shepherdin[79-87]. The gene of Ant-Shepherdin[79-87] was obtained by PCR and T-vector method. After inserted in PBV220-NT4 vector and digested with restricted enzyme, The fusion gene of NT4-Ant-Shepherdin[79-87] was sub-cloned into the shuttle plasmid of adeno-associated virus; the products were co-transferred into HEK-293 cell line with helper plasmid pAAV-Ad and adeno-plasmid pFG140. The recombinant adeno-associated virus was produced by homologous recombination of above 3 plasmids in HEK-293 cells and its titer was measured by Dot-blot hybridization. The effect of rAAV-NT4-Ant-Shepherdin[79-87] on A549 cell line was measured by a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. DNA sequencing results verified that the sequence of Ant-Shepherdin[79-87] was consistent with that we had designed. After transformed E.coli DH5alpha, a fragment of 321 bp was confirmed. High titer of recombinant adeno-associated virus was obtained by homologous recombination in HEK-293 cell lines (3.4x10(13)pfu/L). rAAV-NT4-Ant-Shepherdin[79-87] had strong induce apoptosis effect on A549 cells. The recombinant adeno-associated virus vector encoding fusion gene NT4-Ant-Shepherdin[79-87] is successfully constructed in this experiment by molecular cloning and in vitro recombination techniques, which provided the basis of further research of Survivin for cancer gene therapy.

  2. Rotations with Rodrigues' vector

    International Nuclear Information System (INIS)

    Pina, E


    The rotational dynamics was studied from the point of view of Rodrigues' vector. This vector is defined here by its connection with other forms of parametrization of the rotation matrix. The rotation matrix was expressed in terms of this vector. The angular velocity was computed using the components of Rodrigues' vector as coordinates. It appears to be a fundamental matrix that is used to express the components of the angular velocity, the rotation matrix and the angular momentum vector. The Hamiltonian formalism of rotational dynamics in terms of this vector uses the same matrix. The quantization of the rotational dynamics is performed with simple rules if one uses Rodrigues' vector and similar formal expressions for the quantum operators that mimic the Hamiltonian classical dynamics.

  3. A Genome-Wide Map of AAV-Mediated Human Gene Targeting


    Deyle, David R.; Hansen, R. Scott; Cornea, Anda M.; Li, Li B.; Burt, Amber A.; Alexander, Ian E.; Sandstrom, Richard S.; Stamatoyannopoulos, John A.; Wei, Chia-Lin; Russell, David W.


    To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. An adeno-associated virus vector was used to target identical loci introduced as transcriptionally active retroviral vector proviruses. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats, or DNase I hypersensitive sites. Targeted sit...

  4. MISR Near Real Time (NRT) Level 2 Cloud Motion Vector parameters V001 (United States)

    National Aeronautics and Space Administration — This is the MISR Level 2 Cloud Motion Vector Product containing height-resolved cloud motion vectors with associated data. It is used for MISR Near Real Time...

  5. Supergravity inspired vector curvaton

    International Nuclear Information System (INIS)

    Dimopoulos, Konstantinos


    It is investigated whether a massive Abelian vector field, whose gauge kinetic function is growing during inflation, can be responsible for the generation of the curvature perturbation in the Universe. Particle production is studied and it is shown that the vector field can obtain a scale-invariant superhorizon spectrum of perturbations with a reasonable choice of kinetic function. After inflation the vector field begins coherent oscillations, during which it corresponds to pressureless isotropic matter. When the vector field dominates the Universe, its perturbations give rise to the observed curvature perturbation following the curvaton scenario. It is found that this is possible if, after the end of inflation, the mass of the vector field increases at a phase transition at temperature of order 1 TeV or lower. Inhomogeneous reheating, whereby the vector field modulates the decay rate of the inflaton, is also studied

  6. Orthogonalisation of Vectors

    Indian Academy of Sciences (India)

    The vectors aI, a2 are lin- early independent and each of them has norm 1. How- ever, they are not orthogonal to each other. The Gram-. Schmidt process applied to them gives the vectors ql == (1,0), q2 = (0,1). The distance between the pair {all a2} and the pair {q I' q2} is (t) ~. Can we find another pair of orthonormal vectors ...

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

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


    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. Vectors and their applications

    CERN Document Server

    Pettofrezzo, Anthony J


    Geared toward undergraduate students, this text illustrates the use of vectors as a mathematical tool in plane synthetic geometry, plane and spherical trigonometry, and analytic geometry of two- and three-dimensional space. Its rigorous development includes a complete treatment of the algebra of vectors in the first two chapters.Among the text's outstanding features are numbered definitions and theorems in the development of vector algebra, which appear in italics for easy reference. Most of the theorems include proofs, and coordinate position vectors receive an in-depth treatment. Key concept

  9. The production of viral vectors designed to express large and difficult to express transgenes within neurons. (United States)

    Holehonnur, Roopashri; Lella, Srihari K; Ho, Anthony; Luong, Jonathan A; Ploski, Jonathan E


    Viral vectors are frequently used to deliver and direct expression of transgenes in a spatially and temporally restricted manner within the nervous system of numerous model organisms. Despite the common use of viral vectors to direct ectopic expression of transgenes within the nervous system, creating high titer viral vectors that are capable of expressing very large transgenes or difficult to express transgenes imposes unique challenges. Here we describe the development of adeno-associated viruses (AAV) and lentiviruses designed to express the large and difficult to express GluN2A or GluN2B subunits of the N-methyl-D-aspartate receptor (NMDA) receptor, specifically within neurons. We created a number of custom designed AAV and lentiviral vectors that were optimized for large transgenes, by minimizing DNA sequences that were not essential, utilizing short promoter sequences of 8 widely used promoters (RSV, EFS, TRE3G, 0.4αCaMKII, 1.3αCaMKII, 0.5Synapsin, 1.1Synapsin and CMV) and utilizing a very short (~75 bps) 3' untranslated sequence. Not surprisingly these promoters differed in their ability to express the GluN2 subunits, however surprisingly we found that the neuron specific synapsin and αCaMKII, promoters were incapable of conferring detectable expression of full length GluN2 subunits and detectable expression could only be achieved from these promoters if the transgene included an intron or if the GluN2 subunit transgenes were truncated to only include the coding regions of the GluN2 transmembrane domains. We determined that viral packaging limit, transgene promoter and the presence of an intron within the transgene were all important factors that contributed to being able to successfully develop viral vectors designed to deliver and express GluN2 transgenes in a neuron specific manner. Because these vectors have been optimized to accommodate large open reading frames and in some cases contain an intron to facilitate expression of difficult to express

  10. CNS-directed AAV2-mediated gene therapy ameliorates functional deficits in a murine model of infantile neuronal ceroid lipofuscinosis. (United States)

    Griffey, Megan A; Wozniak, David; Wong, Michael; Bible, Ellen; Johnson, Kendra; Rothman, Steven M; Wentz, Annie E; Cooper, Jonathan D; Sands, Mark S


    The neuronal ceroid lipofuscinoses (Batten disease) are a group of inherited neurodegenerative diseases characterized by the progressive intralysosomal accumulation of autofluorescent material in many cells, visual defects, seizures, cognitive deficits, and premature death. Infantile neuronal ceroid lipofuscinosis (INCL) has the earliest onset ( approximately 1.5 years of age) and is caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Currently there is no effective treatment for children with INCL. In this study, newborn PPT1-deficient mice received two (cortex), four (cortex and hippocampus), or six (cortex, hippocampus, and cerebellum) bilateral intracranial injections of AAV2-PPT1. The AAV-treated animals had localized increases in PPT1 activity, decreased autofluorescent material, improved histologic parameters, and increased brain mass. In addition, the treated animals had dose-dependent improvements in a battery of behavioral tests and improved interictal electroencephalographic tracings. However, there was neither a significant decrease in seizure frequency nor an increase in longevity even in INCL animals receiving six injections. These data suggest that early treatment of INCL using gene transfer techniques can be efficacious. However, higher levels or a broader distribution of PPT1 expression, or both, will be required for more complete correction of this neurodegenerative disease.

  11. High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer. (United States)

    Luo, Yonglun; Li, Juan; Liu, Ying; Lin, Lin; Du, Yutao; Li, Shengting; Yang, Huanming; Vajta, Gábor; Callesen, Henrik; Bolund, Lars; Sørensen, Charlotte Brandt


    Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/∆11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/∆11) fibroblasts. However, all BRCA1(+/∆11) piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.

  12. Low-Fiend Vector Magnetometer (V-400-LF), Phase I (United States)

    National Aeronautics and Space Administration — This 2010 NASA SBIR Phase 1 proposal for an innovative Low-Field Vector Magnetometer (V-400-LF) is a response to subtopic S1.06 Particles and Field Sensors and...

  13. Optimal Thrust Vectoring for an Annular Aerospike Nozzle, Phase I (United States)

    National Aeronautics and Space Administration — Recent success of an annular aerospike flight test by NASA Dryden has prompted keen interest in providing thrust vector capability to the annular aerospike nozzle...

  14. Variable Vector Countermeasure Suit for Space Habitation and Exploration (United States)

    National Aeronautics and Space Administration — The "Variable Vector Countermeasure Suit (V2Suit) for Space Habitation and Exploration" is a visionary system concept that will revolutionize space missions by...

  15. Optimal Thrust Vectoring for an Annular Aerospike Nozzle Project (United States)

    National Aeronautics and Space Administration — Recent success of an annular aerospike flight test by NASA Dryden has prompted keen interest in providing thrust vector capability to the annular aerospike nozzle...

  16. Nitrous Oxide Liquid Injection Thrust Vector Control System Testing Project (United States)

    National Aeronautics and Space Administration — A Nitrous Oxide-fed Liquid Thrust Vector Control system is proposed as an efficient method for vehicle attitude control during powered flight. Pulled from a N2O main...

  17. Self-Calibrating Vector Helium Magnetometer (SVHM), Phase I (United States)

    National Aeronautics and Space Administration — This Phase I SBIR proposal describes proposed development of a conceptual design for a Self-Calibrating Vector Helium Magnetometer (SVHM) for design and fabrication...

  18. Low Power, Self Calibrated Vector Magnetometer, Phase I (United States)

    National Aeronautics and Space Administration — This Phase I SBIR project investigates a novel approach to vector magnetometry based on high precision measurements of the total magnetic field. The calibration is...

  19. Optimal Thrust Vectoring for an Annular Aerospike Nozzle, Phase II (United States)

    National Aeronautics and Space Administration — Recent success of an annular aerospike flight test by NASA Dryden has prompted keen interest in providing thrust vector capability to the annular aerospike nozzle...

  20. Self-Calibrating Vector Helium Magnetometer (SVHM) Project (United States)

    National Aeronautics and Space Administration — This Phase 2 SBIR proposal describes the design, fabrication and calibration of a brass-board Self-Calibrating Vector Helium Magnetometer (SVHM). The SVHM instrument...

  1. Modeling non-Gaussian time-varying vector autoregressive process (United States)

    National Aeronautics and Space Administration — We present a novel and general methodology for modeling time-varying vector autoregressive processes which are widely used in many areas such as modeling of chemical...

  2. Vector-Vector Scattering on the Lattice (United States)

    Romero-López, Fernando; Urbach, Carsten; Rusetsky, Akaki


    In this work we present an extension of the LüScher formalism to include the interaction of particles with spin, focusing on the scattering of two vector particles. The derived formalism will be applied to Scalar QED in the Higgs Phase, where the U(1) gauge boson acquires mass.

  3. De Vector wetenschappelijk bekeken

    NARCIS (Netherlands)

    Slot, D.E.; van der Weijden, F.


    In juni 2008 heeft de sectie Parodontologie van ACTA een systematische review gepubliceerd over het effect van de Vector®. De vraagstelling luidde: Wat is het effect van het gebruik van de Vector® op (menselijke) gebitselementen vergeleken met ultrasone apparatuur of handinstrumentarium op

  4. Brane vector phenomenology

    International Nuclear Information System (INIS)

    Clark, T.E.; Love, S.T.; Nitta, Muneto; Veldhuis, T. ter; Xiong, C.


    Local oscillations of the brane world are manifested as massive vector fields. Their coupling to the Standard Model can be obtained using the method of nonlinear realizations of the spontaneously broken higher-dimensional space-time symmetries, and to an extent, are model independent. Phenomenological limits on these vector field parameters are obtained using LEP collider data and dark matter constraints

  5. Advancing vector biology research

    NARCIS (Netherlands)

    Kohl, Alain; Pondeville, Emilie; Schnettler, Esther; Crisanti, Andrea; Supparo, Clelia; Christophides, George K.; Kersey, Paul J.; Maslen, Gareth L.; Takken, Willem; Koenraadt, Constantianus J.M.; Oliva, Clelia F.; Busquets, Núria; Abad, F.X.; Failloux, Anna Bella; Levashina, Elena A.; Wilson, Anthony J.; Veronesi, Eva; Pichard, Maëlle; Arnaud Marsh, Sarah; Simard, Frédéric; Vernick, Kenneth D.


    Vector-borne pathogens impact public health, animal production, and animal welfare. Research on arthropod vectors such as mosquitoes, ticks, sandflies, and midges which transmit pathogens to humans and economically important animals is crucial for development of new control measures that target

  6. Vector mesons in matter

    Indian Academy of Sciences (India)

    One consequence of the chiral restoration is the mixing of parity partners. We look for a possible signature of the mixing of vector and axial vector mesons in heavy-ion collisions. We suggest an experimental method for its observation. The dynamical evolution of the heavy-ion collision is described by a transport equation of ...

  7. Architecture and Vector Control

    DEFF Research Database (Denmark)

    von Seidlein, Lorenz; Knols, Bart GJ; Kirby, Matthew


    The character of buildings plays a major role in disease control. It is understood that water supply and sanitation are critical for the well-being of residents. Current vector control programs aim to prevent the access of vector to the human host. This can be achieved through screened windows, c...

  8. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    The two branches of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions...... or meromorphic (allowing poles as singularities) functions. There already exists a well-developed theory for iterative holomorphic dynamical systems, and successful relations found between iteration theory and flows of vector fields have been one of the main motivations for the recent interest in holomorphic...... vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...

  9. Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    vector fields. Since the class of complex polynomial vector fields in the plane is natural to consider, it is remarkable that its study has only begun very recently. There are numerous fundamental questions that are still open, both in the general classification of these vector fields, the decomposition...... of parameter spaces into structurally stable domains, and a description of the bifurcations. For this reason, the talk will focus on these questions for complex polynomial vector fields.......The two branches of dynamical systems, continuous and discrete, correspond to the study of differential equations (vector fields) and iteration of mappings respectively. In holomorphic dynamics, the systems studied are restricted to those described by holomorphic (complex analytic) functions...

  10. AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy. (United States)

    Morabito, Giuseppe; Giannelli, Serena G; Ordazzo, Gabriele; Bido, Simone; Castoldi, Valerio; Indrigo, Marzia; Cabassi, Tommaso; Cattaneo, Stefano; Luoni, Mirko; Cancellieri, Cinzia; Sessa, Alessandro; Bacigaluppi, Marco; Taverna, Stefano; Leocani, Letizia; Lanciego, José L; Broccoli, Vania


    The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  11. Adeno-associated virus (AAV-mediated suppression of Ca2+/calmodulin kinase IV activity in the nucleus accumbens modulates emotional behaviour in mice

    Directory of Open Access Journals (Sweden)

    Bading Hilmar


    Full Text Available Abstract Background Calcium/calmodulin-dependent protein kinase IV (CaMKIV controls activity-dependent gene transcription by regulating the activity of the cyclic AMP response element binding protein (CREB. This signaling pathway is involved in gating emotional responses in the CNS but previous studies did not address the potential roles of CaMKIV in discrete brain regions. In the present study, we aimed at specifically dissecting the role of CaMKIV in the nucleus accumbens of adult mice. Results We used recombinant adeno-associated virus (rAAV-mediated gene transfer of a dominant-negative CaMKIV variant (rAAV-dnCaMKIV to inhibit endogenous CaMKIV in the nucleus accumbens. rAAV-dnCaMKIV treated animals were subjected to a battery of tests including, prepulse inhibition of the acoustic startle response, open field, social interaction and anxiety-related behaviour. We found that basal locomotor activity in the open field, and prepulse inhibition or startle performance were unaltered in mice infected with rAAV-dnCaMKIV in the nucleus accumbens. However, anxiogenic effects were revealed in social interaction testing and the light/dark emergence test. Conclusion Our findings suggest a modulatory role of CaMKIV in the nucleus accumbens in anxiety-like behaviour but not sensorimotor gating.

