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Sample records for adeno-associated virus-mediated microrna

  1. Recombinant Adeno-Associated Virus-Mediated microRNA Delivery into the Postnatal Mouse Brain Reveals a Role for miR-134 in Dendritogenesis in Vivo

    Christensen, Mette; Larsen, Lars A; Kauppinen, Sakari; Schratt, Gerhard

    2010-01-01

    delivery of microRNAs in vivo by use of recombinant adeno-associated virus (rAAV). rAAV-mediated overexpression of miR-134 in neurons of the postnatal mouse brain provided evidence for a negative role of miR-134 in dendritic arborization of cortical layer V pyramidal neurons in vivo, thereby confirming...

  2. Adeno-associated Virus Mediated LacZ Gene Transfect to Cultured Human Iris Pigment Epithelium Cells

    Chun Zhang; Shibo Tang; Yan Luo; Xiaoling Liang; Jing Ma; Shaofen Lin

    2003-01-01

    Purpose: To study the feasibility of adeno-associated virus mediated gene transfection tocultured human iris pigment epithelium (IPE) cells in vitro.Methods: Recombinant replication deficient adeno-associated viruses (AAV) expressingLacZ gene were produced without helper virus. The LacZ gene was transduced into culturedhuman IPE cells.Results: Cultured human IPE cells stained positively anticytokeratin, The titer ofrAAV-LacZ was 2.1 × 108 virus particles/ml, 42% cultured human IPE cells expressedβ-galactosidase 7 days after transfection and 67% after 14 days.Conclusions: Recombined AAV produced without helper virus can transfer a foreign geneinto human IPE cells with high efficiency in vitro.

  3. Adeno-Associated Virus-Mediated Cancer Gene Therapy: Current Status

    Luo, Jingfeng; Luo, Yuxuan; Sun, Jihong; Zhou, Yurong; Zhang, Yajing; Yang, Xiaoming

    2014-01-01

    Gene therapy is one of the frontiers of modern medicine. Adeno-associated virus (AAV)-mediated gene therapy is becoming a promising approach to treat a variety of diseases and cancers. AAV-mediated cancer gene therapies have rapidly advanced due to their superiority to other gene-carrying vectors, such as the lack of pathogenicity, the ability to transfect both dividing and non-dividing cells, low host immune response, and long-term expression. This article reviews and provides up to date kno...

  4. Translational data from adeno-associated virus-mediated gene therapy of hemophilia B in dogs.

    Nichols, Timothy C; Whitford, Margaret H; Arruda, Valder R; Stedman, Hansell H; Kay, Mark A; High, Katherine A

    2015-03-01

    Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches. PMID:25675273

  5. Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy.

    Nguyen, J T; Wu, P; Clouse, M E; Hlatky, L; Terwilliger, E F

    1998-12-15

    Antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial targeting, an absence of drug resistance. To promote tumor regression and to maintain dormancy, antiangiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. As a step toward this goal, we have generated recombinant adeno-associated virus (rAAV) vectors that carry genes coding for angiostatin, endostatin, and an antisense mRNA species against vascular endothelial growth factor (VEGF). These rAAVs efficiently transduced three human tumor cell lines tested. Transduction with an rAAV-encoding antisense VEGF mRNA inhibited the production of endogenous tumor cell VEGF. Conditioned media from cells transduced with this rAAV or with rAAV-expressing endostatin or angiostatin inhibited capillary endothelial cell proliferation in vitro. Antiangiogenic rAAVs may offer a novel gene therapy approach to undermining tumor neovascularization and cancer progression. PMID:9865720

  6. Adeno-Associated Virus-Mediated Microdystrophin Expression Protects Young mdx Muscle from Contraction-Induced Injury

    LIU, MINGJU; Yue, Yongping; Harper, Scott Q.; Grange, Robert W.; Jeffrey S. Chamberlain; Duan, Dongsheng

    2005-01-01

    Duchenne muscular dystrophy (DMD) is the most common inherited lethal muscle degenerative disease. Currently there is no cure. Highly abbreviated microdystrophin cDNAs were developed recently for adeno-associated virus (AAV)-mediated DMD gene therapy. Among these, a C-terminal-truncated ΔR4-R23/ΔC microgene (ΔR4/ΔC) has been considered as a very promising therapeutic candidate gene. In this study, we packaged a CMV.ΔR4/ΔC cassette in AAV-5 and evaluated the transduction and muscle contractile...

  7. Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

    Tung-Yu Tsui; Chi-Keung Lau; Jian Ma; Gabriel Glockzin; Aiman Obed; Hans J Schlitt; Sheung-Tat Fan

    2006-01-01

    AIM: To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl4-induced micronodular cirrhosis model.METHODS: Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated. 1x1012 vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis.RESULTS: Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controis, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/HO-1-transduced livers.CONCLUSIONS: Enhancement of HO activity in the livers suppresses the development of cirrhosis.

  8. Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

    Daniel C. Chung

    2011-11-01

    Full Text Available Background/Aims: Gene therapy involves delivery of exogenous DNA to provide a therapeutic protein. Ideally, a gene therapy vector should be non-toxic, non-immunogenic, easy to produce, and efficient in protecting and delivering DNA into target cells. Methods: Adeno-associated virus (AAV offers these advantages and few, if any, disadvantages, and over 100 isolates exist. We previously showed that AAV-mediated gene therapy can be used to restore vision to patients with Leber’s congenital amaurosis, a disease of childhood blindness. Results: Here we show that novel recombinant AAV2/8 and AAV2/9 transduce kidney tubule cells with high efficiency both in vitroin cell culture and in vivoin mice. In addition, we adapted and modified a retrograde approach to allow for optimal transgene delivery to renal tubular cells that further minimizes the risk of an immunogenic reaction. Conclusions: We believe that recombinant AAV2, especially AAV2/8, gene delivery to renal tubule cells via a retrograde approach represents a viable method for gene therapy for a multitude of renal disorders ranging from autosomal dominant polycystic kidney disease to acute kidney injury.

  9. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV) vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy

  10. Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome.

    Jennifer L Daily

    Full Text Available Angelman syndrome (AS, a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286 and Thr(305/306, resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.

  11. Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture

    Kitto Kelley F

    2010-05-01

    Full Text Available Abstract Background Neuronal transduction by adeno-associated viral (AAV vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG and spinal cord offers substantial opportunities to 1 further study mechanisms underlying chronic pain, and 2 develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5- and AAV serotype 8 (AAV8-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture. Results Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP or AAV8 (rAAV8-GFP carrying the green fluorescent protein (GFP gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1 GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2 dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4 -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP. Conclusions The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.

  12. Adeno-associated virus-mediated Bcl-xL prevents aminoglycoside-induced hearing loss in mice

    LIU Yu-he; KE Xiao-mei; QIN Yong; GU Zhi-ping; XIAO Shui-fang

    2007-01-01

    Background Recent studies showed that aminoglycosides destroyed the cochlear cells and induced ototoxicity by producing reactive oxygen species, including free radicals in the mitochondria, damaging the membrane of mitochondria and resulting in apoptotic cell death. Bcl-xL is a well characterized anti-apoptotic member of the Bcl-2 family. The aim of this study was to determine the potential cochlear protective effect of Bcl-xL as a therapeutic agent in the murine model of aminoglycoside ototoxicity.Methods Serotype 2 of adeno-associated virus (AAV2) as a vector encoding the mouse Bcl-xL gene was injected into mice cochleae prior to injection of kanamycin. Bcl-xL expression in vitro and in vivo was examined with Western blotting and immunohistochemistry separately. Cochlear dissection and auditory steady state responses were checked to evaluate the cochlear structure and function.Results The animals in the AAV2-Bcl-xL/kanamycin group displayed better auditory steady state responses hearing thresholds and cochlear structure than those in the artificial perilymph/kanamycin or AAV2-enhanced humanized green fluorescent protein/kanamycin control group at all tested frequencies. The auditory steady state responses hearing thresholds and cochlear structure in the inoculated side were better than that in the contralateral side.Conclusions AAV2-Bcl-xL afforded significant preservation of the cochlear hair cells against ototoxic insults and protected the cochlear function. AAV2-mediated Bcl-xL might be an approach with respect to potential therapeutic application in the cochlear degeneration.

  13. Adeno-associated virus-mediated doxycycline-regulatable TRAIL expression suppresses growth of human breast carcinoma in nude mice

    Zheng Liu

    2012-04-01

    Full Text Available Abstract Background Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. We have showed previous administration of a recombinant adeno-associated virus (rAAV vector expressing soluble TRAIL results in an efficient suppression of human tumor growth in nude mice. In the present study, we introduced Tet-On gene expression system into the rAAV vector to control the soluble TRAIL expression and evaluate the efficiency of the system in cancer gene therapy. Methods Controllability of the Tet-On system was determined by luciferase activity assay, and Western blotting and enzyme-linked immunoabsorbent assay. Cell viability was determined by MTT assay. The breast cancer xenograft animal model was established and recombinant virus was administrated through tail vein injection to evaluate the tumoricidal activity. Results The expression of soluble TRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of human cancer cell lines with rAAV-TRE-TRAIL&rAAV-Tet-On under the presence of inducer doxycycline resulted in a considerable cell death by apoptosis. Intravenous injection of the recombinant virus efficiently suppressed the growth of human breast carcinoma in nude mice when activated by doxycycline. Conclusion These data suggest that rAAV-mediated soluble TRAIL expression under the control of the Tet-On system is a promising strategy for breast cancer therapy.

  14. Recombinant adeno-associated virus-mediated gene transfer for the potential therapy of adenosine deaminase-deficient severe combined immune deficiency.

    Silver, Jared N; Elder, Melissa; Conlon, Thomas; Cruz, Pedro; Wright, Amy J; Srivastava, Arun; Flotte, Terence R

    2011-08-01

    Severe combined immune deficiency due to adenosine deaminase (ADA) deficiency is a rare, potentially fatal pediatric disease, which results from mutations within the ADA gene, leading to metabolic abnormalities and ultimately profound immunologic and nonimmunologic defects. In this study, recombinant adeno-associated virus (rAAV) vectors based on serotypes 1 and 9 were used to deliver a secretory version of the human ADA (hADA) gene to various tissues to promote immune reconstitution following enzyme expression in a mouse model of ADA deficiency. Here, we report that a single-stranded rAAV vector, pTR2-CB-Igκ-hADA, (1) facilitated successful gene delivery to multiple tissues, including heart, skeletal muscle, and kidney, (2) promoted ectopic expression of hADA, and (3) allowed enhanced serum-based enzyme activity over time. Moreover, the rAAV-hADA vector packaged in serotype 9 capsid drove partial, prolonged, and progressive immune reconstitution in ADA-deficient mice. Overview Summary Gene therapies for severe combined immune deficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) over two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoietic progenitor cells. These groundbreaking gene therapies represented an unprecedented revolution in clinical medicine but in most cases did not fully correct the immune deficiency and came with the potential risk of insertional mutagenesis. Alternatively, recombinant adeno-associated virus (rAAV) vectors have gained attention as valuable tools for gene transfer, having demonstrated no pathogenicity in humans, minimal immunogenicity, long-term efficacy, ease of administration, and broad tissue tropism (Muzyczka, 1992 ; Flotte et al., 1993 ; Kessler et al., 1996 ; McCown et al., 1996 ; Lipkowitz et al., 1999 ; Marshall, 2001 ; Chen et al., 2003 ; Conlon and Flotte, 2004 ; Griffey et al., 2005 ; Pacak et al., 2006 ; Stone et al., 2008 ; Liu et al., 2009 ; Choi et al., 2010

  15. Recombinant adeno-associated virus-mediated human kallikrein gene therapy prevents high-salt diet-induced hypertension without effect on basal blood pressure

    Jiang-tao YAN; Tao WANG; Juan LI; Xiao XIAO; Dao-wen WANG

    2008-01-01

    Aim: To investigate the effects of the expression of human kallikrein (HK) on basal level blood pressure and high-salt diet-induced hypertension. Methods: We delivered the recombinant adeno-associated viral (rAAV)-mediated HK (rAAV-HK) gene and rAAV-LacZ (as the control) to normal, adult Sprague-Dawley rats. The animals were administered a normal diet in the first 4 weeks, followed by a high-salt diet. The expression of HK in the rats was assessed by ELISA and RT-PCR. Blood pressure and Na~ and K~ urinary excretion were monitored. Results: Under the normal diet, no obvious changes in blood pressure and Na+ and K+ urinary excretion were observed. When the high-salt diet was administered, sys-tolic blood pressure in the control animals receiving rAAV-LacZ increased from 122.3±1. 13 mmHg to a stable 142.4±1.77 mmHg 8 weeks after the high-salt diet. In contrast, there was no significant increase in the blood pressure in the rAAV-HK-treated group, in which the blood pressure remained at 121.9±1.73 mmHg. In the rAAV-HK-treated group, Na+ and K+ urinary excretion were higher compared to those of the control group. The morphological analysis showed that HK delivery remarkably protected against renal damage induced by a high-salt intake. Conclusion: Our study indicates that rAAV-mediated human tissue kallikrein gene delivery is a potentially safe method for the long-term treatment of hypertension. More importantly, it could be applied in the salt-sensitive population to prevent the occurrence of hypertension.

  16. Optimization of Recombinant Adeno-Associated Virus-Mediated Expression for Large Transgenes, Using a Synthetic Promoter and Tandem Array Enhancers.

    Yan, Ziying; Sun, Xingshen; Feng, Zehua; Li, Guiying; Fisher, John T; Stewart, Zoe A; Engelhardt, John F

    2015-06-01

    The packaging capacity of recombinant adeno-associated viral (rAAV) vectors limits the size of the promoter that can be used to express the 4.43-kb cystic fibrosis transmembrane conductance regulator (CFTR) cDNA. To circumvent this limitation, we screened a set of 100-mer synthetic enhancer elements, composed of ten 10-bp repeats, for their ability to augment CFTR transgene expression from a short 83-bp synthetic promoter in the context of an rAAV vector designed for use in the cystic fibrosis (CF) ferret model. Our initial studies assessing transcriptional activity in monolayer (nonpolarized) cultures of human airway cell lines and primary ferret airway cells revealed that three of these synthetic enhancers (F1, F5, and F10) significantly promoted transcription of a luciferase transgene in the context of plasmid transfection. Further analysis in polarized cultures of human and ferret airway epithelia at an air-liquid interface (ALI), as well as in the ferret airway in vivo, demonstrated that the F5 enhancer produced the highest level of transgene expression in the context of an AAV vector. Furthermore, we demonstrated that increasing the size of the viral genome from 4.94 to 5.04 kb did not significantly affect particle yield of the vectors, but dramatically reduced the functionality of rAAV-CFTR vectors because of small terminal deletions that extended into the CFTR expression cassette of the 5.04-kb oversized genome. Because rAAV-CFTR vectors greater than 5 kb in size are dramatically impaired with respect to vector efficacy, we used a shortened ferret CFTR minigene with a 159-bp deletion in the R domain to construct an rAAV vector (AV2/2.F5tg83-fCFTRΔR). This vector yielded an ∼17-fold increase in expression of CFTR and significantly improved Cl(-) currents in CF ALI cultures. Our study has identified a small enhancer/promoter combination that may have broad usefulness for rAAV-mediated CF gene therapy to the airway. PMID:25763813

  17. 腺相关病毒介导重组血管抑素联合雷公藤红素对大鼠颅内C6胶质瘤的抗血管生成作用%Anti-angiogenesis effect of adeno-associated virus-mediated recombinant angiostatin combined with celastrol on intracranial C6 glioma in rats

    王冠; 周洁; 冯珂珂; 田麒

    2011-01-01

    目的:腺相关病毒(adeno-associated virus,AAV)介导的重组血管抑素(angiostatin,AS)联合应用雷公藤红素( celastrol)治疗大鼠颅内C6胶质瘤,观察其对肿瘤体积、新生血管密度及肿瘤细胞凋亡的影响,探讨抗血管生成重组基因联合雷公藤红素对胶质瘤治疗的前景.方法:建立颅内原位荷C6脑胶质瘤大鼠模型,7d后随机分为4组,分别给予0.9%氯化钠溶液(作为对照)、AAV-AS、雷公藤红素及两者联合用药.每隔7d行头部强化MRI检查,计算肿瘤体积.于22 d后处死动物,检测AS蛋白表达、血管密度及肿瘤细胞凋亡情况.结果:联合治疗组及AAV-AS治疗组均检测到AS蛋白表达,证实基因转导成功.联合治疗组第22天时肿瘤体积、血管密度和凋亡指数均与对照组、雷公藤红素组及AAV-AS治疗组相比差异有统计学意义(P<0.05),联合治疗可以抑制肿瘤生长,降低新生血管密度,促进肿瘤细胞凋亡.结论:基因治疗联合雷公藤红素可通过抑制胶质瘤血管生成而抑制肿瘤生长;两者联合应用具有协同作用,可弥补两者单独应用的不足之处.%Objective: To examine the effects of therapeutic alliance of adeno-associated virus-mediated recombinant angiostatin (AAV-AS) combined with celastrol on tumor growth, microvessel density and apoptosis of intracranial glioma in rats, and to give a prospective of this therapeutic alliance. Methods: A rat intracranial C6 glioma model was established, and then the rats (n=40) were randomly assigned into four groups after 7 days, which were saline control group, AAV-AS group, celastrol group and therapeutic alliance group. The tumor growth was examined by magnetic resonance imaging (MRI) every 7 days, and the volume of tumor was calculated. The rats were killed after 22 days, and the expression of AS protein, the microvessel density and the apoptosis of tumor cells were detected. Results: The expression of AS protein was detectable in AAV

  18. Regression of Schwannomas Induced by Adeno-Associated Virus-Mediated Delivery of Caspase-1

    Prabhakar, Shilpa; Taherian, Mehran; Gianni, Davide; Conlon, Thomas J.; Fulci, Giulia; Brockmann, Jillian; Stemmer-Rachamimov, Anat; Sena-Esteves, Miguel; Breakefield, Xandra O.; Brenner, Gary J.

    2012-01-01

    Schwannomas are tumors formed by proliferation of dedifferentiated Schwann cells. Patients with neurofibromatosis 2 (NF2) and schwannomatosis develop multiple schwannomas in peripheral and cranial nerves. Although benign, these tumors can cause extreme pain and compromise sensory/motor functions, including hearing and vision. At present, surgical resection is the main treatment modality, but it can be problematic because of tumor inaccessibility and risk of nerve damage. We have explored gene...

  19. Adeno-associated virus for cystic fibrosis gene therapy

    S.V. Martini

    2011-11-01

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

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

    Ayşegül Akbay Yarpuzlu

    2009-06-01

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

  1. Adeno-associated virus: from defective virus to effective vector

    Gonçalves Manuel AFV

    2005-05-01

    Full Text Available Abstract The initial discovery of adeno-associated virus (AAV mixed with adenovirus particles was not a fortuitous one but rather an expression of AAV biology. Indeed, as it came to be known, in addition to the unavoidable host cell, AAV typically needs a so-called helper virus such as adenovirus to replicate. Since the AAV life cycle revolves around another unrelated virus it was dubbed a satellite virus. However, the structural simplicity plus the defective and non-pathogenic character of this satellite virus caused recombinant forms to acquire centre-stage prominence in the current constellation of vectors for human gene therapy. In the present review, issues related to the development of recombinant AAV (rAAV vectors, from the general principle to production methods, tropism modifications and other emerging technologies are discussed. In addition, the accumulating knowledge regarding the mechanisms of rAAV genome transduction and persistence is reviewed. The topics on rAAV vectorology are supplemented with information on the parental virus biology with an emphasis on aspects that directly impact on vector design and performance such as genome replication, genetic structure, and host cell entry.

  2. Recombinant adeno-associated virus-mediated inhibiting of interleukin-4 expression in rat model of asthma

    2006-01-01

    @@ Asthma is a chronic disease characterized by reversible airway obstruction, airway hyper- responsiveness, and inflammation of airways. Th2 cells, one sort of CD4+ T lymphocytes, are currently considered to play an important role in the chronic airway inflammation of asthma. Meanwhile, a number of laboratories have clearly established the importance of the Th2-derived cytokine interleukin-4 (IL-4) in mediating the airway inflammatory response. Anti-IL-4 therapy might be beneficial in treatment of chronic asthma.

  3. Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6

    Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2008-01-01

    Adeno-associated virus type 6, a human DNA virus that is being developed as a vector for gene therapy, has been crystallized in a form suitable for structure determination at about 3.2 Å resolution.

  4. Structure of neurotropic adeno-associated virus AAVrh.8.

    Halder, Sujata; Van Vliet, Kim; Smith, J Kennon; Duong, Thao Thi Phuong; McKenna, Robert; Wilson, James M; Agbandje-McKenna, Mavis

    2015-10-01

    Adeno-associated virus rhesus isolate 8 (AAVrh.8) is a leading vector for the treatment of neurological diseases due to its efficient transduction of neuronal cells and reduced peripheral tissue tropism. Toward identification of the capsid determinants for these properties, the structure of AAVrh.8 was determined by X-ray crystallography to 3.5 Å resolution and compared to those of other AAV isolates. The capsid viral protein (VP) structure consists of an αA helix and an eight-stranded anti-parallel β-barrel core conserved in parvoviruses, and large insertion loop regions between the β-strands form the capsid surface topology. The AAVrh.8 capsid exhibits the surface topology conserved in all AAVs: depressions at the icosahedral twofold axis and surrounding the cylindrical channel at the fivefold axis, and three protrusions around the threefold axis. A structural comparison to serotypes AAV2, AAV8, and AAV9, to which AAVrh.8 shares ∼ 84%, ∼ 91%, and ∼ 87% VP sequence identity, respectively, revealed differences in the surface loops known to affect receptor binding, transduction efficiency, and antigenicity. Consistent with this observation, biochemical assays showed that AAVrh.8 is unable to bind heparin and does not cross-react with conformational monoclonal antibodies and human donor serum directed against the other AAVs compared. This structure of AAVrh.8 thus identified capsid surface differences which can serve as template regions for rational design of vectors with enhanced transduction for specific tissues and escape pre-existing antibody recognition. These features are essential for the creation of an AAV vector toolkit that is amenable to personalized disease treatment. PMID:26334681

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

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

    1989-02-01

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

  6. Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies

    Lerch, Thomas F.; Xie, Qing; Ongley, Heather M.; Hare, Joan; Chapman, Michael S.

    2009-01-01

    Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning.

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

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

    2007-01-01

    Mesenchymal stem cells (MSCs) have received considerable attention in the emerging field of regenerative medicine. One aspect of MSC research focuses on genetically modifying the cells with the aim of enhancing their regenerative potential. Adeno-associated virus (AAV) holds promise as a vector for...

  8. Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

    Yakobson, B.; Hrynko, T.A.; Peak, M.J.; Winocour, E.

    1989-03-01

    Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting a requirement for de novo protein synthesis.

  9. Twinned crystals of adeno-associated virus serotype 3b prove suitable for structural studies

    Crystals of adeno-associated virus serotype 3b, a human DNA virus with promise as a vector for gene therapy, have been grown, diffract X-rays to ∼2.6 Å resolution and are suitable for structure determination in spite of twinning. Adeno-associated viruses (AAVs) are leading candidate vectors for gene-therapy applications. The AAV-3b capsid is closely related to the well characterized AAV-2 capsid (87% identity), but sequence and presumably structural differences lead to distinct cell-entry and immune-recognition properties. In an effort to understand these differences and to perhaps harness them, diffraction-quality crystals of purified infectious AAV-3b particles have been grown and several partial diffraction data sets have been recorded. The crystals displayed varying levels of merohedral twinning that in earlier times would have rendered them unsuitable for structure determination, but here is shown to be a tractable complication

  10. Adeno-Associated Virus Serotype-9 Microdystrophin Gene Therapy Ameliorates Electrocardiographic Abnormalities in mdx Mice

    Bostick, Brian; Yue, Yongping; Lai, Yi; Long, Chun; Li, Dejia; Duan, Dongsheng

    2008-01-01

    Adeno-associated virus (AAV)-mediated microdystrophin gene therapy holds great promise for treating Duchenne muscular dystrophy (DMD). Previous studies have revealed excellent skeletal muscle protection. Cardiac muscle is also compromised in DMD patients. Here we show that a single intravenous injection of AAV serotype-9 (AAV-9) microdystrophin vector efficiently transduced the entire heart in neonatal mdx mice, a dystrophin-deficient mouse DMD model. Furthermore, microdystrophin therapy norm...

  11. Feasibility of Generating Adeno-Associated Virus Packaging Cell Lines Containing Inducible Adenovirus Helper Genes

    Qiao, Chunping; Li, Juan; Skold, Anna; Zhang, Xudong; Xiao, Xiao

    2002-01-01

    The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection ...

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

    Yang Lin; Xiao Xiao

    2013-01-01

    Abstract Adeno-associated virus (AAV) is an important vector system for human gene therapy. Although use of AAV serotypes can result in efficient myocardial gene transfer, improvements in the transduction efficiency and specificity are still required. As a method for artificial modification and selection of gene function, directed evolution has been used for diverse applications in genetic engineering of enzymes and proteins. Since 2000, pioneering work has been performed on directed evolutio...

  13. Rapid, simple and versatile manufacturing of recombinant adeno-associated virus vectors at scale

    Lock, Martin; Alvira, Mauricio; Vandenberghe, Luk H.; Samanta, Arabinda; Toelen, Jaan; Debyser, Zeger; Wilson, James M

    2010-01-01

    Adeno-associated virus vector manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. While scalable systems based upon AAV-adenovirus, -herpesvirus and -baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate scale pre-clinical studies in large animals where several combinations of serotype and genome may need to be tested. Recently we observed that d...

  14. Differential Cellular Tropism of Lentivirus and Adeno-Associated Virus in the Brain of Cynomolgus Monkey

    An, Heeyoung; Cho, Doo-Wan; Lee, Seung Eun; Yang, Young-Su; Han, Su-Cheol; Lee, C. Justin

    2016-01-01

    Many researchers are using viruses to deliver genes of interest into the brains of laboratory animals. However, certain target brain cells are not easily infected by viruses. Moreover, the differential tropism of different viruses in monkey brain is not well established. We investigated the cellular tropism of lentivirus and adeno-associated virus (AAV) toward neuron and glia in the brain of cynomolgus monkeys (Macaca fascularis). Lentivirus and AAV were injected into putamen of the monkey br...

  15. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors

    Lai, Yi; Yue, Yongping; LIU, MINGJU; Ghosh, Arkasubhra; Engelhardt, John F.; Jeffrey S. Chamberlain; Duan, Dongsheng

    2005-01-01

    Although adeno-associated virus (AAV)-mediated gene therapy has been hindered by the small viral packaging capacity of the vector, trans-splicing AAV vectors are able to package twice the size of the vector genome. Unfortunately, the efficiency of current trans-splicing vectors is very low. Here we show that rational design of the gene splitting site has a profound influence on trans-splicing vector-mediated gene expression. Using mRNA accumulation as a guide, we generated a set of efficient ...

  16. Production, purification, crystallization and preliminary X-ray analysis of adeno-associated virus serotype 8

    The production, purification, crystallization and preliminary X-ray crystallographic analysis of adeno-associated virus serotype 8 is reported. Adeno-associated viruses (AAVs) are actively being developed for clinical gene-therapy applications and the efficiencies of the vectors could be significantly improved by a detailed understanding of their viral capsid structures and the structural determinants of their tissue-transduction interactions. AAV8 is ∼80% identical to the more widely studied AAV2, but its liver-transduction efficiency is significantly greater than that of AAV2 and other serotypes. The production, purification, crystallization and preliminary X-ray crystallographic analysis of AAV8 viral capsids are reported. The crystals diffract X-rays to 3.0 Å resolution using synchrotron radiation and belong to the hexagonal space group P6322, with unit-cell parameters a = 257.5, c = 443.5 Å. The unit cell contains two viral particles, with ten capsid viral protein monomers per crystallographic asymmetric unit

  17. A role for adeno-associated viral vectors in gene therapy

    Renata dos Santos Coura

    2008-01-01

    Full Text Available Gene therapy constitutes a therapeutic intervention based on modification of the genetic material of living cells, by correcting genetic defects or overexpressing therapeutic proteins. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy. Recent advances on the development of gene delivery systems, particularly on viral vectors engineering and improved gene regulatory systems, have led to marked progress in this field. Although the available vector systems can successfully transfer genes into cells, the ideal delivery vehicle has not been found. In this context, adeno-associated virus vectors (AAV are arising as a promising tool for a wide range of applications, due to a combination of characteristics such as lack of pathogenicity and immunogenicity, wide range of cell tropism and long-term gene expression. Since its isolation, the biological properties of the adeno-associated virus have been increasingly understood, improving our ability to manipulate and use it as a safe and efficient gene therapy vector of wide spectrum. In this work, we review the bases of gene therapy, main types of gene transfer systems and basic properties and use of AAV vectors.

  18. Crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 6

    Adeno-associated virus type 6, a human DNA virus that is being developed as a vector for gene therapy, has been crystallized in a form suitable for structure determination at about 3.2 Å resolution. Adeno-associated viruses are being developed as vectors for gene therapy and have been used in a number of clinical trials. Vectors to date have been based on the type species AAV-2, the structure of which was published in 2002. There is growing interest in modulating the cellular tropism and immune neutralization of AAV-2 with variants inspired by the properties of other serotypes. Towards the determination of a structure for AAV type 6, this paper reports the high-yield production, purification, crystallization and preliminary diffraction studies of infectious AAV-6 virions. The crystals diffracted to 3.2 Å resolution using synchrotron radiation. The most promising crystal form belonged to space group R3 and appeared to be suitable for initial structure determination

  19. Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

    Xiangli Wang; Haili Wang; Baojie Mi

    2007-01-01

    BACKGROUND: Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene.OBJECTIVE: To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN: Gene directed cloning.SETTING: Central Laboratory of Northern China Coal Medical College.MATERIALS: DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University.METHODS: NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus liter.MAIN OUTCOME MEASURES: Size of targeted gene fragments, validity of vehicle construction and virus liter.RESULTS: Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified.β-galactoside staining in situ indicated that LacZ genes were

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

    Aravind Asokan

    2014-09-01

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

  1. Systemic gene delivery to the central nervous system using Adeno-associated virus

    Mathieu eBOURDENX

    2014-06-01

    Full Text Available Adeno-associated virus (AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders. The recent discovery that several serotypes are able to cross the blood-brain-barrier when administered systemically has been a real breakthrough in the field of neurodegenerative diseases. Widespread transgene expression after systemic injection could spark interest as a therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will review the recent results achieved through different systemic routes of injection generated in the last decade using systemic AAV-mediated delivery and propose a brief assessment of their values. In particular, we emphasize how the methods used for virus engineering could improve brain transduction after peripheral delivery.

  2. Size does matter: overcoming the adeno-associated virus packaging limit

    Flotte Terence R

    2000-07-01

    Full Text Available Abstract Recombinant adeno-associated virus (rAAV vectors mediate long-term gene transfer without any known toxicity. The primary limitation of rAAV has been the small size of the virion (20 nm, which only permits the packaging of 4.7 kilobases (kb of exogenous DNA, including the promoter, the polyadenylation signal and any other enhancer elements that might be desired. Two recent reports (D Duan et al: Nat Med 2000, 6:595-598; Z Yan et al: Proc Natl Acad Sci USA 2000, 97:6716-6721 have exploited a unique feature of rAAV genomes, their ability to link together in doublets or strings, to bypass this size limitation. This technology could improve the chances for successful gene therapy of diseases like cystic fibrosis or Duchenne muscular dystrophy that lead to significant pulmonary morbidity.

  3. Adeno-associated virus sensitizes HeLa cell tumors to gamma rays.

    Walz, C; Schlehofer, J R; Flentje, M; Rudat, V; zur Hausen, H

    1992-01-01

    Infection with the helper virus-dependent human parvovirus adeno-associated virus (AAV) is known to interfere with cellular transformation in vitro and oncogenesis in vivo. Here we report on sensitization to gamma irradiation by AAV infection of cells in culture and of tumors established from HeLa cells grafted into immunodeficient (nude) mice: infection of HeLa cells with AAV type 2 enhanced cell killing and reduced plating efficiency after irradiation compared with uninfected cells. Similarly, HeLa cell tumors in nude mice displayed a reduced growth rate and were more sensitive to gamma irradiation when the animals were infected with AAV type 2 prior to or after tumor cell inoculation. Since no pathogenicity is known for AAV, the ability of this virus to render radiotherapy of human tumor cells more efficient may up open novel approaches in cancer treatment. Images PMID:1323717

  4. A novel and highly efficient production system for recombinant adeno-associated virus vector

    WU; Zhijian(伍志坚); WU; Xiaobing(吴小兵); CAO; Hui(曹晖); DONG; Xiaoyan(董小岩); WANG; Hong(王宏); HOU; Yunde(侯云德)

    2002-01-01

    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/△UL2, 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/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 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.

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

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

    2010-01-01

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

  6. Functional analysis of the putative integrin recognition motif on adeno-associated virus 9.

    Shen, Shen; Berry, Garrett E; Castellanos Rivera, Ruth M; Cheung, Roland Y; Troupes, Andrew N; Brown, Sarah M; Kafri, Tal; Asokan, Aravind

    2015-01-16

    Adeno-associated viruses (AAVs) display a highly conserved NGR motif on the capsid surface. Earlier studies have established this tripeptide motif as being essential for integrin-mediated uptake of recombinant AAV serotype 2 (AAV2) in cultured cells. However, functional attributes of this putative integrin recognition motif in other recombinant AAV serotypes displaying systemic transduction in vivo remain unknown. In this study, we dissect the biology of an integrin domain capsid mutant derived from the human isolate AAV9 in mice. The AAV9/NGA mutant shows decreased systemic transduction in mice. This defective phenotype was accompanied by rapid clearance of mutant virions from the blood circulation and nonspecific sequestration by the spleen. Transient vascular hyperpermeability, induced by histamine coinjection, exacerbated AAV9/NGA uptake by the spleen but not the liver. However, such treatment did not affect AAV9 virions, suggesting a potential entry/post-entry defect for the mutant in different tissues. Further characterization revealed modestly decreased cell surface binding but a more pronounced defect in the cellular entry of mutant virions. These findings were corroborated by the observation that blocking multiple integrins adversely affected recombinant AAV9 transduction in different cell types, albeit with variable efficiencies. From a structural perspective, we observed that the integrin recognition motif is located in close proximity to the galactose binding footprint on AAV9 capsids and postulate that this feature could influence cell surface attachment, cellular uptake at the tissue level, and systemic clearance by the reticuloendothelial system. PMID:25404742

  7. Disruption of Microtubules Post-Virus Entry Enhances Adeno-Associated Virus Vector Transduction.

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

    2016-04-01

    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

  8. A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy

    Hangjun Ruan

    2001-01-01

    Full Text Available The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE from the erythropoietin gene (Epo, which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1. Under anoxic conditions, nine copies of HIRE (9XHRE yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy.

  9. Recombinant adeno-associated virus vector expressing angiostatin inhibits preretinal neovascularization in adult rats.

    Lai, Chi-Chun; Wu, Wei-Chi; Chen, Show-Li; Sun, Ming-Hui; Xiao, Xiao; Ma, Lih; Lin, Keng-Kuo; Tsao, Yeou-Ping

    2005-01-01

    Clinically, preretinal neovascularization (PNV) induced by vessel occlusion is one of the leading causes to induce blindness. The present study was designed to determine if a recombinant adeno-associated viral vector expressing mouse angiostatin (rAAV-angiostatin) can inhibit experimental PNV in an adult Sprague-Dawley rat model. rAAV-angiostatin and rAAV-lacZ were delivered by intravitreal injections to the right and left eyes of rats. Transgenetic expression of angiostatin in the retina was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). PNV was established by rose-bengal-assisted laser-induced retinal vein occlusion 21 days after the viral injections. The total number and sizes of the neovascular tufts were analyzed 14 days after venous occlusion using retinal flat mount by fluorescein-isothiocyanate-dextran angiography. Electroretinograms (ERGs) were recorded to study any possibility of retinal toxicity of rAAV-angiostatin 3 months after the injections. Angiostatin gene expression in the retina was detectable by RT-PCR, and ERG analysis showed no reduction of b-waves in the rAAV-angiostatin-injected eyes. The number and size of neovascular tufts were significantly lower in rAAV-angiostatin-injected eyes (p = 0.001) than controls. These findings indicated that rAAV-angiostatin successfully suppressed experimental PNV, and no retinal toxicity of the rAAV-angiostatin injection was observed according to ERG recordings. PMID:15637422

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

    Yang Lin

    2013-02-01

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

  11. Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

    McCraw, Dustin M. [Department of Biochemistry and Molecular Biology, School of Medicine, Mail code L224, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098 (United States); O& #x27; Donnell, Jason K. [Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380 (United States); Taylor, Kenneth A. [Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295 (United States); Stagg, Scott M. [Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4380 (United States); Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306 (United States); Chapman, Michael S., E-mail: chapmami@ohsu.edu [Department of Biochemistry and Molecular Biology, School of Medicine, Mail code L224, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239-3098 (United States)

    2012-09-15

    The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.

  12. Ectopic catalase expression in mitochondria by adeno-associated virus enhances exercise performance in mice.

    Dejia Li

    Full Text Available Oxidative stress is thought to compromise muscle contractility. However, administration of generic antioxidants has failed to convincingly improve performance during exhaustive exercise. One possible explanation may relate to the inability of the supplemented antioxidants to effectively eliminate excessive free radicals at the site of generation. Here, we tested whether delivering catalase to the mitochondria, a site of free radical production in contracting muscle, could improve treadmill performance in C57Bl/6 mice. Recombinant adeno-associated virus serotype-9 (AV.RSV.MCAT was generated to express a mitochondria-targeted catalase gene. AV.RSV.MCAT was delivered to newborn C57Bl/6 mouse circulation at the dose of 10(12 vector genome particles per mouse. Three months later, we observed a approximately 2 to 10-fold increase of catalase protein and activity in skeletal muscle and the heart. Subcellular fractionation western blot and double immunofluorescence staining confirmed ectopic catalase expression in the mitochondria. Compared with untreated control mice, absolute running distance and body weight normalized running distance were significantly improved in AV.RSV.MCAT infected mice during exhaustive treadmill running. Interestingly, ex vivo contractility of the extensor digitorum longus muscle was not altered. Taken together, we have demonstrated that forced catalase expression in the mitochondria enhances exercise performance. Our result provides a framework for further elucidating the underlying mechanism. It also raises the hope of applying similar strategies to remove excessive, pathogenic free radicals in certain muscle diseases (such as Duchenne muscular dystrophy and ameliorate muscle disease.

  13. The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

    WANG An-ping; SUN Huai-chang; WANG Jian-ye; WANG Yong-juan; YUAN Wei-feng

    2007-01-01

    To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus (rAAAV), AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein (GFP) gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast (CEF) or chicken embryonic liver (CEL) cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek's disease virus (MDV), avian adenovirus, chicken embryo lethal orphan (CELO) virus or infectious bursal disease virus (IBDV). Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein (gfp) expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV-mediated transgene expression could be enhanced by super infection with the helper viruses.

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

    Nance, Michael E; Duan, Dongsheng

    2015-12-01

    Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy. PMID:26414293

  15. Directed evolution of novel adeno-associated viruses for therapeutic gene delivery.

    Bartel, M A; Weinstein, J R; Schaffer, D V

    2012-06-01

    Gene therapy vectors based on adeno-associated virus (AAV) are currently in clinical trials for numerous disease targets, such as muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis and macular degeneration. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of neutralizing antibodies in the human population, low transduction of a number of therapeutically relevant cell and tissue types, an inability to overcome physical and cellular barriers in vivo and a relatively limited carrying capacity. These challenges arise as the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral-directed evolution-the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest-offers an approach to address these problems. Here we outline progress in creating novel classes of AAV variant libraries and highlight the successful isolation of variants with novel and advantageous in vitro and in vivo gene delivery properties. PMID:22402323

  16. A Hypoxia-Regulated Adeno-Associated Virus Vector for Cancer-Specific Gene Therapy1

    Ruan, Hangjun; Su, Hua; Hu, Lily; Lamborn, Kathleen R; Kan, YW; Deen, Dennis F

    2001-01-01

    Abstract The presence of hypoxic cells in human brain tumors is an important factor leading to resistance to radiation therapy. However, this physiological difference between normal tissues and tumors also provides the potential for designing cancer-specific gene therapy. We compared the increase of gene expression under anoxia (<0.01% oxygen) produced by 3, 6, and 9 copies of hypoxia-responsive elements (HRE) from the erythropoietin gene (Epo), which are activated through the transcriptional complex hypoxia-inducible factor 1 (HIF-1). Under anoxic conditions, nine copies of HRE (9XHRE) yielded 27- to 37-fold of increased gene expression in U-251 MG and U-87 MG human brain tumor cell lines. Under the less hypoxic conditions of 0.3% and 1% oxygen, gene activation by 9XHRE increased expression 11- to 18-fold in these cell lines. To generate a recombinant adeno-associated virus (rAAV) in which the transgene can be regulated by hypoxia, we inserted the DNA fragment containing 9XHRE and the LacZ reporter gene into an AAV vector. Under anoxic conditions, this vector produced 79- to 110-fold increase in gene expression. We believe this hypoxia-regulated rAAV vector will provide a useful delivery vehicle for cancer-specific gene therapy. PMID:11494119

  17. The adeno-associated virus rep gene suppresses herpes simplex virus-induced DNA amplification.

    Heilbronn, R; Bürkle, A; Stephan, S; zur Hausen, H

    1990-01-01

    Herpes simplex virus (HSV) induces within the host cell genome DNA amplification which can be suppressed by coinfection with adeno-associated virus (AAV). To characterize the AAV functions mediating this effect, cloned AAV type 2 wild-type or mutant genomes were transfected into simian virus 40 (SV40)-transformed hamster cells together with the six HSV replication genes (encoding UL5, UL8, major DNA-binding protein, DNA polymerase, UL42, and UL52) which together are necessary and sufficient for the induction of SV40 DNA amplification (R. Heilbronn and H. zur Hausen, J. Virol. 63:3683-3692, 1989). The AAV rep gene was identified as being responsible for the complete inhibition of HSV-induced SV40 DNA amplification. Likewise, rep inhibited origin-dependent HSV replication. rep neither killed the transfected host cells nor interfered with gene expression from the cotransfected amplification genes. This points to a specific interference with HSV-induced DNA amplification. Images PMID:2159559

  18. Systemic delivery of genes to striated muscles using adeno-associated viral vectors.

    Gregorevic, Paul; Blankinship, Michael J; Allen, James M; Crawford, Robert W; Meuse, Leonard; Miller, Daniel G; Russell, David W; Chamberlain, Jeffrey S

    2004-08-01

    A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant adeno-associated virus pseudotype 6 vectors. The inclusion of vascular endothelium growth factor/vascular permeability factor, to achieve acute permeabilization of the peripheral microvasculature, enhanced tissue transduction at lower vector doses. This technique enabled widespread muscle-specific expression of a functional micro-dystrophin in the skeletal muscles of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy. We propose that these methods may be applicable for systemic delivery of a wide variety of genes to the striated muscles of adult mammals. PMID:15273747

  19. Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia.

    Harrington, Elizabeth A; Sloan, Jennifer L; Manoli, Irini; Chandler, Randy J; Schneider, Mark; McGuire, Peter J; Calcedo, Roberto; Wilson, James M; Venditti, Charles P

    2016-05-01

    Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p gene delivery as a treatment for mut MMA. PMID:26790480

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

    Stoica, Lorelei; Sena-Esteves, Miguel

    2016-01-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of upper and lower motor neurons. Mutations in superoxide dismutase 1 (SOD1) are a leading cause of ALS, responsible for up to 20% of familial cases. Although the exact mechanism by which mutant SOD1 causes disease remains unknown, multiple studies have shown that reduction of the mutant species leads to delayed disease onset and extension of lifespan of animal models. This makes SOD1 an ideal target for gene therapy coupling adeno associated virus vector (AAV) gene delivery with RNAi molecules. In this review we summarize the studies done thus far attempting to decrease SOD1 gene expression, using AAV vectors as delivery tools, and RNAi as therapeutic molecules. Current hurdles to be overcome, such as the need for widespread gene delivery through the entire central nervous system (CNS), are discussed. Continued efforts to improve current AAV delivery methods and capsids will accelerate the application of these therapeutics to the clinic. PMID:27531973

  1. ADENO-ASSOCIATED VIRUS INTRODUCED INTEGRATION AND EXPRESSION OF FOREIGN GENES IN PC12 CELLS

    2001-01-01

    Objective To investigate integration and expression of adeno-associated virus (AAV) vectors in neuronal PC12 cells,the result of which can be applied in further gene therapy of diseases of the central nervous system. Methods Human neurotrophin-3(hNT3)genes were inserted into AAV vectors. Then the recombinat AAV plasmids were encapsidated as recombinant virions. PC12 cells were transfected with the virions and the positive cells were selected by G418. The transfection positive (hNT3 modified)PC12 cells were cultured for several generations and the cellular genomic DNA and total RNA were extracted. We investigated the integration locus of AAV vectors by Southern blot and transcript situation of foreign genes by dot blot. Results The hybridization tests showed that AAV introduced foreign genes were stably integrated, but at random locus, and robustly transcribed in hNT3 modified PC12cells. Conclusion AAV vectors can serve as high efficiency vectors of target genes in neuronal PC12 cells.

  2. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox.

    Senís, Elena; Fatouros, Chronis; Große, Stefanie; Wiedtke, Ellen; Niopek, Dominik; Mueller, Ann-Kristin; Börner, Kathleen; Grimm, Dirk

    2014-11-01

    Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno-associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components - Cas9 and chimeric g(uide)RNA - either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard-to-transfect targets, as shown for human T-cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver-specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR-induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans. PMID:25186301

  3. Proteasome Inhibition Is Partially Effective in Attenuating Pre-Existing Immunity against Recombinant Adeno-Associated Viral Vectors

    Karman, Jozsef; Gumlaw, Nathan K; Zhang, Jinhua; Jiang, Ji-Lei; Cheng, Seng H.; Zhu, Yunxiang

    2012-01-01

    Pre-existing immunity against adeno-associated virus (AAV) remains a major challenge facing the clinical use of systemic administration of recombinant AAV vectors for the treatment of genetic and acquired diseases using gene therapy. In this study, we evaluated the potential of bortezomib (marketed under trade name Velcade) to abrogate a pre-existing immunity to AAV in mice, thereby allowing subsequent transduction by a recombinant AAV vector of the same serotype. We demonstrate that bortezom...

  4. Antibody Neutralization Poses a Barrier to Intravitreal Adeno-Associated Viral Vector Gene Delivery to Non-Human Primates

    Kotterman, Melissa A.; Yin, Lu; Strazzeri, Jennifer M.; Flannery, John G; Merigan, William H.; Schaffer, David V

    2014-01-01

    Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber's congenital amaurosis and choroideremia. One general challenge for AAV is that pre-existing immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizin...

  5. Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression.

    Samulski, R J; Chang, L S; Shenk, T

    1989-01-01

    A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expre...

  6. Persistence, Localization, and External Control of Transgene Expression After Single Injection of Adeno-Associated Virus into Injured Joints

    Lee, Hannah H.; O'Malley, Michael J.; Friel, Nicole A.; Payne, Karin A.; Qiao, Chunping; Xiao, Xiao(Institute for Strings, Cosmology and Astroparticle Physics (ISCAP) and Physics Department, Columbia University, 538 West 120th Street, New York, NY, 10027 U.S.A.); Chu, Constance R.

    2013-01-01

    A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra...

  7. Recombinant adeno-associated viruses (rAAV2) facilitate the intraperitoneal gene delivery to cancer cells

    Malecki, Maciej; PROCZKA, ROBERT; Chorostowska-Wynimko, Joanna; Swoboda, Paweł; DELBANI, ANNA; Pachecka, Jan

    2010-01-01

    Peritoneal dissemination of cancer cells is characteristic of advanced stages of ovarian, breast and lung cancers, and is associated with poor patient survival. The presence of cancer cells in effusions complicates treatment protocols, while cell eradication is seriously limited. One of the novel options available is cancer gene therapy with recombinant adeno-associated viruses. This combination represents the most promising gene delivery vehicles to neoplasmatic cells within serosal cavities...

  8. Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products

    Werling, Natalie Jayne; Satkunanathan, Stifani; Thorpe, Robin; Zhao, Yuan

    2015-01-01

    Abstract Adeno-associated viral (AAV) vectors show great promise for gene therapy because of their excellent safety profile; however, development of robust dose-determining assays for AAV has presented a significant challenge. With the ultimate goal of future harmonization and standardization of AAV dose determination assays, we systematically analyzed the influence of key variables, including sample preparation procedure, the choice of primers, and real-time quantitative PCR (qPCR) target se...

  9. Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges

    Raj, Deepak; Davidoff, Andrew M.; Nathwani, Amit C.

    2011-01-01

    Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated...

  10. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model

    Wang, Bing; Li, Juan; Xiao, Xiao

    2000-01-01

    Duchenne muscular dystrophy (DMD) is the most common and lethal genetic muscle disorder, caused by recessive mutations in the dystrophin gene. One of every 3,500 males suffers from DMD, yet no treatment is currently available. Genetic therapeutic approaches, using primarily myoblast transplantation and adenovirus-mediated gene transfer, have met with limited success. Adeno-associated virus (AAV) vectors, although proven superior for muscle gene transfer, are too sm...

  11. Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation

    Duan, Dongsheng; Yan, Ziying; Yue, Yongping; Ding, Wei; Engelhardt, John F.

    2001-01-01

    Adeno-associated virus (AAV)-based muscle gene therapy has achieved tremendous success in numerous animal models of human diseases. Recent clinical trials with this vector have also demonstrated great promise. However, to achieve therapeutic benefit in patients, large inocula of virus will likely be necessary to establish the required level of transgene expression. For these reasons, efforts aimed at increasing the efficacy of AAV-mediated gene delivery to muscle have the potential for improv...

  12. Long-term Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using Adeno associated Viral Gene Therapy

    Chandler, Randy J.; Venditti, Charles P

    2009-01-01

    Methylmalonic acidemia (MMA) is an organic acidemia caused by deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). This disorder is associated with lethal metabolic instability and carries a poor prognosis for long-term survival. A murine model of MMA that replicates a severe clinical phenotype was used to examine the efficacy of recombinant adeno-associated virus (rAAV) serotype 8 gene therapy as a treatment for MMA. Lifespan extension, body weight, circulating meta...

  13. microRNA-155 Regulates Alpha-Synuclein-Induced Inflammatory Responses in Models of Parkinson Disease.

    Thome, Aaron D; Harms, Ashley S; Volpicelli-Daley, Laura A; Standaert, David G

    2016-02-24

    Increasing evidence points to inflammation as a chief mediator of Parkinson's disease (PD), a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein α-synuclein (α-syn). Recently, microRNAs, small, noncoding RNAs involved in regulating gene expression at the posttranscriptional level, have been recognized as important regulators of the inflammatory environment. Using an array approach, we found significant upregulation of microRNA-155 (miR-155) in an in vivo model of PD produced by adeno-associated-virus-mediated expression of α-syn. Using a mouse with a complete deletion of miR-155, we found that loss of miR-155 reduced proinflammatory responses to α-syn and blocked α-syn-induced neurodegeneration. In primary microglia from miR-155(-/-) mice, we observed a markedly reduced inflammatory response to α-syn fibrils, with attenuation of major histocompatibility complex class II (MHCII) and proinflammatory inducible nitric oxide synthase expression. Treatment of these microglia with a synthetic mimic of miR-155 restored the inflammatory response to α-syn fibrils. Our results suggest that miR-155 has a central role in the inflammatory response to α-syn in the brain and in α-syn-related neurodegeneration. These effects are at least in part due to a direct role of miR-155 on the microglial response to α-syn. These data implicate miR-155 as a potential therapeutic target for regulating the inflammatory response in PD. PMID:26911687

  14. Adeno associated viral-mediated intraosseous labeling of bone marrow derived cells for CNS tracking.

    Selenica, Maj-Linda B; Reid, Patrick; Pena, Gabriela; Alvarez, Jennifer; Hunt, Jerry B; Nash, Kevin R; Morgan, Dave; Gordon, Marcia N; Lee, Daniel C

    2016-05-01

    Inflammation, including microglial activation in the CNS, is an important hallmark in many neurodegenerative diseases. Microglial stimuli not only impact the brain microenvironment by production and release of cytokines and chemokines, but also influence the activity of bone marrow derived cells and blood born macrophage populations. In many diseases including brain disorders and spinal cord injury, researchers have tried to harbor the neuroprotective and repair properties of these subpopulations. Hematopoietic bone marrow derived cells (BMDCs) are of great interest, especially during gene therapy because certain hematopoietic cell subpopulations traffic to the sites of injury and inflammation. The aim of this study was to develop a method of labeling endogenous bone marrow derived cells through intraosseous impregnation of recombinant adeno-associated virus (rAAV) or lentivirus. We utilized rAAV serotype 9 (rAAV-9) or lentivirus for gene delivery of green florescence protein (GFP) to the mouse bone marrow cells. Flow cytometry showed that both viruses were able to efficiently transduce mouse bone marrow cells in vivo. However, the rAAV9-GFP viral construct transduced BMDCs more efficiently than the lentivirus (11.2% vs. 6.8%), as indicated by cellular GFP expression. We also demonstrate that GFP labeled cells correspond to bone marrow cells of myeloid origin using CD11b as a marker. Additionally, we characterized the ability of bone marrow derived, GFP labeled cells to extravasate into the brain parenchyma upon acute and subchronic neuroinflammatory stimuli in the mouse CNS. Viral mediated over expression of chemokine (C-C motif) ligand 2 (CCL2) or intracranial injection of lipopolysaccharide (LPS) recruited GFP labeled BMDCs from the periphery into the brain parenchyma compared to vehicle treated mice. Altogether our findings demonstrate a useful method of labeling endogenous BMDCs via viral transduction and the ability to track subpopulations throughout the body

  15. Supraspinal gene transfer by intrathecal adeno-associated virus serotype 5

    Daniel J. Schuster

    2014-08-01

    Full Text Available We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5. Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir in thalamus was most prominent in medial geniculate nucleus, and otherwise limited to posterior nuclei of the dorsal and lateral margins. Labeling was also observed in neurons and astrocytes of the hippocampal formation and amygdaloid complex. In the hippocampal formation, GFP-ir was found in neuronal cell bodies of the rostral ventral portion, but was largely restricted to fiber-like staining in the molecular layer of dentate gyrus and stratum lacunosum-moleculare of the rostral dorsal region. GFP-ir was seen in neurons and astroglia throughout caudal cortex, whereas in rostral regions of neocortex it was limited to isolated astrocytes and neurons. Labeling was also present in olfactory bulb. These results demonstrate that intrathecal delivery of AAV5 vector leads to transgene expression in discrete CNS regions throughout the rostro-caudal extent of the neuraxis. A caudal-to-rostral gradient of decreasing GFP-ir was present in choroid plexus and Purkinje cells, suggesting that spread of virus through cerebrospinal fluid plays a role in the resulting transduction pattern. Other factors contributing to the observed expression pattern likely include variations in cell-surface receptors and inter-parenchymal space.

  16. Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids.

    Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

    2016-02-01

    Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanisms-result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

  17. Identification of a Heparin-Binding Motif on Adeno-Associated Virus Type 2 Capsids†

    Kern, A.; Schmidt, K.; Leder, C.; Müller, O. J.; Wobus, C. E.; Bettinger, K.; Von der Lieth, C. W.; King, J. A.; Kleinschmidt, J. A.

    2003-01-01

    Infection of cells with adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycan and can be competed by heparin. Mutational analysis of AAV-2 capsid proteins showed that a group of basic amino acids (arginines 484, 487, 585, and 588 and lysine 532) contribute to heparin and HeLa cell binding. These amino acids are positioned in three clusters at the threefold spike region of the AAV-2 capsid. According to the recently resolved atomic structure for AAV-2, arginines 484 and 487 and lysine 532 on one site and arginines 585 and 588 on the other site belong to different capsid protein subunits. These data suggest that the formation of the heparin-binding motifs depends on the correct assembly of VP trimers or even of capsids. In contrast, arginine 475, which also strongly reduces heparin binding as well as viral infectivity upon mutation to alanine, is located inside the capsid structure at the border of adjacent VP subunits and most likely influences heparin binding indirectly by disturbing correct subunit assembly. Computer simulation of heparin docking to the AAV-2 capsid suggests that heparin associates with the three basic clusters along a channel-like cavity flanked by the basic amino acids. With few exceptions, mutant infectivities correlated with their heparin- and cell-binding properties. The tissue distribution in mice of recombinant AAV-2 mutated in R484 and R585 indicated markedly reduced infection of the liver, compared to infection with wild-type recombinant AAV, but continued infection of the heart. These results suggest that although heparin binding influences the infectivity of AAV-2, it seems not to be necessary. PMID:14512555

  18. Structural characterization of the dual glycan binding adeno-associated virus serotype 6.

    Ng, Robert; Govindasamy, Lakshmanan; Gurda, Brittney L; McKenna, Robert; Kozyreva, Olga G; Samulski, R Jude; Parent, Kristin N; Baker, Timothy S; Agbandje-McKenna, Mavis

    2010-12-01

    The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization. PMID:20861247

  19. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  20. Developing immunologically inert adeno-associated virus (AAV) vectors for gene therapy: possibilities and limitations.

    Selot, Ruchita S; Hareendran, Sangeetha; Jayandharan, Giridhara R

    2014-01-01

    Gene therapy has become a clinical reality as demonstrated by remarkable benefits seen in Phase I/II clinical trials for hemophilia B, lipoprotein lipase deficiency and Leber's congenital amarousis. The choice of, and the improved understanding in vector characteristics have contributed significantly to this success. The adeno-associated virus (AAV) vectors used in these trials have been long known to be relatively safe and efficacious. However, certain factors, most notably host immunity to the vector, prevent their widespread use. In patients who have pre-existing antibodies to AAV, these vectors will be rapidly cleared. Administration of a relatively high initial dose of vector to achieve and sustain a higher margin of therapeutic benefit is limited by concerns of vector dose-dependent T cell response. Frequent vector administration necessitated by the non-integrating nature of the virus is difficult due to the variable, yet significant host immunological memory. Thus generation of AAV vectors that are immunologically inert is pivotal for the long-term success with this promising vector system. Several strategies, that aim targeted disruption of antigenic sites or those that chemically modify the vectors have been proposed for host immune evasion. While these approaches have been successful in the pre-clinical model systems, this continues to be a field of intense experimentation and constant improvisation due to limited information available on vector immunology or data from human studies. This review forms a comprehensive report on current strategies available to generate immunologically inert AAV vectors and their potential in mediating longterm gene transfer. PMID:24678652

  1. Virus-mediated EpoR76E Therapy Slows Optic Nerve Axonopathy in Experimental Glaucoma.

    Bond, Wesley S; Hines-Beard, Jessica; GoldenMerry, Y Paul L; Davis, Mara; Farooque, Alma; Sappington, Rebecca M; Calkins, David J; Rex, Tonia S

    2016-02-01

    Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)-lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus-mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment. PMID:26502777

  2. Establishment of a recombinant adeno-associated virus expressing hVEGF165

    Xianghui Huang; Zhibin Shi; Xiaoqian Dang; Chen Zhang; Pengbo Yu; Kunzheng Wang

    2008-01-01

    BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion

  3. Adeno-associated viral vectors engineered for macrolide-adjustable transgene expression In mammalian cells and mice

    Fussenegger Martin

    2007-11-01

    Full Text Available Abstract Background Adjustable gene expression is crucial in a number of applications such as de- or transdifferentiation of cell phenotypes, tissue engineering, various production processes as well as gene-therapy initiatives. Viral vectors, based on the Adeno-Associated Virus (AAV type 2, have emerged as one of the most promising types of vectors for therapeutic applications due to excellent transduction efficiencies of a broad variety of dividing and mitotically inert cell types and due to their unique safety features. Results We designed recombinant adeno-associated virus (rAAV vectors for the regulated expression of transgenes in different configurations. We integrated the macrolide-responsive E.REX systems (EON and EOFF into rAAV backbones and investigated the delivery and expression of intracellular as well as secreted transgenes for binary set-ups and for self- and auto-regulated one-vector configurations. Extensive quantitative analysis of an array of vectors revealed a high level of adjustability as well as tight transgene regulation with low levels of leaky expression, both crucial for therapeutical applications. We tested the performance of the different vectors in selected biotechnologically and therapeutically relevant cell types (CHO-K1, HT-1080, NHDF, MCF-7. Moreover, we investigated key characteristics of the systems, such as reversibility and adjustability to the regulating agent, to determine promising candidates for in vivo studies. To validate the functionality of delivery and regulation we performed in vivo studies by injecting particles, coding for compact self-regulated expression units, into mice and adjusting transgene expression. Conclusion Capitalizing on established safety features and a track record of high transduction efficiencies of mammalian cells, adeno- associated virus type 2 were successfully engineered to provide new powerful tools for macrolide-adjustable transgene expression in mammalian cells as well as

  4. Impact of Pre-Existing Immunity on Gene Transfer to Nonhuman Primate Liver with Adeno-Associated Virus 8 Vectors

    Wang, Lili; Calcedo, Roberto; Bell, Peter; Lin, Jianping; Grant, Rebecca L.; Siegel, Don L.; James M Wilson

    2011-01-01

    Vectors based on the primate-derived adeno-associated virus serotype 8 (AAV8) are being evaluated in preclinical and clinical models. Natural infections with related AAVs activate memory B cells that produce antibodies capable of modulating the efficacy and safety of the vector. We have evaluated the biology of AAV8 gene transfer in macaque liver, with a focus on assessing the impact of pre-existing humoral immunity. Twenty-one macaques with various levels of AAV neutralizing antibody (NAb) w...

  5. Adeno-Associated Virus Site-Specific Integration Is Mediated by Proteins of the Nonhomologous End-Joining Pathway▿

    Daya, Shyam; Cortez, Nenita; Berns, Kenneth I.

    2009-01-01

    Adeno-associated virus type 2 (AAV 2) is the only eukaryotic virus capable of site-specific integration; the target site is at chromosome 19q13.4, a site termed AAVS1. The biology of AAV latency has been extensively studied in cell culture, yet the precise mechanism and the required cellular factors are not known. In this study, we assessed the relative frequencies of stable site-specific integration by characterization of cell clones containing integrated AAV vectors. By this assay, two prot...

  6. Novel Transcriptional Regulatory Signals in the Adeno-Associated Virus Terminal Repeat A/D Junction Element

    Haberman, Rebecca P.; McCown, Thomas J.; Samulski, Richard Jude

    2000-01-01

    Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604–1611, 1998) demonstrated the use of the tetracycline-responsive system for long...

  7. Therapeutic Liabilities of in Vivo Viral Vector Tropism: Adeno-Associated Virus Vectors, NMDAR1 Antisense, and Focal Seizure Sensitivity

    Haberman, Rebecca P.; Criswell, Hugh E.; Snowdy, Stephen; Ming, Zhen; Breese, George R.; Samulski, R. Jude; McCown, Thomas J.

    2002-01-01

    The N-methyl-d-aspartic acid (NMDA) receptor provides a potential target for gene therapy of focal seizure disorders. To test this approach, we cloned a 729-bp NMDA receptor (NMDAR1) cDNA fragment in the antisense orientation into adeno-associated virus (AAV) vectors, where expression was driven by either a tetracycline-off regulatable promoter (AAV-tTAK-NR1A) or a cytomegalovirus (CMV) promoter (AAV-CMV-NR1A). After infection of primary cultured cortical neurons with recombinant AAV-tTAK-NR1...

  8. Reduction of experimental diabetic vascular leakage by delivery of angiostatin with a recombinant adeno-associated virus vector

    Shyong, Mong-Ping; Lee, Fenq-Lih; Kuo, Ping-Chang; Wu, Ai-Ching; Cheng, Huey-Chung; Chen, Show-Li; Tung, Tao-Hsin; Tsao, Yeou-Ping

    2007-01-01

    Purpose To evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage in an experimental diabetic rat model. Methods rAAV-angiostatin was delivered by intravitreal injection to the right eyes of Sprague-Dawley rats. As a control, the contralateral eye received an intravitreal injection of rAAV-lacZ. Gene delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). D...

  9. Adeno-associated Virus 9 Mediated FKRP Gene Therapy Restores Functional Glycosylation of α-dystroglycan and Improves Muscle Functions

    Xu, Lei; Lu, Pei Juan; Wang, Chi-Hsien; Keramaris, Elizabeth; Qiao, Chunping; Xiao, Bin; Blake, Derek J.; Xiao, Xiao; Lu, Qi Long

    2013-01-01

    Mutations in the FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker–Warburg syndrome and muscle-eye-brain disease. The characteristic biochemical feature of these diseases is the hypoglycosylation of α-dystroglycan (α-DG). Currently there is no effective treatment available. In this study, we examined the adeno-associated virus serotype 9 vector (AAV9)-mediated gene therapy in the FKRP mutant mouse model with ...