  12. Revisiting the vector and axial-vector vacuum susceptibilities

    International Nuclear Information System (INIS)

    Chang Lei; Liu Yuxin; Sun Weimin; Zong Hongshi


    We re-investigate the vector and axial-vector vacuum susceptibilities by taking advantage of the vector and axial-vector Ward-Takahashi identities. We show analytically that, in the chiral limit, the vector vacuum susceptibility is zero and the axial-vector vacuum susceptibility equals three fourths of the square of the pion decay constant. Besides, our analysis reproduces the Weinberg sum rule

  13. Antitumor activity and inhibitory effects on cancer stem cell-like properties of Adeno-associated virus (AAV) -mediated Bmi-1 interference driven by Bmi-1 promoter for gastric cancer (United States)

    Wang, Xiaofeng; Liu, Xinyang; Huang, Mingzhu; Gan, Lu; Cheng, Yufan; Li, Jin


    Bmi-1 is aberrantly activated in various cancers and plays a vital role in maintaining the self-renewal of stem cells. Our previous research revealed that Bmi-1 was overexpressed in gastric cancer (GC) and it's overexpression was an independent negative prognostic factor, suggesting it can be a therapeutic target. The main purpose of this investigation was to explore the antitumor activity of Bmi-1 interference driven by its own promoter (Ad-Bmi-1i) for GC. In this study, we used adenoviral vector to deliver Bmi-1 shRNA driven by its own promoter to treat GC. Our results revealed that Ad-Bmi-1i could selectively silence Bmi-1 in GC cells which overexpress Bmi-1 and suppress the malignant phenotypes and stem-like properties of GC cells in vitro and in vivo. Moreover, direct injection of Ad-Bmi-1i into xenografts suppressed tumor growth and destroyed cancer cells in vivo. Ad-Bmi-1i inhibited the proliferation of GC cells mainly via inducing senescence in vitro, but it suppressed tumor through inducing senescence and apoptosis, and inhibiting angiogenesis in vivo. Bmi-1 knockdown by Ad-Bmi-1i downregulated VEGF via inhibiting AKT activity. These results suggest that Ad-Bmi-1i not only inhibits tumor growth and stem cell-like phenotype by inducing cellular senescence directly, but also has an indirect anti-tumor activity by anti-angiogenesis effects via regulating PTEN/AKT/VEGF pathway. Transfer of gene interference guided by its own promoter by an adeno-associated virus (AAV) vector might be a potent antitumor approach for cancer therapy. PMID:27009837

  14. Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases. (United States)

    Luo, Yongquan; Liu, Chengyu; Cerbini, Trevor; San, Hong; Lin, Yongshun; Chen, Guokai; Rao, Mahendra S; Zou, Jizhong


    Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a "safe harbor" locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken β-actin/rabbit β-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo. ©AlphaMed Press.

  15. Beyond Gene Delivery: Strategies to Engineer the Surfaces of Viral Vectors

    Directory of Open Access Journals (Sweden)

    Cristian Capasso


    Full Text Available Viral vectors have been extensively studied due to their great transduction efficiency compared to non-viral vectors. These vectors have been used extensively in gene therapy, enabling the comprehension of, not only the advantages of these vectors, but also the limitations, such as the activation of the immune system after vector administration. Moreover, the need to control the target of the vector has led to the development of chemical and non-chemical modifications of the vector surface, allowing researchers to modify the tropism and biodistribution profile of the vector, leading to the production of viral vectors able to target different tissues and organs. This review describes recent non-genetic modifications of the surfaces of viral vectors to decrease immune system activation and to control tissue targeting. The developments described herein provide opportunities for applications of gene therapy to treat acquired disorders and genetic diseases and to become useful tools in regenerative medicine.

  16. Noncausal Bayesian Vector Autoregression

    DEFF Research Database (Denmark)

    Lanne, Markku; Luoto, Jani

    We propose a Bayesian inferential procedure for the noncausal vector autoregressive (VAR) model that is capable of capturing nonlinearities and incorporating effects of missing variables. In particular, we devise a fast and reliable posterior simulator that yields the predictive distribution...

  17. Tagged Vector Contour (TVC) (United States)

    Kansas Data Access and Support Center — The Kansas Tagged Vector Contour (TVC) dataset consists of digitized contours from the 7.5 minute topographic quadrangle maps. Coverage for the state is incomplete....

  18. Vector hysteresis models

    Czech Academy of Sciences Publication Activity Database

    Krejčí, Pavel


    Roč. 2, - (1991), s. 281-292 ISSN 0956-7925 Keywords : vector hysteresis operator * hysteresis potential * differential inequality Subject RIV: BA - General Mathematics

  19. Support vector machines applications

    CERN Document Server

    Guo, Guodong


    Support vector machines (SVM) have both a solid mathematical background and good performance in practical applications. This book focuses on the recent advances and applications of the SVM in different areas, such as image processing, medical practice, computer vision, pattern recognition, machine learning, applied statistics, business intelligence, and artificial intelligence. The aim of this book is to create a comprehensive source on support vector machine applications, especially some recent advances.

  20. Vector financial rogue waves

    International Nuclear Information System (INIS)

    Yan, Zhenya


    The coupled nonlinear volatility and option pricing model presented recently by Ivancevic is investigated, which generates a leverage effect, i.e., stock volatility is (negatively) correlated to stock returns, and can be regarded as a coupled nonlinear wave alternative of the Black–Scholes option pricing model. In this Letter, we analytically propose vector financial rogue waves of the coupled nonlinear volatility and option pricing model without an embedded w-learning. Moreover, we exhibit their dynamical behaviors for chosen different parameters. The vector financial rogue wave (rogon) solutions may be used to describe the possible physical mechanisms for the rogue wave phenomena and to further excite the possibility of relative researches and potential applications of vector rogue waves in the financial markets and other related fields. -- Highlights: ► We investigate the coupled nonlinear volatility and option pricing model. ► We analytically present vector financial rogue waves. ► The vector financial rogue waves may be used to describe the extreme events in financial markets. ► This results may excite the relative researches and potential applications of vector rogue waves.

  1. Administrating Solr

    CERN Document Server

    Mohan, Surendra


    A fast-paced, example-based guide to learning how to administrate, monitor, and optimize Apache Solr.""Administrating Solr"" is for developers and Solr administrators who have a basic knowledge of Solr and who are looking for ways to keep their Solr server healthy and well maintained. A basic working knowledge of Apache Lucene is recommended, but this is not mandatory.

  2. Administrative Synergy (United States)

    Hewitt, Kimberly Kappler; Weckstein, Daniel K.


    One of the biggest obstacles to overcome in creating and sustaining an administrative professional learning community (PLC) is time. Administrators are constantly deluged by the tyranny of the urgent. It is a Herculean task to carve out time for PLCs, but it is imperative to do so. In this article, the authors describe how an administrative PLC…

  3. Space administration


    Worthington, Scott; Worthington, Scott


    My dissertation consists of two parts. The larger portion is an hour-long piece for double bass, electronics, and projected text called Space Administration. The second portion, this essay, discusses my musical background leading up to Space Administration, details of the composition itself, and what new directions I see in my work that in part stem from creating the piece Space Administration

  4. Recombinant Adeno-associated virus (rAAV)-mediated transduction and optogenetic manipulation of cortical neurons in vitro (United States)

    Lange, Wienke; Jin, Lei; Maybeck, Vanessa; Meisenberg, Annika; Baumann, Arnd; Offenhäusser, Andreas


    Genetically encoded light-sensitive proteins can be used to manipulate and observe cellular functions. According to different modes of action, these proteins are divided into actuators like the blue-light gated cation channel Channelrhodopsin-2 (ChR2) and detectors like the calcium sensor GCaMP. In order to optogenetically control and study the activity of rat primary cortical neurons, we established a transduction procedure using recombinant Adeno-associated viruses (rAAVs) as gene-ferries. Thereby, we achieved high transduction rates of these neurons with ChR2. In ChR2 expressing neurons, action potentials could be repeatedly and precisely elicited with laser pulses and measured via patch clamp recording.

  5. Adeno-Associated Virus (AAV) Mediated Dystrophin Gene Transfer Studies and Exon Skipping Strategies for Duchenne Muscular Dystrophy (DMD). (United States)

    Kawecka, Klaudia; Theodoulides, Michael; Hasoglu, Yalin; Jarmin, Susan; Kymalainen, Hanna; Le-Heron, Anita; Popplewell, Linda; Malerba, Alberto; Dickson, George; Athanasopoulos, Takis


    Duchenne muscular dystrophy (DMD), an X-linked inherited musclewasting disease primarily affecting young boys with prevalence of between1:3,500- 1:5,000, is a rare genetic disease caused by defects in the gene for dystrophin. Dystrophin protein is critical to the stability of myofibers in skeletal and cardiac muscle. There is currently no cure available to ameliorate DMD and/or its patho-physiology. A number of therapeutic strategies including molecular-based therapeutics that replace or correct the missing or nonfunctional dystrophin protein have been devised to correct the patho-physiological consequences induced by dystrophin absence. We will review the current in vivo experimentation status (including preclinical models and clinical trials) for two of these approaches, namely: 1) Adeno-associated virus (AAV) mediated (micro) dystrophin gene augmentation/ supplementation and 2) Antisense oligonucleotide (AON)-mediated exon skipping strategies.

  6. AAV-mediated, optogenetic ablation of Müller Glia leads to structural and functional changes in the mouse retina.

    Directory of Open Access Journals (Sweden)

    Leah C Byrne

    Full Text Available Müller glia, the primary glial cell in the retina, provide structural and metabolic support for neurons and are essential for retinal integrity. Müller cells are closely involved in many retinal degenerative diseases, including macular telangiectasia type 2, in which impairment of central vision may be linked to a primary defect in Müller glia. Here, we used an engineered, Müller-specific variant of AAV, called ShH10, to deliver a photo-inducibly toxic protein, KillerRed, to Müller cells in the mouse retina. We characterized the results of specific ablation of these cells on visual function and retinal structure. ShH10-KillerRed expression was obtained following intravitreal injection and eyes were then irradiated with green light to induce toxicity. Induction of KillerRed led to loss of Müller cells and a concomitant decrease of Müller cell markers glutamine synthetase and cellular retinaldehyde-binding protein, reduction of rhodopsin and cone opsin, and upregulation of glial fibrillary acidic protein. Loss of Müller cells also resulted in retinal disorganization, including thinning of the outer nuclear layer and the photoreceptor inner and outer segments. High resolution imaging of thin sections revealed displacement of photoreceptors from the ONL, formation of rosette-like structures and the presence of phagocytic cells. Furthermore, Müller cell ablation resulted in increased area and volume of retinal blood vessels, as well as the formation of tortuous blood vessels and vascular leakage. Electrophysiologic measures demonstrated reduced retinal function, evident in decreased photopic and scotopic electroretinogram amplitudes. These results show that loss of Müller cells can cause progressive retinal degenerative disease, and suggest that AAV delivery of an inducibly toxic protein in Müller cells may be useful to create large animal models of retinal dystrophies.

  7. Adenoviral vector immunity: its implications and circumvention strategies. (United States)

    Ahi, Yadvinder S; Bangari, Dinesh S; Mittal, Suresh K


    Adenoviral (Ad) vectors have emerged as a promising gene delivery platform for a variety of therapeutic and vaccine purposes during last two decades. However, the presence of preexisting Ad immunity and the rapid development of Ad vector immunity still pose significant challenges to the clinical use of these vectors. Innate inflammatory response following Ad vector administration may lead to systemic toxicity, drastically limit vector transduction efficiency and significantly abbreviate the duration of transgene expression. Currently, a number of approaches are being extensively pursued to overcome these drawbacks by strategies that target either the host or the Ad vector. In addition, significant progress has been made in the development of novel Ad vectors based on less prevalent human Ad serotypes and nonhuman Ad. This review provides an update on our current understanding of immune responses to Ad vectors and delineates various approaches for eluding Ad vector immunity. Approaches targeting the host and those targeting the vector are discussed in light of their promises and limitations.

  8. Administrative Circulars

    CERN Multimedia

    Département des Ressources humaines


    Administrative Circular N° 2 (Rev. 2) - May 2004 Guidelines and procedures concerning recruitment and probation period of staff members This circular has been revised. It cancels and replaces Administrative Circular N° 2 (Rev. 1) - March 2000. Administrative Circular N° 9 (Rev. 3) - May 2004 Staff members contracts This circular has been revised. It cancels and replaces Administrative Circular N° 9 (Rev. 2) - March 2000. Administrative Circular N° 26 (Rev. 4) - May 2004 Procedure governing the career evolution of staff members This circular has also been revised. It Administrative Circulars Administrative Circular N° 26 (Rev. 3) - December 2001 and brings up to date the French version (Rev. 4) published on the HR Department Web site in January 2004. Operational Circular N° 7 - May 2004 Work from home This circular has been drawn up. Operational Circular N° 8 - May 2004 Dealing with alcohol-related problems...

  9. GFAP-driven GFP expression in activated mouse Muller glial cells aligning retinal blood vessels following intravitreal injection of AAV2/6 vectors.

    NARCIS (Netherlands)

    Aartsen, W.M.; Cleef, K.W.R. van; Pellissier, L.P.; Hoek, R.M.; Vos, R.M.; Blits, B.; Ehlert, E.M.; Balaggan, K.S.; Ali, R.R.; Verhaagen, J.; Wijnholds, J.


    BACKGROUND: Muller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Muller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy. METHODOLOGY/PRINCIPAL

  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, Marc J; Verhaagen, J; Oudega, M.


    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. Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2 (United States)

    Hiller, Thomas; Röhrs, Viola; Dehne, Eva-Maria; Wagner, Anke; Fechner, Henry; Lauster, Roland; Kurreck, Jens


    This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models. In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects. PMID:27805597

  12. Political administration


    Åkerstrøm Andersen, Niels


    One of the major discussions of the 1990s has been about the relation between politics and administration. The themes of the discussions have been many and varied. It has been suggested that the level of politics should concentrate on the general political outlining and entrust the remaining to the administration. It has been criticised that politicians make their decisions on the basis of single cases, which ought to be an administrative matter entirely. It has been a theme that efficient op...

  13. Administrative Reform

    DEFF Research Database (Denmark)

    Plum, Maja

    Through the example of a Danish reform of educational plans in early childhood education, the paper critically addresses administrative educational reforms promoting accountability, visibility and documentation. Drawing on Foucaultian perspectives, the relation between knowledge and governing...... of administrative technology, tracing how the humanistic values of education embed and are embedded within ‘the professional nursery teacher' as an object and subject of administrative practice. Rather than undermining the humanistic potential of education, it is argued that the technology of accounting...

  14. Multithreading in vector processors

    Energy Technology Data Exchange (ETDEWEB)

    Evangelinos, Constantinos; Kim, Changhoan; Nair, Ravi


    In one embodiment, a system includes a processor having a vector processing mode and a multithreading mode. The processor is configured to operate on one thread per cycle in the multithreading mode. The processor includes a program counter register having a plurality of program counters, and the program counter register is vectorized. Each program counter in the program counter register represents a distinct corresponding thread of a plurality of threads. The processor is configured to execute the plurality of threads by activating the plurality of program counters in a round robin cycle.

  15. Twisting of paramodular vectors


    Johnson-Leung, Jennifer; Roberts, Brooks


    Let $F$ be a non-archimedean local field of characteristic zero, let $(\\pi,V)$ be an irreducible, admissible representation of $\\GSp(4,F)$ with trivial central character, and let $\\chi$ be a quadratic character of $F^\\times$ with conductor $c(\\chi)>1$. We define a twisting operator $T_\\chi$ from paramodular vectors for $\\pi$ of level $n$ to paramodular vectors for $\\chi \\otimes \\pi$ of level $\\max(n+2c(\\chi),4c(\\chi))$, and prove that this operator has properties analogous to the well-known $...

  16. Matrix vector analysis

    CERN Document Server

    Eisenman, Richard L


    This outstanding text and reference applies matrix ideas to vector methods, using physical ideas to illustrate and motivate mathematical concepts but employing a mathematical continuity of development rather than a physical approach. The author, who taught at the U.S. Air Force Academy, dispenses with the artificial barrier between vectors and matrices--and more generally, between pure and applied mathematics.Motivated examples introduce each idea, with interpretations of physical, algebraic, and geometric contexts, in addition to generalizations to theorems that reflect the essential structur

  17. Sums and Gaussian vectors

    CERN Document Server

    Yurinsky, Vadim Vladimirovich


    Surveys the methods currently applied to study sums of infinite-dimensional independent random vectors in situations where their distributions resemble Gaussian laws. Covers probabilities of large deviations, Chebyshev-type inequalities for seminorms of sums, a method of constructing Edgeworth-type expansions, estimates of characteristic functions for random vectors obtained by smooth mappings of infinite-dimensional sums to Euclidean spaces. A self-contained exposition of the modern research apparatus around CLT, the book is accessible to new graduate students, and can be a useful reference for researchers and teachers of the subject.

  18. Nuclear medium effects via quasielastic (p vector, p vector') and (p vector, n vector) scattering

    International Nuclear Information System (INIS)

    Hillhouse, G.C.; Ventel, B.I.S. van der; Wyngaardt, S.M.; De Kock, P.R.


    For a 40 Ca target at a fixed momentum transfer of 1.97 fm -1 , and incident energies between 135 and 300 MeV, we quantitatively investigate the sensitivity of complete sets of quasielastic (p vector, p vector') and (p vector, n vector) spin observables to medium effects, pseudoscalar versus pseudovector forms of the πNN vertex, and exchange contributions to the NN amplitudes using a relativistic PWIA. The qualitative nature of previous studies failed to give an indication of the experimental statistical uncertainty required for distinguishing between the various model predictions. New Horowitz-Love-Franey relativistic NN amplitudes have been generated in order to yield improved and more quantitative spin observable values than before. This study indicates at which energies particular spin observables need to be measured in order to test current nuclear models. A comparison to the limited available data indicates that the relativistic parametrization of the NN scattering amplitudes in terms of only the five Fermi invariants (the SVPAT form) is questionable. (author)

  19. Orthogonalisation of Vectors

    Indian Academy of Sciences (India)

    converting these vectors to orthonormal ones demanded by the model. The process of finding the closest or- thonormal basis is called the Lowdin orthogonalisation after the Swedish chemist P 0 Lowdin who introduced it. This is related to one of the basic theorems in linear algebra as we will see. Matrix Approximation ...

  20. Calculus with vectors

    CERN Document Server

    Treiman, Jay S


    Calculus with Vectors grew out of a strong need for a beginning calculus textbook for undergraduates who intend to pursue careers in STEM. fields. The approach introduces vector-valued functions from the start, emphasizing the connections between one-variable and multi-variable calculus. The text includes early vectors and early transcendentals and includes a rigorous but informal approach to vectors. Examples and focused applications are well presented along with an abundance of motivating exercises. All three-dimensional graphs have rotatable versions included as extra source materials and may be freely downloaded and manipulated with Maple Player; a free Maple Player App is available for the iPad on iTunes. The approaches taken to topics such as the derivation of the derivatives of sine and cosine, the approach to limits, and the use of "tables" of integration have been modified from the standards seen in other textbooks in order to maximize the ease with which students may comprehend the material. Additio...

  1. Estimation of vector velocity

    DEFF Research Database (Denmark)


    Using a pulsed ultrasound field, the two-dimensional velocity vector can be determined with the invention. The method uses a transversally modulated ultrasound field for probing the moving medium under investigation. A modified autocorrelation approach is used in the velocity estimation. The new...

  2. Sesquilinear uniform vector integral

    Indian Academy of Sciences (India)

    absolute integral which generalizes the Lebesgue integral and is more easy to manipulate. We speak so far about scalar integrals (scalar functions integrated with respect to positive measures-speaking approximately). The idea of integrating vector functions with respect to scalar measures came naturally on the scene of ...

  3. Vector-borne Infections

    Centers for Disease Control (CDC) Podcasts


    This podcast discusses emerging vector-borne pathogens, their role as prominent contributors to emerging infectious diseases, how they're spread, and the ineffectiveness of mosquito control methods.  Created: 4/18/2011 by National Center for Emerging Zoonotic and Infectious Diseases (NCEZID).   Date Released: 4/27/2011.

  4. Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction (United States)

    Xiao, Ping-Jie; Mitchell, Angela M.; Huang, Lu; Li, Chengwen; Samulski, R. Jude


    Perinuclear retention of viral particles is a poorly understood phenomenon observed during many virus infections. In this study, we investigated whether perinuclear accumulation acts as a barrier to limit recombinant adeno-associated virus (rAAV) transduction. After nocodazole treatment to disrupt microtubules at microtubule-organization center (MT-MTOC) after virus entry, we observed higher rAAV transduction. To elucidate the role of MT-MTOC in rAAV infection and study its underlying mechanisms, we demonstrated that rAAV's perinuclear localization was retained by MT-MTOC with fluorescent analysis, and enhanced rAAV transduction from MT-MTOC disruption was dependent on the rAAV capsid's nuclear import signals. Interestingly, after knocking down RhoA or inhibiting its downstream effectors (ROCK and Actin), MT-MTOC disruption failed to increase rAAV transduction or nuclear entry. These data suggest that enhancement of rAAV transduction is the result of increased trafficking to the nucleus via the RhoA-ROCK-Actin pathway. Ten-fold higher rAAV transduction was also observed by disrupting MT-MTOC in brain, liver, and tumor in vivo. In summary, this study indicates that virus perinuclear accumulation at MT-MTOC is a barrier-limiting parameter for effective rAAV transduction and defines a novel defense mechanism by which host cells restrain viral invasion. PMID:26942476

  5. SAT administrator

    International Nuclear Information System (INIS)

    Havas, A.


    SAT Administrator is the Information System for Nuclear Power Plant Personnel Training Program Design. It supports the design of training programs in the following phases: job analysis; task analysis; competency analysis; task competency association; definition of learning objectives to competencies; training program design; definition of test items. The general structure of the database and management software supports application of the SAT Administrator in any nuclear power installation

  6. Offentlig administration

    DEFF Research Database (Denmark)

    Nielsen, Elof Nellemann; Rehr, Preben René

    En undervisningsbog der henvender sig til administrationsbacheloruddannelsen. Kapitlerne er inddelt efter modulerne på uddannelsen og indeholder derfor elementer af administration, forvaltning, økonomistyring, innovation, samfundsvidenskabelige metoder og politisk styrede organisationer.......En undervisningsbog der henvender sig til administrationsbacheloruddannelsen. Kapitlerne er inddelt efter modulerne på uddannelsen og indeholder derfor elementer af administration, forvaltning, økonomistyring, innovation, samfundsvidenskabelige metoder og politisk styrede organisationer....

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


    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, 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 rAAV

  8. Vector-borne diseases

    DEFF Research Database (Denmark)

    More, Simon J.; Bicout, Dominique; Bøtner, Anette


    After a request from the Europea n Commission, EFSA’s Panel on Animal Health and Welfaresummarised the main characteristics of 36 vector-borne disease s (VBDs) in 36 web-based storymaps.The risk of introduction in the EU through movement of livestock or pets was assessed for eac h of the36 VBDs...... individually, using a semiquantitative Metho d to INTegrate all relevant RISK aspects(MINTRI SK model), which was further modified to a European scale into the EFSA-VBD-RISK-m odel .Only eight of the 36 VBD-agents had an overall rate of introduction in the EU (being the combinationof the rate of entry, vector...... transmission and establishment) which was estimated to be above 0.001introductions per year. These were Crimean-Congo haemorrhagic fever virus, bluetongue virus, WestNile virus, Schmallenberg virus, Hepatozoon canis, Leishmania infantum, Bunyamwera virus andHighlands J. virus. For these eight dise ases...

  9. Tensor Calculus: Unlearning Vector Calculus (United States)

    Lee, Wha-Suck; Engelbrecht, Johann; Moller, Rita


    Tensor calculus is critical in the study of the vector calculus of the surface of a body. Indeed, tensor calculus is a natural step-up for vector calculus. This paper presents some pitfalls of a traditional course in vector calculus in transitioning to tensor calculus. We show how a deeper emphasis on traditional topics such as the Jacobian can…

  10. Propagating Gateway Vectors. (United States)

    Reece-Hoyes, John S; Walhout, Albertha J M


    Generating stocks of Entry and Destination vectors for use in the Gateway recombinatorial cloning system requires transforming them into Escherichia coli strain DB3.1, where they can replicate because this strain is immune to the effects of the ccdB gene carried in the Gateway cassette. However, mutations in the ccdB gene can arise at low frequency, and these mutant plasmids will consequently allow growth of standard cloning strains of E. coli (e.g., DH5α). Therefore, after making new stocks of Gateway plasmids, their ability to grow in cloning strains of E. coli must be tested. This involves obtaining multiple stocks of vector, each arising from a single plasmid grown in a single DB3.1 bacterial colony, and transforming each stock into both DB3.1 and the preferred cloning strain of E. coli in a controlled fashion. Only vector stocks that effectively kill the standard cloning strain (i.e., no or few colonies are obtained after transformation) should be used in Gateway cloning reactions. The sequence can be performed in 3 d. © 2018 Cold Spring Harbor Laboratory Press.

  11. Administrative Reform

    DEFF Research Database (Denmark)

    Plum, Maja

    Through the example of a Danish reform of educational plans in early childhood education, the paper critically addresses administrative educational reforms promoting accountability, visibility and documentation. Drawing on Foucaultian perspectives, the relation between knowledge and governing......, implied in the reform, is analysed as a technology of accounting. A technology producing ‘the professional nursery teacher' as a reflective daily researcher, who outlives her pedagogical desire as an analytical care of the optimisation of ‘the learning child'. Thus, the paper analyses the micro physics...... of administrative technology, tracing how the humanistic values of education embed and are embedded within ‘the professional nursery teacher' as an object and subject of administrative practice. Rather than undermining the humanistic potential of education, it is argued that the technology of accounting...

  12. Leishmaniasis vector behaviour in Kenya

    International Nuclear Information System (INIS)

    Mutinga, M.J.


    Leishmaniasis in Kenya exists in two forms: cutaneous and visceral. The vectors of visceral leishmaniasis have been the subject of investigation by various researchers since World War II, when the outbreak of the disease was first noticed. The vectors of cutaneous leishmaniasis were first worked on only a decade ago after the discovery of the disease focus in Mt. Elgon. The vector behaviour of these diseases, namely Phlebotomus pedifer, the vector of cutaneous leishmaniasis, and Phlebotomus martini, the vector of visceral leishmaniasis, are discussed in detail. P. pedifer has been found to breed and bite inside caves, whereas P. martini mainly bites inside houses. (author)

  13. Webb Administration


    Prahl, Hampus


    I’ve built an Internet based administration front-end for the company Allt I Brand in Jamjö. Allt I Brand’s main goals are maintenance and sale of fire preventive equipment. This front-end is programmed mostly in PHP, connected to a MySQL database. Because both Allt I Brand’s main page and my administration front-end use the same database and the database is designed to virtually fit any type of company, the result is both dynamic and powerful. The front-end design is made to make it easy to ...

  14. Database Administrator (United States)

    Moore, Pam


    The Internet and electronic commerce (e-commerce) generate lots of data. Data must be stored, organized, and managed. Database administrators, or DBAs, work with database software to find ways to do this. They identify user needs, set up computer databases, and test systems. They ensure that systems perform as they should and add people to the…

  15. ASTEROID SPIN VECTORS (United States)

    National Aeronautics and Space Administration — This is a tabulation of 526 determinations of asteroid pole orientations, covering 104 numbered asteroids, gathered from the literature from 1932 through 1995.

  16. Supernova: A Versatile Vector System for Single-Cell Labeling and Gene Function Studies in vivo. (United States)

    Luo, Wenshu; Mizuno, Hidenobu; Iwata, Ryohei; Nakazawa, Shingo; Yasuda, Kosuke; Itohara, Shigeyoshi; Iwasato, Takuji


    Here we describe "Supernova" series of vector systems that enable single-cell labeling and labeled cell-specific gene manipulation, when introduced by in utero electroporation (IUE) or adeno-associated virus (AAV)-mediated gene delivery. In Supernova, sparse labeling relies on low TRE leakage. In a small population of cells with over-threshold leakage, initial tTA-independent weak expression is enhanced by tTA/TRE-positive feedback along with a site-specific recombination system (e.g., Cre/loxP, Flpe/FRT). Sparse and bright labeling by Supernova with little background enables the visualization of the morphological details of individual neurons in densely packed brain areas such as the cortex and hippocampus, both during development and in adulthood. Sparseness levels are adjustable. Labeled cell-specific gene knockout was accomplished by introducing Cre/loxP-based Supernova vectors into floxed mice. Furthermore, by combining with RNAi, TALEN, and CRISPR/Cas9 technologies, IUE-based Supernova achieved labeled cell-specific gene knockdown and editing/knockout without requiring genetically altered mice. Thus, Supernova system is highly extensible and widely applicable for single-cell analyses in complex organs, such as the mammalian brain.

  17. On vector equilibrium problem

    Indian Academy of Sciences (India)

    Let X be a real topological vector space; K & X be a nonempty closed convex set; ЕYY PЖ be a real ordered ... induced by a solid pointed closed convex cone P, thus x P y@Ay └ x P P, VxY y P Y; f : X ┬ X└3Y with .... tone; VxY y P K, the function t P Й0Y 1К└3gЕty З Е1 └ tЖxY yЖ is continuous at 0З; g is a P-convex and ...

  18. Scalar and vector Galileons (United States)

    Rodríguez, Yeinzon; Navarro, Andrés A.


    An alternative for the construction of fundamental theories is the introduction of Galileons. These are fields whose action leads to non higher than second-order equations of motion. As this is a necessary but not sufficient condition to make the Hamiltonian bounded from below, as long as the action is not degenerate, the Galileon construction is a way to avoid pathologies both at the classical and quantum levels. Galileon actions are, therefore, of great interest in many branches of physics, specially in high energy physics and cosmology. This proceedings contribution presents the generalities of the construction of both scalar and vector Galileons following two different but complimentary routes.

  19. A family of promoter probe vectors incorporating autofluorescent and chromogenic reporter proteins for studying gene expression in Gram-negative bacteria. (United States)

    Karunakaran, R; Mauchline, T H; Hosie, A H F; Poole, P S


    A series of promoter probe vectors for use in Gram-negative bacteria has been made in two broad-host-range vectors, pOT (pBBR replicon) and pJP2 (incP replicon). Reporter fusions can be made to gfpUV, gfpmut3.1, unstable gfpmut3.1 variants (LAA, LVA, AAV and ASV), gfp+, dsRed2, dsRedT.3, dsRedT.4, mRFP1, gusA or lacZ. The two vector families, pOT and pJP2, are compatible with one another and share the same polylinker for facile interchange of promoter regions. Vectors based on pJP2 have the advantage of being ultra-stable in the environment due to the presence of the parABCDE genes. As a confirmation of their usefulness, the dicarboxylic acid transport system promoter (dctA(p)) was cloned into a pOT (pRU1097)- and a pJP2 (pRU1156)-based vector and shown to be expressed by Rhizobium leguminosarum in infection threads of vetch. This indicates the presence of dicarboxylates at the earliest stages of nodule formation.

  20. Vertical vector face lift. (United States)

    Somoano, Brian; Chan, Joanna; Morganroth, Greg


    Facial rejuvenation using local anesthesia has evolved in the past decade as a safer option for patients seeking fewer complications and minimal downtime. Mini- and short-scar face lifts using more conservative incision lengths and extent of undermining can be effective in the younger patient with lower face laxity and minimal loose, elastotic neck skin. By incorporating both an anterior and posterior approach and using an incision length between the mini and more traditional face lift, the Vertical Vector Face Lift can achieve longer-lasting and natural results with lesser cost and risk. Submentoplasty and liposuction of the neck and jawline, fundamental components of the vertical vector face lift, act synergistically with superficial musculoaponeurotic system plication to reestablish a more youthful, sculpted cervicomental angle, even in patients with prominent jowls. Dramatic results can be achieved in the right patient by combining with other procedures such as injectable fillers, chin implants, laser resurfacing, or upper and lower blepharoplasties. © 2011 Wiley Periodicals, Inc.

  1. Proteomic and transcriptomic studies of HBV-associated liver fibrosis of an AAV-HBV-infected mouse model. (United States)

    Kan, Fangming; Ye, Lei; Yan, Tao; Cao, Jiaqi; Zheng, Jianhua; Li, Wuping


    Human hepatitis B virus (HBV) infection is an important public health issue in the Asia-Pacific region and is associated with chronic hepatitis, liver fibrosis, cirrhosis and even liver cancer. However, the underlying mechanisms of HBV-associated liver fibrosis remain incompletely understood. In the present study, proteomic and transcriptomic approaches as well as biological network analyses were performed to investigate the differentially expressed molecular signature and key regulatory networks that were associated with HBV-mediated liver fibrosis. RNA sequencing and 2DE-MALDI-TOF/TOF were performed on liver tissue samples obtained from HBV-infected C57BL/6 mouse generated via AAV8-HBV virus. The results showed that 322 genes and 173 proteins were differentially expressed, and 28 HBV-specific proteins were identified by comprehensive proteomic and transcriptomic analysis. GO analysis indicated that the differentially expressed proteins were predominantly involved in oxidative stress, which plays a key role in HBV-related liver fibrosis. Importantly, CAT, PRDX1, GSTP1, NXN and BLVRB were shown to be associated with oxidative stress among the differentially expressed proteins. The most striking results were validated by Western blot and RT-qPCR. The RIG-I like receptor signaling pathway was found to be the major signal pathway that changed during HBV-related fibrosis. This study provides novel insights into HBV-associated liver fibrosis and reveals the significant role of oxidative stress in liver fibrosis. Furthermore, CAT, BLVRB, NXN, PRDX1, and IDH1 may be candidates for detection of liver fibrosis or therapeutic targets for the treatment of liver fibrosis.

  2. Optimality Conditions in Vector Optimization

    CERN Document Server

    Jiménez, Manuel Arana; Lizana, Antonio Rufián


    Vector optimization is continuously needed in several science fields, particularly in economy, business, engineering, physics and mathematics. The evolution of these fields depends, in part, on the improvements in vector optimization in mathematical programming. The aim of this Ebook is to present the latest developments in vector optimization. The contributions have been written by some of the most eminent researchers in this field of mathematical programming. The Ebook is considered essential for researchers and students in this field.

  3. Symmetric vectors and algebraic classification

    International Nuclear Information System (INIS)

    Leibowitz, E.