  10. Complete Correction of Hemophilia A with Adeno-Associated Viral Vectors Containing a Full-Size Expression Cassette

    Lu, Hui; Chen, Lingxia; Wang, Jinhui; Huack, Bernd; Sarkar, Rita; Zhou, Shangzhen; Xu, Ray; Ding, Qiulan; Wang, Xuefeng; WANG, HONGLI; Xiao, Weidong

    2008-01-01

    Hemophilia A is caused by a deficiency in the factor VIII (FVIII) gene. Constrained by limited packaging capacity, even the 4.3-kb B domain-deleted FVIII remained a challenge for delivery by a single adeno-associated viral (AAV) vector. Studies have shown that up to a 6.6-kb vector sequence may be packaged into AAV virions, which suggested an alternative strategy for hemophilia A gene therapy. To explore the usefulness of AAV vectors carrying an oversized FVIII gene, we constructed the AAV-FV...

  11. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.; Gurda-Whitaker, Brittney; Kalina, Amy [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Kohlbrenner, Erik [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Chiorini, John A. [GTTB, NIDCR, National Institutes of Health, Bethesda, MD 20892 (United States); McKenna, Robert [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States); Muzyczka, Nicholas [Department of Molecular Genetics and Microbiology and Powell Gene Therapy Center, College of Medicine, University of Florida, Gainesville, FL 32610 (United States); Zolotukhin, Sergei [Division of Cell and Molecular Therapy, University of Florida, Gainesville, FL 32610 (United States); Agbandje-McKenna, Mavis, E-mail: mckenna@ufl.edu [Department of Biochemistry and Molecular Biology, McKnight Brain Institute, Center for Structural Biology, University of Florida, Gainesville, FL 32610 (United States)

    2005-10-01

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

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

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

    2012-01-06

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

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

    Highlights: ► Adeno-associated virus (AAV) is capable of targeted integration in human cells. ► Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. ► A targeted integration system of IDRV DNA using the AAV integration mechanism. ► 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.

  14. Production, purification, crystallization and preliminary X-ray structural studies of adeno-associated virus serotype 5

    The production, purification, crystallization and preliminary crystallographic analysis of empty adeno-associated virus serotype 5 capsids are reported. Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Å resolution using synchrotron radiation and belong to the orthorhombic space group P212121, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Å. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress

  15. Productive life cycle of adeno-associated virus serotype 2 in the complete absence of a conventional polyadenylation signal.

    Wang, Lina; Yin, Zifei; Wang, Yuan; Lu, Yuan; Zhang, Daniel; Srivastava, Arun; Ling, Changquan; Aslanidi, George V; Ling, Chen

    2015-09-01

    We showed that WT adeno-associated virus serotype 2 (AAV2) genome devoid of a conventional polyadenylation [poly(A)] signal underwent complete genome replication, encapsidation and progeny virion production in the presence of adenovirus. The infectivity of the progeny virion was also retained. Using recombinant AAV2 vectors devoid of a human growth hormone poly(A) signal, we also demonstrated that a subset of mRNA transcripts contained the inverted terminal repeat (ITR) sequence at the 3' end, which we designated ITR in RNA (ITRR). Furthermore, AAV replication (Rep) proteins were able to interact with the ITRR. Taken together, our studies suggest a new function of the AAV2 ITR as an RNA element to mediate transgene expression from poly(A)-deleted mRNA. PMID:26297494

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

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

    2015-01-01

    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. PMID:26159373

  17. Induction of differentiation-associated changes in established human cells by infection with adeno-associated virus type 2.

    Klein-Bauernschmitt, P; zur Hausen, H; Schlehofer, J R

    1992-01-01

    The nonpathogenic human defective parvovirus adeno-associated virus (AAV) type 2 induced differentiation-associated antigens in cells of the human leukemia cell line HL60 (CD 67), as well as in two different lines of immortalized human keratinocytes, HaCaT and HPK Ia cells (involucrin and cytokeratin 10). Simultaneously, expression of the c-myc and c-myb oncogenes and the retinoblastoma gene was down regulated whereas c-fos expression increased in infected cells. These data point to the potential of AAV to induce functions related to the differentiation pathway in different types of human cells. This phenomenon may be involved in the reported oncosuppressive properties of AAV infections. Images PMID:1318400

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

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

    2012-05-24

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

  19. A phase 1 study to evaluate the safety and immunogenicity of a recombinant HIV type 1 subtype C adeno-associated virus vaccine

    Mehendale, Sanjay; van Lunzen, Jan; Clumeck, Nathan; Rockstroh, Jurgen; Vets, Eva; Johnson, Philip R.; Anklesaria, Pervin; Barin, Burc; Boaz, Mark; Kochhar, Sonali; Lehrman, Jennifer; Schmidt, Claudia; Peeters, Mathieu; Schwarze-Zander, Carolynne; Kabamba, Kabeya; Glaunsinger, Tobias; Sahay, Seema; Thakar, Madhuri; Paranjape, Ramesh; Gilmour, Jill; Excler, Jean-Louis; Fast, Patricia; Heald, A1lison E.

    2008-01-01

    A novel prophylactic AIDS vaccine candidate, consisting of single-stranded DNA for HIV-1 subtype C gag, protease, and part of reverse transcriptase genes, enclosed within a recombinant adeno-associated virus serotype-2 protein capsid (tgAAC09) induced T cell responses and antibodies in nonhuman prim

  20. Immunological inhibition of transplanted liver allografts by adeno-associated virus vector encoding CTLA4Ig in rats

    Sen Lu; Yue Yu; Yun Gao; Guo-Qiang Li; Xue-Hao Wang

    2008-01-01

    BACKGROUND: Blockade interaction between CD28 and B7 with CTLA4Ig has been shown to induce experimental transplantation tolerance. In order to prolong the inhibitory effect of CTLA4Ig, a recombinant adeno-associated virus vector pSNAV expressing CTLA4Ig was constructed, and its effects on transplanted liver allografts were investigated. METHODS:The pSNAV-CTLA4Ig construct was infused into partial liver allografts of rats via the portal vein during transplantation. CTLA4Ig expression in the transplanted livers was detected with reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Furthermore, real-time quantita-tive PCR was used to measure the expression of IL-2, IFN-γ, IL-4 and IL-10 in the allografts. RESULTS:The expression of CTLA4Ig in the partial allograft was detected successfully and pSNAV-CTLA4Ig improved the survival rate of rats after liver transplantation. Agarose gel analysis of RT-PCR products indicated the presence of CTLA4Ig in the pSNAV-CTLA4Ig treatment group. Cytokines expressed in allografts on day 7 after orthotopic liver transplantation showed that IL-2, IFN-γ, IL-4 and IL-10 mRNA levels decreased in transplant recipients treated with pSNAV-CTLA4Ig compared with those treated with pSNAV-LacZ (1.62±0.09, 1.52±0.11, 1.50± 0.07 and 1.43±0.07 versus 1.29±0.09, 1.32±0.07, 1.34±0.06 and 1.35±0.04, respectively). CONCLUSIONS:pSNAV-CTLA4Ig effectively expressed CTLA4Ig in liver allografts. CTLA4Ig improved the pathological ifndings after liver transplantation. CTLA4Ig induced immune tolerance of liver transplantation, and the mechanism involved induced alteration of Th1 and Th2 cytokine transcripts. The adeno-associated virus vector encoding CTLA4Ig may be useful in the clinical study of transplantation tolerance.

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

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

    2016-02-01

    Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. PMID:26284700

  2. Recombinant Adeno-Associated Virus Vector Genomes Take the Form of Long-Lived, Transcriptionally Competent Episomes in Human Muscle.

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

    2016-01-01

    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

  3. Production, Purification, Crystallization and Preliminary X-ray Structural Studies of Adeno-Associated Virus Serotype 5

    DiMattia,M.; Govindasamy, L.; Levy, H.; Whitaker-Gurda, B.; Kohlbrenner, E.; Chiorini, J.; McKenna, R.; Muzyczka, N.; Zolotukhin, S.; Agbandje-McKenna, M.

    2005-01-01

    Adeno-associated virus serotype 5 (AAV5) is under development for gene-therapy applications for the treatment of cystic fibrosis. To elucidate the structural features of AAV5 that control its enhanced transduction of the apical surface of airway epithelia compared with other AAV serotypes, X-ray crystallographic studies of the viral capsid have been initiated. The production, purification, crystallization and preliminary crystallographic analysis of empty AAV5 viral capsids are reported. The crystals diffract X-rays to beyond 3.2 Angstroms resolution using synchrotron radiation and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 264.7, b = 447.9, c = 629.7 Angstroms. There is one complete T = 1 viral capsid per asymmetric unit. The orientation and position of the viral capsid in the asymmetric unit have been determined by rotation and translation functions, respectively, and the AAV5 structure determination is in progress.

  4. Stable transduction of large DNA by high-capacity adeno-associated virus/adenovirus hybrid vectors

    Viral vectors with high cloning capacity and host chromosomal integration ability are in demand for the efficient and permanent genetic modification of target cells with large DNA molecules. We have generated a hybrid gene transfer vehicle consisting of recombinant adeno-associated virus (AAV) replicative intermediates packaged in adenovirus (Ad) capsids. This arrangement allows cell cycle-independent nuclear delivery of recombinant AAV genomes with lengths considerably above the maximum size (i.e., 4.7 kb) that can be accommodated within AAV capsids. Here we show that high-capacity AAV/Ad hybrid vector gene transfer mediates cellular genomic integration of large fragments of foreign DNA and accomplishes stable long-term transgene expression in rapidly proliferating cells. Southern blot and polymerase chain reaction analyses of chromosomal DNA extracted from clones of stably transduced cells revealed that most of them contained a single copy of the full-length hybrid vector genome with AAV inverted terminal repeat (ITR) sequences at both ends. The high-capacity AAV/Ad hybrid vector system can thus be used for the transfer and expression of transgenes that cannot be delivered by conventional integrating viral vectors

  5. Safe and bodywide muscle transduction in young adult Duchenne muscular dystrophy dogs with adeno-associated virus.

    Yue, Yongping; Pan, Xiufang; Hakim, Chady H; Kodippili, Kasun; Zhang, Keqing; Shin, Jin-Hong; Yang, Hsiao T; McDonald, Thomas; Duan, Dongsheng

    2015-10-15

    The ultimate goal of muscular dystrophy gene therapy is to treat all muscles in the body. Global gene delivery was demonstrated in dystrophic mice more than a decade ago using adeno-associated virus (AAV). However, translation to affected large mammals has been challenging. The only reported attempt was performed in newborn Duchenne muscular dystrophy (DMD) dogs. Unfortunately, AAV injection resulted in growth delay, muscle atrophy and contracture. Here we report safe and bodywide AAV delivery in juvenile DMD dogs. Three ∼2-m-old affected dogs received intravenous injection of a tyrosine-engineered AAV-9 reporter or micro-dystrophin (μDys) vector at the doses of 1.92-6.24 × 10(14) viral genome particles/kg under transient or sustained immune suppression. DMD dogs tolerated injection well and their growth was not altered. Hematology and blood biochemistry were unremarkable. No adverse reactions were observed. Widespread muscle transduction was seen in skeletal muscle, the diaphragm and heart for at least 4 months (the end of the study). Nominal expression was detected in internal organs. Improvement in muscle histology was observed in μDys-treated dogs. In summary, systemic AAV gene transfer is safe and efficient in young adult dystrophic large mammals. This may translate to bodywide gene therapy in pediatric patients in the future. PMID:26264580

  6. Adeno-associated viral-mediated LARGE gene therapy rescues the muscular dystrophic phenotype in mouse models of dystroglycanopathy.

    Yu, Miao; He, Yonglin; Wang, Kejian; Zhang, Peng; Zhang, Shengle; Hu, Huaiyu

    2013-03-01

    Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, and fukutin, creating the possibilities of a one-for-all gene therapy. To determine the feasibility of LARGE gene therapy, a serotype 9 adeno-associated viral vector for overexpressing LARGE (AAV9-LARGE) was injected intracardially into newborns of two mouse models of CMD: the natural LARGE mutant Large(myd) mice and protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice. AAV9-LARGE virus treatment yielded partial restoration of α-DG glycosylation and ligand-binding activity. The muscular dystrophy phenotype in skeletal muscles was ameliorated as revealed by significantly reduced fibrosis, necrosis, and numbers of centrally located nuclei with improved motor function. These results indicate that LARGE overexpression in vivo by AAV9-mediated gene therapy is effective at restoring functional glycosylation of α-DG and rescuing the muscular dystrophy phenotype in deficiency of not only LARGE but also POMGnT1, providing evidence that in vivo LARGE gene therapy may be broadly useful in dystroglycanopathies. PMID:23379513

  7. Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

    The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68

  8. Human Adeno-Associated Virus Type 5 Is Only Distantly Related to Other Known Primate Helper-Dependent Parvoviruses

    Bantel-Schaal, Ursula; Delius, Hajo; Schmidt, Rainer; zur Hausen, Harald

    1999-01-01

    We have characterized 95% (4,404 nucleotides) of the genome of adeno-associated virus type 5 (AAV5), including part of the terminal repeats and the terminal resolution site. Our results show that AAV5 is different from all other described AAV serotypes at the nucleotide level and at the amino acid level. The sequence homology to AAV2, AAV3B, AAV4, and AAV6 at the nucleotide level is only between 54 and 56%. The positive strand contains two large open reading frames (ORFs). The left ORF encodes the nonstructural (Rep) proteins, and the right ORF encodes the structural (Cap) proteins. At the amino acid level the identities with the capsid proteins of other AAVs range between 51 and 59%, with a high degree of heterogeneity in regions which are considered to be on the exterior surface of the viral capsid. The overall identity for the nonstructural Rep proteins at the amino acid level is 54.4%. It is lowest at the C-terminal 128 amino acids (10%). There are only two instead of the common three putative Zn fingers in the Rep proteins. The Cap protein data suggest differences in capsid surfaces and raise the possibility of a host range distinct from those of other parvoviruses. This may have important implications for AAV vectors used in gene therapy. PMID:9882294

  9. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing

    Adachi, Kei; Enoki, Tatsuji; Kawano, Yasuhiro; Veraz, Michael; Nakai, Hiroyuki

    2014-01-01

    Adeno-associated virus (AAV) capsid engineering is an emerging approach to advance gene therapy. However, a systematic analysis on how each capsid amino acid contributes to multiple functions remains challenging. Here we show proof-of-principle and successful application of a novel approach, termed AAV Barcode-Seq, that allows us to characterize phenotypes of hundreds of different AAV strains in a high-throughput manner and therefore overcomes technical difficulties in the systematic analysis. In this approach, we generate DNA barcode-tagged AAV libraries and determine a spectrum of phenotypes of each AAV strain by Illumina barcode sequencing. By applying this method to AAV capsid mutant libraries tagged with DNA barcodes, we can draw a high-resolution map of AAV capsid amino acids important for the structural integrity and functions including receptor binding, tropism, neutralization and blood clearance. Thus, Barcode-Seq provides a new tool to generate a valuable resource for virus and gene therapy research. PMID:24435020

  10. Targeted Genome Editing by Recombinant Adeno-Associated Virus (rAAV) Vectors for Generating Genetically Modified Pigs

    Yonglun Luo; Emil Kofod-Olsen; Rikke Christensen; Charlotte Brandt S(φ)rensen; Lars Bolund

    2012-01-01

    Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases.Several advantages,such as simple vector construction,high targeting frequency by homologous recombination,and applicability to many cell types,make rAAV an attractive approach for targeted genome editing.Combined with cloning by somatic cell nuclear transfer (SCNT),this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis,hereditary tyrosinemia type 1,and breast cancer.This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination.We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts,which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

  11. Genetic Manipulation of Brown Fat Via Oral Administration of an Engineered Recombinant Adeno-associated Viral Serotype Vector.

    Huang, Wei; McMurphy, Travis; Liu, Xianglan; Wang, Chuansong; Cao, Lei

    2016-06-01

    Recombinant adeno-associated virus (rAAV) vectors are attractive vehicles for gene therapy. Gene delivery to the adipose tissue using naturally occurring AAV serotypes is less successful compared to liver and muscle. Here, we demonstrate that oral administration of an engineered serotype Rec2 led to preferential transduction of brown fat with absence of transduction in the gastrointestinal track. Among the six natural and engineered serotypes being compared, Rec2 was the most efficient serotype achieving high level transduction at a dose 1~2 orders lower than reported doses for systemic administration. Overexpressing vascular endothelial growth factor (VEGF) in brown fat via oral administration of Rec2-VEGF vector increased the brown fat mass and enhanced thermogenesis. In contrast, knockdown VEGF in brown fat of VEGF (loxP) mice via Rec2-Cre vector hampered cold response and decreased brown fat mass. Oral administration of Rec2 vector provides a novel tool to genetically manipulate brown fat for research and therapeutic applications. PMID:26857843

  12. Preparation of a recombinant adeno-associated virus vector encoding the human NIS gene and its expression in thyroid cancer cell lines

    Objective: To demonstrate the feasibility of thyroid gene therapy by using a adeno-associated virus vector to deliver the sodium/iodide symporter (NIS) gene into the thyroid tumor cells. Methods: The recombinant adeno-associated virus encoding the human NIS gene (rAAV-NIS, the immunofluorescence and iodide uptake studies as well as inhibited iodide uptake tests were carried out. Results: A rAAV encoding the human NIS gene was successfully prepared. Immunofluorescence analysis confirmed the expression of the NIS protein in the tumor cells, and it was localized at the cell surface and possessed a function of iodine uptake as well as suppression by perchlorates similar to the function and character with normal thyroid cell. Conclusion: rAAV-NIS is very efficient in triggering iodide uptake by infected tumor cells, and it outline the potential of this novel cancer gene therapy approach for a targted radiotherapy. (authors)

  13. Efficient Replication of Adeno-Associated Virus Type 2 Vectors: a cis-Acting Element outside of the Terminal Repeats and a Minimal Size

    Tullis, Gregory E.; Shenk, Thomas

    2000-01-01

    Recombinant adeno-associated virus type 2 (AAV2) can be produced in adenovirus-infected cells by cotransfecting a plasmid containing the recombinant AAV2 genome, which is generally comprised of the viral terminal repeats flanking a transgene, together with a second plasmid expressing the AAV2 rep and cap genes. However, recombinant viruses generally replicate inefficiently, often producing 100-fold fewer virus particles per cell than can be obtained after transfection with a plasmid containin...

  14. Intraparenchymal spinal cord delivery of adeno-associated virus IGF-1 is protective in the SOD1G93A model of ALS

    Lepore, Angelo C.; Haenggeli, Christine; Gasmi, Mehdi; Bishop, Kathie M.; Bartus, Raymond T.; Maragakis, Nicholas J.; Rothstein, Jeffrey D.

    2007-01-01

    The potent neuroprotective activities of neurotrophic factors, including insulin-like growth factor 1 (IGF-1), make them promising candidates for treatment of amyotrophic lateral sclerosis (ALS). In an effort to maximize rate of motor neuron transduction, achieve high levels of spinal IGF-1, and thus enhance therapeutic benefit, we injected an adeno-associated virus 2 (AAV2)-based vector encoding human IGF-1 (CERE-130) into lumbar spinal cord parenchyma of SOD1G93A mice. We observed robust an...

  15. A single injection of recombinant adeno-associated virus into the lumbar cistern delivers transgene expression throughout the whole spinal cord

    Guo, Yansu; Wang, Dan; Qiao, Tao; Yang, Chunxing; Su, Qin; Gao, Guangping; Xu, Zuoshang

    2015-01-01

    The lack of methods to deliver transgene expression in spinal cord has hampered investigation of gene function and therapeutic targets for spinal cord diseases. Here we report that a single intrathecal injection of recombinant adeno-associated virus rhesus-10 (rAAVrh10) into the lumbar cistern led to transgene expression in sixty to ninety percent of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells and endothelial cells. Add...

  16. Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1

    Arruda, Valder R.; Schuettrumpf, Joerg; Herzog, Roland W; Nichols, Timothy C.; Robinson, Nancy; Lotfi, Yasmin; Mingozzi, Federico; Xiao, Weidong; Couto, Linda B.; High, Katherine A.

    2003-01-01

    Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observe...

  17. High-Throughput Functional MicroRNAs Profiling by Recombinant AAV-Based MicroRNA Sensor Arrays

    Tian, Wenhong; Dong, Xiaoyan; Liu, Xuerong; Wang, Gang; Dong, Zheyue; Shen, Wei; Zheng, Gang; Lu, Jianxin; Chen, Jinzhong; Wang, Yue; Wu, Zhijian; Wu, Xiaobing

    2012-01-01

    Background microRNAs (miRNAs) are small and non-coding RNAs which play critical roles in physiological and pathological processes. A number of methods have been established to detect and quantify miRNA expression. However, method for high-throughput miRNA function detection is still lacking. Principal Findings We describe an adeno-associated virus (AAV) vector-based microRNA (miRNA) sensor (Asensor) array for high-throughput functional miRNA profiling. Each Asensor contains a Gaussia lucifera...

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

    Luo, Xiaoyan

    2011-11-01

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

  19. Persistence, localization, and external control of transgene expression after single injection of adeno-associated virus into injured joints.

    Lee, Hannah H; O'Malley, Michael J; Friel, Nicole A; Payne, Karin A; Qiao, Chunping; Xiao, Xiao; Chu, Constance R

    2013-04-01

    A single intra-articular injection of adeno-associated virus (AAV) results in stable and controllable transgene expression in normal rat knees. Because undamaged joints are unlikely to require treatment, the study of AAV delivery in joint injury models is crucial to potential therapeutic applications. This study tests the hypotheses that persistent and controllable AAV-transgene expression are (1) highly localized to the cartilage when AAV is injected postinjury and (2) localized to the intra-articular soft tissues when AAV is injected preinjury. Two AAV injection time points, postinjury and preinjury, were investigated in osteochondral defect and anterior cruciate ligament transection models of joint injury. Rats injected with AAV tetracycline response element (TRE)-luciferase received oral doxycycline for 7 days. Luciferase expression was evaluated longitudinally for 6 months. Transgene expression was persistent and controllable with oral doxycycline for 6 months in all groups. However, the location of transgene expression was different: postinjury AAV-injected knees had luciferase expression highly localized to the cartilage, while preinjury AAV-injected knees had more widespread signal from intra-articular soft tissues. The differential transgene localization between preinjury and postinjury injection can be used to optimize treatment strategies. Highly localized postinjury injection appears advantageous for treatments targeting repair cells. The more generalized and controllable reservoir of transgene expression following AAV injection before anterior cruciate ligament transection (ACLT) suggests an intriguing concept for prophylactic delivery of joint protective factors to individuals at high risk for early osteoarthritis (OA). Successful external control of intra-articular transgene expression provides an added margin of safety for these potential clinical applications. PMID:23496155

  20. A new genetic vaccine platform based on an adeno-associated virus isolated from a rhesus macaque.

    Lin, Jianping; Calcedo, Roberto; Vandenberghe, Luk H; Bell, Peter; Somanathan, Suryanarayan; Wilson, James M

    2009-12-01

    We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8(+) T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8(+) T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses. PMID:19812149

  1. In vivo evaluation of adeno-associated virus gene transfer in airways of mice with acute or chronic respiratory infection.

    Myint, Melissa; Limberis, Maria P; Bell, Peter; Somanathan, Suryanarayan; Haczku, Angela; Wilson, James M; Diamond, Scott L

    2014-11-01

    Patients with cystic fibrosis (CF) often suffer chronic lung infection with concomitant inflammation, a setting that may reduce the efficacy of gene transfer. While gene therapy development for CF often involves viral-based vectors, little is known about gene transfer in the context of an infected airway. In this study, three mouse models were established to evaluate adeno-associated virus (AAV) gene transfer in such an environment. Bordetella bronchiseptica RB50 was used in a chronic, nonlethal respiratory infection in C57BL/6 mice. An inoculum of ∼10(5) CFU allowed B. bronchiseptica RB50 to persist in the upper and lower respiratory tracts for at least 21 days. In this infection model, administration of an AAV vector on day 2 resulted in 2.8-fold reduction of reporter gene expression compared with that observed in uninfected controls. Postponement of AAV administration to day 14 resulted in an even greater (eightfold) reduction of reporter gene expression, when compared with uninfected controls. In another infection model, Pseudomonas aeruginosa PAO1 was used to infect surfactant protein D (SP-D) or surfactant protein A (SP-A) knockout (KO) mice. With an inoculum of ∼10(5) CFU, infection persisted for 2 days in the nasal cavity of either mouse model. Reporter gene expression was approximately ∼2.5-fold lower compared with uninfected mice. In the SP-D KO model, postponement of AAV administration to day 9 postinfection resulted in only a two fold reduction in reporter gene expression, when compared with expression seen in uninfected controls. These results confirm that respiratory infections, both ongoing and recently resolved, decrease the efficacy of AAV-mediated gene transfer. PMID:25144316

  2. Liver-Specific Allergen Gene Transfer by Adeno-Associated Virus Suppresses Allergic Airway Inflammation in Mice.

    Chan, Cheng-Chi; Lai, Chin-Wen; Wu, Chia-Jen; Chen, Li-Chen; Tao, Mi-Hua; Kuo, Ming-Ling

    2016-08-01

    Allergic airway inflammation driven by T helper 2 (Th2)-type immunity is characterized by airway hyperresponsiveness, eosinophilic infiltration, and elevated IgE production. Various novel strategies for managing asthma have been explored, such as DNA vaccines, T-cell peptides, and allergen-specific immunotherapy. A principal goal of most immunotherapeutic approaches is active and long-term allergen-specific tolerance. Liver-specific gene transfer using adeno-associated virus (AAV) has been shown to favorably induce tolerogenic responses to therapeutic products in various experimental models. AAV8 has strong liver tropism and induces immune tolerance in mice. The present study aimed to determine whether hepatocyte-specific allergen expression by pseudotyped AAV2/8 alleviates asthmatic symptoms in ovalbumin (OVA)-sensitized mice. Mice were intravenously injected with AAV2/8 vector carrying membrane-bound OVA transgene under transcriptional control of a hepatocyte-specific alpha 1 antitrypsin promoter (AAV2/8-OVA) and then sensitized with OVA. AAV2/8-OVA specifically transduced the OVA transgene in the liver. Airway hyperresponsiveness, eosinophilia, mucus hypersecretion, and Th2 cytokines were significantly suppressed in both the lungs and secondary lymphoid organs of asthmatic mice infected with AAV2/8-OVA. Significant reduction of OVA-specific antibodies was detected in the bronchoalveolar lavage fluid from AAV2/8-OVA-treated mice. Moreover, AAV2/8-OVA treatment prominently promoted the expression of Foxp3, IL-10, and TGF-β in the liver. Enhanced Foxp3 expression was also detected in the lungs of asthmatic mice after AAV2/8-OVA treatment. Taken together, these results suggest that the induction of immune tolerance by hepatic AAV gene transfer may be beneficial for modulating allergic asthma. PMID:27178525

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

    Flotte, Terence R

    2011-10-01

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

  4. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity

    Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process

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

    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

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

    Vidović, Dragana; Gijsbers, Rik; Jimenez, Ana Quiles; Dooley, James; Van den Haute, Chris; Van der Perren, Anke; Liston, Adrian; Baekelandt, Veerle; Debyser, Zeger; Carlon, Marianne Sylvia

    2016-01-01

    Gene therapy holds promise to cure a wide range of genetic and acquired diseases. Recent successes in recombinant adeno-associated viral vector (rAAV)-based gene therapy in the clinic for hereditary disorders such as Leber's congenital amaurosis and hemophilia B encouraged us to reexplore an rAAV approach for pulmonary gene transfer. Only limited clinical successes have been achieved for airway gene transfer so far, underscoring the need for further preclinical development of rAAV-based gene therapy for pulmonary disorders. We sought to determine the preclinical potential of an airway-tropic serotype, rAAV2/5, encoding reporter genes when delivered to mouse airways. Although several groups have assessed the stability of gene transfer using a nonintegrating rAAV in mouse airways, long-term stability for more than a year has not been reported. Additionally, an extensive quantitative analysis of the specific cell types targeted by rAAV2/5 using cell-specific markers is lacking. We obtained sustained gene expression in upper and lower airways up to 15 months after vector administration, a substantial proportion of the lifespan of a laboratory mouse. In addition, we demonstrated that readministration of rAAV2/5 to the airways is feasible and increases gene expression 14 months after primary vector administration, despite the presence of circulating neutralizing antibodies. Finally, identification of transduced cell types revealed different subpopulations being targeted by rAAV2/5, with 64% of β-galactosidase-positive cells being ciliated cells, 34% club cells in the conducting airways, and 75% alveolar type II cells in the alveoli at 1 month postinjection. This underscores the therapeutic potential of a nonintegrating rAAV vector to develop a gene therapeutic drug for a variety of pulmonary disorders, such as cystic fibrosis, primary ciliary dyskinesia, and surfactant deficiencies. PMID:26567984

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

    Maohua Cao

    Full Text Available 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.

  8. Construction of a recombinant human parvovirus B19: adeno-associated virus 2 (AAV) DNA inverted terminal repeats are functional in an AAV-B19 hybrid virus.

    Srivastava, C H; Samulski, R J; L. Lu; Larsen, S H; A Srivastava

    1989-01-01

    To facilitate genetic analysis of the human pathogenic parvovirus B19, we constructed a hybrid B19 viral genome in which the defective B19 inverted terminal repeats were replaced with the full-length inverted terminal repeats from a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The hybrid AAV-B19 genome was rescued from a recombinant plasmid and then the DNA was replicated upon transfection into adenovirus 2-infected human KB cells in the presence of AAV genes coding for...

  9. Activation of the NF-κB pathway by adeno-associated virus (AAV) vectors and its implications in immune response and gene therapy

    Jayandharan, Giridhara R.; Aslanidi, George; Martino, Ashley T.; Jahn, Stephan C.; Perrin, George Q.; Herzog, Roland W.; Srivastava, Arun

    2011-01-01

    Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold...

  10. Cloning of adeno-associated virus into pBR322: Rescue of intact virus from the recombinant plasmid in human cells

    1982-01-01

    We have cloned intact duplex adeno-associated virus (AAV) DNA into the bacterial plasmid pBR322. The AAV genome could be rescued from the recombinant plasmid by transfection of the plasmid DNA into human cells with adenovirus 5 as helper. The efficiency of rescue from the plasmid was sufficiently high to produce yields of AAV DNA comparable to those observed after transfection with equal amounts of purified virion DNA. Thus, the recombinant plasmid itself may be a model for studying the rescu...

  11. A Novel Gene Expression Control System and Its Use in Stable, High-Titer 293 Cell-Based Adeno-Associated Virus Packaging Cell Lines

    Qiao, Chunping; Bing WANG; Zhu, Xiaodong; Li, Juan; Xiao, Xiao

    2002-01-01

    Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene ex...

  12. Full Functional Rescue of a Complete Muscle (TA) in Dystrophic Hamsters by Adeno-Associated Virus Vector-Directed Gene Therapy

    Xiao, Xiao; Li, Juan; Tsao, Yeou-Ping; Dressman, Devin; Hoffman, Eric P; Watchko, Jon F.

    2000-01-01

    Limb girdle muscular dystrophy (LGMD) 2F is caused by mutations in the δ-sarcoglycan (SG) gene. Previously, we have shown successful application of a recombinant adeno-associated virus (AAV) vector for genetic and biochemical rescue in the Bio14.6 hamster, a homologous animal model for LGMD 2F (J. Li et al., Gene Ther. 6:74–82, 1999). In this report, we show efficient and long-term δ-SG expression accompanied by nearly complete recovery of physiological function deficits after a single-dose A...

  13. Preparation of a recombinant adeno-associated viral vector with a mutation of human factor IX in large scale and its expression in vitro and in vivo

    2001-01-01

    A series of adeno-associated viral vectors conraining a mutation of human factor IX (hFIXR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFIXR338Awere obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFIXR338Awas prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFIXR338A viral stocks on a large scale and higher fiter was established,which can be used for industrial purpose. The titer of rAAV-hFIXR338A was more than 1.25x1012 particle/mL, and then, a mammalian cell line, C2C12 and the factor IXknock-out mice were transfected with the rAAV-hFIXR338Ain vitro and in vivo. The results show that the high-level expression of rAAV-hFIXR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng@ (106cells)-1 @ (24 h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFIX R338A, which was as high as the expression of rAAV-hFIX -wt (2565.76±64.36) ng@ (106 cells)-1@ (24 h)-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFIX-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFIXR338A is about 2.46times higher than that of hFIX-wt. It was first reported that a mutation of human factor IX was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFIX R338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFIX.``

  14. High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.

    Jin, Lei; Lange, Wienke; Kempmann, Annika; Maybeck, Vanessa; Günther, Anne; Gruteser, Nadine; Baumann, Arnd; Offenhäusser, Andreas

    2016-09-10

    In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons. PMID:27416794

  15. Silencing of T lymphocytes by antigen-driven programmed death in recombinant adeno-associated virus vector–mediated gene therapy

    Velazquez, Victoria M.; Bowen, David G.