    The concept of symmetric vector field in Riemannian manifolds, which arises in the study of relativistic cosmological models, is analyzed. Symmetric vectors are tied up with the algebraic properties of the manifold curvature. A procedure for generating a congruence of symmetric fields out of a given pair is outlined. The case of a three-dimensional manifold of constant curvature (''isotropic universe'') is studied in detail, with all its symmetric vector fields being explicitly constructed

  4. Zinc finger nuclease-expressing baculoviral vectors mediate targeted genome integration of reprogramming factor genes to facilitate the generation of human induced pluripotent stem cells. (United States)

    Phang, Rui-Zhe; Tay, Felix Chang; Goh, Sal-Lee; Lau, Cia-Hin; Zhu, Haibao; Tan, Wee-Kiat; Liang, Qingle; Chen, Can; Du, Shouhui; Li, Zhendong; Tay, Johan Chin-Kang; Wu, Chunxiao; Zeng, Jieming; Fan, Weimin; Toh, Han Chong; Wang, Shu


    Integrative gene transfer using retroviruses to express reprogramming factors displays high efficiency in generating induced pluripotent stem cells (iPSCs), but the value of the method is limited because of the concern over mutagenesis associated with random insertion of transgenes. Site-specific integration into a preselected locus by engineered zinc-finger nuclease (ZFN) technology provides a potential way to overcome the problem. Here, we report the successful reprogramming of human fibroblasts into a state of pluripotency by baculoviral transduction-mediated, site-specific integration of OKSM (Oct3/4, Klf4, Sox2, and c-myc) transcription factor genes into the AAVS1 locus in human chromosome 19. Two nonintegrative baculoviral vectors were used for cotransduction, one expressing ZFNs and another as a donor vector encoding the four transcription factors. iPSC colonies were obtained at a high efficiency of 12% (the mean value of eight individual experiments). All characterized iPSC clones carried the transgenic cassette only at the ZFN-specified AAVS1 locus. We further demonstrated that when the donor cassette was flanked by heterospecific loxP sequences, the reprogramming genes in iPSCs could be replaced by another transgene using a baculoviral vector-based Cre recombinase-mediated cassette exchange system, thereby producing iPSCs free of exogenous reprogramming factors. Although the use of nonintegrating methods to generate iPSCs is rapidly becoming a standard approach, methods based on site-specific integration of reprogramming factor genes as reported here hold the potential for efficient generation of genetically amenable iPSCs suitable for future gene therapy applications.

  5. Vector continued fractions using a generalized inverse

    International Nuclear Information System (INIS)

    Haydock, Roger; Nex, C M M; Wexler, Geoffrey


    A real vector space combined with an inverse (involution) for vectors is sufficient to define a vector continued fraction whose parameters consist of vector shifts and changes of scale. The choice of sign for different components of the vector inverse permits construction of vector analogues of the Jacobi continued fraction. These vector Jacobi fractions are related to vector and scalar-valued polynomial functions of the vectors, which satisfy recurrence relations similar to those of orthogonal polynomials. The vector Jacobi fraction has strong convergence properties which are demonstrated analytically, and illustrated numerically

  6. Covariant Lyapunov vectors

    International Nuclear Information System (INIS)

    Ginelli, Francesco; Politi, Antonio; Chaté, Hugues; Livi, Roberto


    Recent years have witnessed a growing interest in covariant Lyapunov vectors (CLVs) which span local intrinsic directions in the phase space of chaotic systems. Here, we review the basic results of ergodic theory, with a specific reference to the implications of Oseledets’ theorem for the properties of the CLVs. We then present a detailed description of a ‘dynamical’ algorithm to compute the CLVs and show that it generically converges exponentially in time. We also discuss its numerical performance and compare it with other algorithms presented in the literature. We finally illustrate how CLVs can be used to quantify deviations from hyperbolicity with reference to a dissipative system (a chain of Hénon maps) and a Hamiltonian model (a Fermi–Pasta–Ulam chain). This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Lyapunov analysis: from dynamical systems theory to applications’. (paper)

  7. Covariant Lyapunov vectors (United States)

    Ginelli, Francesco; Chaté, Hugues; Livi, Roberto; Politi, Antonio


    Recent years have witnessed a growing interest in covariant Lyapunov vectors (CLVs) which span local intrinsic directions in the phase space of chaotic systems. Here, we review the basic results of ergodic theory, with a specific reference to the implications of Oseledets’ theorem for the properties of the CLVs. We then present a detailed description of a ‘dynamical’ algorithm to compute the CLVs and show that it generically converges exponentially in time. We also discuss its numerical performance and compare it with other algorithms presented in the literature. We finally illustrate how CLVs can be used to quantify deviations from hyperbolicity with reference to a dissipative system (a chain of Hénon maps) and a Hamiltonian model (a Fermi-Pasta-Ulam chain). This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Lyapunov analysis: from dynamical systems theory to applications’.

  8. Chameleon vector bosons

    International Nuclear Information System (INIS)

    Nelson, Ann E.; Walsh, Jonathan


    We show that for a force mediated by a vector particle coupled to a conserved U(1) charge, the apparent range and strength can depend on the size and density of the source, and the proximity to other sources. This chameleon effect is due to screening from a light charged scalar. Such screening can weaken astrophysical constraints on new gauge bosons. As an example we consider the constraints on chameleonic gauged B-L. We show that although Casimir measurements greatly constrain any B-L force much stronger than gravity with range longer than 0.1 μm, there remains an experimental window for a long-range chameleonic B-L force. Such a force could be much stronger than gravity, and long or infinite range in vacuum, but have an effective range near the surface of the earth which is less than a micron.

  9. Administrative contracts

    Directory of Open Access Journals (Sweden)

    Vukićević-Petković Milica


    Full Text Available Administrative contracts are a special type of contract where usually one of the contracting parties is a public law body and which is concluded for the performance of public service and the realization of a public interest. They go a long way since its inception to its eventual final acceptance of all the legal systems. One of the enduring characteristics of this type of contract is their disquised or unnoticed existence. This is why only monitoring their development may lead to a complete understanding of the importance and essence of this institution as well as the need for its complete legal regulation.

  10. Administrative contracts


    Vukićević-Petković Milica


    Administrative contracts are a special type of contract where usually one of the contracting parties is a public law body and which is concluded for the performance of public service and the realization of a public interest. They go a long way since its inception to its eventual final acceptance of all the legal systems. One of the enduring characteristics of this type of contract is their disquised or unnoticed existence. This is why only monitoring their development may lead to a complete u...

  11. Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression (United States)

    Mueller, Christian; Chulay, Jeffrey D.; Trapnell, Bruce C.; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A.; McElvaney, Noel G.; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N.; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R.; Ye, Guo-jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P.; Vonderheide, Robert H.; Hulme, Maigan A.; Brusko, Todd M.; Wilson, James M.; Flotte, Terence R.


    Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy. PMID:24231351

  12. Human Treg responses allow sustained recombinant adeno-associated virus-mediated transgene expression. (United States)

    Mueller, Christian; Chulay, Jeffrey D; Trapnell, Bruce C; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A; McElvaney, Noel G; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R; Ye, Guo-Jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P; Vonderheide, Robert H; Hulme, Maigan A; Brusko, Todd M; Wilson, James M; Flotte, Terence R


    Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1-AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.

  13. Polar Pathfinder Daily 25 km EASE-Grid Sea Ice Motion Vectors (United States)

    National Aeronautics and Space Administration — Daily ice motion vectors are computed from a wide variety of sensors ranging from passive microwave radiometers, such as the Scanning Multichannel Microwave...

  14. Effectiveness of Nitrous Oxide as a Liquid Injection Thrust Vector Control Fluid, Phase I (United States)

    National Aeronautics and Space Administration — Nitrous Oxide is proposed as an energetic liquid injection thrust vector control fluid for vehicle attitude control during dynamic vehicle maneuvers. Pulled from the...

  15. MISR Level 3 Cloud Motion Vector monthly Product in netCDF format V002 (United States)

    National Aeronautics and Space Administration — The MISR Level 3 Monthly Cloud Motion Vector Product contains retrievals of cloud motion determined by geometrically triangulating the position and motion of cloud...

  16. MISR Level 3 Cloud Motion Vector yearly Product in netCDF format V001 (United States)

    National Aeronautics and Space Administration — The MISR Level 3 Yearly Cloud Motion Vector Product contains retrievals of cloud motion determined by geometrically triangulating the position and motion of cloud...

  17. Variable Vector Countermeasure Suit (V2Suit) for Space Habitation and Exploration Project (United States)

    National Aeronautics and Space Administration — The Variable Vector Countermeasure Suit (V2Suit) is a specialized spacesuit designed to keep astronauts healthy during long-duration space exploration missions and...

  18. Effectiveness of Nitrous Oxide as a Liquid Injection Thrust Vector Control Fluid Project (United States)

    National Aeronautics and Space Administration — Nitrous Oxide is proposed as an energetic liquid injection thrust vector control fluid for vehicle attitude control during dynamic vehicle maneuvers. Pulled from the...

  19. Relative Position Indicator Concept for Managing Mixed RNAV and Vectored Arrival Traffic Project (United States)

    National Aeronautics and Space Administration — Mosaic ATM proposes to study a Relative Position Indicator (RPI) concept for managing mixed RNAV and traditionally vectored arrival traffic, to enable increased...

  20. MISR Level 3 Cloud Motion Vector quarterly Product in netCDF format V001 (United States)

    National Aeronautics and Space Administration — This file contains the MISR Level 3 Cloud Motion Vector quarterly Product in netCDF format (Suggested Usage: This file contains one quarter of MISR Level 2 TC_STEREO...

  1. MISR Level 3 Cloud Motion Vector monthly Product in netCDF format V001 (United States)

    National Aeronautics and Space Administration — The MISR Level 3 Monthly Cloud Motion Vector Product contains retrievals of cloud motion determined by geometrically triangulating the position and motion of cloud...

  2. NOAA Composite Shoreline - Vectorized Shoreline Derived From NOAA-NOS Coastal Survey Maps and Aerial Photographs (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The NOAA Composite Shoreline is primarily intended for high-resolution cartographic representation of the shoreline. It is a high-resolution vector shoreline based...

  3. Simplified Representation of Vector Fields

    NARCIS (Netherlands)

    Telea, Alexandru; Wijk, Jarke J. van


    Vector field visualization remains a difficult task. Although many local and global visualization methods for vector fields such as flow data exist, they usually require extensive user experience on setting the visualization parameters in order to produce images communicating the desired insight. We

  4. The Neural Support Vector Machine

    NARCIS (Netherlands)

    Wiering, Marco; van der Ree, Michiel; Embrechts, Mark; Stollenga, Marijn; Meijster, Arnold; Nolte, A; Schomaker, Lambertus


    This paper describes a new machine learning algorithm for regression and dimensionality reduction tasks. The Neural Support Vector Machine (NSVM) is a hybrid learning algorithm consisting of neural networks and support vector machines (SVMs). The output of the NSVM is given by SVMs that take a

  5. Estimation of Motion Vector Fields

    DEFF Research Database (Denmark)

    Larsen, Rasmus


    This paper presents an approach to the estimation of 2-D motion vector fields from time varying image sequences. We use a piecewise smooth model based on coupled vector/binary Markov random fields. We find the maximum a posteriori solution by simulated annealing. The algorithm generate sample...

  6. Disease Vector Ecology Profile: Peru (United States)


    vector of urban plague in Peru is the Oriental rat flea, Xenopsylla cheopis. The human flea, Pulex irritans, although biologically an inefficient vector...TEL: (215) 688-4400 Peru - 10 Instituto Nacional de Hygiene Lima, Peru Peru – 11 Institutos Nacionales de Salud Departamento de Animales

  7. GPU Accelerated Vector Median Filter (United States)

    Aras, Rifat; Shen, Yuzhong


    Noise reduction is an important step for most image processing tasks. For three channel color images, a widely used technique is vector median filter in which color values of pixels are treated as 3-component vectors. Vector median filters are computationally expensive; for a window size of n x n, each of the n(sup 2) vectors has to be compared with other n(sup 2) - 1 vectors in distances. General purpose computation on graphics processing units (GPUs) is the paradigm of utilizing high-performance many-core GPU architectures for computation tasks that are normally handled by CPUs. In this work. NVIDIA's Compute Unified Device Architecture (CUDA) paradigm is used to accelerate vector median filtering. which has to the best of our knowledge never been done before. The performance of GPU accelerated vector median filter is compared to that of the CPU and MPI-based versions for different image and window sizes, Initial findings of the study showed 100x improvement of performance of vector median filter implementation on GPUs over CPU implementations and further speed-up is expected after more extensive optimizations of the GPU algorithm .

  8. Archimedeanization of ordered vector spaces


    Emelyanov, Eduard Yu.


    In the case of an ordered vector space with an order unit, the Archimedeanization method has been developed recently by V.I Paulsen and M. Tomforde. We present a general version of the Archimedeanization which covers arbitrary ordered vector spaces.

  9. Transcriptional Silencing of Retroviral Vectors

    DEFF Research Database (Denmark)

    Lund, Anders Henrik; Duch, M.; Pedersen, F.S.


    Although retroviral vector systems have been found to efficiently transduce a variety of cell types in vitro, the use of vectors based on murine leukemia virus in preclinical models of somatic gene therapy has led to the identification of transcriptional silencing in vivo as an important problem....... Extinction of long-term vector expression has been observed after implantation of transduced hematopoietic cells as well as fibroblasts, myoblasts and hepatocytes. Here we review the influence of vector structure, integration site and cell type on transcriptional silencing. While down-regulation of proviral...... transcription is known from a number of cellular and animal models, major insight has been gained from studies in the germ line and embryonal cells of the mouse. Key elements for the transfer and expression of retroviral vectors, such as the viral transcriptional enhancer and the binding site for the t...

  10. Vector superconductivity in cosmic strings

    International Nuclear Information System (INIS)

    Dvali, G.R.; Mahajan, S.M.


    We argue that in most realistic cases, the usual Witten-type bosonic superconductivity of the cosmic string is automatically (independent of the existence of superconducting currents) accompanied by the condensation of charged gauge vector bosons in the core giving rise to a new vector type superconductivity. The value of the charged vector condensate is related with the charged scalar expectation value, and vanishes only if the latter goes to zero. The mechanism for the proposed vector superconductivity, differing fundamentally from those in the literature, is delineated using the simplest realistic example of the two Higgs doublet standard model interacting with the extra cosmic string. It is shown that for a wide range of parameters, for which the string becomes scalarly superconducting, W boson condensates (the sources of vector superconductivity) are necessarily excited. (author). 14 refs

  11. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

    Directory of Open Access Journals (Sweden)

    Laura Ordovás


    Full Text Available Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs. We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

  12. Enhancing poxvirus vectors vaccine immunogenicity. (United States)

    García-Arriaza, Juan; Esteban, Mariano


    Attenuated recombinant poxvirus vectors expressing heterologous antigens from pathogens are currently at various stages in clinical trials with the aim to establish their efficacy. This is because these vectors have shown excellent safety profiles, significant immunogenicity against foreign expressed antigens and are able to induce protective immune responses. In view of the limited efficacy triggered by some poxvirus strains used in clinical trials (i.e, ALVAC in the RV144 phase III clinical trial for HIV), and of the restrictive replication capacity of the highly attenuated vectors like MVA and NYVAC, there is a consensus that further improvements of these vectors should be pursuit. In this review we considered several strategies that are currently being implemented, as well as new approaches, to improve the immunogenicity of the poxvirus vectors. This includes heterologous prime/boost protocols, use of co-stimulatory molecules, deletion of viral immunomodulatory genes still present in the poxvirus genome, enhancing virus promoter strength, enhancing vector replication capacity, optimizing expression of foreign heterologous sequences, and the combined use of adjuvants. An optimized poxvirus vector triggering long-lasting immunity with a high protective efficacy against a selective disease should be sought.

  13. Chikungunya Virus–Vector Interactions (United States)

    Coffey, Lark L.; Failloux, Anna-Bella; Weaver, Scott C.


    Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed. PMID:25421891

  14. Chikungunya Virus–Vector Interactions

    Directory of Open Access Journals (Sweden)

    Lark L. Coffey


    Full Text Available Chikungunya virus (CHIKV is a mosquito-borne alphavirus that causes chikungunya fever, a severe, debilitating disease that often produces chronic arthralgia. Since 2004, CHIKV has emerged in Africa, Indian Ocean islands, Asia, Europe, and the Americas, causing millions of human infections. Central to understanding CHIKV emergence is knowledge of the natural ecology of transmission and vector infection dynamics. This review presents current understanding of CHIKV infection dynamics in mosquito vectors and its relationship to human disease emergence. The following topics are reviewed: CHIKV infection and vector life history traits including transmission cycles, genetic origins, distribution, emergence and spread, dispersal, vector competence, vector immunity and microbial interactions, and co-infection by CHIKV and other arboviruses. The genetics of vector susceptibility and host range changes, population heterogeneity and selection for the fittest viral genomes, dual host cycling and its impact on CHIKV adaptation, viral bottlenecks and intrahost diversity, and adaptive constraints on CHIKV evolution are also discussed. The potential for CHIKV re-emergence and expansion into new areas and prospects for prevention via vector control are also briefly reviewed.

  15. Violation of vector dominance in the vector manifestation

    International Nuclear Information System (INIS)

    Sasaki, Chihiro


    The vector manifestation (VM) is a new pattern for realizing the chiral symmetry in QCD. In the VM, the massless vector meson becomes the chiral partner of pion at the critical point, in contrast with the restoration based on the linear sigma model. Including the intrinsic temperature dependences of the parameters of the hidden local symmetry (HLS) Lagrangian determined from the underlying QCD through the Wilsonian matching together with the hadronic thermal corrections, we present a new prediction of the VM on the direct photon-π-π coupling which measures the validity of the vector dominance (VD) of the electromagnetic form factor of the pion. We find that the VD is largely violated at the critical temperature, which indicates that the assumption of the VD made in several analysis on the dilepton spectra in hot matter may need to be weakened for consistently including the effect of the dropping mass of the vector meson. (author)

  16. Vector calculation of particle code

    International Nuclear Information System (INIS)

    Nishiguchi, A.; Yabe, T.; Orii, S.


    The development of vector computer requires the modification of the algorithm into a suitable form for vector calculation. Among many algorithms, the particle code is a typical example which has suffered a damage in the calculation on supercomputer owing to its possibility of recurrent data access in collecting cell-wise quantities from particle's quartities. In this article, we report a new method to liberate the particle code from recurrent calculations. It should be noticed, however, that the method may depend on the architecture of supercomputer, and works well on FACOM VP-100 and VP-200: the indirect data accessing must be vectorized and its speed should be fast. (Mori, K.)

  17. On the Witt vector Frobenius

    DEFF Research Database (Denmark)

    Davis, Christopher James; Kedlaya, Kiran


    We study the kernel and cokernel of the Frobenius map on the p-typical Witt vectors of a commutative ring, not necessarily of characteristic p. We give many equivalent conditions to surjectivity of the Frobenius map on both finite and infinite length Witt vectors. In particular, surjectivity...... on finite Witt vectors turns out to be stable under certain integral extensions; this provides a clean formulation of a strong generalization of Faltings’s almost purity theorem from p-adic Hodge theory, incorporating recent improvements by Kedlaya–Liu and by Scholze....

  18. Synthetic Aperture Vector Flow Imaging

    DEFF Research Database (Denmark)

    Oddershede, Niels


    of the thesis considers a method for estimating the two-dimensional velocity vector within the image plane. This method, called synthetic aperture vector flow imaging, is first shortly reviewed. The main contribution of this work is partly an analysis of the method with respect to focusing effects, motion...... estimation. The method can be used for increasing the frame rate of color flow maps or alternatively for a new imaging modality entitled quadroplex imaging, featuring a color flow map and two independent spectrograms at a high frame rate. The second is an alternative method for ultrasonic vector velocity...

  19. Vector control of induction machines

    CERN Document Server

    Robyns, Benoit


    After a brief introduction to the main law of physics and fundamental concepts inherent in electromechanical conversion, ""Vector Control of Induction Machines"" introduces the standard mathematical models for induction machines - whichever rotor technology is used - as well as several squirrel-cage induction machine vector-control strategies. The use of causal ordering graphs allows systematization of the design stage, as well as standardization of the structure of control devices. ""Vector Control of Induction Machines"" suggests a unique approach aimed at reducing parameter sensitivity for

  20. Are Bred Vectors The Same As Lyapunov Vectors? (United States)

    Kalnay, E.; Corazza, M.; Cai, M.

    Regional loss of predictability is an indication of the instability of the underlying flow, where small errors in the initial conditions (or imperfections in the model) grow to large amplitudes in finite times. The stability properties of evolving flows have been studied using Lyapunov vectors (e.g., Alligood et al, 1996, Ott, 1993, Kalnay, 2002), singular vectors (e.g., Lorenz, 1965, Farrell, 1988, Molteni and Palmer, 1993), and, more recently, with bred vectors (e.g., Szunyogh et al, 1997, Cai et al, 2001). Bred vectors (BVs) are, by construction, closely related to Lyapunov vectors (LVs). In fact, after an infinitely long breeding time, and with the use of infinitesimal ampli- tudes, bred vectors are identical to leading Lyapunov vectors. In practical applications, however, bred vectors are different from Lyapunov vectors in two important ways: a) bred vectors are never globally orthogonalized and are intrinsically local in space and time, and b) they are finite-amplitude, finite-time vectors. These two differences are very significant in a dynamical system whose size is very large. For example, the at- mosphere is large enough to have "room" for several synoptic scale instabilities (e.g., storms) to develop independently in different regions (say, North America and Aus- tralia), and it is complex enough to have several different possible types of instabilities (such as barotropic, baroclinic, convective, and even Brownian motion). Bred vectors share some of their properties with leading LVs (Corazza et al, 2001a, 2001b, Toth and Kalnay, 1993, 1997, Cai et al, 2001). For example, 1) Bred vectors are independent of the norm used to define the size of the perturba- tion. Corazza et al. (2001) showed that bred vectors obtained using a potential enstro- phy norm were indistinguishable from bred vectors obtained using a streamfunction squared norm, in contrast with singular vectors. 2) Bred vectors are independent of the length of the rescaling period as long as the


    DEFF Research Database (Denmark)

    Glud, Andreas Nørgaard; Landau, A.M.; Johnsen, Erik Lisbjerg


    Animal models towards understanding and treating Parkinson’s disease (PD) are important translational steps toward clinical applications. The Göttingen minipig(GM), fits progressional neurological models due to an relative low adult weight between 20-40 kg, and has a large gyrencephalic brain (6x 5...... x 4 cm) that can be examined at sufficient resolution using both conventional clinical scanning modalities and preclinical testing of deep brain stimulation, stem cell grafting and other neuromodulatory devices. Aim: Using inoculating of human or pig alpha-synuclein(aSYN) fibrils or overexpressing a......SYN using Lenti virus(LV) and Adeno Assosiated Virus(AAV) vectors in the nigrostriatal system, we hope to create a new porcine model for PD. Methods: Using conventional human-intended stereotaxic neurosurgery methods, we apply aSYN in the catecholamine nigrostriatal system of 13 GM. The changes...

  2. Vectorization at the KENO-IV code

    International Nuclear Information System (INIS)

    Asai, K.; Higuchi, K.; Katakura, J.


    The multigroup criticality safety code KENO-IV has been vectorized and tested on the FACOM VP-100 vector processor. At first, the vectorized KENO-IV on a scalar processor was slower than the original one by a factor of 1.4 because of the overhead introduced by vectorization. Making modifications of algorithms and techniques for vectorization, the vectorized version has become faster than the original one by a factor of 1.4 on the vector processor. For further speedup of the code, some improvements on compiler and hardware, especially on addition of Monte Carlo pipelines to the vector processor, are discussed

  3. Introduction to matrices and vectors

    CERN Document Server

    Schwartz, Jacob T


    In this concise undergraduate text, the first three chapters present the basics of matrices - in later chapters the author shows how to use vectors and matrices to solve systems of linear equations. 1961 edition.

  4. Disease Vector Ecology Profile: Ecuador (United States)


    biologically an inefficient vector, has an insatiable feeding preference for Rattus spp., Cavia porcellus (guinea pigs), domestic animals (swine...71 Peru – 11 Institutos Nacionales de Salud Departamento de Animales Venenosos Calle Capac Yupanqui 1400 Apartado 451 Lima, Peru TEL

  5. GRE Enzymes for Vector Analysis (United States)

    U.S. Environmental Protection Agency — Microbial enzyme data that were collected during the 2004-2006 EMAP-GRE program. These data were then used by Moorhead et al (2016) in their ecoenzyme vector...

  6. Anxiolytic-like effects after vector-mediated overexpression of neuropeptide Y in the amygdala and hippocampus of mice

    DEFF Research Database (Denmark)

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


    , injections of rAAV-NPY caused significant anxiolytic-like effect in the open field, elevated plus maze, and light-dark transition tests. In the hippocampus, rAAV-NPY treatment was associated with anxiolytic-like effect only in the elevated plus maze. No additive effect was observed after combined r......AAV-NPY injection into both the amygdala and hippocampus where anxiolytic-like effect was found in the elevated plus maze and light-dark transition tests. Antidepressant-like effects were not detected in any of the rAAV-NPY injected groups. Immobility was even increased in the tail suspension and forced swim tests...... after intra-amygdaloid rAAV-NPY. Taken together, the present data show that rAAV-NPY treatment may confer non-additive anxiolytic-like effect after injection into the amygdala or hippocampus, being most pronounced in the amygdala...

  7. Challenges and Prospects for Helper-Dependent Adenoviral Vector-Mediated Gene Therapy

    Directory of Open Access Journals (Sweden)

    Pasquale Piccolo


    Full Text Available Helper-dependent adenoviral (HDAd vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types in vivo, have a large cloning capacity, and drive long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration and result in acute toxicity, the severity of which is dose dependent. Intense efforts have been focused on elucidating adenoviral vector–host interactions and the factors involved in the acute toxicity. This review focuses on the recent acquisition of data on such interactions and on strategies investigated to improve the therapeutic index of HDAd vectors.

  8. Pre-clinical Safety and Off-Target Studies to Support Translation of AAV-Mediated RNAi Therapy for FSHD

    Directory of Open Access Journals (Sweden)

    Lindsay M. Wallace


    Full Text Available RNAi emerged as a prospective molecular therapy nearly 15 years ago. Since then, two major RNAi platforms have been under development: oligonucleotides and gene therapy. Oligonucleotide-based approaches have seen more advancement, with some promising therapies that may soon reach market. In contrast, vector-based approaches for RNAi therapy have remained largely in the pre-clinical realm, with limited clinical safety and efficacy data to date. We are developing a gene therapy approach to treat the autosomal-dominant disorder facioscapulohumeral muscular dystrophy. Our strategy involves silencing the myotoxic gene DUX4 using adeno-associated viral vectors to deliver targeted microRNA expression cassettes (miDUX4s. We previously demonstrated proof of concept for this approach in mice, and we are now taking additional steps here to assess safety issues related to miDUX4 overexpression and sequence-specific off-target silencing. In this study, we describe improvements in vector design and expansion of our miDUX4 sequence repertoire and report differential toxicity elicited by two miDUX4 sequences, of which one was toxic and the other was not. This study provides important data to help advance our goal of translating RNAi gene therapy for facioscapulohumeral muscular dystrophy.

  9. Molecular Therapy of Melanocortin-4-Receptor Obesity by an Autoregulatory BDNF Vector

    Directory of Open Access Journals (Sweden)

    Jason J. Siu


    Full Text Available Mutations in the melanocortin-4-receptor (MC4R comprise the most common monogenic form of severe early-onset obesity, and conventional treatments are either ineffective long-term or contraindicated. Immediately downstream of MC4R—in the pathway for regulating energy balance—is brain-derived neurotrophic factor (BDNF. Our previous studies show that adeno-associated virus (AAV-mediated hypothalamic BDNF gene transfer alleviates obesity and diabetes in both diet-induced and genetic models. To facilitate clinical translation, we developed a built-in autoregulatory system to control therapeutic gene expression mimicking the body’s natural feedback systems. This autoregulatory approach leads to a sustainable plateau of body weight after substantial weight loss is achieved. Here, we examined the efficacy and safety of autoregulatory BDNF gene therapy in Mc4r heterozygous mice, which best resemble MC4R obese patients. Mc4r heterozygous mice were treated with either autoregulatory BDNF vector or YFP control and monitored for 30 weeks. BDNF gene therapy prevented the development of obesity and metabolic syndromes characterized by decreasing body weight and adiposity, suppressing food intake, alleviating hyperleptinemia and hyperinsulinemia, improving glucose and insulin tolerance, and increasing energy expenditure, without adverse cardiovascular function or behavioral disturbances. These safety and efficacy data provide preclinical evidence that BDNF gene therapy is a compelling treatment option for MC4R-deficient obese patients.

  10. Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the “safe harbor” AAVS1 locus

    Directory of Open Access Journals (Sweden)

    Julio Castaño


    Full Text Available We report the generation-characterization of a fetal liver (FL B-cell progenitor (BCP-derived human induced pluripotent stem cell (hiPSC line CRISPR/Cas9-edited to carry/express a single copy of doxycycline-inducible Cas9 gene in the “safe locus” AAVS1 (iCas9-FL-BCP-hiPSC. Gene-edited iPSCs remained pluripotent after CRISPR/Cas9 genome-edition. Correct genomic integration of a unique copy of Cas9 was confirmed by PCR and Southern blot. Cas9 was robustly and specifically expressed on doxycycline exposure. T7-endonuclease assay demonstrated that iCas9 induces robust gene-edition when gRNAs against hematopoietic transcription factors were tested. This iCas9-FL-BCP-hiPSC will facilitate gene-editing approaches for studies on developmental biology, drug screening and disease modeling.

  11. Decays of the vector glueball (United States)

    Giacosa, Francesco; Sammet, Julia; Janowski, Stanislaus


    We calculate two- and three-body decays of the (lightest) vector glueball into (pseudo)scalar, (axial-)vector, as well as pseudovector and excited vector mesons in the framework of a model of QCD. While absolute values of widths cannot be predicted because the corresponding coupling constants are unknown, some interesting branching ratios can be evaluated by setting the mass of the yet hypothetical vector glueball to 3.8 GeV as predicted by quenched lattice QCD. We find that the decay mode ω π π should be one of the largest (both through the decay chain O →b1π →ω π π and through the direct coupling O →ω π π ). Similarly, the (direct and indirect) decay into π K K*(892 ) is sizable. Moreover, the decays into ρ π and K*(892 )K are, although subleading, possible and could play a role in explaining the ρ π puzzle of the charmonium state ψ (2 S ) thanks to a (small) mixing with the vector glueball. The vector glueball can be directly formed at the ongoing BESIII experiment as well as at the future PANDA experiment at the FAIR facility. If the width is sufficiently small (≲100 MeV ) it should not escape future detection. It should be stressed that the employed model is based on some inputs and simplifying assumptions: the value of glueball mass (at present, the quenched lattice value is used), the lack of mixing of the glueball with other quarkonium states, and the use of few interaction terms. It then represents a first step toward the identification of the main decay channels of the vector glueball, but shall be improved when corresponding experimental candidates and/or new lattice results will be available.

  12. Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery. (United States)

    Giannelli, Serena G; Luoni, Mirko; Castoldi, Valerio; Massimino, Luca; Cabassi, Tommaso; Angeloni, Debora; Demontis, Gian Carlo; Leocani, Letizia; Andreazzoli, Massimiliano; Broccoli, Vania


    P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

  13. Local Patch Vectors Encoded by Fisher Vectors for Image Classification

    Directory of Open Access Journals (Sweden)

    Shuangshuang Chen


    Full Text Available The objective of this work is image classification, whose purpose is to group images into corresponding semantic categories. Four contributions are made as follows: (i For computational simplicity and efficiency, we directly adopt raw image patch vectors as local descriptors encoded by Fisher vector (FV subsequently; (ii For obtaining representative local features within the FV encoding framework, we compare and analyze three typical sampling strategies: random sampling, saliency-based sampling and dense sampling; (iii In order to embed both global and local spatial information into local features, we construct an improved spatial geometry structure which shows good performance; (iv For reducing the storage and CPU costs of high dimensional vectors, we adopt a new feature selection method based on supervised mutual information (MI, which chooses features by an importance sorting algorithm. We report experimental results on dataset STL-10. It shows very promising performance with this simple and efficient framework compared to conventional methods.

  14. Learning with LOGO: Logo and Vectors. (United States)

    Lough, Tom; Tipps, Steve


    This is the first of a two-part series on the general concept of vector space. Provides tool procedures to allow investigation of vector properties, vector addition and subtraction, and X and Y components. Lists several sources of additional vector ideas. (JM)

  15. Administrative Appeals and ADR in Danish Administrative Law

    DEFF Research Database (Denmark)

    Conradsen, Inger Marie; Gøtze, Michael


    Administrative Appeals, review, administrative tribunals, ombudsman, alternative dispute resolution......Administrative Appeals, review, administrative tribunals, ombudsman, alternative dispute resolution...

  16. Toward lattice fractional vector calculus (United States)

    Tarasov, Vasily E.


    An analog of fractional vector calculus for physical lattice models is suggested. We use an approach based on the models of three-dimensional lattices with long-range inter-particle interactions. The lattice analogs of fractional partial derivatives are represented by kernels of lattice long-range interactions, where the Fourier series transformations of these kernels have a power-law form with respect to wave vector components. In the continuum limit, these lattice partial derivatives give derivatives of non-integer order with respect to coordinates. In the three-dimensional description of the non-local continuum, the fractional differential operators have the form of fractional partial derivatives of the Riesz type. As examples of the applications of the suggested lattice fractional vector calculus, we give lattice models with long-range interactions for the fractional Maxwell equations of non-local continuous media and for the fractional generalization of the Mindlin and Aifantis continuum models of gradient elasticity.

  17. Gauge Theories of Vector Particles (United States)

    Glashow, S. L.; Gell-Mann, M.


    The possibility of generalizing the Yang-Mills trick is examined. Thus we seek theories of vector bosons invariant under continuous groups of coordinate-dependent linear transformations. All such theories may be expressed as superpositions of certain "simple" theories; we show that each "simple theory is associated with a simple Lie algebra. We may introduce mass terms for the vector bosons at the price of destroying the gauge-invariance for coordinate-dependent gauge functions. The theories corresponding to three particular simple Lie algebras - those which admit precisely two commuting quantum numbers - are examined in some detail as examples. One of them might play a role in the physics of the strong interactions if there is an underlying super-symmetry, transcending charge independence, that is badly broken. The intermediate vector boson theory of weak interactions is discussed also. The so-called "schizon" model cannot be made to conform to the requirements of partial gauge-invariance.