    2009-01-01

    Recombinant adeno-associated virus (rAAV) vectors are considered promising for human gene replacement because they facilitate stable expression of therapeutic proteins in transduced tissues. Whether the success of gene therapy will be influenced by cellular immune responses targeting transgene-encoded proteins that are potentially immunogenic is unknown. Here we characterized CD8+ T-cell activity against β-galactosidase and enhanced green fluorescent protein, model antigens containing major histocompatibility complex (MHC) class I epitopes that are constitutively produced in murine skeletal muscle after rAAV vector transduction. Antigen-specific CD8+ T cells were detected in the spleen and liver of mice within 7 days of muscle transduction. CD8+ T-cell frequencies in these organs were stable, and effector functions were intact for months despite ongoing antigen production in muscle. CD8+ T cells also infiltrated transduced muscle, where frequencies were at least 5-fold higher than in untransduced spleen and liver. Significantly, the majority of antigen-specific CD8+ T cells in vector-transduced muscle were not functional. Loss of function in the muscle was associated with programmed death of the effector cells. Stable gene expression therefore depended on selective death of CD8+ T cells at the site of antigen production, an effective mechanism for subverting immunity that is also potentially reversible. PMID:18566327

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

    Lina Li

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

  17. Ultracentrifugation-free chromatography-mediated large-scale purification of recombinant adeno-associated virus serotype 1 (rAAV1)

    Tomono, Taro; Hirai, Yukihiko; Okada, Hironori; Adachi, Kumi; Ishii, Akiko; Shimada, Takashi; Onodera, Masafumi; Tamaoka, Akira; Okada, Takashi

    2016-01-01

    Recombinant adeno-associated virus (rAAV) is an attractive tool for gene transfer and shows potential for use in human gene therapies. The current methods for the production and purification of rAAV from the transfected cell lysate are mainly based on cesium chloride and iodixanol density ultracentrifugation, although those are not scalable. Meanwhile, chromatography-based systems are more scalable. Therefore, in this study, we developed a novel method for the production and purification of rAAV serotype 1 (rAAV1) from serum-free culture supernatant based on ion-exchange and gel-filtration chromatography to obtain highly purified products with an ultracentrifugation-free technique towards Good Manufacturing Practice (GMP) production. The purified rAAV1 displayed three clear and sharp bands (VP1, VP2, and VP3) following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and more than 90% of rAAV1 particles contained fully packaged viral genomes according to negative-stain electron micrographic analysis. Consequently, the resultant genomic titer of the purified rAAV1 was 3.63 × 1013 v.g./ml (the total titer was 4.17 × 1013 v.g.) from the 4 × 109 HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy. PMID:26913289

  18. Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

    Heller, Kristin N; Montgomery, Chrystal L; Shontz, Kimberly M; Clark, K Reed; Mendell, Jerry R; Rodino-Klapac, Louise R

    2015-10-01

    Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD. PMID:26076707

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

    Adamson-Small, Laura; Potter, Mark; Falk, Darin J; Cleaver, Brian; Byrne, Barry J; Clément, Nathalie

    2016-01-01

    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. PMID:27222839

  20. Adeno-associated virus type 2 rep protein inhibits human papillomavirus type 16 E2 recruitment of the transcriptional coactivator p300.

    Marcello, A; Massimi, P; Banks, L; Giacca, M

    2000-10-01

    Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1- and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300. PMID:10982355

  1. HoxD10 gene delivery using adenovirus/adeno-associate hybrid virus inhibits the proliferation and tumorigenicity of GH4 pituitary lactotrope tumor cells

    Prolactinoma is one of the most common types of pituitary adenoma. It has been reported that a variety of growth factors and cytokines regulating cell growth and angiogenesis play an important role in the growth of prolactinoma. HoxD10 has been shown to impair endothelial cell migration, block angiogenesis, and maintain a differentiated phenotype of cells. We investigated whether HoxD10 gene delivery could inhibit the growth of prolactinoma. Rat GH4 lactotrope tumor cells were infected with adenovirus/adeno-associated virus (Ad/AAV) hybrid vectors carrying the mouse HoxD10 gene (Hyb-HoxD10) or the β-galactosidase gene (Hyb-Gal). Hyb-HoxD10 expression inhibited GH4 cell proliferation in vitro. The expression of FGF-2 and cyclin D2 was inhibited in GH4 cells infected with Hyb-HoxD10. GH4 cells transduced with Hyb-HoxD10 did not form tumors in nude mice. These results indicate that the delivery of HoxD10 could potentially inhibit the growth of PRL-secreting tumors. This approach may be a useful tool for targeted therapy of prolactinoma and other neoplasms

  2. Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor.

    Ponnazhagan, Selvarangan; Mahendra, Gandham; Kumar, Sanjay; Shaw, Denise R; Stockard, Cecil R; Grizzle, William E; Meleth, Sreelatha

    2004-03-01

    Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy. PMID:14996740

  3. Pulmonary Targeting of Adeno-associated Viral Vectors by Next-generation Sequencing-guided Screening of Random Capsid Displayed Peptide Libraries.

    Körbelin, Jakob; Sieber, Timo; Michelfelder, Stefan; Lunding, Lars; Spies, Elmar; Hunger, Agnes; Alawi, Malik; Rapti, Kleopatra; Indenbirken, Daniela; Müller, Oliver J; Pasqualini, Renata; Arap, Wadih; Kleinschmidt, Jürgen A; Trepel, Martin

    2016-06-01

    Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine. PMID:27018516

  4. Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors.

    Van der Perren, Anke; Toelen, Jaan; Casteels, Cindy; Macchi, Francesca; Van Rompuy, Anne-Sophie; Sarre, Sophie; Casadei, Nicolas; Nuber, Silke; Himmelreich, Uwe; Osorio Garcia, Maria Isabel; Michotte, Yvette; D'Hooge, Rudi; Bormans, Guy; Van Laere, Koen; Gijsbers, Rik; Van den Haute, Chris; Debyser, Zeger; Baekelandt, Veerle

    2015-03-01

    Testing of new therapeutic strategies for Parkinson's disease (PD) is currently hampered by the lack of relevant and reproducible animal models. Here, we developed a robust rat model for PD by injection of adeno-associated viral vectors (rAAV2/7) encoding α-synuclein into the substantia nigra, resulting in reproducible nigrostriatal pathology and behavioral deficits in a 4-week time period. Progressive dopaminergic dysfunction was corroborated by histopathologic and biochemical analysis, motor behavior testing and in vivo microdialysis. L-DOPA treatment was found to reverse the behavioral phenotype. Non-invasive positron emission tomography imaging and magnetic resonance spectroscopy allowed longitudinal monitoring of neurodegeneration. In addition, insoluble α-synuclein aggregates were formed in this model. This α-synuclein rat model shows improved face and predictive validity, and therefore offers the possibility to reliably test novel therapeutics. Furthermore, it will be of great value for further research into the molecular pathogenesis of PD and the importance of α-synuclein aggregation in the disease process. PMID:25599874

  5. Pre-existing immunity to adeno-associated virus (AAV)2 limits transgene expression following intracerebral AAV2-based gene delivery in a 6-hydroxydopamine model of Parkinson's disease.

    Janelidze, Shorena; Nordström, Ulrika; Kügler, Sebastian; Brundin, Patrik

    2014-01-01

    BACKGROUND: Adeno-associated virus (AAV) vectors are used to deliver potentially therapeutic genes in clinical trials in Parkinson's disease (PD). Pre-existing immunity to AAV and a local neuroinflammatory response might negatively affect the efficacy of such AAV-mediated gene delivery. METHODS: We pre-immunized rats with wild-type AAV-2. Three months later, we created PD-like lesions by intrastriatal injections of 6-hydroxydopamine (6-OHDA) in 50% of the animals. One month later, we inj...

  6. Trans-Splicing Adeno-Associated Viral Vector-Mediated Gene Therapy Is Limited by the Accumulation of Spliced mRNA but Not by Dual Vector Coinfection Efficiency

    XU, ZHUPING; Yue, Yongping; Lai, Yi; Ye, Chaoyang; Qiu, Jianming; Pintel, David J.; Duan, Dongsheng

    2004-01-01

    Therapeutic application of recombinant adeno-associated virus (AAV) has been limited by its small carrying capacity. To overcome this limitation trans-splicing vectors were developed recently. However, the transduction efficiency of trans-splicing vectors is considerably lower than that of a single intact vector in skeletal muscle. To improve trans-splicing vectors for skeletal muscle gene therapy, we examined whether coinfection efficiency is a rate-limiting factor in the mdx mouse, a model ...

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

    Hélène Hall

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

  8. Recombinant adeno-associated virus 2-mediated transfer of the human superoxide-dismutase gene does not confer radioresistance on HeLa cervical carcinoma cells

    Background and purpose: The success rate of any therapeutic approach depends on the therapeutic window, which can be increased by either raising the resistance of the normal tissue without protecting the tumor cells or by sensitizing the tumor cells but not the normal cells. Two promising candidate genes for normal tissue protection against radiation-induced damage may be the copper-zinc (CuZnSOD) and manganese superoxide-dismutase genes (MnSOD). The recombinant adeno-associated virus 2 (rAAV-2) offers attractive advantages over other vector systems: low immunogenicity, ability to infect dividing and non-dividing tissues and a low chance of insertional mutagenesis, due to extra-chromosomal localization. We report the production of novel rAAV-2-SOD vectors and the investigation of their modulating effects on HeLa-RC cells after irradiation. Material and methods: rAAV-2 vectors were cloned containing the human CuZnSOD or MnSOD as transgene and vector stocks were produced. In the initial experiments human cervix carcinoma (HeLa-RC) cells were chosen for their susceptibility to rAAV-2. On day 0, cells were seeded and transduced with the rAAV-2-SOD vectors. On day 3, cells were harvested, irradiated (0.5-8 Gy) and reseeded in different assays (FACS, SOD, MTT and colony assays). Results: Although >70% of all cells expressed SOD and significant amounts of functional SOD protein were detected, no radioprotective effect of SOD was observed after transduction of HeLa-RC cells. Conclusions: Novel rAAV-2-SOD vectors that could be produced at high titer, were able to efficiently infect cells and express the SOD genes. The absence of a radioprotective effect in HeLa-RC cancer cells indicates an additional safety feature and suggests that rAAV-mediated MnSOD overexpression might contribute to increasing the therapeutic index when applied for normal tissue protection

  9. Development of Recombinant Adeno-Associated Virus Serotype 2/8 Carrying Kringle Domains of Human Plasminogen for Sustained Expression and Cancer Therapy.

    Kuo, Cheng-Hsiang; Chang, Bi-Ing; Lee, Fang-Tzu; Chen, Po-Ku; Lee, Jeng-Shin; Shi, Guey-Yueh; Wu, Hua-Lin

    2015-09-01

    Angiostatin and other plasminogen derivatives exhibit antitumor activities directly or indirectly, have demonstrated promising anticancer effects in preclinical studies, but have mostly failed in clinical trials partly due to their short serum half-lives. Our previous studies demonstrated that recombinant human plasminogen kringle 1-5 (K1-5) has superior antitumor activity compared with angiostatin. In addition, optimization of recombinant K1-5 with three amino acid substitutions enhances its antitumor effect. The current study was thus undertaken to evaluate prolonged expression of optimized K1-5 as cancer gene therapy. The recombinant adeno-associated virus (AAV) vector was used to express a secreted form of the optimized K1-5 (AAV-sK15tm) to improve its pharmacokinetic profile, which was considered to be the hurdle in angiostatin treatment of cancer. We successfully generated high-titer recombinant AAV vectors and observed sustained transgene expression for 567 days after a single injection of virus. The treated animals did not display any visible signs of abnormalities and showed normal serum biochemistry. The therapeutic potential of this treatment modality was demonstrated by both a strong inhibition of lung metastasis in the mouse B16F10 melanoma model and significant growth retardation of Lewis lung carcinoma xenografts in C57BL/6N mice as well as human A2058 melanoma xenografts in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Taken together, our results suggested that AAV-sK15tm produced long-term suppressive effects on cancer growth in vivo and should warrant serious consideration for clinical development. PMID:25950911

  10. Light-Activated Nuclear Translocation of Adeno-Associated Virus Nanoparticles Using Phytochrome B for Enhanced, Tunable, and Spatially Programmable Gene Delivery.

    Gomez, Eric J; Gerhardt, Karl; Judd, Justin; Tabor, Jeffrey J; Suh, Junghae

    2016-01-26

    Gene delivery vectors that are activated by external stimuli may allow improved control over the location and the degree of gene expression in target populations of cells. Light is an attractive stimulus because it does not cross-react with cellular signaling networks, has negligible toxicity, is noninvasive, and can be applied in space and time with unparalleled precision. We used the previously engineered red (R)/far-red (FR) light-switchable protein phytochrome B (PhyB) and its R light dependent interaction partner phytochrome interacting factor 6 (PIF6) from Arabidopsis thaliana to engineer an adeno-associated virus (AAV) platform whose gene delivery efficiency is controlled by light. Upon exposure to R light, AAV engineered to display PIF6 motifs on the capsid bind to PhyB tagged with a nuclear localization sequence (NLS), resulting in significantly increased translocation of viruses into the host cell nucleus and overall gene delivery efficiency. By modulating the ratio of R to FR light, the gene delivery efficiency can be tuned to as little as 35% or over 600% of the unengineered AAV. We also demonstrate spatial control of gene delivery using projected patterns of codelivered R and FR light. Overall, our successful use of light-switchable proteins in virus capsid engineering extends these important optogenetic tools into the adjacent realm of nucleic acid delivery and enables enhanced, tunable, and spatially controllable regulation of viral gene delivery. Our current light-triggered viral gene delivery prototype may be broadly useful for genetic manipulation of cells ex vivo or in vivo in transgenic model organisms, with the ultimate prospect of achieving dose- and site-specific gene expression profiles for either therapeutic (e.g., regenerative medicine) or fundamental discovery research efforts. PMID:26618393

  11. Intracerebral adeno-associated virus gene delivery of apolipoprotein E2 markedly reduces brain amyloid pathology in Alzheimer's disease mouse models.

    Zhao, Lingzhi; Gottesdiener, Andrew J; Parmar, Mayur; Li, Mingjie; Kaminsky, Stephen M; Chiuchiolo, Maria J; Sondhi, Dolan; Sullivan, Patrick M; Holtzman, David M; Crystal, Ronald G; Paul, Steven M

    2016-08-01

    The common apolipoprotein E alleles (ε4, ε3, and ε2) are important genetic risk factors for late-onset Alzheimer's disease, with the ε4 allele increasing risk and reducing the age of onset and the ε2 allele decreasing risk and markedly delaying the age of onset. Preclinical and clinical studies have shown that apolipoprotein E (APOE) genotype also predicts the timing and amount of brain amyloid-β (Aβ) peptide deposition and amyloid burden (ε4 >ε3 >ε2). Using several administration protocols, we now report that direct intracerebral adeno-associated virus (AAV)-mediated delivery of APOE2 markedly reduces brain soluble (including oligomeric) and insoluble Aβ levels as well as amyloid burden in 2 mouse models of brain amyloidosis whose pathology is dependent on either the expression of murine Apoe or more importantly on human APOE4. The efficacy of APOE2 to reduce brain Aβ burden in either model, however, was highly dependent on brain APOE2 levels and the amount of pre-existing Aβ and amyloid deposition. We further demonstrate that a widespread reduction of brain Aβ burden can be achieved through a single injection of vector via intrathalamic delivery of AAV expressing APOE2 gene. Our results demonstrate that AAV gene delivery of APOE2 using an AAV vector rescues the detrimental effects of APOE4 on brain amyloid pathology and may represent a viable therapeutic approach for treating or preventing Alzheimer's disease especially if sufficient brain APOE2 levels can be achieved early in the course of the disease. PMID:27318144

  12. A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear.

    Gomes, Michelle M; Wang, Lingyan; Jiang, Han; Kahl, Christoph A; Brigande, John V

    2016-01-01

    There is keen interest to define gene therapies aimed at restoration of auditory and vestibular function in the diseased or damaged mammalian inner ear. A persistent limitation of regenerative medical strategies that seek to correct or modify gene expression in the sensory epithelia of the inner ear involves efficacious delivery of a therapeutic genetic construct. Our approach is to define methodologies that enable fetal gene transfer to the developing mammalian inner ear in an effort to correct defective gene expression during formation of the sensory epithelia or during early postnatal life. Conceptually, the goal is to atraumatically introduce the genetic construct into the otocyst-staged mouse inner ear and transfect otic progenitors that give rise to sensory hair cells and supporting cells. Our long-term goal is to define therapeutic interventions for congenital deafness and balance disorders with the expectation that the approach may also be exploited for therapeutic intervention postnatally.In the inaugural volume of this series, we introduced electroporation-mediated gene transfer to the developing mouse inner ear that encompassed our mouse survival surgery and transuterine microinjection protocols (Brigande et al., Methods Mol Biol 493:125-139, 2009). In this chapter, we first briefly update our use of sodium pentobarbital anesthesia, our preferred anesthetic for mouse ventral laparotomy, in light of its rapidly escalating cost. Next, we define a rapid, cost-effective method to produce recombinant adeno-associated virus (rAAV) for efficient gene transfer to the developing mouse inner ear. Our immediate goal is to provide a genetic toolkit that will permit the definition and validation of gene therapies in mouse models of human deafness and balance disorders. PMID:27259920

  13. A Novel Gene Expression Control System and Its Use in Stable, High-Titer 293 Cell-Based Adeno-Associated Virus Packaging Cell Lines

    Qiao, Chunping; Wang, Bing; Zhu, Xiaodong; Li, Juan; Xiao, Xiao

    2002-01-01

    Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene expression since its promoter is located within the coding sequence of Rep78/68. To tightly regulate all four Rep proteins by using their own promoters, we have developed a novel gene control paradigm termed “dual splicing switch,” which disrupts all four Rep genes by inserting into their shared coding region an intron that harbors transcription termination sequences flanked the LoxP sites. As a result, the structure and activities of the Rep gene promoters, both p5 and p19, are not affected; however, all of the Rep transcripts are prematurely terminated and the genes were inactivated. Removal of the terminator by Cre protein reactivates the transcription of all four Rep proteins derived from their own promoters. This switch system was initially tested in the lacZ gene and a 600-fold induction of β-galactosidase activity was observed. Using the dual splicing switch strategy, we have subsequently established a number of AAV packaging cell lines from 293 cells, which showed a normal growth rate, high stability, and more importantly, high yields of AAV vectors. Such a gene control paradigm is also useful for other viruses, e.g., autonomous parvoviruses. Finally, the high-titer 293-based AAV packaging cell lines should greatly reduce the risk of wild-type adenovirus contamination and provide a scalable AAV vector production method for both preclinical and clinical studies. PMID:12438627

  14. Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

    Amin Hajitou

    2013-10-01

    Full Text Available Bacteriophage (phage, viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV. This novel AAV/phage hybrid (AAVP specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

  15. Infection of primary cells by adeno-associated virus type 2 results in a modulation of cell cycle-regulating proteins.

    Hermanns, J; Schulze, A; Jansen-Db1urr, P; Kleinschmidt, J A; Schmidt, R; zur Hausen, H

    1997-01-01

    It has been demonstrated that infection of primary human cells with adeno-associated viruses (AAV) leads to a decrease in cellular proliferation and to growth arrest. We analyzed the molecular basis of this phenomenon and observed that infection with AAV type 2 (AAV2) had an effect on several factors engaged in the control of the mammalian cell cycle. In particular, all of the pRB family members, pRB, p107, and p130, which are involved in G1 cell cycle checkpoint control, were affected. After infection, a shift from hyper- to hypophosphorylated forms was observed. Cyclins A and B1, which are required for G1/S transition and progression into mitosis, respectively, were downregulated at the transcriptional level as well as at the protein level, whereas the G1 cyclins D1 and E remained unaffected. In addition, the steady-state levels of cyclin-dependent kinases CDK1 and CDK2 and of transcription factor E2F-1 were diminished. Of all the factors known to be involved in phosphorylation of pRB family proteins, only the CDK inhibitor p21WAF1 exhibited a response to AAV2 infection. p21WAF1 mRNA was quickly and progressively upregulated in a p53-independent manner over at least 72 h. Consistent with the increased p21WAF1 protein levels, cyclin E- and cyclin A-dependent kinase activities declined to low levels and E2F-p130-cyclin-CDK2 complexes were disrupted. From these data, we conclude that the major effect of AAV2 infection on primary human fibroblasts appears to be upregulation of p21WAF1 gene expression and thus cell cycle arrest by the suppression of pRB family protein phosphorylation. PMID:9223493

  16. Partial correction of the CFTR-dependent ABPA mouse model with recombinant adeno-associated virus gene transfer of truncated CFTR gene.

    Mueller, Christian; Torrez, Daniel; Braag, Sofia; Martino, Ashley; Clarke, Tracy; Campbell-Thompson, Martha; Flotte, Terence R

    2008-01-01

    Recently, we have developed a model of airway inflammation in a CFTR knockout mouse utilizing Aspergillus fumigatus crude protein extract (Af-cpe) to mimic allergic bronchopulmonary aspergillosis (ABPA) 1, an unusual IgE-mediated hypersensitivity syndrome seen in up to 15% of cystic fibrosis (CF) patients and rarely elsewhere. We hypothesized that replacement of CFTR via targeted gene delivery to airway epithelium would correct aberrant epithelial cytokine signaling and ameliorate the ABPA phenotype in CFTR-deficient (CFTR 489X - /-, FABP-hCFTR + / +) mice. CFTR knockout mice underwent intra-tracheal (IT) delivery of recombinant adeno-associated virus serotype 5 (rAAV5Delta-264CFTR) or rAAV5-GFP at 2.58 x 10(12) viral genomes/mouse. All mice were then sensitized with two serial injections (200 microg) of crude Af antigen via the intra-peritoneal (IP) route. Untreated mice were sensitized without virus exposure. Challenges were performed 2 weeks after final sensitization, using a 0.25% solution containing Aspergillus fumigatus crude protein extract delivered by inhalation on three consecutive days. The rAAV5Delta-264CFTR-treated mice had lower total serum IgE levels (172513 ng/ml +/- 1312) than rAAV5-GFP controls (26 892 ng/ml +/- 3715) (p = 0.037) and non-treated, sensitized controls (24 816 +/- 4219 ng/ml). Serum IgG1 levels also were lower in mice receiving the CFTR vector. Interestingly, splenocytes from rAAV5Delta-264CFTR-treated mice secreted less IL-13, INFg, TNFa, RANTES and GM-CSF after ConA stimulation. Gene therapy with rAAV5Delta-264CFTR attenuated the hyper-IgE response in this reproducible CF mouse model of ABPA, with systemic effects also evident in the cytokine response of stimulated splenocytes. PMID:18023072

  17. Effect of nuclear factor κB inhibition on serotype 9 adeno-associated viral (AAV9) minidystrophin gene transfer to the mdx mouse.

    Reay, Daniel P; Niizawa, Gabriela A; Watchko, Jon F; Daood, Molly; Reay, Ja'Nean C; Raggi, Eugene; Clemens, Paula R

    2012-01-01

    Gene therapy studies for Duchenne muscular dystrophy (DMD) have focused on viral vector-mediated gene transfer to provide therapeutic protein expression or treatment with drugs to limit dystrophic changes in muscle. The pathological activation of the nuclear factor (NF)-κB signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy. Furthermore, activation of NF-κB may inhibit gene transfer by promoting inflammation in response to the transgene or vector. Therefore, we hypothesized that inhibition of pathological NF-κB activation in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD. Systemic gene transfer using serotype 9 adeno-associated viral (AAV9) vectors is promising for treatment of preclinical models of DMD because of vector tropism to cardiac and skeletal muscle. In quadriceps of C57BL/10ScSn-Dmd(mdx)/J (mdx) mice, the addition of octalysine (8K)-NF-κB essential modulator (NEMO)-binding domain (8K-NBD) peptide treatment to AAV9 minidystrophin gene delivery resulted in increased levels of recombinant dystrophin expression suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of expression. Indices of necrosis and regeneration were diminished with AAV9 gene delivery alone and to a greater degree with the addition of 8K-NBD treatment. In diaphragm muscle, high-level transgene expression was achieved with AAV9 minidystoophin gene delivery alone; therefore, improvements in histological and physiological indices were comparable in the two treatment groups. The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer, which may be highly significant for clinical applications of muscle gene delivery. PMID:22231732

  18. An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

    Abdelwahed Chtarto

    Full Text Available Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS. This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

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

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

    1984-10-01

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

  20. Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells

    Tandon Apurva

    2011-08-01

    Full Text Available Abstract Background In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2 induces apoptosis in Human Papillomavirus (HPV positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive, as well as MDA-MB-231 (highly invasive human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs isolated from tissue biopsies of patients undergoing breast reduction surgery. Results AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. Conclusion AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated

  1. Expression of human nerve growth factor β gene in central nervous system mediated by recombinant adeno-associated viruses type-2 vector

    高凯; 吴勇杰; 吴小兵; 饶春明; 王军志

    2004-01-01

    Background Neurone atrophy and loss are major causes of chronic neurodegenerative disorders such as Alzheimer's disease. Despite many pharmacotherapies for neurodegeneration, there are no accepted treatments. We investigated the feasibility of human nerve growth factor β (hNGFβ) gene expression mediated by recombinant adeno-associated viruses type-2 (rAAV-2) vector in the central nervous system (CNS) after blood brain barrier (BBB) disruption.Methods rAAV-2 containing hNGFβ gene was constructed. The ability of hNGFβ gene mediated by rAAV-2 vector (rAAV-2/hNGFβ) to transfect cells in vitro was confirmed by both ELISA and bioassay of hNGFβ in the culture supernatant of BHK-21 cells infected by rAAV-2/hNGFβ. rAAV-2/hNGFβ and rAAV-2/green fluorescence protein (GFP) were administrated separately to rat brains through internal carotid intubation after BBB disruption with hypertonic mannitol. Brain hNGFβ concentration was measured by ELISA and GFP in brain sections was examined by laser scan confocal microscope.Results After 48 hours, hNGFβ content in supernatant was up to (188.0±28.6) pg/ml when BHK-21 cells were infected by rAAV-2/hNGFβ at multiplicity of infection (MOI)1.0×106 vector genome. Neurone fibre outgrowths were obvious in dorsal root ganglion neurone assays by adding serum free culture medium harvested from BHK-21 cells exposed to rAAV-2/hNGFβ. Whole brain hNGFβ content in rAAV-2/hNGFβ transferred group was up to (636.2±140.6) pg/ml. hNGFβ content of BBB disruption in rAAV-2/hNGFβ infused group increased significantly compared to the control group (P<0.05). GFP expression was clearly observed in brain sections of rAAV-2/GFP transferred group.Conclusion rAAV-2/hNGFβ successfully expresses in the CNS after BBB disruption induced by hypertonic mannitol.

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

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

    2016-07-01

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

  3. Capsid Mutated Adeno-Associated Virus Delivered to the Anterior Chamber Results in Efficient Transduction of Trabecular Meshwork in Mouse and Rat.

    Barbara Bogner

    Full Text Available Adeno associated virus (AAV is well known for its ability to deliver transgenes to retina and to mediate improvements in animal models and patients with inherited retinal disease. Although the field is less advanced, there is growing interest in AAV's ability to target cells of the anterior segment. The purpose of our study was to fully articulate a reliable and reproducible method for injecting the anterior chamber (AC of mice and rats and to investigate the transduction profiles of AAV2- and AAV8-based capsid mutants containing self-complementary (sc genomes in the anterior segment of the eye.AC injections were performed in C57BL/6 mice and Sprague Dawley rats. The cornea was punctured anterior of the iridocorneal angle. To seal the puncture site and to prevent reflux an air bubble was created in the AC. scAAVs expressing GFP were injected and transduction was evaluated by immunohistochemistry. Both parent serotype and capsid modifications affected expression. scAAV2- based vectors mediated efficient GFP-signal in the corneal endothelium, ciliary non-pigmented epithelium (NPE, iris and chamber angle including trabecular meshwork, with scAAV2(Y444F and scAAV2(triple being the most efficient.This is the first study to semi quantitatively evaluate transduction of anterior segment tissues following injection of capsid-mutated AAV vectors. scAAV2- based vectors transduced corneal endothelium, ciliary NPE, iris and trabecular meshwork more effectively than scAAV8-based vectors. Mutagenesis of surface-exposed tyrosine residues greatly enhanced transduction efficiency of scAAV2 in these tissues. The number of Y-F mutations was not directly proportional to transduction efficiency, however, suggesting that proteosomal avoidance alone may not be sufficient. These results are applicable to the development of targeted, gene-based strategies to investigate pathological processes of the anterior segment and may be applied toward the development of gene

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

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

    2016-01-01

    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. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    LU; Huazhong; (

    2001-01-01

    [1]Chang, J., Jin, J., Lollar, P. et al., Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity, J. Bio. Chem., 1998, 273 (20): 12089-12094.[2]Lai, L., Chen, L., Zhou, H. et al., Clinical phenotype and genetic stability of factor IX gene knock out mice, J. Fudan Uni., 1999, 38 (4): 435-438.[3]Wu, Z. J., Wu, X. B., Hou, Y. D., Generation of a recombinant herps simplex virus which can provide packaging function for recombinant adeno-associated virus, Chinese Sci. Bull., 1999, 44 (8): 715-719.[4]Snyder, R. O., Miao, C. H., Patijn, G. A. et al., Persistent and therapeutic concentrations of human factor IX in mice after hepatic gene transfer of recombinant AAV vectors, Nat. Genet., 1997, 16 (3): 270-276.[5]Lai, L. H., Chen, L., Wang, J. M. et al., Skeletal muscle-specific expression of human blood coagulation factor IX rescues factor IX deficiency mouse by AAV-mediated gene transfer, Science in China, Ser. C, 1999, 42 (6): 628-634.[6]Snyder, R. O., Miao, C., Meuse, L. et al., Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors, Nat. Med., 1999, 5 (1): 64-70.[7]Kung, S. H., Hagstrom, J. N., Cass, D. et al., Human factor IX corrects the bleeding diathesis of mice with hemophilia B, Blood, 1998, 91(3): 784-790.[8]Hirt, B., Selective extraction of polyoma DNA from infected mouse cell culture, J. Mol. Biol., 1967, 26: 365-369.[9]Sambrook, J., Fritsch, E., Maniatis, T., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989, 6, 20-21.[10]Chao, H., Samulski, R. J., Bellinger, D. A. et al., Persistent expression of canine factor IX in hemophilia B canines, Gene Ther., 1999, 6: 1695-1704.[11]Kaufman, R. J., Advances toward gene therapy for hemophilia at the millennium, Hum. Gene Ther., 1999, 10 (13): 2091-2107.[12]Lu, D. R., Zhou, J. M., Zheng, B. et al., Stage I clinical trial of gene

  6. Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

    陆华中; 陈立; 王红卫; 伍志坚; 吴小兵; 王学峰; 王鸿利; 卢大儒; 邱信芳; 薛京伦

    2001-01-01

    A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.

  7. A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine.

    Fengqin Zhu

    Full Text Available More than 170 million individuals worldwide are infected with hepatitis C virus (HCV, and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

  8. Intracranial delivery of interleukin-17A via adeno-associated virus fails to induce physical and learning disabilities and neuroinflammation in mice but improves glucose metabolism through AKT signaling pathway.

    Yang, Junling; Kou, Jinghong; Lim, Jeong-Eun; Lalonde, Robert; Fukuchi, Ken-Ichiro

    2016-03-01

    Interleukin-17A (IL-17A) is generally considered as one of the pathogenic factors involved in multiple sclerosis (MS). Indirect evidence for this is that IL-17A-producing T helper 17 (Th17) cells preferentially accumulate in lesions of MS and experimental autoimmune encephalomyelitis (EAE). However, a direct involvement of IL-17A in MS pathogenesis is still an open question. In this study, we overexpressed IL-17A in the brains of mice (IL-17A-in-Brain mice) via recombinant adeno-associated virus serotype 5 (rAAV5)-mediated gene delivery. In spite of high levels of IL-17A expression in the brain and blood, IL-17A-in-Brain mice exhibit no inflammatory responses and no abnormalities in motor coordination and spatial orientation. Unexpectedly, IL-17A-in-Brain mice show decreases in body weight and adipose tissue mass and an improvement in glucose tolerance and insulin sensitivity. IL-17A enhances glucose uptake in PC12 cells by activation of AKT. Our results provide direct evidence for the first time that IL-17A overexpression in the central nervous system does not cause physical and learning disabilities and neuroinflammation and suggest that IL-17A may regulate glucose metabolism through the AKT signaling pathway. PMID:26562537

  9. Comparison of Efficacy of the Disease-Specific LOX1- and Constitutive Cytomegalovirus-Promoters in Expressing Interleukin 10 through Adeno-Associated Virus 2/8 Delivery in Atherosclerotic Mice

    Zhu, Hongqing; Cao, Maohua; Mirandola, Leonardo; Figueroa, Jose A.; Cobos, Everardo; Chiriva-Internati, Maurizio; Hermonat, Paul L.