  18. Introduction to vector velocity imaging

    DEFF Research Database (Denmark)

    Jensen, Jørgen Arendt; Udesen, Jesper; Hansen, Kristoffer Lindskov

    it virtually impossible to compensate for the factor and obtain correct velocity estimates for either CFM or spectral velocity estimation. This talk will describe methods for finding the correct velocity by estimating both the axial and lateral component of the velocity vector. The transverse oscillation...... method introduces an ultrasound field that oscillation not only along the ultrasound beam both also transverse to it to estimate both the lateral and axial velocity for the full velocity vector. The correct velocity magnitude can be found from this as well as the instantaneous angle. This can be obtained...... over the full region of interest and a real time image at a frame rate of 20 Hz can be displayed. Real time videos have been obtained from both our research systems and from commercial BK Medical scanners. The vector velocity images reveal the full complexity of the human blood flow. It is easy to see...

  19. 3D vector flow imaging

    DEFF Research Database (Denmark)

    Pihl, Michael Johannes

    The main purpose of this PhD project is to develop an ultrasonic method for 3D vector flow imaging. The motivation is to advance the field of velocity estimation in ultrasound, which plays an important role in the clinic. The velocity of blood has components in all three spatial dimensions, yet...... conventional methods can estimate only the axial component. Several approaches for 3D vector velocity estimation have been suggested, but none of these methods have so far produced convincing in vivo results nor have they been adopted by commercial manufacturers. The basis for this project is the Transverse...... on the TO fields are suggested. They can be used to optimize the TO method. In the third part, a TO method for 3D vector velocity estimation is proposed. It employs a 2D phased array transducer and decouples the velocity estimation into three velocity components, which are estimated simultaneously based on 5...

  20. Topological vector spaces and distributions

    CERN Document Server

    Horvath, John


    ""The most readable introduction to the theory of vector spaces available in English and possibly any other language.""-J. L. B. Cooper, MathSciNet ReviewMathematically rigorous but user-friendly, this classic treatise discusses major modern contributions to the field of topological vector spaces. The self-contained treatment includes complete proofs for all necessary results from algebra and topology. Suitable for undergraduate mathematics majors with a background in advanced calculus, this volume will also assist professional mathematicians, physicists, and engineers.The precise exposition o

  1. Synthetic Aperture Vector Flow Imaging

    DEFF Research Database (Denmark)

    Villagómez Hoyos, Carlos Armando

    The main objective of this project was to continue the development of a synthetic aperture vector flow estimator. This type of estimator is capable of overcoming two of the major limitations in conventional ultrasound systems: 1) the inability to scan large region of interest with high temporal......, this thesis showed that novel information can be obtained with vector velocity methods providing quantitative estimates of blood flow and insight into the complexity of the hemodynamics dynamics. This could give the clinician a new tool in assessment and treatment of a broad range of diseases....

  2. Generation of arbitrary vector beams (United States)

    Perez-Garcia, Benjamin; López-Mariscal, Carlos; Hernandez-Aranda, Raul I.; Gutiérrez-Vega, Julio C.


    Optical vector beams arise from point to point spatial variations of the electric component of an electromagnetic field over the transverse plane. In this work, we present a novel experimental technique to generate arbitrary vec- tor beams, and provide sufficient evidence to validate their state of polarization. This technique takes advantage of the capability of a Spatial Light Modulator to simultaneously generate two components of an electromagnetic field by halving the screen of the device and subsequently recombining them in a Sagnac interferometer. Our experimental results show the versatility and robustness of this technique for the generation of vector beams.

  3. Learning with Support Vector Machines

    CERN Document Server

    Campbell, Colin


    Support Vectors Machines have become a well established tool within machine learning. They work well in practice and have now been used across a wide range of applications from recognizing hand-written digits, to face identification, text categorisation, bioinformatics, and database marketing. In this book we give an introductory overview of this subject. We start with a simple Support Vector Machine for performing binary classification before considering multi-class classification and learning in the presence of noise. We show that this framework can be extended to many other scenarios such a

  4. Senior Administrators Should Have Administrative Contracts. (United States)

    Posner, Gary J.


    Recognizing that termination is viewed by the employee as the equivalent to capital punishment of a career, an administrative contract can reduce the emotional and financial entanglements that often result. Administrative contracts are described. (MLW)

  5. The consequences of poor vectorization

    CERN Multimedia

    CERN. Geneva


    This talk briefly discusses the vectorization problem and how it impacts scientific and engineering systems. A simple cost model of designing such system in context of different phases of software lifetime is considered. Finally a concept for scalable solution is presented.

  6. Parallel Sparse Matrix - Vector Product

    DEFF Research Database (Denmark)

    Alexandersen, Joe; Lazarov, Boyan Stefanov; Dammann, Bernd

    This technical report contains a case study of a sparse matrix-vector product routine, implemented for parallel execution on a compute cluster with both pure MPI and hybrid MPI-OpenMP solutions. C++ classes for sparse data types were developed and the report shows how these class can be used...

  7. Phlebotomine Vectors of Human Disease. (United States)


    different. We refrain from naming this specimen until more material becomes available. 12. Lutzomyia olmeca bicolor Fairchild and Theodor 1971...Castillo (1958) and Arzube (1960). Lutzomyia olmeca bicolor is the suspected vector of Leishmania mexicana aristedesi among rodents and marsupials in

  8. Scalar Calibration of Vector Magnetometers

    DEFF Research Database (Denmark)

    Merayo, José M.G.; Brauer, Peter; Primdahl, Fritz


    The calibration parameters of a vector magnetometer are estimated only by the use of a scalar reference magnetometer. The method presented in this paper differs from those previously reported in its linearized parametrization. This allows the determination of three offsets or signals in the absence...

  9. Vector-meson dominance revisited

    Directory of Open Access Journals (Sweden)

    Terschlüsen Carla


    Full Text Available The interaction of mesons with electromagnetism is often well described by the concept of vector-meson dominance (VMD. However, there are also examples where VMD fails. A simple chiral Lagrangian for pions, rho and omega mesons is presented which can account for the respective agreement and disagreement between VMD and phenomenology in the sector of light mesons.

  10. Vector fields on nonorientable surfaces

    Directory of Open Access Journals (Sweden)

    Ilie Barza


    X, and the space of vector fields on X are proved by using a symmetrisation process. An example related to the normal derivative on the border of the Möbius strip supports the nontriviality of the concepts introduced in this paper.

  11. Portfolio Analysis for Vector Calculus (United States)

    Kaplan, Samuel R.


    Classic stock portfolio analysis provides an applied context for Lagrange multipliers that undergraduate students appreciate. Although modern methods of portfolio analysis are beyond the scope of vector calculus, classic methods reinforce the utility of this material. This paper discusses how to introduce classic stock portfolio analysis in a…

  12. Vector ecology of equine piroplasmosis (United States)

    Equine piroplasmosis (EP) is a disease of equidae including horses, donkeys, mules and zebras caused by either of two protozoan parasites, Theileria equi or Babesia caballi. These parasites are biologically transmitted between hosts via tick-vectors and although they have inherent differences, they ...

  13. Vector variational inequalities and their relations with vector optimization

    Directory of Open Access Journals (Sweden)

    Surjeet Kaur Suneja


    Full Text Available In this paper, K- quasiconvex, K- pseudoconvex and other related functions have been introduced in terms of their Clarke subdifferentials, where is an arbitrary closed convex, pointed cone with nonempty interior. The (strict, weakly -pseudomonotonicity, (strict K- naturally quasimonotonicity and K- quasimonotonicity of Clarke subdifferential maps have also been defined. Further, we introduce Minty weak (MVVIP and Stampacchia weak (SVVIP vector variational inequalities over arbitrary cones. Under regularity assumption, we have proved that a weak minimum solution of vector optimization problem (VOP is a solution of (SVVIP and under the condition of K- pseudoconvexity we have obtained the converse for MVVIP (SVVIP. In the end we study the interrelations between these with the help of strict K-naturally quasimonotonicity of Clarke subdifferential map.

  14. Toxicity and biodistribution of the serotype 2 recombinant adeno-associated viral vector, encoding Aquaporin-1, after retroductal delivery to a single mouse parotid gland.

    Directory of Open Access Journals (Sweden)

    Dariya Momot

    Full Text Available In preparation for testing the safety of using serotype 2 recombinant adeno-associated vector, encoding Aquaporin-1 to treat radiation-induced salivary gland damage in a phase 1 clinical trial, we conducted a 13 week GLP biodistribution and toxicology study using Balb/c mice. To best assess the safety of rAAV2hAQP1 as well as resemble clinical delivery, vector (10(8, 10(9, 10(10, or 4.4 × 10(10 vector particles/gland or saline was delivered to the right parotid gland of mice via retroductal cannulation. Very mild surgically induced inflammation was caused by this procedure, seen in 3.6% of animals for the right parotid gland, and 5.3% for the left parotid gland. Long term distribution of vector appeared to be localized to the site of cannulation as well as the right and left draining submandibular lymph nodes at levels >50 copies/μg in some animals. As expected, there was a dose-related increase in neutralizing antibodies produced by day 29. Overall, animals appeared to thrive, with no differences in mean body weight, food or water consumption between groups. There were no significant adverse effects due to treatment noted by clinical chemistry and pathology evaluations. Hematology assessment of serum demonstrated very limited changes to the white blood cell, segmented neutrophils, and hematocrit levels and were concluded to not be vector-associated. Indicators for liver, kidney, cardiac functions and general tissue damage showed no changes due to treatment. All of these indicators suggest the treatment is clinically safe.

  15. Nonseparable closed vector subspaces of separable topological vector spaces

    Czech Academy of Sciences Publication Activity Database

    Kąkol, Jerzy; Leiderman, A. G.; Morris, S. A.


    Roč. 182, č. 1 (2017), s. 39-47 ISSN 0026-9255 R&D Projects: GA ČR GF16-34860L Institutional support: RVO:67985840 Keywords : locally convex topological vector space * separable topological space Subject RIV: BA - General Mathematics OBOR OECD: Pure mathematics Impact factor: 0.716, year: 2016

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


    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.

  17. Transversals of Complex Polynomial Vector Fields

    DEFF Research Database (Denmark)

    Dias, Kealey

    Vector fields in the complex plane are defined by assigning the vector determined by the value P(z) to each point z in the complex plane, where P is a polynomial of one complex variable. We consider special families of so-called rotated vector fields that are determined by a polynomial multiplied...... by rotational constants. Transversals are a certain class of curves for such a family of vector fields that represent the bifurcation states for this family of vector fields. More specifically, transversals are curves that coincide with a homoclinic separatrix for some rotation of the vector field. Given...... a concrete polynomial, it seems to take quite a bit of work to prove that it is generic, i.e. structurally stable. This has been done for a special class of degree d polynomial vector fields having simple equilibrium points at the d roots of unity, d odd. In proving that such vector fields are generic...

  18. Administrative Data Repository (ADR) (United States)

    Department of Veterans Affairs — The Administrative Data Repository (ADR) was established to provide support for the administrative data elements relative to multiple categories of a person entity...

  19. Philippines - Revenue Administration Reform (United States)

    Millennium Challenge Corporation — The Millennium Challenge Account-Philippines' (MCA-P) implementation of the Revenue Administration Reform Project (RARP) is expected to improve tax administration,...

  20. Problems of vector Lagrangians in field theories

    International Nuclear Information System (INIS)

    Krivsky, I.Yu.; Simulik, V.M.


    A vector Lagrange approach to the Dirac spinor field and the relationship between the vector Lagrangians for the spinor and electromagnetic fields are considered. A vector Lagrange approach for the system of interacting electromagnetic B=(B μ υ)=(E-bar,H-bar) and spinor Ψ fields is constructed. New Lagrangians (scalar and vector) for electromagnetic field in terms of field strengths are found. The foundations of two new QED models are formulated

  1. Active set support vector regression. (United States)

    Musicant, David R; Feinberg, Alexander


    This paper presents active set support vector regression (ASVR), a new active set strategy to solve a straightforward reformulation of the standard support vector regression problem. This new algorithm is based on the successful ASVM algorithm for classification problems, and consists of solving a finite number of linear equations with a typically large dimensionality equal to the number of points to be approximated. However, by making use of the Sherman-Morrison-Woodbury formula, a much smaller matrix of the order of the original input space is inverted at each step. The algorithm requires no specialized quadratic or linear programming code, but merely a linear equation solver which is publicly available. ASVR is extremely fast, produces comparable generalization error to other popular algorithms, and is available on the web for download.

  2. 3-D Vector Flow Imaging

    DEFF Research Database (Denmark)

    Holbek, Simon

    ultrasonic vector flow estimation and bring it a step closer to a clinical application. A method for high frame rate 3-D vector flow estimation in a plane using the transverse oscillation method combined with a 1024 channel 2-D matrix array is presented. The proposed method is validated both through phantom...... studies and in vivo. Phantom measurements are compared with their corresponding reference value, whereas the in vivo measurement is validated against the current golden standard for non-invasive blood velocity estimates, based on magnetic resonance imaging (MRI). The study concludes, that a high precision...... are introduced with the array. The major disadvantage with an RC transducer, is the limited field-of-view, which is restricted to the forward looking array. It is discussed, that this drawback may be solved with a diverging lens, providing a larger field-of-view, due the the dispersion of the energy. Based...

  3. Lentiviral vectors in cancer immunotherapy. (United States)

    Oldham, Robyn Aa; Berinstein, Elliot M; Medin, Jeffrey A


    Basic science advances in cancer immunotherapy have resulted in various treatments that have recently shown success in the clinic. Many of these therapies require the insertion of genes into cells to directly kill them or to redirect the host's cells to induce potent immune responses. Other analogous therapies work by modifying effector cells for improved targeting and enhanced killing of tumor cells. Initial studies done using γ-retroviruses were promising, but safety concerns centered on the potential for insertional mutagenesis have highlighted the desire to develop other options for gene delivery. Lentiviral vectors (LVs) have been identified as potentially more effective and safer alternative delivery vehicles. LVs are now in use in clinical trials for many different types of inherited and acquired disorders, including cancer. This review will discuss current knowledge of LVs and the applications of this viral vector-based delivery vehicle to cancer immunotherapy.

  4. Vector fields on singular varieties

    CERN Document Server

    Brasselet, Jean-Paul; Suwa, Tatsuo


    Vector fields on manifolds play a major role in mathematics and other sciences. In particular, the Poincaré-Hopf index theorem gives rise to the theory of Chern classes, key manifold-invariants in geometry and topology. It is natural to ask what is the ‘good’ notion of the index of a vector field, and of Chern classes, if the underlying space becomes singular. The question has been explored by several authors resulting in various answers, starting with the pioneering work of M.-H. Schwartz and R. MacPherson. We present these notions in the framework of the obstruction theory and the Chern-Weil theory. The interplay between these two methods is one of the main features of the monograph.

  5. Microneedle-mediated delivery of viral vectored vaccines. (United States)

    Zaric, Marija; Ibarzo Yus, Bárbara; Kalcheva, Petya Petrova; Klavinskis, Linda Sylvia


    Microneedle array platforms are a promising technology for vaccine delivery, due to their ease of administration with no sharp waste generated, small size, possibility of targeted delivery to the specified skin depth and efficacious delivery of different vaccine formulations, including viral vectors. Areas covered: Attributes and challenges of the most promising viral vector candidates that have advanced to the clinic and that have been leveraged for skin delivery by microneedles; The importance of understanding the immunobiology of antigen-presenting cells in the skin, in particular dendritic cells, in order to generate further improved skin vaccination strategies; recent studies where viral vectors expressing various antigens have been coupled with microneedle technology to examine their potential for improved vaccination. Expert opinion: Simple, economic and efficacious vaccine delivery methods are needed to improve health outcomes and manage possible outbreaks of new emerging viruses. Understanding what innate/inflammatory signals are required to induce both immediate and long-term responses remains a major hurdle in the development of the effective vaccines. One approach to meet these needs is microneedle-mediated viral vector vaccination. In order for this technology to fulfil this potential the industry must invest significantly to further develop its design, production, biosafety, delivery and large-scale manufacturing.


    Energy Technology Data Exchange (ETDEWEB)

    STEINWART, INGO [Los Alamos National Laboratory; HUSH, DON [Los Alamos National Laboratory; SCOVEL, CLINT [Los Alamos National Laboratory; LIST, NICOLAS [Los Alamos National Laboratory


    We show that the stopping criteria used in many support vector machine (SVM) algorithms working on the dual can be interpreted as primal optimality bounds which in turn are known to be important for the statistical analysis of SVMs. To this end we revisit the duality theory underlying the derivation of the dual and show that in many interesting cases primal optimality bounds are the same as known dual optimality bounds.