    2014-01-01

    The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of “disease-specific promoters” has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2) using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery. PMID:24736312

  10. Comparison of efficacy of the disease-specific LOX1- and constitutive cytomegalovirus-promoters in expressing interleukin 10 through adeno-associated virus 2/8 delivery in atherosclerotic mice.

    Hongqing Zhu

    Full Text Available The development of gene therapy vectors for treating diseases of the cardiovascular system continues at a steady pace. Moreover, in the field of gene therapy the utility of "disease-specific promoters" has strong appeal. Many therapeutic genes, including transforming growth factor beta 1 or interleukin 10, are associated to adverse effects. The use of a disease-specific promoter might minimize toxicity. The lectin-like oxidized low density lipoprotein receptor 1 is a marker of cardiovascular disease and a potential therapeutic target. The lectin-like oxidized low density lipoprotein receptor 1 is known to be up-regulated early during disease onset in a number of cell types at the sites where the disease will be clinically evident. In this study an adeno-associated virus-2 DNA vector (AAV2 using the AAV8 capsid, and containing the full length The lectin-like oxidized low density lipoprotein receptor 1 promoter, was generated and assayed for its ability to express human interleukin 10 in low density lipoprotein receptor knockout mice on high cholesterol diet. The cytomegalovirus early promoter was used for comparison in a similarly structured vector. The two promoters were found to have equal efficacy in reducing atherogenesis as measured by aortic systolic blood velocity, aortic cross sectional area, and aortic wall thickness. This is the first head-to-head comparison of a constitutive with a disease-specific promoter in a therapeutic context. These data strongly suggest that the use of a disease-specific promoter is appropriate for therapeutic gene delivery.

  11. In vitro evaluation of mitochondrial dysfunction and treatment with adeno-associated virus vector in fibroblasts from Doberman Pinschers with dilated cardiomyopathy and a pyruvate dehydrogenase kinase 4 mutation.

    Sosa, Ivan; Estrada, Amara H; Winter, Brandy D; Erger, Kirsten E; Conlon, Thomas J

    2016-02-01

    OBJECTIVE To compare mitochondrial oxygen consumption rate (OCR) of fibroblasts from Doberman Pinschers with and without dilated cardiomyopathy (DCM) and mutation of the gene for pyruvate dehydrogenase kinase isozyme 4 (PDK4) and to evaluate in vitro whether treatment with adeno-associated virus (AAV) vector (ie, gene therapy) would alter metabolic efficiency. ANIMALS 10 Doberman Pinschers screened for DCM and PDK4 mutation. PROCEDURES Fibroblasts were harvested from skin biopsy specimens obtained from Doberman Pinschers, and dogs were classified as without DCM or PDK4 mutation (n = 3) or with occult DCM and heterozygous (4) or homozygous (3) for PDK4 mutation. Fibroblasts were or were not treated with tyrosine mutant AAV type 2 vector containing PDK4 at multiplicities of infection of 1,000. Mitochondrial OCR was measured to evaluate mitochondrial metabolism. The OCR was compared among dog groups and between untreated and treated fibroblasts within groups. RESULTS Mean ± SD basal OCR of fibroblasts from heterozygous (74 ± 8 pmol of O2/min) and homozygous (58 ± 12 pmol of O2/min) dogs was significantly lower than that for dogs without PDK4 mutation (115 ± 9 pmol of O2/min). After AAV transduction, OCR did not increase significantly in any group (mutation-free group, 121 ± 26 pmol of O2/min; heterozygous group, 88 ± 6 pmol of O2/min; homozygous group, 59 ± 3 pmol of O2/min). CONCLUSIONS AND CLINICAL RELEVANCE Mitochondrial function was altered in skin fibroblasts of Doberman Pinschers with DCM and PDK4 mutation. Change in mitochondrial function after in vitro gene therapy at the multiplicities of infection used in this study was not significant. (Am J Vet Res 2016;77:156-161). PMID:27027709

  12. Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector.

    Grieger, Joshua C; Soltys, Stephen M; Samulski, Richard Jude

    2016-02-01

    Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 10(5) vector genome containing particles (vg)/cell or greater than 1 × 10(14) vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1-6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 10(13) vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal

  13. [Establishment of hepatitis B virus (HBV) chronic infection mouse model by in vivo transduction with a recombinant adeno-associated virus 8 carrying 1. 3 copies of HBV genome (rAAN8-1. 3HBV)].

    Dong, Xiao-Yan; Yu, Chi-Jie; Wang, Gang; Tian, Wen-Hong; Lu, Yue; Zhang, Feng-Wei; Wang, Wen; Wang, Yue; Tan, Wen-Jie; Wu, Xiao-Bing

    2010-11-01

    In this report, we developed a HBV infection model in C57BL/6 mouse line by in vivo injection of a recombinant adeno-associated virus 8 vector carrying 1. 3 copies of HBV genome (ayw subtype) (rAAV8-1. 3HBV). We firstly prepared and purified the rAAV8-1. 3HBV and then injected it into three C57BL/6 mice with the dose of 2 x 10e11vg, respectively. HBsAg and HBeAg were assayed in sera collected at different time points post injection. Ten weeks post injection, the three mice were sacrificed and blood and liver tissue were taken for assay. Copies of HBV DNA were detected by real time PCR and the way of HBV DNA replication was identified by PCR. Subsequently, detection of HBV antigen by immunohistochemistry and pathology analysis of liver tissue of mice were performed. The results suggested that expression of HBsAg and HBeAg lasted for at least 10 weeks in mice sera. Among mice injected with rAAV8-1. 3HBV, HBsAg levels were showed an 'increasing-decreasing-increasing' pattern (the lowest level at the 4th week post injection), while HBeAg levels were kept high and relatively stable. HBV DNA copies were 4.2 x 10(3), 3.6 x 10(3), 2.5 x 10(3) copies/mL in sera and 8.0 x 10(6), 5.7 x 10(6), 2.6 x 10(6) copies/g in hepatic tissues of three mice, respectively. We found that the linear 1. 3HBV DNA in the rAAV8-1. 3HBV could self form into circular HBV genome and replicate in livers of HBV transfected mice. HBsAg and HBcAg were both positive in liver tissue of mice injected with rAAV8-1. 3HBV and no obvious pathological characters were found in liver of mice injected with rAAV8-1. 3HBV. In conclusion, we successfully developed a HBV chronic infection model in C57BL/6 mouse line by in vivo transduction with the recombinant virus rAAV8-1. 3HBV, in which HBV genes could be continuously expressed and replicated over 10 weeks, and paved a way for further characterization of the human chronic hepatitis B virus infection and evaluation of vaccine and anti-HBV agents. PMID:21344744

  14. 重组腺相关病毒2型/人凝血因子IX的质量研究%Quality control of recombinant adeno-associated virus type 2/human blood coagulation factor IX

    高凯; 王军志; 饶春明; 吴小兵

    2003-01-01

    目的研究并建立重组腺相关病毒2型/人凝血因子IX(recombinant adeno-associated virus type 2/human blood coagulation factor IX,rAAV-2/hFIX)的质量标准.方法采用PCR法确认病毒所携带的重组核酸结构和测定辅助病毒(helper virus)和野生型腺相关病毒(wtAAV)的残留片段.SDS-PAGE电泳测定病毒外壳蛋白分子量及纯度,TSK gel SP-NPR阳离子交换柱系统测定病毒颗粒纯度.以斑点杂交法测定病毒颗粒数.一期法于IX因子基因剔除小鼠体内测定rAAV-2/hFIX生物学活性,并通过ELISA法测定感染BHK-21细胞后hFIX的表达量.结果 PCR法确证病毒的重组核酸结构与构建预期相同;在1×107 VG的rAAV-2/hFIX颗粒中,残留辅助病毒的基因片段数少于1个拷贝;在1×108 VG的rAAV-2/hFIX颗粒中,野生型AAV-2基因片段数少于1个拷贝.病毒颗粒及外壳蛋白纯度均大于98%,病毒颗粒数大于1.0×1015 VG*L-1(virus genome*L-1).IX因子剔除小鼠肌肉注射病毒后21 d,小鼠血液中人凝血因子IX活性达到大于正常人因子IX活性的15%,IX因子的体外表达水平大于20.0 μg*L-1.其他各项检测指标均符合规定.结论建立了rAAV-2/hFIX的质量标准,用于控制产品质量.

  15. MicroRNAs and drug addiction

    Paul J Kenny

    2013-05-01

    Full Text Available Drug addiction is considered a disorder of neuroplasticity in brain reward and cognition systems resulting from aberrant activation of gene expression programs in response to prolonged drug consumption. Noncoding RNAs are key regulators of almost all aspects of cellular physiology. MicroRNAs (miRNAs are small (~21–23 nucleotides noncoding RNA transcripts that regulate gene expression at the post-transcriptional level. Recently, microRNAs were shown to play key roles in the drug-induced remodeling of brain reward systems that likely drives the emergence of addiction. Here, we review evidence suggesting that one particular miRNA, miR-212, plays a particularly prominent role in vulnerability to cocaine addiction. We review evidence showing that miR-212 expression is increased in the dorsal striatum of rats that show compulsive-like cocaine-taking behaviors. Increases in miR-212 expression appear to protect against cocaine addiction, as virus-mediated striatal miR-212 over-expression decreases cocaine consumption in rats. Conversely, disruption of striatal miR-212 signaling using an antisense oligonucleotide increases cocaine intake. We also review data that identify two mechanisms by which miR-212 may regulate cocaine intake. First, miR-212 has been shown to amplify striatal CREB signaling through a mechanism involving activation of Raf1 kinase. Second, miR-212 was also shown to regulate cocaine intake by repressing striatal expression of methyl CpG binding protein 2 (MeCP2, consequently decreasing protein levels of brain-derived neurotrophic factor (BDNF. The concerted actions of miR-212 on striatal CREB and MeCP2/BDNF activity greatly attenuate the motivational effects of cocaine. These findings highlight the unique role for miRNAs in simultaneously controlling multiple signaling cascades implicated in addiction.

  16. 重组8型腺相关病毒介导HBV急性感染树鼩模型建立%Establishment of a tree shrew model of acute hepatitis B virus infection by transduction with a recombinant adeno-associated virus 8 carrying 1.3 copies of HBV genome

    曾扬; 吴小红; 胡靓雅; 刘晨风; 于虹; 郭彦; 周勇; 孙世惠; 周育森

    2013-01-01

    目的 利用重组8型腺相关病毒介导1.3拷贝HBV基因组(1.3HBV,ayw亚型)在树鼩肝脏表达,建立HBV急性感染树鼩模型.方法 通过大腿内侧静脉注射将携带有1.3 HBV的重组8型腺相关病毒(recombinant adeno-associated virus 8,rAAV8-1.3HBV)导入树鼩肝脏,通过ELISA检测树鼩血清中HBsAg、HBeAg、HBsAb、HBeAb、HBcAb,荧光定量PCR检测树鼩肝脏和血清中HBV DNA,全自动生化分析仪检测血清中ALT水平,并观察感染后肝脏的病变情况.结果 HBV感染主要血清标志物1~2周内均检测阳性;30 d后肝组织仍可检测到病毒抗原阳性细胞;55 d时肝组织HBV DNA拷贝数仍可达到104~105;树鼩血清中HBV DNA拷贝数持续一个月高于正常组;肝组织炎细胞略增多,血清ALT水平持续升高.结论 rAAV8所携带的HBV基因组高效专一导入树鼩肝细胞并复制表达,成功建立HBV急性感染树鼩模型,为进一步探索rAAV8树鼩慢性感染模型打下一定的基础.%Objective To establish a tree shrew model of acute hepatitis B virus infection by injection of a recombinant adeno-associated virus 8 vector carrying 1.3 copies of HBV genome (ayw subtype) (rAAV8-1.3 HBV)into the liver of tree shrews.Methods Serum and liver tissues were collected at indicated times after i.v.injection of rAAV8-1.3 HBV into the tree shrews.The HBsAg,BeAg,HBsAb,HBeAb,HBcAb,ALT and HBV virus load were examined by ELISA and real-time PCR,respectively.The expression of HBcAg and pathological changes in the liver were also observed after the rAAV8-1.3 HBV infection.Results Markers of serum HBV were all positive 2 weeks after and HBcAg-positive hepatocytes were even detected in the liver 55 days after rAAV8-1.3 HBV injection.The copies of HBV DNA in liver reached 104-105 at 55 days after rAAV8-1.3HBV injection.Serum HBV DNA could be detected for over one month.Mild pathological changes with elevated ALT were observed after rAAV8-1.3 HBV injection.Conclusions A tree shrew

  17. MicroRNAs

    Devaux, Yvan; Stammet, Pascal; Friberg, Hans; Hassager, Christian; Kuiper, Michael A; Wise, Matt P; Nielsen, Niklas

    2015-01-01

    cardiac arrest would allow subsequent health care delivery to be tailored to individual patients. However, currently available predictive methods and biomarkers lack sufficient accuracy and therefore cannot be generally recommended in clinical practice. MicroRNAs have recently emerged as potential...... biomarkers of cardiovascular diseases. While the biomarker value of microRNAs for myocardial infarction or heart failure has been extensively studied, less attention has been devoted to their prognostic value after cardiac arrest. This review highlights the recent discoveries suggesting that microRNAs may be...

  18. Micro-RNAs

    Taipaleenmäki, H.; Hokland, L. B.; Chen, Li;

    2012-01-01

    Osteoblast differentiation and bone formation (osteogenesis) are regulated by transcriptional and post-transcriptional mechanisms. Recently, a novel class of regulatory factors termed microRNAs has been identified as playing an important role in the regulation of many aspects of osteoblast biology...... including proliferation, differentiation, metabolism and apoptosis. Also, preliminary data from animal disease models suggest that targeting miRNAs in bone can be a novel approach to increase bone mass. This review highlights the current knowledge of microRNA biology and their role in bone formation and...

  19. 重组8型腺相关病毒介导双荧光素酶基因在小鼠体内的表达%Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse

    王刚; 尉迟捷; 董小岩; 田文洪; 吴小兵

    2012-01-01

    目的 利用共表达的分泌型荧光素酶Gluc(gaussia princeps luciferase)和非分泌型荧光素酶Fluc(firefly luciferase)研究重组8型腺相关病毒(recombinant adeno-associated virus type 8,rAAV8)介导的转基因在小鼠体内的表达特点.方法 制备携带双荧光素酶基因的重组8型腺相关病毒rAAV8-Gluc/Fluc,体外感染HEK293细胞并检测上清和胞内Gluc和Fluc活性;将不同剂量的rAAV8-Gluc/Fluc尾静脉注射或肌内注射至BALB/c小鼠,通过尾静脉采血检测Gluc活性,通过活体成像和裂解组织检测Fluc活性.结果 成功制备了rAAV8-Gluc/Fluc,可以有效感染HEK293细胞,同时分泌表达Gluc和胞内表达Fluc;尾静脉注射或肌内注射rAAV8-Gluc/Fluc至小鼠后,外周血Gluc活性均在注射后10 ~20 d达到高峰并稳定持续120 d以上,Gluc活性随注射剂量增加而增高;静脉注射rAAV8-Gluc/Fluc时Fluc主要在肝脏表达,在骨骼肌和心肌有少量表达,而肌内注射时Fluc既在肌内注射局部表达同时也在肝脏中表达.结论 本研究成功制备了携带双荧光素酶基因rAAV8-Gluc/Fluc,研究了其介导的转基因在小鼠体内的表达特点,为rAAV8的临床前应用打下基础.%Objective Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc).Methods rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro.BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im).Then Gluc activities in blood were measured,the whole-body images for Fluc activities were performed and Fluc activities of tissue lysate were also detected.Results rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells.The Gluc was mainly detected in the culture media while the Fluc was mainly

  20. Phenotypic MicroRNA Microarrays

    Veronica Soloveva

    2013-04-01

    Full Text Available Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

  1. MicroRNA and cancer

    Jansson, Martin D; Lund, Anders H

    2012-01-01

    biological phenomena and pathologies. The best characterized non-coding RNA family consists in humans of about 1400 microRNAs for which abundant evidence have demonstrated fundamental importance in normal development, differentiation, growth control and in human diseases such as cancer. In this review, we...... summarize the current knowledge and concepts concerning the involvement of microRNAs in cancer, which have emerged from the study of cell culture and animal model systems, including the regulation of key cancer-related pathways, such as cell cycle control and the DNA damage response. Importantly, microRNA...

  2. Overview of MicroRNA Biology

    Mott, Justin L.; Mohr, Ashley M

    2015-01-01

    In considering an overview of microRNA biology, it is useful to consider microRNAs as a part of cellular communication. At the simplest level, microRNAs act to decrease the expression of mRNAs that contain stretches of sequence complementary to the microRNA. This function can be likened to the function of endogenous or synthetic short interfering RNA (siRNA). However, microRNA function is more complicated and nuanced than this ‘on-off’ model would suggest. Further, many microRNA targets are t...

  3. Isolation of microRNA targets using biotinylated synthetic microRNAs

    Ørom, Ulf Andersson; Lund, Anders H

    2007-01-01

    MicroRNAs are small regulatory RNAs found in multicellular organisms where they post-transcriptionally regulate gene expression. In animals, microRNAs bind mRNAs via incomplete base pairings making the identification of microRNA targets inherently difficult. Here, we present a detailed method for...... experimental identification of microRNA targets based on affinity purification of tagged microRNAs associated with their targets. Udgivelsesdato: 2007-Oct...

  4. 重组腺相关病毒转导人树突状细胞体外诱导抗肝癌免疫应答%Generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells with adeno-associated virus expressing α-fetoprotein

    杜文贞; 于天霞

    2011-01-01

    Objective To investigate the generation of antitumor response against hepatocellular carcinoma by in vitro transduction of dendritic cells (DC)with recombinant adeno-associated virus expressing α-fetoprotein (rAAV-AFP). Methods Peripheral blood mononuclear cells were isolated from healthy volunteers. Adherent peripheral blood mononuclear cells were transduced with AAV-AFP and cultured in the presence of granulocyte macrophage colony stimulating factor and interleukin-4 to generate dendritic cells.MTS assay was used to measure the ability of DC transduced with AAV-AFP ( AAV-AFP + DC) to stimulate the proliferation of T cell. The phenotype and AFP protein expression of DC and the secretion of IFN (interferon)-γ and IL (interleukin)-4 by T cells were detected by flow cytometry. The killing efficacy of cytotoxic T lymphocytes (CTL) activated by AAV-AFP + DC against AFP positive hepatocellular carcinoma cell lines was detected by lactate dehydrogenase (LDH) release assay. Results AAV-AFP + DC expressed HLA Ⅰ (97. 12%), HLAⅡ (97.32%), CD80(38.94%), CD83(60.84%)and CD86(98. 14%). AFP was secreted by 81.2% of AAV-AFP + DC. And it could stimulate effectively the proliferation of T cell.19. 84% of CD4 + T cells and 18.65% of CD8 + T cells activated by AAV-AFP + DC produced IFN-γbut not IL-4 and showed distinct killing activities against AFP positive hepatocellular carcinoma cell lines HepG2 (56. 45% ) and BEL7402 (78. 84% ). Conclusion AAV-AFP + DC can elicit distinct antitumor responses against AFP positive hepatocellular carcinoma cell lines so as to provide a basis for further researches on the clinical application of AAV-AFP + DC in the treatment of hepatocellular carcinoma.%目的 探讨携带甲胎蛋白基因的重组腺相关病毒(rAAV-AFP)转导人树突状细胞(DC)体外诱导抗肝癌免疫应答.方法 分离健康志愿者外周血单核细胞,贴壁细胞转导rAAV-AFP后,在粒细胞巨噬细胞集落刺激因子(GMCSF)和白细胞介素4(IL-4)的联

  5. MicroRNAs in Ewing Sarcoma

    PaulJedlicka; LayneDylla

    2013-01-01

    MicroRNAs have emerged recently as important regulators of gene expression in the cell. Frequently dysregulated in cancer, microRNAs have shed new light on molecular mechanisms of oncogenesis, and have generated substantial interest as biomarkers, and novel therapeutic agents and targets. Recently, a number of studies have examined microRNA biology in Ewing Sarcoma. Findings indicate that alterations in microRNA expression in Ewing Sarcoma are widespread, involve both EWS/Ets oncogenic fusion...

  6. MicroRNA Regulation of Lipid Metabolism

    Flowers, Elena; Froelicher, Erika Sivarajan; Aouizerat, Bradley E.

    2012-01-01

    MicroRNA are structural components of an epigenetic mechanism of post-transcriptional regulation of messenger RNA translation. Recently, there is significant interest in the application of microRNA as a blood-based biomarker of underlying physiologic conditions, and the therapeutic administration of microRNA inhibitors and mimics. The purpose of this review is to describe the current body of knowledge on microRNA regulation of genes involved in lipid metabolism, and to introduce the role of m...

  7. Study of adeno-associated virus carrying the HGFK1 gene(AAV-HGFK1) in treating rat hepatocellular carcinoma%腺相关病毒介导的HGFK1对大鼠肝细胞癌的治疗作用研究

    顾春荣; 郭跃武; 赵晖; 孙元珏; 姚阳; 沈赞; 林李家宓

    2009-01-01

    -angiogenesis molecule than angiostatin. In this study, we observed the effects and mechanisms of HGFK1 gene on the HCC. Methods: A recombinant adeno-associated vires carrying the HGFK1 gene (rAAV-HGFK1) was constructed.HCC of rat was induced by McA-RH7777. rAAV-HGFK1 was used to treat the rat, median survival time and metastasis rate were observed. Results: Ten days after tumor cell inoculation, surgery were performed to confirm the tumor formation, PBS, rAAV-EGFP or rAAV-HGFK1 was injected directly into the tumor nodule followed by portal vein injection. Results from our study demonstrated that rAAV-HGFK1 treatment significantly prolonged the median survival time of the HCC bearing rats from 30 days (PBS and rAAV-EGFP groups) to 49 days (rAAV-HGFK1 group). More importantly rAAV-HGFK1 inhibited tumor growth and completely prevented liver, lung and peritoneal metastasis. In the controlled PBS and AAV-EGFP group, liver and peritoneal metastasis rate were both 100%, and lung metastasis rate was 100% and 83%, respectively. While there was no metastasis found in treatment group, with only 33% of ascites happened. This was most possibly due to the primary tumor in liver but not due to the metastasis. Moreover, at a higher magnification (1000×), it was clear that the HGFK1 protein was expressed mainly in the cytoplasma of liver cells. In parallel, IHC staining of CD31 also demonstrated a significantly lower level of microvessel density (MVD) (6.21±1.6) in the liver tumor of the AAV-HGFK1 treatment group, as compared to the two control PBS and AAV-EGFP groups (25.1±2.1 and 26.8±2.5, respectively, P<0.01). HE staining showed that AAV-HGFK1 treatment induced large areas of necrosis in the tumor tissues, while minimal areas of necrosis were observed in the tumor tissue in the control groups. In addition, no toxicity appeared when high dosage (4.8× 1012 vg/rat) of rAAV-HGFK1 was administered in rats. Conclusion: Results from this study demonstrated that HGFK1 inhibited the growth and

  8. Combinatorial microRNA target predictions

    Krek, Azra; Grün, Dominic; Poy, Matthew N.;

    2005-01-01

    MicroRNAs are small noncoding RNAs that recognize and bind to partially complementary sites in the 3' untranslated regions of target genes in animals and, by unknown mechanisms, regulate protein production of the target transcript1, 2, 3. Different combinations of microRNAs are expressed in...... different cell types and may coordinately regulate cell-specific target genes. Here, we present PicTar, a computational method for identifying common targets of microRNAs. Statistical tests using genome-wide alignments of eight vertebrate genomes, PicTar's ability to specifically recover published micro......RNA targets, and experimental validation of seven predicted targets suggest that PicTar has an excellent success rate in predicting targets for single microRNAs and for combinations of microRNAs. We find that vertebrate microRNAs target, on average, roughly 200 transcripts each. Furthermore, our results...

  9. Seed microRNA Research

    MicroRNAs (miRNAs) are key regulatory molecules that play critical roles in gene expression. The biological functions of miRNAs are important for developmental processes in plants and animals. Little is known about the functions of miRNAs in seeds. To gain a better understand-ing of the regulation o...

  10. Enhanced viral production and virus-mediated mortality of bacterioplankton in a natural iron-fertilized bloom event above the Kerguelen Plateau

    Malits, A.; Christaki, U.; Obernosterer, I.; Weinbauer, M. G.

    2014-12-01

    Above the Kerguelen Plateau in the Southern Ocean natural iron fertilization sustains a large phytoplankton bloom over 3 months during austral summer. During the KEOPS1 project (KErguelen Ocean and Plateau compared Study1) we sampled this phytoplankton bloom during its declining phase along with the surrounding high-nutrient-low-chlorophyll (HNLC) waters to study the effect of natural iron fertilization on the role of viruses in the microbial food web. Bacterial and viral abundances were 1.7 and 2.1 times, respectively, higher within the bloom than in HNLC waters. Viral production and virus-mediated mortality of bacterioplankton were 4.1 and 4.9 times, respectively, higher in the bloom, while the fraction of infected cells (FIC) and the fraction of lysogenic cells (FLC) showed no significant differences between environments. The present study suggests viruses to be more important for bacterial mortality within the bloom and dominate over grazing of heterotrophic nanoflagellates (HNFs) during the late bloom phase. As a consequence, at least at a late bloom stage, viral lysis shunts part of the photosynthetically fixed carbon in iron-fertilized regions into the dissolved organic matter (DOM) pool with potentially less particulate organic carbon transferred to larger members of the food web or exported.

  11. Enhanced viral production and virus-mediated mortality of bacterioplankton in a natural iron-fertilized bloom event above the Kerguelen Plateau

    A. Malits

    2014-07-01

    Full Text Available Above the Kerguelen Plateau in the Southern Ocean natural iron fertilization sustains a large phytoplankton bloom over three months during austral summer. During the KEOPS1 project (KErguelen Ocean and Plateau compared Study1 we sampled this phytoplankton bloom during its declining phase along with the surrounding HNLC waters to study the effect of natural iron fertilization on the role of viruses in the microbial food web. Bacterial and viral abundances were 1.7 and 2.1 times, respectively, higher within the bloom than in HNLC waters. Viral production and virus-mediated mortality of bacterioplankton was 4.1 and 4.9 times, respectively, higher in the bloom, while the fraction of infected cells (FIC and the fraction of lysogenic cells (FLC showed no significant differences between environments. The present study suggests viruses to be more important for bacterial mortality within the bloom and dominate over protozoan grazing during the late bloom phase. As a consequence, at least at a late bloom stage, viral lysis shunts part of the photosynthetically fixed carbon in iron-fertilized regions into the dissolved organic matter (DOM pool with potentially less particulate organic carbon transfered to larger members of the food web or exported.

  12. microRNAs and microRNA Targets Involved in Alfalfa Stem Development

    To examine the possible involvement of microRNAs in alfalfa stem development, we hybridized 32P-labled total microRNAs purified from elongating and post-elongation stem internodes (ES and PES, respectively) of the alfalfa Clone 252 to a microRNA dot blot that contains a total of 70 reference anti-mi...

  13. microRNAs and Endometrial Pathophysiology.

    Chill, Henry H; Dior, Uri P; Kogan, Liron; Revel, Ariel

    2015-01-01

    Embryo implantation requires a reciprocal interaction between the blastocyst and endometrium and is associated with complex regulatory mechanisms. Since their discovery, microRNAs became prominent candidates providing missing links for many biological pathways. In recent years, microRNAs were implicated as one of the important players in regulation of various biological and physiological endometrial related processes. This chapter aims to present recent knowledge pertaining to the diverse aspects of microRNAs in the embryo-endometrial relationship. We will focus on the role of microRNAs in decidualization and their part in natural and stimulated cycles. Next, we will present recent studies deliberating the role of microRNAs in recurrent pregnancy loss and in the important phenomenon of recurrent implantation failure. Lastly, demonstrating an important aspect of embryo implantation and invasion, we will outline few microRNA related shared pathways of implantation and carcinogenesis. PMID:26662990

  14. Experimental identification of microRNA targets

    Ørom, Ulf Andersson; Lund, Anders H

    microRNAs are small RNAs that regulate protein synthesis post-transcriptionally. Animal microRNAs recognize their targets by incomplete base pairing to sequence motifs most often present in the 3' untranslated region of their target mRNAs. This partial complementarity vastly expands the repertoire...... of potential targets and constitutes a problem for computational target prediction. Although computational analyses have shed light on important aspects of microRNA target recognition, several questions remain regarding how microRNAs can recognize and regulate their targets. Forward experimental...... approaches allow for an unbiased study of microRNA target recognition and may unveil novel, rare or uncommon target binding patterns. In this review we focus on animal microRNAs and the experimental approaches that have been described for identification of their targets....

  15. MicroRNA in the immune system, microRNA as an immune system

    Lu, Li-Fan; Liston, Adrian

    2009-01-01

    The advent of microRNA has potentially uncovered a new level of complexity to be considered for every biological process. Through the modulation of transcription and translation, microRNA alter the basal state of cells and the outcome of stimulatory events. The exact effect of the microRNA network and individual microRNA on cellular processes is only just starting to be dissected. In the immune system, microRNA appear to have a key role in the early differentiation and effector differentiatio...

  16. MicroRNA involvement in glioblastoma pathogenesis

    MicroRNAs are endogenously expressed regulatory noncoding RNAs. Altered expression levels of several microRNAs have been observed in glioblastomas. Functions and direct mRNA targets for these microRNAs have been relatively well studied over the last years. According to these data, it is now evident, that impairment of microRNA regulatory network is one of the key mechanisms in glioblastoma pathogenesis. MicroRNA deregulation is involved in processes such as cell proliferation, apoptosis, cell cycle regulation, invasion, glioma stem cell behavior, and angiogenesis. In this review, we summarize the current knowledge of miRNA functions in glioblastoma with an emphasis on its significance in glioblastoma oncogenic signaling and its potential to serve as a disease biomarker and a novel therapeutic target in oncology.

  17. Phenotypic MicroRNA Microarrays

    Veronica Soloveva; Michel Liuzzi; Jin Yeop Kim; Hi Chul Kim; Jin Yeong Heo; Yong-Jun Kwon

    2013-01-01

    Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the bio...

  18. MicroRNAs - Biology and clinical applications

    Kannan Ranganathan; Vaishnavi Sivasankar

    2014-01-01

    MicroRNAs are a highly conserved group of small, non-coding RNA molecules, which are 19-25 nucleotides in size. Previously thought to be evolutionary debris with no evident function, these small RNAs have been found to control gene expression primarily by silencing the gene. MicroRNAs are critical to cell physiology and development. They are also implicated in pathological processes such as autoimmune diseases, viral infections and carcinogenesis.

  19. MicroRNA Targets - How to predict?

    Das, Nabajit

    2012-01-01

    A number of web tools are available for the prediction and identification of target microRNAs (miRNAs). The choice, availability, validity and selection of an optimal yet appropriate tool are a challenge for the design of high throughput assays with promising miRNA targets. The current trends and challenges for target microRNAs (miRNAs) prediction, identification and selection is described in this review.

  20. 两种不同病毒载体携带靶向大鼠金属蛋白酶组织抑制因子(TIMP)-1小干扰RNA抗肝纤维化作用的比较%Comparison between the antifibrotic effects of adeno-associated virus and lentivirus carrying small interfering RNA of TIMP-1 in rat liver fibrosis

    马雪梅; 张群; 庞国进; 丛敏

    2013-01-01

    Objective To construct recombinant adeno-associated virus and lentivirus carrying siRNA of TIMP-1 and to investigate their antifibrotic effects on CCl4-induced liver fibrosis in rats.Methods One pair of siRNA which could effectively inhibit expression of the TIMP-1 gene in HSC-T6 was screened and cloned into AAV vector and lentiviral vector to construct the recombinant AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1.AAV/EGFP and Lenti/EGFP as negative control were also obtained.Fifty-eight male Wistar rats were randomly divided into six groups:control group (n =8),CCl4 group,AAV/EGFP,Lenti/EGFP,AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups (all n =10).After the administration of CCl4 for four weeks,liver samples were collected for the immunohistochemical staining and detection of TIMP-1 expression.Results Livers from the control rats showed normal lobular structure around vessels (HE and Masson staining).In contrast,livers from the model,AAV/EGFP and Lenti/EGFP groups showed severe fibrosis,including septal fibrosis,extensive bridging,and fatty degeneration.The expressions of TIMP-1 mRNA and protein were also elevated in the livers from these groups.Compared with the fibrosis model group,the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups showed good preservation of liver lobular architecture and only mild bridging fibrosis,accompanied by decreased expression of TIMP-1 mRNA and protein.Semi-quantitative analysis of the fibrosis stage indicated that most rats in the model,AAV/EGFP and Lenti/EGFP groups were of S3 and S4 (80%),while 20% of the rats were of S5.In contrast,most rats (90%) in the AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 groups were of stages S2 and S3,with only one rat of S4.There was no significant difference between these recombinant virus therapy groups.Conclusions Both AAV/siRNA-TIMP-1 and Lenti/siRNA-TIMP-1 can suppress the expression of TIMP-1 in rat fibrotic liver,playing an effective antifibrotic role in the rat liver.%目的 观察以腺相关病

  1. 9型重组腺相关病毒介导抗核转录因子-κB核酶基因体外转染大鼠心肌细胞及对核转录因子-κB活性的影响%Transfection of rats H9C2 cells with recombinant adeno-associated virus Serotype 9 mediated AntiNF-κB ribozyme in vitro and effects on nuclear factor-κB activity

    高霞; 马依彤; 杨毅宁; 向阳; 陈邦党; 刘芬

    2010-01-01

    Objective To evaluate the transfection efficiency using recombinant adeno-associated virus serotype 9 (rAAV9) mediated anti-nuclear factor-κB (NF) -κB ribozyme and enhanced green fluorescent protein (rAAV9-EGFP-R65) to rats H9C2 cells and the effect on NF-κB activity. Methods rAAV9EGFP-R65 was transfected into H9C2 ceils at multiplicities of infection ( MOI = 1 x 106 v. g./cell). EGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of EGFP positive cells was determined by flow cytometry. Alamar Blue assay was used to assess the proliferation of the transfected cells. H9C2 ceils were treated with tumor necrosis factor (TNF)-α, rAAV9-EGFP-R65 and PDTC. The DNA binding activity of NF-KF-κB was examined by electrophoretic mobility shift assay (EMSA). Results The cells began to exhibit EGFP expression one day after transfection. The fluorescence intensity was increased with the time of transfection. EGFP expression reached the maximum on the day 5, at the point of which the transduction efficiency was (32.27 + 3.19)%. Alamar Blue assay did not reveal significant difference in the absorbance between the transfected cells and the control cells. TNF-α could activate NF-κB, and rAAV9-EGFP-R65 and PDCT could efficiently decrease NF-κB activation in rats H9C2 cells. Conclusion rAAV9-EGFP-R65 can be stably and efficiently expressed in H9C2 cells without causing cell growth inhibition, rAAVg-EGFP-R65 can availably inhibit NF-κB activation in rats H9C2 cells in vitro.%目的 观察9型重组腺相关病毒(rAAV9)介导抗核转录因子-κB(NF-κB)核酶基因(rAAV9-ECFP-R65)对大鼠心肌H9C2细胞的转染及对NF-κB活性的影响.方法 rAAV9-EGFP-R65按转染复数(MOI)1×106v.g./cell转染H9C2细胞,在倒置荧光显微镜下观察增强型绿色荧光蛋白(EGFP)阳性表达,采用流式细胞仪检测转染效率.Alamar Blue法检测rAAV9-EGFP-R65对H9C2细胞增殖影响.肿瘤坏死因子-α(TNF-α)、rAAV9

  2. Computational methods for the identification of microRNA targets

    Dai, Yang; ZHOU, Xiaofeng

    2010-01-01

    MicroRNAs are pivotal regulators of development and cellular homeostasis. They act as post-transcriptional regulators, which control the stability and translation efficiency of their target mRNAs. The prediction of microRNA targets and detection of microRNA-mRNA regulatory modules (MRMs) are crucial components for understanding of microRNA functions. Numerous computational methods for microRNA target prediction have been developed. Computationally-predicted targets have been recently used in ...

  3. Integrated analysis of microRNA and mRNA expression: Adding biological significance to microRNA target predictions

    M. van Iterson (Mat); S. Bervoets (Sander); E.J. de Meijer (Emile); H.P. Buermans (Henk); P.A.C. 't Hoen (Peter); R.X. Menezes (Renée); J.M. Boer (Judith)

    2013-01-01

    textabstractCurrent microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, mi

  4. Resveratrol, MicroRNAs, Inflammation, and Cancer

    Esmerina Tili

    2011-01-01

    Full Text Available MicroRNAs are short noncoding RNAs that regulate the expression of many target genes posttranscriptionally and are thus implicated in a wide array of cellular and developmental processes. The expression of miR-155 or miR-21 is upregulated during the course of the inflammatory response, but these microRNAs are also considered oncogenes due to their upregulation of expression in several types of tumors. Furthermore, it is now well established that inflammation is associated with the induction or the aggravation of nearly 25% of cancers. Therefore, the above microRNAs are thought to link inflammation and cancer. Recently, resveratrol (trans-3,4′,5-trihydroxystilbene, a natural polyphenol with antioxidant, anti-inflammatory, and anticancer properties, currently at the stage of preclinical studies for human cancer prevention, has been shown to induce the expression of miR-663, a tumor-suppressor and anti-inflammatory microRNA, while downregulating miR-155 and miR-21. In this paper we will discuss how the use of resveratrol in therapeutics may benefit from the preanalyses on the status of expression of miR-155 or miR-21 as well as of TGFβ1. In addition, we will discuss how resveratrol activity might possibly be enhanced by simultaneously manipulating the levels of its key target microRNAs, such as miR-663.

  5. microRNAs in mycobacterial disease: friend or foe?

    Mehta, Manali D.; Liu, Philip T.

    2014-01-01

    As the role of microRNA in all aspects of biology continues to be unraveled, the interplay between microRNAs and human disease is becoming clearer. It should come of no surprise that microRNAs play a major part in the outcome of infectious diseases, since early work has implicated microRNAs as regulators of the immune response. Here, we provide a review on how microRNAs influence the course of mycobacterial infections, which cause two of humanity’s most ancient infectious diseases: tuberculos...

  6. MicroRNA Expression Profiling of the Porcine Developing Brain

    Podolska, Agnieszka; Kaczkowski, Bogumil; Busk, Peter Kamp; Søkilde, Rolf; Litman, Thomas; Fredholm, Merete; Cirera, Susanna

    2011-01-01

    MicroRNAs are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in the control of developmental and physiological processes. In particular, the developing brain contains an impressive diversity of microRNAs. Most microRNA...... the growth curve when compared to humans. Considering these similarities, studies examining microRNA expression during porcine brain development could potentially be used to predict the expression profile and role of microRNAs in the human brain....

  7. MicroRNA and Metastasis.

    Ma, L

    2016-01-01

    Noncoding RNAs are important regulatory molecules of cellular processes. MicroRNAs (miRNAs) are small noncoding RNAs that bind to complementary sequences in the 3' untranslated region of target mRNAs, leading to degradation of the target mRNAs and/or inhibition of their translation. Some miRNAs are essential for normal animal development; however, many other miRNAs are dispensable for development but play a critical role in pathological conditions, including tumorigenesis and metastasis. miRNA genes often reside at fragile chromosome sites and are deregulated in cancer. Some miRNAs function as oncogenes or tumor suppressors, collectively termed "oncomirs." Specific metastasis-regulating miRNAs, collectively termed "metastamirs," govern molecular processes and pathways in malignant progression in either a tumor cell-autonomous or a cell-nonautonomous manner. Recently, exosome-transferred miRNAs have emerged as mediators of the tumor-stroma cross talk. In this chapter, we focus on the functions, mechanisms of action, and therapeutic potential of miRNAs, particularly oncomirs and metastamirs. PMID:27613133

  8. Novel role of microRNAs in prostate cancer

    YU Jun-jie; XIA Shu-jie

    2013-01-01

    Objective To discuss the novel biomarkers of microRNAs in prostate cancer.Data sources The literatures about microRNAs and prostate cancer cited in this review were obtained mainly from Pubmed published in English from 2004 to 2012.Study selection Original articles regarding the novel role of microRNAs in prostate cancer were selected.Results MicroRNAs play an important role in prostate cancer such as cell differentiation,proliferation,apoptosis,and invasion.Especially microRNAs correlate with prostate cancer cell epithelial-mesenchymal transition (EMT),cancer stem cells (CSCs),drug sensitivity,cancer microenvironment,energy metabolism,androgen independence transformation,and diagnosis prediction.Conclusions MicroRNAs are involved in various aspects of prostate cancer biology.The role of microRNA in the initiation and development of prostate cancer deserves further study.

  9. microRNAs in mycobacterial disease: friend or foe?

    Manali D Mehta

    2014-07-01

    Full Text Available As the role of microRNA in all aspects of biology continues to be unraveled, the interplay between microRNAs and human disease is becoming clearer. It should come of no surprise that microRNAs play a major part in the outcome of infectious diseases, since early work has implicated microRNAs as regulators of the immune response. Here, we provide a review on how microRNAs influence the course of mycobacterial infections, which cause two of humanity’s most ancient infectious diseases: tuberculosis and leprosy. Evidence derived from profiling and functional experiments suggests that regulation of specific microRNAs during infection can either enhance the immune response or facilitate pathogen immune evasion. Now, it remains to be seen if the manipulation of host cell microRNA profiles can be an opportunity for therapeutic intervention for these difficult-to-treat diseases.

  10. Targeting microRNAs in heart failure.

    Duygu, Burcu; de Windt, Leon J; da Costa Martins, Paula A

    2016-02-01

    MicroRNAs play pivotal roles in cardiac disease, and their therapeutic modulation raises exciting and unique opportunities, as well as challenges in the path toward clinical development and implementation. In this review, we provide a detailed overview of recent studies highlighting the important role of microRNAs in heart failure (HF) and the potential use of microRNA-based technology for diagnosis, prevention, and treatment of HF. We will focus on the strategies presently used for microRNA-based therapy by discussing their use and drawbacks, as well as the challenges and future directions for their development in the context of human HF. PMID:26119078

  11. Adeno-associated virus–targeted disruption of the CFTR gene in cloned ferrets

    Sun, Xingshen; Yan, Ziying; Yi, Yaling; Li, Ziyi; Lei, Diana; Rogers, Christopher S.; Chen, Juan; Zhang, Yulong; Welsh, Michael J.; Leno, Gregory H.; Engelhardt, John F.

    2008-01-01

    Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model...

  12. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates

    Marrero Luis

    2003-09-01

    Full Text Available Abstract Background Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes. Methods Recombinant AAV2 carrying green fluorescent protein (GFP was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor. Results Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year. Conclusions Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.

  13. Generation of Insulin-Producing Human Mesenchymal Stem Cells Using Recombinant Adeno-Associated Virus

    Kim, Jeong Hwan; Park, Si-Nae; Suh, Hwal

    2007-01-01

    The purpose of current experiment is the generation of insulin-producing human mesenchymal stem cells as therapeutic source for the cure of type 1 diabetes. Type 1 diabetes is generally caused by insulin deficiency accompanied by the destruction of islet β-cells. In various trials for the treatment of type 1 diabetes, cell-based gene therapy using stem cells is considered as one of the most useful candidate for the treatment. In this experiment, human mesenchymal stem cells were transduced wi...

  14. Engineered Adeno-Associated Viral Vectors for Gene Therapy in the Retina

    Byrne, Leah

    2011-01-01

    Inherited retinal degenerations are genetically heterogeneous conditions affecting roughly 1:3000 people and are characterized by the loss of photoreceptors. Progressive retinal degenerative disease is the leading cause of vision loss in industrialized countries, and is the result of a wide range of mutations, mostly in rod-specific transcripts. Over 140 disease-causing genes have been identified to date. As the genetic mechanisms underlying inherited forms of retinal degeneration are identif...

  15. Gene replacement therapies for Duchenne muscular dystrophy using adeno-associated viral vectors

    Seto, Jane T.; Ramos, Julian N.; Muir, Lindsey; Jeffrey S. Chamberlain; Odom, Guy L.

    2012-01-01

    The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise...

  16. Restriction Factors Against Recombinant Adeno-associated Virus Vectormediated Gene Transfer in Dystrophin-deficient Muscles.

    Dupont, Jean-Baptiste

    2016-01-01

    Despite the unprecedented beneficial effects of rAAV gene therapy in animal models of Duchenne muscular dystrophy (DMD), the need to inject large amounts of vector in vivo to improve phenotype raises obvious biosafety concerns. While rAAV vectors generally exhibit a good safety profile, specific pathological phenotypes such as those observed in dystrophin-deficient muscles may promote immunotoxic/genotoxic effects. Increasing the therapeutic index of rAAV in DMD muscles by reducing the effective dose could be a pivotal means of ensuring efficient clinical translation. This requires a comprehensive understanding of the rAAV transduction process, which is almost always studied in non-pathological tissues or in vitro. In this review, we focus on the molecular fate of rAAV after injection, and how the individual stages of transduction could be affected in the context of DMD. PMID:27121109

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

    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.

  18. A role for adeno-associated viral vectors in gene therapy

    Renata dos Santos Coura; Nance Beyer Nardi

    2008-01-01

    Gene therapy constitutes a therapeutic intervention based on modification of the genetic material of living cells, by correcting genetic defects or overexpressing therapeutic proteins. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy. Recent advances on the development of gene delivery systems, particularly on viral vectors engineering and improved gene regulatory systems, ha...

  19. Systemic delivery of genes to striated muscles using adeno-associated viral vectors

    Gregorevic, Paul; Blankinship, Michael J; Allen, James M.; Robert W Crawford; Meuse, Leonard; Miller, Daniel G.; Russell, David W.; Jeffrey S. Chamberlain

    2004-01-01

    A major obstacle limiting gene therapy for diseases of the heart and skeletal muscles is an inability to deliver genes systemically to muscles of an adult organism. Systemic gene transfer to striated muscles is hampered by the vascular endothelium, which represents a barrier to distribution of vectors via the circulation. Here we show the first evidence of widespread transduction of both cardiac and skeletal muscles in an adult mammal, after a single intravenous administration of recombinant ...

  20. Studying microRNAs in lymphoma

    Kluiver, Joost; Slezak-Prochazka, Izabella; van den Berg, Anke

    2013-01-01

    MicroRNAs (miRNAs) play important roles in development, differentiation, homeostasis, and also in diseases such as lymphoma. This chapter describes methods to study the role of miRNAs in lymphoma. First, we describe a multiplex RT reaction followed by qPCR that can be used to determine differential

  1. Beginning to understand microRNA function

    Tingting Du; Phillip D Zamore

    2007-01-01

    @@ MicroRNAs (miRNAs) are -22 nt small RNAs expressed by plants, animals, viruses and at least one unicellular organism, the green alga, Chlamydomonas reinhardtii [1]. Most miRNAs are transcribed as primary miRNAs (pri-miRNAs) by RNA polymerase Ⅱ, although a few are transcribed by RNA polymerase Ⅲ.

  2. Targeting of microRNAs for therapeutics

    Stenvang, Jan; Lindow, Morten; Kauppinen, Sakari

    miRNAs (microRNAs) comprise a class of small endogenous non-coding RNAs that post-transcriptionally repress gene expression by base-pairing with their target mRNAs. Recent evidence has shown that miRNAs play important roles in a wide variety of human diseases, such as viral infections, cancer and...

  3. MicroRNAs in the Hypothalamus

    Meister, Björn; Herzer, Silke; Silahtaroglu, Asli

    2013-01-01

    MicroRNAs (miRNAs) are short (∼22 nucleotides) non-coding ribonucleic acid (RNA) molecules that negatively regulate the expression of protein-coding genes. Posttranscriptional silencing of target genes by miRNA is initiated by binding to the 3'-untranslated regions of target mRNAs, resulting in...

  4. MicroRNA biomarkers in glioblastoma

    Hermansen, Simon Kjær; Kristensen, Bjarne Winther

    2013-01-01

    Recent research suggests that deregulation of microRNAs (miRNAs) is involved in initiation and progression of many cancers, including gliomas and that miRNAs hold great potential as future diagnostic and therapeutic tools in cancer. MiRNAs are a class of short non-coding RNA sequences (18...

  5. MicroRNA: Mechanism of Gene Regulation

    MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts through activation of a specific cellular pathway. The small RNA classified as miR are short sequences of 18-26 nucleotide long, encoded by nuclear genes with distinctive...

  6. MicroRNA regulation of Autophagy

    Frankel, Lisa B; Lund, Anders H

    2012-01-01

    progress has recently contributed to our understanding of the molecular mechanisms of the autophagy machinery, yet several gaps remain in our knowledge of this process. The discovery of microRNAs (miRNAs) established a new paradigm of post-transcriptional gene regulation and during the past decade these...

  7. microRNAs in CNS disorders

    Kocerha, Jannet; Kauppinen, Sakari; Wahlestedt, Claes

    2009-01-01

    In recent years, there has been a shift in the conventional paradigms for transcriptional and translational regulation as extensive sequencing efforts have yielded new insights into the landscape of the human genome and transcriptome. Hundreds of non-coding regulatory RNA molecules called microRNAs...

  8. MicroRNAs in cardiac arrhythmia

    Hedley, Paula L; Carlsen, Anting L; Christiansen, Kasper M; Kanters, Jørgen K.; Behr, Elijah R; Corfield, Valerie A; Christiansen, Michael

    2014-01-01

    LQTS-causing mutations have been identified in 13 genes worldwide. Despite this, the genetic cause of 30-50% of LQTS is presently unknown. MicroRNAs (miRNAs) are small (∼ 22 nucleotides) noncoding RNAs which post-transcriptionally regulate gene expression by binding complementary sequences within...

  9. MicroRNAs in cardiovascular disease

    Elton, Terry S.; Khan, Mahmood; Terentyev, Dmitry

    2011-01-01

    Rapid and accurate diagnosis of heart attacks—and the assessment of damage—are critical for improving coronary care. Mature microRNAs (miRNAs) are abundant, easily measured, and relatively stable in blood plasma. If they prove indicative of disease states, miRNAs measured from peripheral blood may be a particularly attractive source for routine clinical assessments.

  10. Profile of microRNA in Blood Plasma of Healthy Humans.

    Skurnikov, M Yu; Makarova, Yu A; Knyazev, E N; Fomicheva, K A; Nyushko, K M; Saribekyan, E K; Alekseev, B Ya; Kaprin, A D

    2016-03-01

    Analysis of the plasma microRNA profile can be used for the diagnosis of various pathological and physiological conditions. Complete microRNA microprofiling is an extremely important task. Here we used microarray analysis allowing measurement of the expression of 2500 microRNA (MirBase, version 20). About 10% known microRNA were found in the plasma. Most of the detected microRNA (69 microRNA; ~30%) were encoded by mirtrons. PMID:27021098

  11. MicroRNA signature of the human developing pancreas

    Correa-Medina Mayrin

    2010-09-01

    Full Text Available Abstract Background MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. Results The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga, was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase altgorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. Conclusions We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in

  12. Dual Role of MicroRNAs in NAFLD

    Valerio Nobili

    2013-04-01

    Full Text Available MicroRNAs are important post-transcriptional regulators in different pathophysiological processes. They typically affect the mRNA stability or translation finally leading to the repression of target gene expression. Notably, it is thought that microRNAs are crucial for regulating gene expression during metabolic-related disorders, such as nonalcoholic fatty liver disease (NAFLD. Several studies identify specific microRNA expression profiles associated to different histological features of NAFLD, both in animal models and in patients. Therefore, specific assortments of certain microRNAs could have enormous diagnostic potentiality. In addition, microRNAs have also emerged as possible therapeutic targets for the treatment of NAFLD-related liver damage. In this review, we discuss the experimental evidence about microRNAs both as potential non-invasive early diagnostic markers and as novel therapeutic targets in NAFLD and its more severe liver complications.

  13. Pervasive roles of microRNAs in cardiovascular biology

    Small, Eric M.; Olson, Eric N.

    2011-01-01

    First recognized as regulators of development in worms and fruitflies, microRNAs are emerging as pivotal modulators of mammalian cardiovascular development and disease. Individual microRNAs modulate the expression of collections of messenger RNA targets that often have related functions, thereby governing complex biological processes. The wide-ranging functions of microRNAs in the cardiovascular system have provided new perspectives on disease mechanisms and have revealed intriguing therapeut...

  14. MicroRNA function in NK cell biology

    Beaulieu, AM; Bezman, NA; Lee, JE; Matloubian, M; Sun, JC; Lanier, LL

    2013-01-01

    The important role of microRNAs in directing immune responses has become increasingly clear. Here, we highlight discoveries uncovering the role of specific microRNAs in regulating the development and function of natural killer (NK) cells. Furthermore, we discuss the impact of NK cells on the entire immune system during global and specific microRNA ablation in the settings of inflammation, infection, and immune dysregulation. © 2013 John Wiley & Sons A/S.

  15. MicroRNA from tuberculosis RNA: A bioinformatics study

    Wiwanitkit, Somsri; Wiwanitkit, Viroj

    2012-01-01

    The role of microRNA in the pathogenesis of pulmonary tuberculosis is the interesting topic in chest medicine at present. Recently, it was proposed that the microRNA can be a useful biomarker for monitoring of pulmonary tuberculosis and might be the important part in pathogenesis of disease. Here, the authors perform a bioinformatics study to assess the microRNA within known tuberculosis RNA. The microRNA part can be detected and this can be important key information in further study of the p...

  16. [Progress in microRNAs associated with major avian viruses].

    Man, Chaolai; Mu, Weitao; Zhao, Dongxue; Chang, Yang

    2015-09-01

    Recently, avian viral diseases have become one of the main models to study mechanisms of viral infections and pathogenesis. The study of regulatory relationships and mechanisms between viruses and microRNAs has also become the focus. In this review, we briefly summarize the general situations of microRNAs encoded by avian herpesviruses. Also, we analyze the regulatory relationships between tumorigenicity of avian herpesviruses and microRNAs. Additionally, the possible applications for prevention and treatment of viral diseases (such as infectious bursal disease, avian influenza and avian leucosis) using the regulatory mechanisms of microRNAs are also discussed. PMID:26955707

  17. High-throughput assessment of microRNA activity and function using microRNA sensor and decoy libraries

    Mullokandov, Gavriel; Baccarini, Alessia; Ruzo, Albert; Jayaprakash, Anitha D.; Tung, Navpreet; Israelow, Benjamin; Evans, Matthew J.; Sachidanandam, Ravi; Brown, Brian D.

    2012-01-01

    We introduce two large-scale resources for functional analysis of microRNA—a decoy/sponge library for inhibiting microRNA function and a sensor library for monitoring microRNA activity. To take advantage of the sensor library, we developed a high-throughput assay called Sensor-seq, which permits the activity of hundreds of microRNAs to be quantified simultaneously. Using this approach, we show that only the most abundant microRNAs within a cell mediate significant target suppression. Over 60%...

  18. MicroRNA Methylation in Colorectal Cancer.

    Kaur, Sippy; Lotsari-Salomaa, Johanna E; Seppänen-Kaijansinkko, Riitta; Peltomäki, Päivi

    2016-01-01

    Epigenetic alterations such as DNA methylation, histone modifications and non-coding RNA (including microRNA) associated gene silencing have been identified as a major characteristic in human cancers. These alterations may occur more frequently than genetic mutations and play a key role in silencing tumor suppressor genes or activating oncogenes, thereby affecting multiple cellular processes. In recent years, studies have shown that microRNAs, that act as posttranscriptional regulators of gene expression are frequently deregulated in colorectal cancer (CRC), via aberrant DNA methylation. Over the past decade, technological advances have revolutionized the field of epigenetics and have led to the identification of numerous epigenetically dysregulated miRNAs in CRC, which are regulated by CpG island hypermethylation and DNA hypomethylation. In addition, aberrant DNA methylation of miRNA genes holds a great promise in several clinical applications such as biomarkers for early screening, prognosis, and therapeutic applications in CRC. PMID:27573897

  19. MicroRNAs, epigenetics and disease

    Silahtaroglu, Asli; Stenvang, Jan

    2010-01-01

    Epigenetics is defined as the heritable chances that affect gene expression without changing the DNA sequence. Epigenetic regulation of gene expression can be through different mechanisms such as DNA methylation, histone modifications and nucleosome positioning. MicroRNAs are short RNA molecules...... which do not code for a protein but have a role in post-transcriptional silencing of multiple target genes by binding to their 3' UTRs (untranslated regions). Both epigenetic mechanisms, such as DNA methylation and histone modifications, and the microRNAs are crucial for normal differentiation......, development and maintenance of tissue-specific gene expression. These mechanisms also explain how cells with the same DNA content can differentiate into cells with different functions. Changes in epigenetic processes can lead to changes in gene function, cancer formation and progression, as well as other...

  20. MicroRNAs in Common Human Diseases

    Li, Yu; Kowdley, Kris V.

    2012-01-01

    MicroRNAs (miRNAs) are a class of short non-coding RNA molecules that have attracted tremendous attention from the biological and biomedical research communities over the past decade. With over 1900 miRNAs discovered in humans to date, many of them have already been implicated in common human disorders. Facilitated by high-throughput genomics and bioinformatics in conjunction with traditional molecular biology techniques and animal models, miRNA research is now positioned to make the transiti...

  1. MicroRNAs in Pulmonary Arterial Hypertension

    Zhou, Guofei; Chen, Tianji; Raj, J. Usha

    2015-01-01

    Pulmonary arterial hypertension (PAH) is a devastating disease without effective treatment. Despite decades of research and the development of novel treatments, PAH remains a fatal disease, suggesting an urgent need for better understanding of the pathogenesis of PAH. Recent studies suggest that microRNAs (miRNAs) are dysregulated in patients with PAH and in experimental pulmonary hypertension. Furthermore, normalization of a few miRNAs is reported to inhibit experimental pulmonary hypertensi...

  2. MicroRNAs in Rice Innate Immunity

    Baldrich, Patricia; San Segundo, Blanca

    2016-01-01

    MicroRNAs (miRNAs) are short regulatory non-coding RNAs that guide gene silencing in most eukaryotes. They regulate gene expression by triggering sequence-specific cleavage or translational repression of target transcripts. Plant miRNAs are known to play important roles in a wide range of developmental processes. Increasing evidence also supports that the modulation of miRNA levels plays an important role in reprogramming plant responses to abiotic stress (drought, cold, salinity and nutrient...

  3. MicroRNA expression profiling of the porcine developing brain.

    Agnieszka Podolska

    Full Text Available BACKGROUND: MicroRNAs are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level and play an important role in the control of developmental and physiological processes. In particular, the developing brain contains an impressive diversity of microRNAs. Most microRNA expression profiling studies have been performed in human or rodents and relatively limited knowledge exists in other mammalian species. The domestic pig is considered to be an excellent, alternate, large mammal model for human-related neurological studies, due to its similarity in both brain development and the growth curve when compared to humans. Considering these similarities, studies examining microRNA expression during porcine brain development could potentially be used to predict the expression profile and role of microRNAs in the human brain. METHODOLOGY/PRINCIPAL FINDINGS: MicroRNA expression profiling by use of microRNA microarrays and qPCR was performed on the porcine developing brain. Our results show that microRNA expression is regulated in a developmentally stage-specific, as well as a tissue-specific manner. Numerous developmental stage or tissue-specific microRNAs including, miR-17, miR-18a, miR-29c, miR-106a, miR-135a and b, miR-221 and miR-222 were found by microarray analysis. Expression profiles of selected candidates were confirmed by qPCR. CONCLUSIONS/SIGNIFICANCE: The differential expression of specific microRNAs in fetal versus postnatal samples suggests that they likely play an important role in the regulation of developmental and physiological processes during brain development. The data presented here supports the notion that microRNAs act as post-transcriptional switches which may regulate gene expression when required.

  4. Many novel mammalian microRNA candidates identified by extensive cloning and RAKE analysis

    Berezikov, Eugene; van Tetering, Geert; Verheul, Mark; van de Belt, Jose; van Laake, Linda; Vos, Joost; Verloop, Robert; van de Wetering, Marc; Guryev, Victor; Takada, Shuji; van Zonneveld, Anton Jan; Mano, Hiroyuki; Plasterk, Ronald; Cuppen, Edwin

    2006-01-01

    MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA cand

  5. Dynamics of microRNAs in bull spermatozoa

    Govindaraju Aruna

    2012-09-01

    Full Text Available Abstract Background MicroRNAs are small non-coding RNAs that regulate gene expression and thus play important roles in mammalian development. However, the comprehensive lists of microRNAs, as well as, molecular mechanisms by which microRNAs regulate gene expression during gamete and embryo development are poorly defined. The objectives of this study were to determine microRNAs in bull sperm and predict their functions. Methods To accomplish our objectives we isolated miRNAs from sperm of high and low fertility bulls, conducted microRNA microarray experiments and validated expression of a panel of microRNAs using real time RT-PCR. Bioinformatic approaches were carried out to identify regulated targets. Results We demonstrated that an abundance of microRNAs were present in bovine spermatozoa, however, only seven were differentially expressed; hsa-aga-3155, -8197, -6727, -11796, -14189, -6125, -13659. The abundance of miRNAs in the spermatozoa and the differential expression in sperm from high vs. low fertility bulls suggests that the miRNAs possibly play important functions in the regulating mechanisms of bovine spermatozoa. Conclusion Identification of specific microRNAs expressed in spermatozoa of bulls with different fertility phenotypes will help better understand mammalian gametogenesis and early development.

  6. MicroRNA-126 inhibits the proliferation of lung cancer cell line A549

    Xun Yang; Bei-Bei Chen; Ming-Hua Zhang; Xin-Rong Wang

    2015-01-01

    Objective:To study the role of microRNA-126 in the development of lung cancer.Methods:The biological function of microRNA-126 was detected using EdU assay and CCK-8 assay;the target gene of microRNA-126 was analyzed using real time RT-PCR and Western blot assay.Results: In A549 cell line, overexpression of microRNA-126 inhibits the proliferation rate; VEGF is the target gene of microRNA-126; microRNA-126 exerts its function via regulating VEGF protein level.Conclusions: microRNA-126 inhibits the proliferation in A549 cell line.

  7. Regulation of cardiac microRNAs by serum response factor

    Wei Jeanne Y

    2011-02-01

    Full Text Available Abstract Serum response factor (SRF regulates certain microRNAs that play a role in cardiac and skeletal muscle development. However, the role of SRF in the regulation of microRNA expression and microRNA biogenesis in cardiac hypertrophy has not been well established. In this report, we employed two distinct transgenic mouse models to study the impact of SRF on cardiac microRNA expression and microRNA biogenesis. Cardiac-specific overexpression of SRF (SRF-Tg led to altered expression of a number of microRNAs. Interestingly, downregulation of miR-1, miR-133a and upregulation of miR-21 occurred by 7 days of age in these mice, long before the onset of cardiac hypertrophy, suggesting that SRF overexpression impacted the expression of microRNAs which contribute to cardiac hypertrophy. Reducing cardiac SRF level using the antisense-SRF transgenic approach (Anti-SRF-Tg resulted in the expression of miR-1, miR-133a and miR-21 in the opposite direction. Furthermore, we observed that SRF regulates microRNA biogenesis, specifically the transcription of pri-microRNA, thereby affecting the mature microRNA level. The mir-21 promoter sequence is conserved among mouse, rat and human; one SRF binding site was found to be in the mir-21 proximal promoter region of all three species. The mir-21 gene is regulated by SRF and its cofactors, including myocardin and p49/Strap. Our study demonstrates that the downregulation of miR-1, miR-133a, and upregulation of miR-21 can be reversed by one single upstream regulator, SRF. These results may help to develop novel therapeutic interventions targeting microRNA biogenesis.

  8. MicroRNAs in tumor stem cells

    Xiaochen Hu; Junqiang Yang; Ruijie Yang; Ruina Yang; Xinshuai Wang; Shegan Gao

    2015-01-01

    MicroRNAs (miRNAs) are a class of non-coding RNAs that are believed to have a significant role in tumori-genesis and cancer metastasis. Cancer stem cel s play a major role in tumor recurrence, metastasis, and drug resistance. Research has shown that miRNAs can promote or inhibit the stemness of cancer stem cel s and regulate the dif erentiation and self-renewal of cancer stem cel s. In this article, the phenotype and regulatory mechanisms of miRNAs in cancer stem cel s wil be described, together with an explanation of their potential role in tumor diagnosis and treatment.