  7. Arthropods: Vectors of Disease Agents (United States)


    African trypanosomiasis (sleep- Relationships between pathogens and a cycle of development or multipli- ing sickness) and American try- their vectors are...introduced cases of arthropods directly affect human of malaria. This is just a small per- this and other exotic diseases con- health. This article...diseases that are occasionally education credits, earned by complet- transmitted to humans . Tick-borne ing the Laboratory Medicine CE From the

  8. Disease Vector Ecology Profile: Colombia (United States)


    collected in human dwellings using a mouth or powered aspirator and aided by a flashlight. Collections from whatever source are suitable for...Ecuadorian lowland provinces of Sucumbios and Napo bordering the Colombian departments of Putumayo and eastern Amazonas . Otherwise, little is known Appendix A.4. 4. Vector Surveillance and Suppression. Surveillance of triatomines is best conducted with the aid of a flashlight during

  9. A ocean bottom vector magnetometer (United States)

    Wang, Xiaomei; Teng, Yuntian; Wang, Chen; Ma, Jiemei


    The new development instrument with a compact spherical coil system and Overhauser magnetometer for measuring the total strength of the magnetic field and the vectors of strength, Delta inclination - Delta declination, meanwhile we also use a triaxial fluxgate instrument of the traditional instrument for geomagnetic vector filed measurement. The advantages of this method are be calibrated by each other and get good performances with automatic operation, good stability and high resolution. Firstly, a brief description of the instrument measurement principles and the key technologies are given. The instrument used a spherical coil system with 34 coils to product the homogeneous volume inside the coils which is large enough to accommodate the sensor of Overhauser total field sensor; the rest of the footlocker-sized ocean-bottom vector magnetometer consists of equipment to run the sensors and records its data (batteries and a data logger), weight to sink it to the sea floor, a remote-controlled acoustic release and flotation to bring the instrument back to the surface. Finally, the accuracy of the instrument was tested in the Geomagnetic station, and the measurement accuracies of total strength and components were better than 0.2nT and 1nT respectively. The figure 1 shows the development instrument structure. it includes six thick glass spheres which protect the sensor, data logger and batteries from the pressures of the deep sea, meanwhile they also provide recycling positive buoyancy; To cushion the glass, the spheres then go inside yellow plastic "hardhats". The triaxial fluxgate is inside No.1 glass spheres, data logger and batteries are inside No.2 glass spheres, the new vector sensor is inside No.3 glass spheres, acoustic communication unit is inside No.4 glass spheres, No.5 and No.6 glass spheres are empty which only provide recycling positive buoyancy. The figure 2 shows the development instrument Physical photo.

  10. Cre Activated and Inactivated Recombinant Adeno-Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation. (United States)

    Saunders, Arpiar; Sabatini, Bernardo L


    Recombinant adeno-associated viruses (rAAVs) transcriptionally activated by Cre recombinase (Cre-On) are powerful tools for determining the anatomy and function of genetically defined neuronal types in transgenic Cre driver mice. Here we describe how rAAVs transcriptionally inactivated by Cre (Cre-Off) can be used in conjunction with Cre-On rAAVs or genomic Cre-reporter alleles to study brain circuits. Intracranial injection of Cre-On/Cre-Off rAAVs into spatially intermingled Cre(+) and Cre(-) neurons allows these populations to be differentially labeled or manipulated within individual animals. This comparison helps define the unique properties of Cre(+) neurons, highlighting the specialized role they play in their constituent brain circuits. This protocol touches on the conceptual and experimental background of Cre-Off rAAV systems, including caveats and methods of validation. Copyright © 2015 John Wiley & Sons, Inc.

  11. Vector Fields and Flows on Differentiable Stacks

    DEFF Research Database (Denmark)

    A. Hepworth, Richard


    This paper introduces the notions of vector field and flow on a general differentiable stack. Our main theorem states that the flow of a vector field on a compact proper differentiable stack exists and is unique up to a uniquely determined 2-cell. This extends the usual result on the existence...... of vector fields....

  12. Construction of expression vectors carrying mouse peroxisomal ...

    African Journals Online (AJOL)

    The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in ... pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively.

  13. SOLIS vector spectromagnetograph: status and science

    NARCIS (Netherlands)

    Henney, C.J.; Keller, C.U.; Harvey, J.W.; Georgoulis, M.K.; Hadder, N.L.; Norton, A.A.; Raouafi, N.-E.; Toussaint, R.M.


    The Vector Spectromagnetograph (VSM) instrument has recorded photospheric and chromospheric magnetograms daily since August 2003. Fulldisk photospheric vector magnetograms are observed at least weekly and, since November 2006, area-scans of active regions daily. Quick-look vector magnetic images,

  14. Vector and tensor analysis with applications

    CERN Document Server

    Borisenko, A I; Silverman, Richard A


    Concise and readable, this text ranges from definition of vectors and discussion of algebraic operations on vectors to the concept of tensor and algebraic operations on tensors. It also includes a systematic study of the differential and integral calculus of vector and tensor functions of space and time. Worked-out problems and solutions. 1968 edition.

  15. Vector fields and gravity on the lattice

    International Nuclear Information System (INIS)

    Khatsymovsky, V.M.


    The problem of discretization of vector field on Regge lattice is considered. Our approach is based on geometrical interpretation of the vector field as the field of infinitesimal coordinate transformation. A discrete version of the vector field action is obtained as a particular case of the continuum action, and it is shown to have the true continuum limit

  16. Extremal vectors and rectifiability | Enflo | Quaestiones Mathematicae

    African Journals Online (AJOL)

    Extremal vectors and rectifiability. ... The concept of extremal vectors of a linear operator with a dense range but not onto on a Hilbert space was introduced by P. Enflo in 1996 as a new approach to study invariant subspaces ... We show that in general curves that map numbers to backward minimal vectors are not rectifiable.

  17. Toward lattice fractional vector calculus

    International Nuclear Information System (INIS)

    Tarasov, Vasily E


    An analog of fractional vector calculus for physical lattice models is suggested. We use an approach based on the models of three-dimensional lattices with long-range inter-particle interactions. The lattice analogs of fractional partial derivatives are represented by kernels of lattice long-range interactions, where the Fourier series transformations of these kernels have a power-law form with respect to wave vector components. In the continuum limit, these lattice partial derivatives give derivatives of non-integer order with respect to coordinates. In the three-dimensional description of the non-local continuum, the fractional differential operators have the form of fractional partial derivatives of the Riesz type. As examples of the applications of the suggested lattice fractional vector calculus, we give lattice models with long-range interactions for the fractional Maxwell equations of non-local continuous media and for the fractional generalization of the Mindlin and Aifantis continuum models of gradient elasticity. (papers)

  18. Consciousness disintegrates without conscious vectors. (United States)

    Bodovitz, Steven


    Consciousness is discontinuous. The transition from the raw output of parallel, distributed processors into the unified, serial content of consciousness is not instantaneous. Consciousness is divided into discrete cycles, yet appears to be continuous. The continuity requires temporal integration. The simplest mechanism is the calculation of the direction and magnitude of change from one conscious cycle to the next and then the fusion of these conscious vectors with the content of subsequent cycles. This mechanism, while putative, has supporting evidence. It is based on the same mechanism as motion vision, in which motion vectors are calculated in MT/V5 and then fused with discrete images. Moreover, it is based on the known separation of cognitive timing in the brain, in which temporal integrity is maintained in the prefrontal cortex (PFC) and the rest of the content of consciousness is maintained in the rest of the cortex. In fact, limited activity of the PFC matches the predicted effects of the absence of conscious vectors: thoughts strobe and consciousness disintegrates into a series of discrete cycles. The PFC, for example, is one of the primary brain regions deactivated during dreaming, when thoughts shift without any awareness of the transitions. In addition, the PFC is immature in infants, when there is no memory of the experience of consciousness because there is no temporal integrity to assemble the discrete cycles into a coherent experience. Without temporal integration, the human brain is an advanced biological computer, but is not sentient.

  19. Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice. (United States)

    Moda, Fabio; Vimercati, Chiara; Campagnani, Ilaria; Ruggerone, Margherita; Giaccone, Giorgio; Morbin, Michela; Zentilin, Lorena; Giacca, Mauro; Zucca, Ileana; Legname, Giuseppe; Tagliavini, Fabrizio


    Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

  20. Effects of FVIII immunity on hepatocyte and hematopoietic stem cell–directed gene therapy of murine hemophilia A (United States)

    Lytle, Allison M; Brown, Harrison C; Paik, Na Yoon; Knight, Kristopher A; Wright, J Fraser; Spencer, H Trent; Doering, Christopher B


    Immune responses to coagulation factors VIII (FVIII) and IX (FIX) represent primary obstacles to hemophilia treatment. Previously, we showed that hematopoietic stem cell (HSC) retroviral gene therapy induces immune nonresponsiveness to FVIII in both naive and preimmunized murine hemophilia A settings. Liver-directed adeno-associated viral (AAV)-FIX vector gene transfer achieved similar results in preclinical hemophilia B models. However, as clinical immune responses to FVIII and FIX differ, we investigated the ability of liver-directed AAV-FVIII gene therapy to affect FVIII immunity in hemophilia A mice. Both FVIII naive and preimmunized mice were administered recombinant AAV8 encoding a liver-directed bioengineered FVIII expression cassette. Naive animals receiving high or mid-doses subsequently achieved near normal FVIII activity levels. However, challenge with adjuvant-free recombinant FVIII induced loss of FVIII activity and anti-FVIII antibodies in mid-dose, but not high-dose AAV or HSC lentiviral (LV) vector gene therapy cohorts. Furthermore, unlike what was shown previously for FIX gene transfer, AAV-FVIII administration to hemophilia A inhibitor mice conferred no effect on anti-FVIII antibody or inhibitory titers. These data suggest that functional differences exist in the immune modulation achieved to FVIII or FIX in hemophilia mice by gene therapy approaches incorporating liver-directed AAV vectors or HSC-directed LV. PMID:26909355

  1. AAV-mediated gene therapy in Dystrophin-Dp71 deficient mouse leads to blood-retinal barrier restoration and oedema reabsorption. (United States)

    Vacca, Ophélie; Charles-Messance, Hugo; El Mathari, Brahim; Sene, Abdoulaye; Barbe, Peggy; Fouquet, Stéphane; Aragón, Jorge; Darche, Marie; Giocanti-Aurégan, Audrey; Paques, Michel; Sahel, José-Alain; Tadayoni, Ramin; Montañez, Cecilia; Dalkara, Deniz; Rendon, Alvaro


    Dystrophin-Dp71 being a key membrane cytoskeletal protein, expressed mainly in Müller cells that provide a mechanical link at the Müller cell membrane by direct binding to actin and a transmembrane protein complex. Its absence has been related to blood-retinal barrier (BRB) permeability through delocalization and down-regulation of the AQP4 and Kir4.1 channels (1). We have previously shown that the adeno-associated virus (AAV) variant, ShH10, transduces Müller cells in the Dp71-null mouse retina efficiently and specifically (2,3). Here, we use ShH10 to restore Dp71 expression in Müller cells of Dp71 deficient mouse to study molecular and functional effects of this restoration in an adult mouse displaying retinal permeability. We show that strong and specific expression of exogenous Dp71 in Müller cells leads to correct localization of Dp71 protein restoring all protein interactions in order to re-establish a proper functional BRB and retina homeostasis thus preventing retina from oedema. This study is the basis for the development of new therapeutic strategies in dealing with diseases with BRB breakdown and macular oedema such as diabetic retinopathy (DR). © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail:

  2. Problems and worked solutions in vector analysis

    CERN Document Server

    Shorter, LR


    ""A handy book like this,"" noted The Mathematical Gazette, ""will fill a great want."" Devoted to fully worked out examples, this unique text constitutes a self-contained introductory course in vector analysis for undergraduate and graduate students of applied mathematics.Opening chapters define vector addition and subtraction, show how to resolve and determine the direction of two or more vectors, and explain systems of coordinates, vector equations of a plane and straight line, relative velocity and acceleration, and infinitely small vectors. The following chapters deal with scalar and vect

  3. Stability and compatibility of recombinant adeno-associated virus under conditions commonly encountered in human gene therapy trials. (United States)

    Gruntman, Alisha M; Su, Lin; Su, Qin; Gao, Guangping; Mueller, Christian; Flotte, Terence R


    Recombinant adeno-associated virus (rAAV) vectors are rapidly becoming the first choice for human gene therapy studies, as clinical efficacy has been demonstrated in several human trials and proof-of-concept data have been demonstrated for correction of many others. When moving into human use under the auspices of an FDA Investigational New Drug (IND) application, it is necessary to demonstrate the stability of vector material under various conditions of storage, dilution, and administration when used in humans. Limited data are currently available in the literature regarding vector compatibility and stability, leading most IND sponsors to repeat all necessary studies. The current study addresses this issue with an rAAV vector (rAAV1-CB-chAATmyc) containing AAV2-inverted terminal repeat sequences packaged into an AAV1 capsid. Aliquots of vector were exposed to a variety of temperatures, diluents, container constituents, and other environmental conditions, and its functional biological activity (after these various treatments) was assessed by measuring transgene expression after intramuscular injection in mice. rAAV was found to be remarkably stable at temperatures ranging from 4°C to 55°C (with only partial loss of potency after 20 min at 70°C), at pH ranging from 5.5 to 8.5, after contact with mouse or human serum (with or without complement depletion) or with gadolinium and after contact with glass, polystyrene, polyethylene, polypropylene, and stainless steel. The only exposure resulting in near-total loss of vector activity (10,000-fold loss) was UV exposure for 10 min. The stability of rAAV1 preparations bodes well for future dissemination of this therapeutic modality.

  4. Multiscale vector fields for image pattern recognition (United States)

    Low, Kah-Chan; Coggins, James M.


    A uniform processing framework for low-level vision computing in which a bank of spatial filters maps the image intensity structure at each pixel into an abstract feature space is proposed. Some properties of the filters and the feature space are described. Local orientation is measured by a vector sum in the feature space as follows: each filter's preferred orientation along with the strength of the filter's output determine the orientation and the length of a vector in the feature space; the vectors for all filters are summed to yield a resultant vector for a particular pixel and scale. The orientation of the resultant vector indicates the local orientation, and the magnitude of the vector indicates the strength of the local orientation preference. Limitations of the vector sum method are discussed. Investigations show that the processing framework provides a useful, redundant representation of image structure across orientation and scale.

  5. Protamine/DNA/Niosome Ternary Nonviral Vectors for Gene Delivery to the Retina: The Role of Protamine. (United States)

    Puras, G; Martínez-Navarrete, G; Mashal, M; Zárate, J; Agirre, M; Ojeda, E; Grijalvo, S; Eritja, R; Diaz-Tahoces, A; Avilés-Trigueros, M; Fernández, E; Pedraz, J L


    The present study aimed to evaluate the incorporation of protamine into niosome/DNA vectors to analyze the potential application of this novel ternary formulation to deliver the pCMS-EGFP plasmid into the rat retina. Binary vectors based on niosome/DNA and ternary vectors based on protamine/DNA/niosomes were prepared and physicochemically characterized. In vitro experiments were performed in ARPE-19 cells. At 1:1:5 protamine/DNA/niosome mass ratio, the resulted ternary vectors had 150 nm size, positive charge, spherical morphology, and condensed, released, and protected the DNA against enzymatic digestion. The presence of protamine in the ternary vectors improved transfection efficiency, cell viability, and DNA condensation. After ocular administration, the EGFP expression was detected in different cell layers of the retina depending on the administration route without any sign of toxicity associated with the formulations. While subretinal administration transfected mainly photoreceptors and retinal pigment epithelial cells at the site of injection, intravitreal administration produced a more uniform distribution of the protein expression through the inner layers of the retina. The protein expression in the retina persisted for at least one month after both administrations. Our study highlights the flattering properties of protamine/DNA/niosome ternary vectors for efficient and safe gene delivery to the rat retina.

  6. Determination of key parameters of vector multifractal vector fields (United States)

    Schertzer, D. J. M.; Tchiguirinskaia, I.


    For too long time, multifractal analyses and simulations have been restricted to scalar-valued fields (Schertzer and Tchiguirinskaia, 2017a,b). For instance, the wind velocity multifractality has been mostly analysed in terms of scalar structure functions and with the scalar energy flux. This restriction has had the unfortunate consequences that multifractals were applicable to their full extent in geophysics, whereas it has inspired them. Indeed a key question in geophysics is the complexity of the interactions between various fields or they components. Nevertheless, sophisticated methods have been developed to determine the key parameters of scalar valued fields. In this communication, we first present the vector extensions of the universal multifractal analysis techniques to multifractals whose generator belong to a Levy-Clifford algebra (Schertzer and Tchiguirinskaia, 2015). We point out further extensions noting the increased complexity. For instance, the (scalar) index of multifractality becomes a matrice. Schertzer, D. and Tchiguirinskaia, I. (2015) `Multifractal vector fields and stochastic Clifford algebra', Chaos: An Interdisciplinary Journal of Nonlinear Science, 25(12), p. 123127. doi: 10.1063/1.4937364. Schertzer, D. and Tchiguirinskaia, I. (2017) `An Introduction to Multifractals and Scale Symmetry Groups', in Ghanbarian, B. and Hunt, A. (eds) Fractals: Concepts and Applications in Geosciences. CRC Press, p. (in press). Schertzer, D. and Tchiguirinskaia, I. (2017b) `Pandora Box of Multifractals: Barely Open ?', in Tsonis, A. A. (ed.) 30 Years of Nonlinear Dynamics in Geophysics. Berlin: Springer, p. (in press).

  7. On Weighted Support Vector Regression

    DEFF Research Database (Denmark)

    Han, Xixuan; Clemmensen, Line Katrine Harder


    We propose a new type of weighted support vector regression (SVR), motivated by modeling local dependencies in time and space in prediction of house prices. The classic weights of the weighted SVR are added to the slack variables in the objective function (OF‐weights). This procedure directly...... the differences and similarities of the two types of weights by demonstrating the connection between the Least Absolute Shrinkage and Selection Operator (LASSO) and the SVR. We show that an SVR problem can be transformed to a LASSO problem plus a linear constraint and a box constraint. We demonstrate...

  8. Behavioral Public Administration

    DEFF Research Database (Denmark)

    Grimmelikhuijsen, Stephan; Jilke, Sebastian; Olsen, Asmus Leth


    Behavioral public administration is the analysis of public administration from the micro-level perspective of individual behavior and attitudes by drawing on insights from psychology on the behavior of individuals and groups. The authors discuss how scholars in public administration currently draw...... theories. As such, behavioral public administration complements traditional public administration. Furthermore, it could be a two-way street for psychologists who want to test the external validity of their theories in a political-administrative setting. Finally, four principles are proposed to narrow...


    Directory of Open Access Journals (Sweden)

    Liana Teodora PASCARIU


    Full Text Available Article examines whether all contracts of public persons are administrative contracts; in other words, if the administration may conclude contracts that, according to their legal nature, are not administrative. If we start from the definition of administrative contracts as it appears in Law no. 554/2004, these include contracts by public authorities which concern the enhancement of public property execution of works of public interest, public services, public procurement and other administrative contracts provided by special laws and subject to the jurisdiction of the administrative courts.

  10. Estimation of pure autoregressive vector models for revenue series ...

    African Journals Online (AJOL)

    This paper aims at applying multivariate approach to Box and Jenkins univariate time series modeling to three vector series. General Autoregressive Vector Models with time varying coefficients are estimated. The first vector is a response vector, while others are predictor vectors. By matrix expansion each vector, whether ...

  11. [A recombinant adenovirus vector carrying murine interleukin-21 gene controls chronic HBV infection in mice]. (United States)

    Gao, Xue-Ping; Zhou, Yang; Zheng, Xin-Chun; Yi, Xuan; Tang, Li-Bo; Hou, Jin-Lin; Li, Yong-Yin


    To investigate the effect of an adenovirus vector containing murine interleukin-21 gene (Ad-GFP-mIL-21) in virus clearance and on the production of HBV-specific antibodies in mice with persistent HBV infection. ELISA and Western blot analysis were used to detect the expression of mIL-21 in the supernatant and cytoplasm of cultured HepG2.2.15 cells after infection by Ad-GFP-mIL-21. Mouse models of chronic HBV infection established by in vivo transduction with rAAV8-1.3HBV were divided into 3 groups for treatment 12 weeks later with injection of Ad-GFP-mIL-21, GFP recombinant adenovirus or PBS via the tail vein. Serum levels of HBsAg, HBsAb, HBcAb, and mIL-21 in the mice were detected using ELISA, and the expression of Ad-GFP-mIL-21 in the organs was observed by fluorescent microscopy at different time points after the injection. Ad-GFP-mIL-21 was capable of infecting HepG2.2.15 cells in vitro, and the levels of mIL-21 in the supernatant were correlated with the titers of adenovirus administered and the infection time. In the mice with persistent HBV infection, green fluorescence expression was observed almost exclusively in the liver on day 4 after injection of Ad-GFP-mIL21, and serum levels of IL-21 increased significantly compared with the level before treatment (PHBcAb was detected in the mice with Ad-GFP-mIL21 injection (PHBcAb production, suggesting its efficacy in controlling chronic HBV infection.

  12. Vector-tensor and vector-vector decay amplitude analysis of B0-->phiK*0. (United States)

    Aubert, B; Bona, M; Boutigny, D; Couderc, F; Karyotakis, Y; Lees, J P; Poireau, V; Tisserand, V; Zghiche, A; Grauges, E; Palano, A; Chen, J C; Qi, N D; Rong, G; Wang, P; Zhu, Y S; Eigen, G; Ofte, I; Stugu, B; Abrams, G S; Battaglia, M; Brown, D N; Button-Shafer, J; Cahn, R N; Charles, E; Gill, M S; Groysman, Y; Jacobsen, R G; Kadyk, J A; Kerth, L T; Kolomensky, Yu G; Kukartsev, G; Pegna, D Lopes; Lynch, G; Mir, L M; Orimoto, T J; Pripstein, M; Roe, N A; Ronan, M T; Wenzel, W A; Sanchez, P del Amo; Barrett, M; Ford, K E; Harrison, T J; Hart, A J; Hawkes, C M; Watson, A T; Held, T; Koch, H; Lewandowski, B; Pelizaeus, M; Peters, K; Schroeder, T; Steinke, M; Boyd, J T; Burke, J P; Cottingham, W N; Walker, D; Asgeirsson, D J; Cuhadar-Donszelmann, T; Fulsom, B G; Hearty, C; Knecht, N S; Mattison, T S; McKenna, J A; Khan, A; Kyberd, P; Saleem, M; Sherwood, D J; Teodorescu, L; Blinov, V E; Bukin, A D; Druzhinin, V P; Golubev, V B; Onuchin, A P; Serednyakov, S I; Skovpen, Yu I; Solodov, E P; Todyshev, K Yu; Best, D S; Bondioli, M; Bruinsma, M; Chao, M; Curry, S; Eschrich, I; Kirkby, D; Lankford, A J; Lund, P; Mandelkern, M; Roethel, W; Stoker, D P; Abachi, S; Buchanan, C; Foulkes, S D; Gary, J W; Long, O; Shen, B C; Wang, K; Zhang, L; Hadavand, H K; Hill, E J; Paar, H P; Rahatlou, S; Sharma, V; Berryhill, J W; Campagnari, C; Cunha, A; Dahmes, B; Hong, T M; Kovalskyi, D; Richman, J D; Beck, T W; Eisner, A M; Flacco, C J; Heusch, C A; Kroseberg, J; Lockman, W S; Nesom, G; Schalk, T; Schumm, B A; Seiden, A; Spradlin, P; Williams, D C; Wilson, M G; Albert, J; Chen, E; Cheng, C H; Dvoretskii, A; Fang, F; Hitlin, D G; Narsky, I; Piatenko, T; Porter, F C; Mancinelli, G; Meadows, B T; Mishra, K; Sokoloff, M D; Blanc, F; Bloom, P C; Chen, S; Ford, W T; Hirschauer, J F; Kreisel, A; Nagel, M; Nauenberg, U; Olivas, A; Ruddick, W O; Smith, J G; Ulmer, K A; Wagner, S R; Zhang, J; Chen, A; Eckhart, E A; Soffer, A; Toki, W H; Wilson, R J; Winklmeier, F; Zeng, Q; Altenburg, D D; Feltresi, E; Hauke, A; Jasper, H; Merkel, J; Petzold, A; Spaan, B; Brandt, T; Klose, V; Lacker, H M; Mader, W F; Nogowski, R; Schubert, J; Schubert, K R; Schwierz, R; Sundermann, J E; Volk, A; Bernard, D; Bonneaud, G R; Latour, E; Thiebaux, Ch; Verderi, M; Clark, P J; Gradl, W; Muheim, F; Playfer, S; Robertson, A I; Xie, Y; Andreotti, M; Bettoni, D; Bozzi, C; Calabrese, R; Cibinetto, G; Luppi, E; Negrini, M; Petrella, A; Piemontese, L; Prencipe, E; Anulli, F; Baldini-Ferroli, R; Calcaterra, A; de Sangro, R; Finocchiaro, G; Pacetti, S; Patteri, P; Peruzzi, I M; Piccolo, M; Rama, M; Zallo, A; Buzzo, A; Contri, R; Vetere, M Lo; Macri, M M; Monge, M R; Passaggio, S; Patrignani, C; Robutti, E; Santroni, A; Tosi, S; Brandenburg, G; Chaisanguanthum, K S; Lee, C L; Morii, M; Wu, J; Dubitzky, R S; Marks, J; Schenk, S; Uwer, U; Bard, D J; Bhimji, W; Bowerman, D A; Dauncey, P D; Egede, U; Flack, R L; Nash, J A; Nikolich, M B; Vazquez, W Panduro; Behera, P K; Chai, X; Charles, M J; Mallik, U; Meyer, N T; Ziegler, V; Cochran, J; Crawley, H B; Dong, L; Eyges, V; Meyer, W T; Prell, S; Rosenberg, E I; Rubin, A E; Gao, Y; Gritsan, A V; Guo, Z J; Denig, A G; Fritsch, M; Schott, G; Arnaud, N; Davier, M; Grosdidier, G; Höcker, A; Lepeltier, V; Diberder, F Le; Lutz, A M; Oyanguren, A; Pruvot, S; Rodier, S; Roudeau, P; Schune, M H; Serrano, J; Stocchi, A; Wang, W F; Wormser, G; Lange, D J; Wright, D M; Chavez, C A; Forster, I J; Fry, J R; Gabathuler, E; Gamet, R; George, K A; Hutchcroft, D E; Payne, D J; Schofield, K C; Touramanis, C; Bevan, A J; Clarke, C K; Lodovico, F Di; Menges, W; Sacco, R; Cowan, G; Flaecher, H U; Hopkins, D A; Jackson, P S; McMahon, T R; Salvatore, F; Wren, A C; Brown, D N; Davis, C L; Allison, J; Barlow, N R; Barlow, R J; Chia, Y M; Edgar, C L; Lafferty, G D; Naisbit, M T; Williams, J C; Yi, J I; Chen, C; Hulsbergen, W D; Jawahery, A; Lae, C K; Roberts, D A; Simi, G; Blaylock, G; Dallapiccola, C; Hertzbach, S S; Li, X; Moore, T B; Saremi, S; Staengle, H; Cowan, R; Sciolla, G; Sekula, S J; Spitznagel, M; Taylor, F; Yamamoto, R K; Kim, H; McLachlin, S E; Patel, P M; Robertson, S H; Lazzaro, A; Lombardo, V; Palombo, F; Bauer, J M; Cremaldi, L; Eschenburg, V; Godang, R; Kroeger, R; Sanders, D A; Summers, D J; Zhao, H W; Brunet, S; Côté, D; Simard, M; Taras, P; Viaud, F B; Nicholson, H; Cavallo, N; Nardo, G De; Fabozzi, F; Gatto, C; Lista, L; Monorchio, D; Paolucci, P; Piccolo, D; Sciacca, C; Baak, M A; Raven, G; Snoek, H L; Jessop, C P; Losecco, J M; Benelli, G; Corwin, L A; Gan, K K; Honscheid, K; Hufnagel, D; Jackson, P D; Kagan, H; Kass, R; Rahimi, A M; Regensburger, J J; Ter-Antonyan, R; Wong, Q K; Blount, N L; Brau, J; Frey, R; Igonkina, O; Kolb, J A; Lu, M; Potter, C T; Rahmat, R; Sinev, N B; Strom, D; Strube, J; Torrence, E; Gaz, A; Margoni, M; Morandin, M; Pompili, A; Posocco, M; Rotondo, M; Simonetto, F; Stroili, R; Voci, C


    We perform an amplitude analysis of the decays B(0)-->phiK*(2)(1430)(0), phiK*(892)(0), and phi(Kpi)(0)(S-wave) with a sample of about 384x10(6) BB[over ] pairs recorded with the BABAR detector. The fractions of longitudinal polarization f(L) of the vector-tensor and vector-vector decay modes are measured to be 0.853(-0.069+0.061)+/-0.036 and 0.506+/-0.040+/-0.015, respectively. Overall, twelve parameters are measured for the vector-vector decay and seven parameters for the vector-tensor decay, including the branching fractions and parameters sensitive to CP violation.

  13. Meta-Analyses of Japanese Encephalitis Virus Infection, Dissemination, and Transmission Rates in Vectors. (United States)

    Oliveira, Ana R S; Cohnstaedt, Lee W; Strathe, Erin; Etcheverry, Luciana; McVey, D Scott; Piaggio, José; Cernicchiaro, Natalia


    The objective of this work was to summarize and quantify Japanese encephalitis virus (JEV) infection, dissemination, and transmission rates in mosquitoes, using a meta-analysis approach. Data were obtained from experimental studies, gathered by means of a systematic review of the literature. Random-effects subgroup meta-analysis models by mosquito species were fitted to estimate pooled estimates and to calculate the variance between studies for three outcomes of interest: JEV infection, dissemination, and transmission rates in mosquitoes. To identify sources of heterogeneity among studies and to assess the association between different predictors (mosquito species, virus administration route, incubation period, and diagnostic method) with the outcome JEV infection rate in vectors, we fitted univariable meta-regression models. Mosquito species and administration route represented the main sources of heterogeneity associated with JEV infection rate in vectors. This study provided summary effect size estimates to be used as reference for other investigators when assessing transmission efficiency of vectors and explored sources of variability for JEV infection rates in vectors. Because transmission efficiency, as part of vector competence assessment, is an important parameter when studying the relative contribution of vectors to JEV transmission, our findings contribute to further our knowledge, potentially moving us toward more informed and targeted actions to prevent and control JEV in both affected and susceptible regions worldwide.

  14. Dengue Vectors and their Spatial Distribution. (United States)

    Higa, Yukiko


    The distribution of dengue vectors, Ae. aegypti and Ae. albopictus, is affected by climatic factors. In addition, since their life cycles are well adapted to the human environment, environmental changes resulting from human activity such as urbanization exert a great impact on vector distribution. The different responses of Ae. aegypti and Ae albopictus to various environments result in a difference in spatial distribution along north-south and urban-rural gradients, and between the indoors and outdoors. In the north-south gradient, climate associated with survival is an important factor in spatial distribution. In the urban-rural gradient, different distribution reflects a difference in adult niches and is modified by geographic and human factors. The direct response of the two species to the environment around houses is related to different spatial distribution indoors and outdoors. Dengue viruses circulate mainly between human and vector mosquitoes, and the vector presence is a limiting factor of transmission. Therefore, spatial distribution of dengue vectors is a significant concern in the epidemiology of the disease.Current technologies such as GIS, satellite imagery and statistical models allow researchers to predict the spatial distribution of vectors in the changing environment. Although it is difficult to confirm the actual effect of environmental and climate changes on vector abundance and vector-borne diseases, environmental changes caused by humans and human behavioral changes due to climate change can be expected to exert an impact on dengue vectors. Longitudinal monitoring of dengue vectors and viruses is therefore necessary.

  15. Administration of Anesthesia

    Medline Plus

    Full Text Available ... OMSs) are trained in all aspects of anesthesia administration. Following dental school, they complete at least four ... complications and emergencies that may arise during the administration of anesthesia. Before your surgery, your OMS will ...

  16. Administration for Community Living (United States)

    ... Public Input Working Together, in Our Communities The Administration for Community Living was created around the fundamental ... Meals on Wheels America - November 16, 2017 The Administration for Community Living recently awarded a three-year ...

  17. Vectorization of phase space Monte Carlo code in FACOM vector processor VP-200

    International Nuclear Information System (INIS)

    Miura, Kenichi


    This paper describes the vectorization techniques for Monte Carlo codes in Fujitsu's Vector Processor System. The phase space Monte Carlo code FOWL is selected as a benchmark, and scalar and vector performances are compared. The vectorized kernel Monte Carlo routine which contains heavily nested IF tests runs up to 7.9 times faster in vector mode than in scalar mode. The overall performance improvement of the vectorized FOWL code over the original scalar code reaches 3.3. The results of this study strongly indicate that supercomputer can be a powerful tool for Monte Carlo simulations in high energy physics. (Auth.)

  18. Cloudera administration handbook

    CERN Document Server

    Menon, Rohit


    An easy-to-follow Apache Hadoop administrator's guide filled with practical screenshots and explanations for each step and configuration. This book is great for administrators interested in setting up and managing a large Hadoop cluster. If you are an administrator, or want to be an administrator, and you are ready to build and maintain a production-level cluster running CDH5, then this book is for you.

  19. Vector assembly of colloids on monolayer substrates (United States)

    Jiang, Lingxiang; Yang, Shenyu; Tsang, Boyce; Tu, Mei; Granick, Steve


    The key to spontaneous and directed assembly is to encode the desired assembly information to building blocks in a programmable and efficient way. In computer graphics, raster graphics encodes images on a single-pixel level, conferring fine details at the expense of large file sizes, whereas vector graphics encrypts shape information into vectors that allow small file sizes and operational transformations. Here, we adapt this raster/vector concept to a 2D colloidal system and realize `vector assembly' by manipulating particles on a colloidal monolayer substrate with optical tweezers. In contrast to raster assembly that assigns optical tweezers to each particle, vector assembly requires a minimal number of optical tweezers that allow operations like chain elongation and shortening. This vector approach enables simple uniform particles to form a vast collection of colloidal arenes and colloidenes, the spontaneous dissociation of which is achieved with precision and stage-by-stage complexity by simply removing the optical tweezers.

  20. Legal and Administrative Language (United States)

    Schwarz, Hans


    A discussion of legal and administrative language, and the necessity for accurate translation of this language in the field of international relations. Topics treated are: characteristic features of legal and administrative terminology; the interpretation of it; and the technique of translating legal and administrative texts. (AMH)