  9. MicroRNA mimicry blocks pulmonary fibrosis

    Montgomery, Rusty L.; Yu, Guoying; Latimer, Paul A.; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

    2014-01-01

    Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of ...

  10. MicroRNAs that target RGS5

    Amir Banaei-Esfahani; Hamidreza Moazzeni; Pooya Naseri Nosar; Sadaf Amin; Ehsan Arefian; Masoud Soleimani; Shahin Yazdani; Elahe Elahi

    2015-01-01

    Objective(s):An earlier meta-analysis on gene expression data derived from four microarray, two cDNA library, and one SAGE experiment had identified RGS5 as one of only ten non-housekeeping genes that were reported to be expressed in human trabecular meshwork (TM) cells by all studies. RGS5 encodes regulator of G-protein signaling-5. The TM tissue is the route of aqueous fluid outflow, and is relevant to the pathology of glaucoma. MicroRNAs constitute the most recently identified components o...

  11. MicroRNA gene expression in malignant lymphoproliferative disorders

    XU Wei; LI Jian-yong

    2007-01-01

    Objective To review the recent studies about microRNAs and advances in malignant lymphoproliferative disorders.Data sources Published articles (2001-2006) about microRNAs and malignant iymphoproliferative disorders were selected using MEDLINE.Study selection After independent review by two observers, 43 of 421 originally identified articles were selected that specifically addressed the stated purpose.Results Two observers independently assessed studies using explicit methodological criteria for evaluating microRNAs in malignant lymphoproliferative disorders. Recent work has revealed a class of small noncoding RNA species,microRNAs, which affect various biological processes. MicroRNAs inhibit the expression of protein encoding genes at the posttranscriptional level in a variety of eukaryotic organisms. In this review, we focused on the biogenetic pathways of microRNAs (miR-15a, miR-16-1, miR-155, miR-17-92 cluster, miR-142) and discussed the implications for human malignant lymphoproliferative disorders.Conclusions microRNAs are involved in tumorigenesis and mediate gene regulation as a fundamental genetic program at the posttranscriptional level. Further study of microRNAs may lead to novel concepts in the diagnosis and treatment of malignant lymphoproliferative disorders.

  12. Bio-barcode gel assay for microRNA

    Lee, Hyojin; Park, Jeong-Eun; Nam, Jwa-Min

    2014-02-01

    MicroRNA has been identified as a potential biomarker because expression level of microRNA is correlated with various cancers. Its detection at low concentrations would be highly beneficial for cancer diagnosis. Here, we develop a new type of a DNA-modified gold nanoparticle-based bio-barcode assay that uses a conventional gel electrophoresis platform and potassium cyanide chemistry and show this assay can detect microRNA at aM levels without enzymatic amplification. It is also shown that single-base-mismatched microRNA can be differentiated from perfectly matched microRNA and the multiplexed detection of various combinations of microRNA sequences is possible with this approach. Finally, differently expressed microRNA levels are selectively detected from cancer cells using the bio-barcode gel assay, and the results are compared with conventional polymerase chain reaction-based results. The method and results shown herein pave the way for practical use of a conventional gel electrophoresis for detecting biomolecules of interest even at aM level without polymerase chain reaction amplification.

  13. MicroRNAs in sensorineural diseases of the ear

    Kathy eUshakov

    2013-12-01

    Full Text Available Non-coding microRNAs have a fundamental role in gene regulation and expression in almost every multicellular organism. Only discovered in the last decade, microRNAs are already known to play a leading role in many aspects of disease. In the vertebrate inner ear, microRNAs are essential for controlling development and survival of hair cells. Moreover, dysregulation of microRNAs has been implicated in sensorineural hearing impairment, as well as in other ear diseases such as cholesteatomas, vestibular schwannomas and otitis media. Due to the inaccessibility of the ear in humans, animal models have provided the optimal tools to study microRNA expression and function, in particular mice and zebrafish. A major focus of current research has been to discover the targets of the microRNAs expressed in the inner ear, in order to determine the regulatory pathways of the auditory and vestibular systems. The potential for microRNA manipulation in development of therapeutic tools for hearing impairment is as yet unexplored, paving the way for future work in the field.

  14. MicroRNA expression profiles associated with pancreatic adenocarcinoma and ampullary adenocarcinoma

    Schultz, Nicolai A; Werner, Jens; Willenbrock, Hanni;

    2012-01-01

    MicroRNAs have potential as diagnostic cancer biomarkers. The aim of this study was (1) to define microRNA expression patterns in formalin-fixed parafin-embedded tissue from pancreatic ductal adenocarcinoma, ampullary adenocarcinoma, normal pancreas and chronic pancreatitis without using micro......, normal pancreas and duodenal adenocarcinoma. In all, 43 microRNAs had higher and 41 microRNAs reduced expression in pancreatic cancer compared with normal pancreas. In all, 32 microRNAs were differently expressed in pancreatic adenocarcinoma compared with chronic pancreatitis (17 higher; 15 reduced......-dissection and (2) to discover new diagnostic microRNAs and combinations of microRNAs in cancer tissue. The expression of 664 microRNAs in tissue from 170 pancreatic adenocarcinomas and 107 ampullary adenocarcinomas were analyzed using a commercial microRNA assay. Results were compared with chronic pancreatitis...

  15. MicroRNAs in rheumatoid arthritis.

    Salehi, Eisa; Eftekhari, Rahil; Oraei, Mona; Gharib, Alvand; Bidad, Katayoon

    2015-04-01

    The role of genetic and epigenetic factors in the development of rheumatic diseases has been an interesting field of research over the past decades all around the world. Research on the role of microRNAs (miRNAs) in rheumatoid arthritis (RA) has been active and ongoing, and investigations have attempted to use miRNAs as biomarkers in disease diagnosis, prognosis, and treatment. This review focuses on experimental researches in the field of miRNAs and RA to present the data available up to this date and includes researches searched by keywords "microRNA" and "rheumatoid arthritis" in PubMed from 2008 to January 2015. All references were also searched for related papers. miRNAs are shown to act as proinflammatory or anti-inflammatory agents in diverse cell types, and their role seems to be regulatory in most instances. Researchers have evaluated miRNAs in patients compared to controls or have investigated their role by overexpressing or silencing them. Multiple targets have been identified in vivo, in vitro, or in silico, and the researches still continue to show their efficacy in clinical settings. PMID:25736037

  16. MicroRNA and Brain Tumors

    2007-01-01

    MicroRNAs (miRNAs) were first described in 1993 by Lee and colleagues, and the term microRNA was only introduced in 2001 in a set of three articles in Science[1]. One of the biggest surprises in the past few years has been the emergence of miRNAs as a major new class of gene expression regulators. Recent studies suggest that miRNA alterations are involved in the initiation and progression of human cancer. The brain tumor,glioblastoma multiforme, is the most malignant and deadly form of gliomas.The prognosis is poor and the median survival with combined radiotherapy and chemotherapy is only 14.6 months. With the discovery of miRNA, the miRNA profiles may become useful biomarkers for brain tumor diagnostics,and miRNA therapy could be a powerful tool for brain tumor prevention and therapeutics. This review outlines the background of miRNA and its expression and therapeutic potential for brain tumors.

  17. Evaluation of microRNA alignment techniques.

    Ziemann, Mark; Kaspi, Antony; El-Osta, Assam

    2016-08-01

    Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing. PMID:27284164

  18. Identification of Loop Nucleotide Polymorphisms Affecting MicroRNA Processing and Function

    Xiong, Xiaoxing; Kang, Xianhui; Zheng, Yueying; Yue, Sibiao; Zhu, Shengmei

    2013-01-01

    MicroRNAs are short 21–22 nucleotide single strand RNAs that are involved in post-transcriptional regulation of gene expression. Most microRNAs are first transcribed as long primary microRNAs and then undergo a two step-wise sequential processing to yield single-stranded mature microRNAs. It has been suggested that the loop region of primary microRNAs plays an important role in regulating microRNA biogenesis and target recognition. However, despite the fact that several single nucleotide poly...

  19. MicroRNA Clusters in the Adult Mouse Heart: Age-Associated Changes

    Xiaomin Zhang; Gohar Azhar; Williams, Emmanuel D.; Rogers, Steven C.; Wei, Jeanne Y.

    2015-01-01

    The microRNAs and microRNA clusters have been implicated in normal cardiac development and also disease, including cardiac hypertrophy, cardiomyopathy, heart failure, and arrhythmias. Since a microRNA cluster has from two to dozens of microRNAs, the expression of a microRNA cluster could have a substantial impact on its target genes. In the present study, the configuration and distribution of microRNA clusters in the mouse genome were examined at various inter-microRNA distances. Three import...

  20. The locus of microRNA-10b

    Biagioni, Francesca; Bossel Ben-Moshe, Noa; Fontemaggi, Giulia; Yarden, Yosef; Domany, Eytan; Blandino, Giovanni

    2013-01-01

    Contemporary microRNA research has led to significant advances in our understanding of the process of tumorigenesis. MicroRNAs participate in different events of a cancer cell’s life, through their ability to target hundreds of putative transcripts involved in almost every cellular function, including cell cycle, apoptosis, and differentiation. The relevance of these small molecules is even more evident in light of the emerging linkage between their expression and both prognosis and clinical outcome of many types of human cancers. This identifies microRNAs as potential therapeutic modifiers of cancer phenotypes. From this perspective, we overview here the miR-10b locus and its involvement in cancer, focusing on its role in the establishment (miR-10b*) and spreading (miR-10b) of breast cancer. We conclude that targeting the locus of microRNA 10b holds great potential for cancer treatment. PMID:23839045

  1. Novel microRNA families expanded in the human genome

    Du, Zhi-Qiang; Yang, Cai-Xia; Rothschild, Max F.; Ross, Jason W.

    2013-01-01

    Background Most studies on the origin and evolution of microRNA in the human genome have been focused on its relationship with repetitive elements and segmental duplications. However, duplication events at a smaller scale (

  2. Detection and quantification of microRNA in cerebral microdialysate

    Bache, Søren; Rasmussen, Rune; Rossing, Maria; Hammer, Niels Risør; Juhler, Marianne; Friis-Hansen, Lennart; Nielsen, Finn Cilius; Møller, Kirsten

    2015-01-01

    BACKGROUND: Secondary brain injury accounts for a major part of the morbidity and mortality in patients with spontaneous aneurysmal subarachnoid hemorrhage (SAH), but the pathogenesis and pathophysiology remain controversial. MicroRNAs (miRNAs) are important posttranscriptional regulators of...

  3. MicroRNA expression profiles and functions in the brain

    Yanting Qi; Yu Zhao; Zhuyin Chen; Xiaona Chen; Marie C. Lin; Xiangfu Kong; Lihui Lai

    2008-01-01

    MicroRNAs are abundant in the brains of vertebrates and some show a brain-specific or brain-enriched expression pattern. Because microRNAs regulate the expression of hundreds of target genes, it is not surprising that they have profoundly important functions in brain development and pathological processes. For example, miR-124 plays an important role in inducing and maintaining neuronal identity through targeting at least two anti-neural factors. MicroRNAs have also been implicated in brain disorders, including brain tumors and neurodegenerative diseases. This review aims to present an overview of the expression profiles and functions of microRNAs in the developing brains of vertebrates.

  4. The role of microRNAs in enteroviral infections

    Jing Wu; Li Shen; Jianguo Chen; Huaxi Xu; Lingxiang Mao

    2015-01-01

    ABSTRACTThe genus Enterovirus, a member of thePicornavirus family, are RNA viruses that can cause poliomyelitis, hand-food-mouth disease, viral meningitis or meningoencephalitis, viral myocarditis and so on. MicroRNAs are a class of highly conserved, small noncoding RNAs recognized as important regulators of gene expression. Recent studies found that MicroRNAs play a significant role in the infection ofEnterovirus, such as enterovirus 71, coxsackievirus B3 and other Enterovirus. Enteroviral i...

  5. The miR-10 microRNA precursor family

    Tehler, Disa; Høyland-Kroghsbo, Nina Molin; Lund, Anders H

    2011-01-01

    The miR-10 microRNA precursor family encodes a group of short non-coding RNAs involved in gene regulation. The miR-10 family is highly conserved and has sparked the interest of many research groups because of the genomic localization in the vicinity of, coexpression with and regulation of the Hox...... gene developmental regulators. Here, we review the current knowledge of the evolution, physiological function and involvement in cancer of this family of microRNAs....

  6. Identification and validation of human papillomavirus encoded microRNAs.

    Kui Qian

    Full Text Available We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.

  7. Effects of myostatin propeptide gene tranfection on glucose metabolism in cultured C2C12 cells

    张莎莎

    2014-01-01

    Objective To investigate the effects of recombinant adeno-associated virus-mediated myostatin propeptide(MPRO)on uptake and oxidation of glucose,and glycogen synthesis in C2C12 myotubes,as well as the associated molecular mechanism.Methods Mature C2C12myotubes were assigned to the following 6 groups:control,insulin,green fluorescent protein(GFP),insulin+

  8. Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity

    Chao, Pei-Ting; Liang YANG; Aja, Susan; Moran, Timothy H.; Bi, Sheng

    2011-01-01

    Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympa...

  9. Characteristics of microRNA co-target networks

    Lee, Chang-Yong

    2011-07-01

    The database of microRNAs and their predicted target genes in humans were used to extract a microRNA co-target network. Based on the finding that more than two miRNAs can target the same gene, we constructed a microRNA co-target network and analyzed it from the perspective of the complex network. We found that a network having a positive assortative mixing can be characterized by small-world and scale-free characteristics which are found in most complex networks. The network was further analyzed by the nearest-neighbor average connectivity, and it was shown that the more assortative a microRNA network is, the wider the range of increasing average connectivity. In particular, an assortative network has a power-law relationship of the average connectivity with a positive exponent. A percolation analysis of the network showed that, although the network is diluted, there is no percolation transition in the network. From these findings, we infer that the microRNAs in the network are clustered together, forming a core group. The same analyses carried out on different species confirmed the robustness of the main results found in the microRNA networks of humans.

  10. MicroRNA in Human Glioma

    Li, Mengfeng, E-mail: limf@mail.sysu.edu.cn [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Li, Jun [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Lei; Li, Wei [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Yang, Yi [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Yuan, Jie [Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Chinese Ministry of Education, Guangzhou 510080 (China); Key Laboratory of Functional Molecules from Oceanic Microorganisms (Sun Yat-sen University), Department of Education of Guangdong Province, Guangzhou 510080 (China)

    2013-10-23

    Glioma represents a serious health problem worldwide. Despite advances in surgery, radiotherapy, chemotherapy, and targeting therapy, the disease remains one of the most lethal malignancies in humans, and new approaches to improvement of the efficacy of anti-glioma treatments are urgently needed. Thus, new therapeutic targets and tools should be developed based on a better understanding of the molecular pathogenesis of glioma. In this context, microRNAs (miRNAs), a class of small, non-coding RNAs, play a pivotal role in the development of the malignant phenotype of glioma cells, including cell survival, proliferation, differentiation, tumor angiogenesis, and stem cell generation. This review will discuss the biological functions of miRNAs in human glioma and their implications in improving clinical diagnosis, prediction of prognosis, and anti-glioma therapy.

  11. MicroRNA in Human Glioma

    Glioma represents a serious health problem worldwide. Despite advances in surgery, radiotherapy, chemotherapy, and targeting therapy, the disease remains one of the most lethal malignancies in humans, and new approaches to improvement of the efficacy of anti-glioma treatments are urgently needed. Thus, new therapeutic targets and tools should be developed based on a better understanding of the molecular pathogenesis of glioma. In this context, microRNAs (miRNAs), a class of small, non-coding RNAs, play a pivotal role in the development of the malignant phenotype of glioma cells, including cell survival, proliferation, differentiation, tumor angiogenesis, and stem cell generation. This review will discuss the biological functions of miRNAs in human glioma and their implications in improving clinical diagnosis, prediction of prognosis, and anti-glioma therapy

  12. MicroRNA: Biological and Computational Perspective

    Yong Kong; Jin-Hua Han

    2005-01-01

    MicroRNAs (miRNAs) are endogenously expressed non-coding RNAs of 20-24nucleotides, which post-transcriptionally regulate gene expression in plants and animals. Recently it has been recognized that miRNAs comprise one of the abundant gene families in multicellular species, and their regulatory functions in various biological processes are widely spread. There has been a surge in the research activities in this field in the past few years. From the very beginning, computational methods have been utilized as indispensable tools, and many discoveries have been obtained through combination of experimental and computational approaches. In this review, both biological and computational aspects of miRNA will be discussed.A brief history of the discovery of miRNA and discussion of microarray applications in miRNA research are also included.

  13. MicroRNA Dysregulation in Multiple Sclerosis

    Omar ede Faria Jr.

    2013-01-01

    Full Text Available Multiple Sclerosis (MS is a chronic inflammatory disease characterized by central nervous system (CNS demyelination and axonal degeneration. Although the cause of MS is still unknown, it is widely accepted that novel drug targets need to focus on both decreasing inflammation and promoting CNS repair. In MS and experimental autoimmune encephalomyelitis (EAE non-coding small microRNAs (miRNAs are dysregulated in the immune and central nervous systems. Since individual miRNAs are able to downregulate multiple targeted mRNA transcripts, even minor changes in miRNA expression may lead to significant alterations in post-transcriptional gene expression. Herein, we review miRNA signatures reported in CNS tissue and immune cells of MS patients and consider how altered miRNA expression may influence MS pathology.

  14. MicroRNAs and Endothelial (Dys) Function.

    Santulli, Gaetano

    2016-08-01

    Accumulating evidence indicates that microRNAs (miRs)-non-coding RNAs that can regulate gene expression via translational repression and/or post-transcriptional degradation-are becoming one of the most fascinating areas of physiology, given their fundamental roles in countless pathophysiological processes. The relative roles of different miRs in vascular biology as direct or indirect post-transcriptional regulators of fundamental genes implied in vascular remodeling designate miRs as potential biomarkers and/or promising drug targets. The mechanistic importance of miRs in modulating endothelial cell (EC) function in physiology and in disease is addressed here. Drawbacks of currently available therapeutic options are also discussed, pointing at the challenges and clinical opportunities provided by miR-based treatments. J. Cell. Physiol. 231: 1638-1644, 2016. © 2015 Wiley Periodicals, Inc. PMID:26627535

  15. MicroRNAs in pulmonary arterial hypertension.

    Zhou, Guofei; Chen, Tianji; Raj, J Usha

    2015-02-01

    Pulmonary arterial hypertension (PAH) is a devastating disease without effective treatment. Despite decades of research and the development of novel treatments, PAH remains a fatal disease, suggesting an urgent need for better understanding of the pathogenesis of PAH. Recent studies suggest that microRNAs (miRNAs) are dysregulated in patients with PAH and in experimental pulmonary hypertension. Furthermore, normalization of a few miRNAs is reported to inhibit experimental pulmonary hypertension. We have reviewed the current knowledge about miRNA biogenesis, miRNA expression pattern, and their roles in regulation of pulmonary artery smooth muscle cells, endothelial cells, and fibroblasts. We have also identified emerging trends in our understanding of the role of miRNAs in the pathogenesis of PAH and propose future studies that might lead to novel therapeutic strategies for the treatment of PAH. PMID:25192340

  16. MicroRNAs in nasopharyngeal carcinoma

    Jeff P Bruce; Fei-Fei Liu

    2014-01-01

    MicroRNAs (miRNAs) provide insight into both the biology and clinical behavior of many human cancers, including nasopharyngeal carcinoma (NPC). The dysregulation of miRNAs in NPC results in a variety of tumor-promoting effects. Furthermore, several miRNAs are prognostic markers for NPC. In addition to celular miRNAs, NPC samples also often contain miRNAs encoded by Epstein-Barr virus, and these miRNAs may impact NPC biology by targeting both celular and viral genes. Given their numerous putative roles in NPC development and progression, a thorough understanding of the impact of miRNA dysregulation in NPC is expected to shed light on useful biomarkers and therapeutic targets for the clinical management of this disease. In this review, we describe the efforts to date to identify and characterize such miRNAs in the context of NPC.

  17. Towards a complete description of the microRNA complement of animal genomes

    Brennecke, Julius; Cohen, Stephen M.

    2003-01-01

    Recent cloning and computational studies have sought to catalog all the microRNA genes encoded in animal genomes. Here, we highlight recent advances in identifying Caenorhabditis elegans and Drosophila melanogaster microRNAs.

  18. Prognostic microRNAs in high-grade glioma reveal a link to oligodendrocyte precursor differentiation

    Hayes, Josie; Thygesen, Helene; Droop, Alastair; Hughes, Thomas A; Westhead, David; Lawler, Sean E.; Wurdak, Heiko; Short, Susan C.

    2015-01-01

    MicroRNA expression can be exploited to define tumor prognosis and stratification for precision medicine. It remains unclear whether prognostic microRNA signatures are exclusively tumor grade and/or molecular subtype-specific, or whether common signatures of aggressive clinical behavior can be identified. Here, we defined microRNAs that are associated with good and poor prognosis in grade III and IV gliomas using data from The Cancer Genome Atlas. Pathway analysis of microRNA targets that are...

  19. MicroRNA characterize genetic diversity and drug resistance in pediatric acute lymphoblastic leukemia

    Schotte, Diana; Menezes, Renee; Akbari Moqadam, Farhad; Khankahdani, Leila Mohammadi; Lange-Turenhout, Ellen; Chen, Caifu; Pieters, Rob; de Boer, Monique

    2011-01-01

    textabstractBackground MicroRNA regulate the activity of protein-coding genes including those involved in hematopoietic cancers. The aim of the current study was to explore which microRNA are unique for seven different subtypes of pediatric acute lymphoblastic leukemia. Design and Methods Expression levels of 397 microRNA (including novel microRNA) were measured by quantitative real-time polymerase chain reaction in 81 cases of pediatric leukemia and 17 normal hematopoietic control cases. Res...

  20. Myogenic MicroRNA Expression Requires ATP-Dependent Chromatin Remodeling Enzyme Function▿ †

    Mallappa, Chandrashekara; Nasipak, Brian T.; Etheridge, Letitiah; Androphy, Elliot J.; Jones, Stephen N.; Sagerström, Charles G; Ohkawa, Yasuyuki; Imbalzano, Anthony N.

    2010-01-01

    Knockdown of the Brg1 ATPase subunit of SWI/SNF chromatin remodeling enzymes in developing zebrafish caused stunted tail formation and altered sarcomeric actin organization, which phenocopies the loss of the microRNA processing enzyme Dicer, or the knockdown of myogenic microRNAs. Furthermore, myogenic microRNA expression and differentiation was blocked in Brg1 conditional myoblasts differentiated ex vivo. The binding of Brg1 upstream of myogenic microRNA sequences correlated with MyoD bindin...

  1. The terminal loop region controls microRNA processing by Drosha and Dicer

    Zhang, Xiaoxiao; Zeng, Yan

    2010-01-01

    microRNAs are widely expressed, ∼22-nt-long regulatory RNAs. They are first transcribed as much longer primary transcripts, which then undergo a series of processing steps to yield the single-stranded, mature microRNAs, although the mechanisms are incompletely understood. Here, we show that the terminal loop region of human primary microRNA transcripts is an important determinant of microRNA biogenesis. Mutations that restrain the terminal loop region inhibit Drosha processing of primary micr...

  2. Differentially expressed microRNA in multiple sclerosis: A window into pathogenesis?

    Martin, Nellie Anne; Illés, Zsolt

    2014-01-01

    MicroRNA are small non-coding RNA that mediate mRNA translation repression or mRNA degradation, and thereby refine protein expression levels. More than 30–60% of all genes are regulated by microRNA. Exploring disease-related microRNA signatures is an emerging tool in biomarker discovery, and sile...

  3. Nasal mucosal microRNA expression in children with respiratory syncytial virus infection

    Inchley, Christopher S; Sonerud, Tonje; Fjærli, Hans O; Nakstad, Britt

    2015-01-01

    Background Respiratory syncytial virus (RSV) infection is a common cause of pediatric hospitalization. microRNA, key regulators of the immune system, have not previously been investigated in respiratory specimens during viral infection. We investigated microRNA expression in the nasal mucosa of 42 RSV-positive infants, also comparing microRNA expression between disease severity subgroups. Methods Nasa...

  4. Hypoxia-regulated microRNAs in human cancer

    Guomin SHEN; Xiaobo LI; Yong-feng JIA; Gary A PIAZZA; Yaguang XI

    2013-01-01

    Hypoxia plays an important role in the tumor microenvironment by allowing the development and maintenance of cancer cells,but the regulatory mechanisms by which tumor cells adapt to hypoxic conditions are not yet well understood.MicroRNAs are recognized as a new class of master regulators that control gene expression and are responsible for many normal and pathological cellular processes.Studies have shown that hypoxia inducible factor 1 (HIF1) regulates a panel of microRNAs,whereas some of microRNAs target HIF1.The interaction between microRNAs and HIF1 can account for many vital events relevant to tumorigenesis,such as angiogenesis,metabolism,apoptosis,cell cycle regulation,proliferation,metastasis,and resistance to anticancer therapy.This review will summarize recent findings on the roles of hypoxia and microRNAs in human cancer and illustrate the machinery by which microRNAs interact with hypoxia in tumor cells,It is expected to update our knowledge about the regulatory roles of microRNAs in regulating tumor microenvironments and thus benefit the development of new anticancer drugs.

  5. MicroRNA target prediction in glaucoma.

    Romano, Giovanni Luca; Platania, Chiara Bianca Maria; Forte, Stefano; Salomone, Salvatore; Drago, Filippo; Bucolo, Claudio

    2015-01-01

    Glaucoma is a progressive optic neuropathy and is one of the leading causes of blindness in the industrialized countries. The aim of this study is to investigate microRNA (miRNA) regulation in glaucoma and other neurodegenerative diseases, that share similar pathways, by means of in silico approaches such as bibliographic search and access to bioinformatic resources. First of all, data mining was carried out on Human miRNA Disease Database (HMDD) and miR2Disease databases. Then, predictions of deregulated miRNAs were carried out accessing to microrna.org database. Finally, the potential combinatorial effect of miRNAs, on regulation of biochemical pathways, was studied by an enrichment analysis performed by DIANA-miRPath v.2.0. We found, from literature search, 8 deregulated miRNAs in glaucoma and 9 and 23 in age-related macular degeneration (AMD) and Alzheimer's disease (AD), respectively. One miRNA is commonly deregulated in glaucoma and AMD (miR-23a). Two miRNAs (miR-29a, miR-29b) are common to glaucoma and AD, and four miRNAs were identified to be commonly deregulated in AMD and AD (miR-9, miR-21, miR-34a, miR-146a). The match of the miRNA common to glaucoma and the other two neurodegenerative diseases (AMD and AD) did not generate any output. Enrichment of information has been reached through miRNAs prediction: 88 predicted miRNAs are common to glaucoma and AMD, 19 are common to glaucoma and AD, and 9 are common to AMD and AD. Indeed, predicted miRNAs common to the three neurodegenerative diseases are nine (miR-107, miR-137, miR-146a, miR-181c, miR-197, miR-21, miR-22, miR-590, miR-9). DIANA-miRPath predicted that those nine miRNAs might regulate pathways involved in inflammation. The findings hereby obtained provide a valuable hint to assess deregulation of specific miRNA, as potential biomarkers and therapeutic targets, in glaucoma and other neurodegenerative diseases by means of preclinical and clinical studies. PMID:26497793

  6. MicroRNAs in Cardiometabolic Diseases

    Anna Meiliana

    2013-08-01

    Full Text Available BACKGROUND: MicroRNAs (miRNAs are ~22-nucleotide noncoding RNAs with critical functions in multiple physiological and pathological processes. An explosion of reports on the discovery and characterization of different miRNA species and their involvement in almost every aspect of cardiac biology and diseases has established an exciting new dimension in gene regulation networks for cardiac development and pathogenesis. CONTENT: Alterations in the metabolic control of lipid and glucose homeostasis predispose an individual to develop cardiometabolic diseases, such as type 2 diabetes mellitus and atherosclerosis. Work over the last years has suggested that miRNAs play an important role in regulating these physiological processes. Besides a cell-specific transcription factor profile, cell-specific miRNA-regulated gene expression is integral to cell fate and activation decisions. Thus, the cell types involved in atherosclerosis, vascular disease, and its myocardial sequelae may be differentially regulated by distinct miRNAs, thereby controlling highly complex processes, for example, smooth muscle cell phenotype and inflammatory responses of endothelial cells or macrophages. The recent advancements in using miRNAs as circulating biomarkers or therapeutic modalities, will hopefully be able to provide a strong basis for future research to further expand our insights into miRNA function in cardiovascular biology. SUMMARY: MiRNAs are small, noncoding RNAs that function as post-transcriptional regulators of gene expression. They are potent modulators of diverse biological processes and pathologies. Recent findings demonstrated the importance of miRNAs in the vasculature and the orchestration of lipid metabolism and glucose homeostasis. MiRNA networks represent an additional layer of regulation for gene expression that absorbs perturbations and ensures the robustness of biological systems. A detailed understanding of the molecular and cellular mechanisms of mi

  7. Progress, challenges and new concepts in microRNAs

    ZHANG YouYi

    2011-01-01

    MicroRNAs are a group of small noncoding RNAs.They have rapidly gained attention in the field as novel regulators of cellular morphology and function.Currently hundreds of microRNAs have been described in human genome.MicroRNAs play important roles in posttranscriptional regulation of gene expression by inhibiting protein translation and/or promoting mRNA degradation.MicroRNAs were initially thought to be subtle regulators of gene expression,but increasing evidence demonstrates that the regulatory functions of microRNAs are crucial for the cell.MicroRNAs have been found to be involved in the development,tissue homeostasis as well as diseases.

  8. MicroRNA profiling in the malignant progression of gliomas

    Stupak, E. V.; Veryaskina, Yu. A.; Titov, S. E.; Achmerova, L. G.; Stupak, V. V.; Ivanov, M. K.; Zhimulev, I. F.; Kolesnikov, N. N.

    2016-08-01

    Wealth of data indicates that microRNAs (miRNAs) are directly involved in carcinogenesis and that miRNA can, on their own, act as diagnostic and prognostic markers for various types of cancers, including gliomas. The aim of this study was to conduct a comparative analysis of expression profile for 10 microRNAs (miR-124, -125b, -16, -181b, -191, -21, -221, -223, -31, and -451) in surgical specimens of various hystotypes of glioimatissues vs adjacent normal tissues from the same patient (n = 77). The study identified specific microRNA expression profiles for different histotypes of tumors that are related to their degree of malignancy. We have outlined approaches to development of miRNA-based diagnostic and prognostic panel, which may be used to compensate for the lack of appropriate screening methods.

  9. MicroRNA regulation network in colorectal cancer metastasis

    Jiao-Jiao; Zhou; Shu; Zheng; Li-Feng; Sun; Lei; Zheng

    2014-01-01

    Colorectal cancer is the third most common cancer worldwide. Metastasis is a major cause of colorectal cancer-related death. Mechanisms of metastasis remain largely obscure. MicroRNA is one of the most important epigenetic regulators by targeting mRNAs posttranscriptionally. Accumulated evidence has supported its significant role in the metastasis of colorectal cancer, including epithelial-mesenchymal transition and angiogenesis. Dissecting microRNAome potentially identifies specific microRNAs as biomarkers of colorectal cancer metastasis. Better understanding of the complex network of microRNAs in colorectal cancer metastasis provide new insights in the biological process of metastasis and in the potential targets for colorectal cancer therapies and for diagnosis of recurrent and metastatic colorectal cancer.

  10. Analysis of Plasma microRNA Associated with Hemolysis.

    Shkurnikov, M Yu; Knyazev, E N; Fomicheva, K A; Mikhailenko, D S; Nyushko, K M; Saribekyan, E K; Samatov, T R; Alekseev, B Ya

    2016-04-01

    We analyzed the effect of hemolysis on microRNA profi le of blood plasma. It was found that hemolysis of ~0.05% erythrocytes in a sample signifi cantly affected the concentration of 9 microRNA: hsa-miR-486-5p, hsa-miR-16-5p, hsa-miR-451a, hsa-miR-106a-5p, hsa-miR-17-5p, hsa-miR-93-5p, hsa-miR-20a-5p, hsa-miR-107, and hsa-miR-20b-5p. The effect of hemolysis on plasma content of miR-17 family microRNA was demonstrated. PMID:27165077

  11. Transcriptome dynamics of the microRNA inhibition response

    Wen, Jiayu; Leucci, Elenora; Vendramin, Roberto; Kauppinen, Sakari; Lund, Anders H; Krogh, Anders; Parker, Brian J

    2015-01-01

    We report a high-resolution time series study of transcriptome dynamics following antimiR-mediated inhibition of miR-9 in a Hodgkin lymphoma cell-line-the first such dynamic study of the microRNA inhibition response-revealing both general and specific aspects of the physiological response. We show...... validate the key observations with independent time series qPCR and we experimentally validate key predicted miR-9 targets. Methodologically, we developed sensitive functional data analytic predictive methods to analyse the weak response inherent in microRNA inhibition experiments. The methods of this...... study will be applicable to similar high-resolution time series transcriptome analyses and provides the context for more accurate experimental design and interpretation of future microRNA inhibition studies....

  12. Genomic Organization of Zebrafish microRNAs

    Paydar Ima

    2008-05-01

    Full Text Available Abstract Background microRNAs (miRNAs are small (~22 nt non-coding RNAs that regulate cell movement, specification, and development. Expression of miRNAs is highly regulated, both spatially and temporally. Based on direct cloning, sequence conservation, and predicted secondary structures, a large number of miRNAs have been identified in higher eukaryotic genomes but whether these RNAs are simply a subset of a much larger number of noncoding RNA families is unknown. This is especially true in zebrafish where genome sequencing and annotation is not yet complete. Results We analyzed the zebrafish genome to identify the number and location of proven and predicted miRNAs resulting in the identification of 35 new miRNAs. We then grouped all 415 zebrafish miRNAs into families based on seed sequence identity as a means to identify possible functional redundancy. Based on genomic location and expression analysis, we also identified those miRNAs that are likely to be encoded as part of polycistronic transcripts. Lastly, as a resource, we compiled existing zebrafish miRNA expression data and, where possible, listed all experimentally proven mRNA targets. Conclusion Current analysis indicates the zebrafish genome encodes 415 miRNAs which can be grouped into 44 families. The largest of these families (the miR-430 family contains 72 members largely clustered in two main locations along chromosome 4. Thus far, most zebrafish miRNAs exhibit tissue specific patterns of expression.

  13. MicroRNAs in Rice Innate Immunity.

    Baldrich, Patricia; San Segundo, Blanca

    2016-12-01

    MicroRNAs (miRNAs) are short regulatory non-coding RNAs that guide gene silencing in most eukaryotes. They regulate gene expression by triggering sequence-specific cleavage or translational repression of target transcripts. Plant miRNAs are known to play important roles in a wide range of developmental processes. Increasing evidence also supports that the modulation of miRNA levels plays an important role in reprogramming plant responses to abiotic stress (drought, cold, salinity and nutrient deficiency) and biotic stress (antibacterial resistance). Most of these studies were carried out in the model plant Arabidopsis thaliana. During the last years, the adoption of high-throughput sequencing technologies has significantly contributed to uncover multiple miRNAs while allowing miRNA profiling in plants. However, although a plethora of rice miRNAs have been shown to be regulated by pathogen infection, the biological function remains largely unknown for most of them. In this review, we summarize our current understanding on the contribution of miRNAs to rice immunity and discuss their potential applications in rice biotechnology. A better understanding of the miRNA species controlling rice immunity may lead to practical biotechnological applications leading to the development of appropriate strategies for rice protection. PMID:26897721

  14. MicroRNA and Heart Failure

    Lee Lee Wong

    2016-04-01

    Full Text Available Heart failure (HF imposes significant economic and public health burdens upon modern society. It is known that disturbances in neurohormonal status play an important role in the pathogenesis of HF. Therapeutics that antagonize selected neurohormonal pathways, specifically the renin-angiotensin-aldosterone and sympathetic nervous systems, have significantly improved patient outcomes in HF. Nevertheless, mortality remains high with about 50% of HF patients dying within five years of diagnosis thus mandating ongoing efforts to improve HF management. The discovery of short noncoding microRNAs (miRNAs and our increasing understanding of their functions, has presented potential therapeutic applications in complex diseases, including HF. Results from several genome-wide miRNA studies have identified miRNAs differentially expressed in HF cohorts suggesting their possible involvement in the pathogenesis of HF and their potential as both biomarkers and as therapeutic targets. Unravelling the functional relevance of miRNAs within pathogenic pathways is a major challenge in cardiovascular research. In this article, we provide an overview of the role of miRNAs in the cardiovascular system. We highlight several HF-related miRNAs reported from selected cohorts and review their putative roles in neurohormonal signaling.

  15. MicroRNA mimicry blocks pulmonary fibrosis

    Montgomery, Rusty L; Yu, Guoying; Latimer, Paul A; Stack, Christianna; Robinson, Kathryn; Dalby, Christina M; Kaminski, Naftali; van Rooij, Eva

    2014-01-01

    Over the last decade, great enthusiasm has evolved for microRNA (miRNA) therapeutics. Part of the excitement stems from the fact that a miRNA often regulates numerous related mRNAs. As such, modulation of a single miRNA allows for parallel regulation of multiple genes involved in a particular disease. While many studies have shown therapeutic efficacy using miRNA inhibitors, efforts to restore or increase the function of a miRNA have been lagging behind. The miR-29 family has gained a lot of attention for its clear function in tissue fibrosis. This fibroblast-enriched miRNA family is downregulated in fibrotic diseases which induces a coordinate increase of many extracellular matrix genes. Here, we show that intravenous injection of synthetic RNA duplexes can increase miR-29 levels in vivo for several days. Moreover, therapeutic delivery of these miR-29 mimics during bleomycin-induced pulmonary fibrosis restores endogenous miR-29 function whereby decreasing collagen expression and blocking and reversing pulmonary fibrosis. Our data support the feasibility of using miRNA mimics to therapeutically increase miRNAs and indicate miR-29 to be a potent therapeutic miRNA for treating pulmonary fibrosis. PMID:25239947

  16. MicroRNAs horizon in retinoblastoma.

    Mojgan Mirakholi

    2013-12-01

    Full Text Available In the retinoblastoma research, it is of great interest to identify molecular markers associated with the genetics of tumorigenesis. microRNAs (miRNAs are small non-coding RNA molecules that play a regulatory role in many crucial cellular pathways such as differentiation, cell cycle progression, and apoptosis. A body of evidences showed dysregulation of miRNAs in tumor biology and many diseases. They potentially play a significant role in tumorigenesis processes and have been the subject of research in many types of cancers including retinal tumorigenesis. miRNA expression profiling was found to be associated with tumor development, progression and treatment. These associations demonstrate the putative applications of miRNAs in monitoring of different aspect of tumors consisting diagnostic, prognostic and therapeutic. Herein, we review the current literature concerning to the study of miRNA target recognition, function to tumorigenesis and treatment in retinoblastoma. Identification the specific miRNA biomarkers associated with retinoblastoma cancer may help to establish new therapeutic approaches for salvage affected eyes in patients.

  17. MicroRNAs in nasopharyngeal carcinoma.

    Spence, Tara; Bruce, Jeff; Yip, Kenneth W; Liu, Fei-Fei

    2016-04-01

    It is becoming increasingly evident that aberrantly expressed microRNAs (miRNAs) are responsible for a number of disease processes, including cancer initiation and progression. miRNAs have been implicated as key players in numerous neoplasms, including nasopharyngeal carcinoma (NPC). Functionally, deregulation of miRNAs that act either as tumour suppressors or oncogenes results in numerous cancer-associated phenomena, including changes in proliferation, migration, and cell survival. Furthermore, miRNA expression has been associated with chemoresistant or radioresistant phenotypes; highlighting the importance of miRNAs in mediating oncogenic processes. Prognostic and predictive miRNA signatures have been defined for a variety of cancer types, including NPC, whereby these signatures offer a potentially important clinical tool for assessing the disease state, as well as predicting treatment response and clinical outcome. Therefore, further examination and validation of miRNAs that are deregulated in NPC will provide insight into the fundamental drivers of this disease, which will aid in the identification of novel targeted treatments. This review summarizes recent advances in the study of miRNAs in NPC, with specific discussion on the role of miRNAs in NPC pathogenesis and the potential utility of miRNAs as prognostic biomarkers. Our increasing understanding of the role of miRNAs in NPC tumorigenesis and their application as novel biomarkers will undoubtedly prove useful in the stratification of future patients into clinically relevant treatment classifications, thereby improving and personalizing disease management. PMID:27121877

  18. MicroRNA and Heart Failure

    Wong, Lee Lee; Wang, Juan; Liew, Oi Wah; Richards, Arthur Mark; Chen, Yei-Tsung

    2016-01-01

    Heart failure (HF) imposes significant economic and public health burdens upon modern society. It is known that disturbances in neurohormonal status play an important role in the pathogenesis of HF. Therapeutics that antagonize selected neurohormonal pathways, specifically the renin-angiotensin-aldosterone and sympathetic nervous systems, have significantly improved patient outcomes in HF. Nevertheless, mortality remains high with about 50% of HF patients dying within five years of diagnosis thus mandating ongoing efforts to improve HF management. The discovery of short noncoding microRNAs (miRNAs) and our increasing understanding of their functions, has presented potential therapeutic applications in complex diseases, including HF. Results from several genome-wide miRNA studies have identified miRNAs differentially expressed in HF cohorts suggesting their possible involvement in the pathogenesis of HF and their potential as both biomarkers and as therapeutic targets. Unravelling the functional relevance of miRNAs within pathogenic pathways is a major challenge in cardiovascular research. In this article, we provide an overview of the role of miRNAs in the cardiovascular system. We highlight several HF-related miRNAs reported from selected cohorts and review their putative roles in neurohormonal signaling. PMID:27058529

  19. MicroRNA dysregulation in melanoma.

    Latchana, Nicholas; Ganju, Akaansha; Howard, J Harrison; Carson, William E

    2016-09-01

    Melanoma is the deadliest form of skin cancer. Current challenges facing the management of melanoma include accurate prediction of individuals who will respond to adjuvant therapies as well as early detection of recurrences. These and other challenges have prompted investigation into biomarkers that could be used as diagnostic, prognostic and therapeutic aids. MicroRNAs (miRs) are small 19-22 nucleotide RNA inhibitors of protein translation. Over 800 different miRs are present within cells and importantly miR expression profiles may vary across different cells types and stages of malignancy. Unique expression profiles have been described for malignant melanoma; however, this work has yet to be translated into routine clinical practice. We highlight pertinent studies involving common miRs implicated in the oncogenesis of melanoma including miR-21, miR-125b, miR-150, miR-155, miR-205, and miR-211. In particular, emphasis is placed upon differential expression across different stages of melanoma progression, prognostic implications and potential mechanistic involvement. Focused efforts on inhibition of these miRs could be the most efficient method of translating preclinical endeavors into clinically meaningful applications. PMID:27566021

  20. Expression profiles of microRNAs after focal cerebral ischemia/reperfusion injury in rats

    Fengguo Zhai; Xiuping Zhang; Yue Guan; Xudong Yang; Yang Li; Gaochen Song; Lixin Guan

    2012-01-01

    Rat models of focal cerebral ischemia/reperfusion injury were established by occlusion of the middle cerebral artery. Microarray analysis showed that 24 hours after cerebral ischemia, there were nine up-regulated and 27 down-regulated microRNA genes in cortical tissue. Bioinformatic analysis showed that bcl-2 was the target gene of microRNA-384-5p and microRNA-494, and caspase-3 was the target gene of microRNA-129, microRNA-320 and microRNA-326. Real-time PCR and western blot analyses showed that 24 hours after cerebral ischemia, bcl-2 mRNA and protein levels in brain tissue were significantly decreased, while caspase-3 mRNA and protein levels were significantly increased. This suggests that following cerebral ischemia, differentially expressed microRNA-384-5p, microRNA-494, microRNA-320, microRNA-129 and microRNA-326 can regulate bcl-2 and caspase-3 expression in brain tissue.

  1. MicroRNAs as potential therapeutics for treating spinal cord injury

    Hualin Yan; Peiwei Hong; Mei Jiang; Hedong Li

    2012-01-01

    MicroRNAs are a class of recently discovered, small non-coding RNAs that have been shown to play essential roles in a vast majority of biological processes. Very little is known about the role of microRNAs during spinal cord injury. This review summarizes the changes in expression levels of microRNAs after spinal cord injury. These aberrant changes suggest that microRNAs play an important role in inflammation, oxidative stress, apoptosis, glial scar formation and axonal regeneration. Given their small size and specificity of action, microRNAs could be potential therapeutics for treating spinal cord injury in the future. There are rapidly developing techniques for manipulating microRNA levels in animals; we review different chemical modification and delivery strategies. These may provide platforms for designing efficient microRNA delivery protocols for use in the clinic.

  2. A conformation-induced fluorescence method for microRNA detection

    Aw, Sherry S; Tang, Melissa Xm; Teo, Yin Nah; Cohen, Stephen M

    2016-01-01

    MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and...... quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3' and 5' ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA...... sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA...

  3. New wheat microRNA using whole-genome sequence.

    Kurtoglu, Kuaybe Yucebilgili; Kantar, Melda; Budak, Hikmet

    2014-06-01

    MicroRNAs are post-transcriptional regulators of gene expression, taking roles in a variety of fundamental biological processes. Hence, their identification, annotation and characterization are of great significance, especially in bread wheat, one of the main food sources for humans. The recent availability of 5× coverage Triticum aestivum L. whole-genome sequence provided us with the opportunity to perform a systematic prediction of a complete catalogue of wheat microRNAs. Using an in silico homology-based approach, stem-loop coding regions were derived from two assemblies, constructed from wheat 454 reads. To avoid the presence of pseudo-microRNAs in the final data set, transposable element related stem-loops were eliminated by repeat analysis. Overall, 52 putative wheat microRNAs were predicted, including seven, which have not been previously published. Moreover, with distinct analysis of the two different assemblies, both variety and representation of putative microRNA-coding stem-loops were found to be predominant in the intergenic regions. By searching available expressed sequences and small RNA library databases, expression evidence for 39 (out of 52) putative wheat microRNAs was provided. Expression of three of the predicted microRNAs (miR166, miR396 and miR528) was also comparatively quantified with real-time quantitative reverse transcription PCR. This is the first report on in silico prediction of a whole repertoire of bread wheat microRNAs, supported by the wet-lab validation. PMID:24395439

  4. New microRNAs from mouse and human

    Lagos-Quintana, M.; Rauhut, R; Meyer, J.; Borkhardt, A.; Tuschl, T.

    2003-01-01

    MicroRNAs (miRNAs) represent a new class of noncoding RNAs encoded in the genomes of plants, invertebrates, and vertebrates. MicroRNAs regulate translation and stability of target mRNAs based on (partial) sequence complementarity. Although the number of newly identified miRNAs is still increasing, target mRNAs of animal miRNAs remain to be identified. Here we describe 31 novel miRNAs that were identified by cloning from mouse tissues and the human Saos-2 cell line. Fifty-three percent of all ...

  5. Silencing MicroRNA-155 Ameliorates Experimental Autoimmune Encephalomyelitis

    Murugaiyan, Gopal; Beynon, Vanessa; Mittal, Akanksha; Joller, Nicole; Howard L Weiner

    2011-01-01

    IFN-γ–producing Th1 and IL-17–producing Th17 cells are the key participants in various autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Although both of these T cell subsets are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control these two inflammatory T cell subsets and whether targeting microRNAs can have therapeutic effects are not known. In this study, we show that...

  6. NAViGaTing the micronome--using multiple microRNA prediction databases to identify signalling pathway-associated microRNAs.

    Elize A Shirdel

    Full Text Available BACKGROUND: MicroRNAs are a class of small RNAs known to regulate gene expression at the transcript level, the protein level, or both. Since microRNA binding is sequence-based but possibly structure-specific, work in this area has resulted in multiple databases storing predicted microRNA:target relationships computed using diverse algorithms. We integrate prediction databases, compare predictions to in vitro data, and use cross-database predictions to model the microRNA:transcript interactome--referred to as the micronome--to study microRNA involvement in well-known signalling pathways as well as associations with disease. We make this data freely available with a flexible user interface as our microRNA Data Integration Portal--mirDIP (http://ophid.utoronto.ca/mirDIP. RESULTS: mirDIP integrates prediction databases to elucidate accurate microRNA:target relationships. Using NAViGaTOR to produce interaction networks implicating microRNAs in literature-based, KEGG-based and Reactome-based pathways, we find these signalling pathway networks have significantly more microRNA involvement compared to chance (p<0.05, suggesting microRNAs co-target many genes in a given pathway. Further examination of the micronome shows two distinct classes of microRNAs; universe microRNAs, which are involved in many signalling pathways; and intra-pathway microRNAs, which target multiple genes within one signalling pathway. We find universe microRNAs to have more targets (p<0.0001, to be more studied (p<0.0002, and to have higher degree in the KEGG cancer pathway (p<0.0001, compared to intra-pathway microRNAs. CONCLUSIONS: Our pathway-based analysis of mirDIP data suggests microRNAs are involved in intra-pathway signalling. We identify two distinct classes of microRNAs, suggesting a hierarchical organization of microRNAs co-targeting genes both within and between pathways, and implying differential involvement of universe and intra-pathway microRNAs at the disease level.

  7. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

    Vanas, Vanita; Haigl, Barbara; Stockhammer, Verena; Sutterlüty-Fall, Hedwig

    2016-01-01

    Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin. PMID:27513462

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

    Sallach, Jessica

    2014-01-01

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

  9. Adeno-Associated Viral-Mediated LARGE Gene Therapy Rescues the Muscular Dystrophic Phenotype in Mouse Models of Dystroglycanopathy

    Yu, Miao; He, Yonglin; Wang, Kejian; Zhang, Peng; Zhang, Shengle; Hu, Huaiyu

    2013-01-01

    Dystroglycanopathies are a group of congenital muscular dystrophies (CMD) often caused by mutations in genes encoding glycosyltransferases that lead to hypoglycosylation of α-dystroglycan (α-DG) and reduce its extracellular matrix-binding activity. Overexpressing LARGE (formerly known as like-glycosyltransferase) generates an extracellular matrix-binding carbohydrate epitope in cells with CMD-causing mutations in not only LARGE but also other glycosyltransferases, including POMT1, POMGnT1, an...

  10. Adeno-Associated Virus Overexpression of Angiotensin-Converting Enzyme-2 Reverses Diabetic Retinopathy in Type 1 Diabetes in Mice.

    Dominguez, James M; Hu, Ping; Caballero, Sergio; Moldovan, Leni; Verma, Amrisha; Oudit, Gavin Y; Li, Qiuhong; Grant, Maria B

    2016-06-01

    Angiotensin-converting enzyme (ACE)-2 is the primary enzyme of the vasoprotective axis of the renin angiotensin system that regulates the classic renin angiotensin system axis. We aimed to determine whether local retinal overexpression of adenoassociated virus (AAV)-ACE2 prevents or reverses diabetic retinopathy. Green fluorescent protein (GFP)-chimeric mice were generated to distinguish resident (retinal) from infiltrating bone marrow-derived inflammatory cells and were made diabetic using streptozotocin injections. Retinal digestion using trypsin was performed and acellular capillaries enumerated. Capillary occlusion by GFP(+) cells was used to measure leukostasis. Overexpression of ACE2 prevented (prevention cohort: untreated diabetic, 11.3 ± 1.4; ACE2 diabetic, 6.4 ± 0.9 per mm(2)) and partially reversed (reversal cohort: untreated diabetic, 15.7 ± 1.9; ACE2 diabetic, 6.5 ± 1.2 per mm(2)) the diabetes-associated increase of acellular capillaries and the increase of infiltrating inflammatory cells into the retina (F4/80(+)) (prevention cohort: untreated diabetic, 24.2 ± 6.7; ACE2 diabetic, 2.5 ± 1.6 per mm(2); reversal cohort: untreated diabetic, 56.8 ± 5.2; ACE2 diabetic, 5.6 ± 2.3 per mm(2)). In both study cohorts, intracapillary bone marrow-derived cells, indicative of leukostasis, were only observed in diabetic animals receiving control AAV injections. These results indicate that diabetic retinopathy, and possibly other diabetic microvascular complications, can be prevented and reversed by locally restoring the balance between the classic and vasoprotective renin angiotensin system. PMID:27178803

  11. MicroRNA in rectal cancer

    Azizian, Azadeh; Gruber, Jens; Ghadimi, B Michael; Gaedcke, Jochen

    2016-01-01

    In rectal cancer, one of the most common cancers worldwide, the proper staging of the disease determines the subsequent therapy. For those with locally advanced rectal cancer, a neoadjuvant chemoradiotherapy (CRT) is recommended before any surgery. However, response to CRT ranges from complete response (responders) to complete resistance (non-responders). To date we are not able to separate in advance the first group from the second, due to the absence of a valid biomarker. Therefore all patients receive the same therapy regardless of whether they reap benefits. On the other hand almost all patients receive a surgical resection after the CRT, although a watch-and-wait procedure or an endoscopic resection might be sufficient for those who responded well to the CRT. Being highly conserved regulators of gene expression, microRNAs (miRNAs) seem to be promising candidates for biomarkers. Many studies have been analyzing the miRNAs expressed in rectal cancer tissue to determine a specific miRNA profile for the ailment. Unfortunately, there is only a small overlap of identified miRNAs between different studies, posing the question as to whether different methods or differences in tissue storage may contribute to that fact or if the results simply are not reproducible, due to unknown factors with undetected influences on miRNA expression. Other studies sought to find miRNAs which correlate to clinical parameters (tumor grade, nodal stage, metastasis, survival) and therapy response. Although several miRNAs seem to have an impact on the response to CRT or might predict nodal stage, there is still only little overlap between different studies. We here aimed to summarize the current literature on rectal cancer and miRNA expression with respect to the different relevant clinical parameters. PMID:27190581

  12. MicroRNA expression correlated with hygienic behaviour in honeybees

    Francesca Dell'Orco

    2015-07-01

    Full Text Available Honeybees (Apis mellifera play important roles in modern agriculture regarding zootechnical production and crop pollination. Recently, honeybees have received more attention from the public, beekeepers and researchers due to emerging heath issues. Thus, scientific interest for honeybee health and selection resistance to major pathogens is sharply increasing. Honeybees evolved social immunity mechanisms consisting in the cooperation of individuals to control disease level in the hive, and in particular hygienic behavior (HB, as based on the uncapping and removal of dead, diseased or parasitized brood. HB is affected by heritable and environmental factors, and specific neurogenomic states can be inferred based on the coordinated brain expression of transcription factors and their predicted target genes, including Mblk-1 (transcription factor that function in the mushroom body and Obp4 (sensitive olfactory detection in the antennae of adult bees. Besides, microRNAs are known to influence neurological status linked to age-related social behaviour in honeybees7. In order to investigate the relationship between microRNA expression and HB, the present work performed the expression profile of selected honeybee brain microRNA in individual’s honeybee from field colonies with high HB level compared to low HB level, in comparison with the expression profile of Mblk-1 and Obp4. The genetic information resulting from this project could help to understand the role of microRNAs in HB and to drive honeybee selection schemes for production, health, and behavioral traits favoring pathogen control.

  13. Pharmacogenomics genes show varying perceptibility to microRNA regulation

    Rukov, Jakob Lewin; Vinther, Jeppe; Shomron, Noam

    2011-01-01

    The aim of pharmacogenomics is to identify individual differences in genome and transcriptome composition and their effect on drug efficacy. MicroRNAs (miRNAs) are short noncoding RNAs that negatively regulate expression of the majority of animal genes, including many genes involved in drug effic...

  14. Differential microRNA expression in blood in multiple sclerosis

    Søndergaard, Helle Bach; Hesse, Dan; Krakauer, Martin;

    2013-01-01

    microRNAs (miRNAs) regulate the expression of the genome at the post-transcriptional level. They play a role in autoimmunity and inflammation, and show potential for use as therapeutic targets in many diseases. With the recent detection of miRNAs in body fluids, the possibility for using miRNAs a...

  15. Subgenomic analysis of microRNAs in polyploid wheat

    Kantar, M.; Akpinar, B. A.; Valárik, Miroslav; Lucas, S. J.; Doležel, Jaroslav; Hernandez, P.; Budak, H.

    2012-01-01

    Roč. 12, č. 3 (2012), s. 465-479. ISSN 1438-793X Grant ostatní: GA MŠk(CZ) ED0007/01/01 Institutional research plan: CEZ:AV0Z50380511 Keywords : Triticum aestivum * microRNA * miRNA prediction Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.292, year: 2012

  16. Brain expressed microRNAs implicated in schizophrenia etiology

    Hansen, Thomas; Olsen, Line; Lindow, Morten;

    2007-01-01

    Protein encoding genes have long been the major targets for research in schizophrenia genetics. However, with the identification of regulatory microRNAs (miRNAs) as important in brain development and function, miRNAs genes have emerged as candidates for schizophrenia-associated genetic factors. I...

  17. Reproducible pattern of microRNA in normal human skin

    Holst, Line; Kaczkowski, Bogumil; Gniadecki, Robert

    2010-01-01

    MicroRNAs (miRNAs) regulate cell growth, differentiation and apoptosis via specific targeting of messenger RNA (mRNA). Aberrant mRNA expression contributes to pathological processes such as carcinogenesis. To take advantage of miRNA profiling in skin disease it is essential to investigate mi...

  18. Aberrant microRNA expression in multiple myeloma

    Dimopoulos, Konstantinos; Gimsing, Peter; Grønbæk, Kirsten

    2013-01-01

    Multiple myeloma (MM) is a devastating disease with a complex biology, and in spite of improved survivability by novel treatment strategies over the last decade, MM is still incurable by current therapy. MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at a post...

  19. The MicroRNA Repertoire in Enteroendocrine Cells

    Knudsen, Lina A; Petersen, Natalia; Schwartz, Thue W; Egerod, Kristoffer L

    2015-01-01

    Micro-RNAs (miRNAs) are crucial for many biological processes, but their role in the enteroendocrine development and differentiation has been neglected due to the elusive nature of the enteroendocrine cells. However, transgenic mice expressing fluorescent reporter proteins under the control of...

  20. microRNA-101 is a potent inhibitor of autophagy

    Frankel, Lisa B; Wen, Jiayu; Lees, Michael; Høyer-Hansen, Maria; Farkas, Thomas; Krogh, Anders; Jäättelä, Marja; Lund, Anders H

    2011-01-01

    performed a functional screen in search of microRNAs (miRNAs), which regulate the autophagic flux in breast cancer cells. In this study, we identified the tumour suppressive miRNA, miR-101, as a potent inhibitor of basal, etoposide- and rapamycin-induced autophagy. Through transcriptome profiling, we...

  1. Intratumoral Heterogeneity of MicroRNA Expression in Rectal Cancer

    Eriksen, Anne Haahr Mellergaard; Andersen, Rikke Fredslund; Nielsen, Boye Schnack; Sørensen, Flemming Brandt; Appelt, Ane Lindegaard; Jakobsen, Anders; Hansen, Torben Frøstrup

    2016-01-01

    INTRODUCTION: An increasing number of studies have investigated microRNAs (miRNAs) as potential markers of diagnosis, treatment and prognosis. So far, agreement between studies has been minimal, which may in part be explained by intratumoral heterogeneity of miRNA expression. The aim of the present...

  2. Circulating microRNAs as biomarkers of adult Crohn's disease

    Jensen, Michael D; Andersen, Rikke F; Christensen, Henry;

    2015-01-01

    OBJECTIVE: Previous studies have found a differential expression of microRNAs (miRNAs) in the blood of patients with Crohn's disease (CD) compared with healthy controls. The aim of this study was to identify circulating miRNAs expressed in CD and assess their performance as biomarkers in patients...

  3. MicroRNA Related Polymorphisms and Breast Cancer Risk

    Khan, Sofia; Greco, Dario; Michailidou, Kyriaki;

    2014-01-01

    Genetic variations, such as single nucleotide polymorphisms (SNPs) in microRNAs (miRNA) or in the miRNA binding sites may affect the miRNA dependent gene expression regulation, which has been implicated in various cancers, including breast cancer, and may alter individual susceptibility to cancer...

  4. miRBase: integrating microRNA annotation and deep-sequencing data.

    Kozomara, Ana; Griffiths-Jones, Sam

    2011-01-01

    miRBase is the primary online repository for all microRNA sequences and annotation. The current release (miRBase 16) contains over 15,000 microRNA gene loci in over 140 species, and over 17,000 distinct mature microRNA sequences. Deep-sequencing technologies have delivered a sharp rise in the rate of novel microRNA discovery. We have mapped reads from short RNA deep-sequencing experiments to microRNAs in miRBase and developed web interfaces to view these mappings. The user can view all read data associated with a given microRNA annotation, filter reads by experiment and count, and search for microRNAs by tissue- and stage-specific expression. These data can be used as a proxy for relative expression levels of microRNA sequences, provide detailed evidence for microRNA annotations and alternative isoforms of mature microRNAs, and allow us to revisit previous annotations. miRBase is available online at: http://www.mirbase.org/. PMID:21037258

  5. Characterization and identification of microRNA core promoters in four model species.

    Xuefeng Zhou

    2007-03-01

    Full Text Available MicroRNAs are short, noncoding RNAs that play important roles in post-transcriptional gene regulation. Although many functions of microRNAs in plants and animals have been revealed in recent years, the transcriptional mechanism of microRNA genes is not well-understood. To elucidate the transcriptional regulation of microRNA genes, we study and characterize, in a genome scale, the promoters of intergenic microRNA genes in Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, and Oryza sativa. We show that most known microRNA genes in these four species have the same type of promoters as protein-coding genes have. To further characterize the promoters of microRNA genes, we developed a novel promoter prediction method, called common query voting (CoVote, which is more effective than available promoter prediction methods. Using this new method, we identify putative core promoters of most known microRNA genes in the four model species. Moreover, we characterize the promoters of microRNA genes in these four species. We discover many significant, characteristic sequence motifs in these core promoters, several of which match or resemble the known cis-acting elements for transcription initiation. Among these motifs, some are conserved across different species while some are specific to microRNA genes of individual species.

  6. MicroRNA evolution, expression, and function during short germband development in Tribolium castaneum.

    Ninova, Maria; Ronshaugen, Matthew; Griffiths-Jones, Sam

    2016-01-01

    MicroRNAs are well-established players in the development of multicellular animals. Most of our understanding of microRNA function in arthropod development comes from studies in Drosophila. Despite their advantages as model systems, the long germband embryogenesis of fruit flies is an evolutionary derived state restricted to several holometabolous insect lineages. MicroRNA evolution and expression across development in animals exhibiting the ancestral and more widespread short germband mode of embryogenesis has not been characterized. We sequenced small RNA libraries of oocytes and successive intervals covering the embryonic development of the short germband model organism, Tribolium castaneum. We analyzed the evolution and temporal expression of the microRNA complement and sequenced libraries of total RNA to investigate the relationships with microRNA target expression. We show microRNA maternal loading and sequence-specific 3' end nontemplate oligoadenylation of maternally deposited microRNAs that is conserved between Tribolium and Drosophila. We further uncover large clusters encoding multiple paralogs from several Tribolium-specific microRNA families expressed during a narrow interval of time immediately after the activation of zygotic transcription. These novel microRNAs, together with several early expressed conserved microRNAs, target a significant number of maternally deposited transcripts. Comparison with Drosophila shows that microRNA-mediated maternal transcript targeting is a conserved process in insects, but the number and sequences of microRNAs involved have diverged. The expression of fast-evolving and species-specific microRNAs in the early blastoderm of T. castaneum is consistent with previous findings in Drosophila and shows that the unique permissiveness for microRNA innovation at this stage is a conserved phenomenon. PMID:26518483

  7. Computational Characterization of Exogenous MicroRNAs that Can Be Transferred into Human Circulation.

    Jiang Shu

    Full Text Available MicroRNAs have been long considered synthesized endogenously until very recent discoveries showing that human can absorb dietary microRNAs from animal and plant origins while the mechanism remains unknown. Compelling evidences of microRNAs from rice, milk, and honeysuckle transported to human blood and tissues have created a high volume of interests in the fundamental questions that which and how exogenous microRNAs can be transferred into human circulation and possibly exert functions in humans. Here we present an integrated genomics and computational analysis to study the potential deciding features of transportable microRNAs. Specifically, we analyzed all publicly available microRNAs, a total of 34,612 from 194 species, with 1,102 features derived from the microRNA sequence and structure. Through in-depth bioinformatics analysis, 8 groups of discriminative features have been used to characterize human circulating microRNAs and infer the likelihood that a microRNA will get transferred into human circulation. For example, 345 dietary microRNAs have been predicted as highly transportable candidates where 117 of them have identical sequences with their homologs in human and 73 are known to be associated with exosomes. Through a milk feeding experiment, we have validated 9 cow-milk microRNAs in human plasma using microRNA-sequencing analysis, including the top ranked microRNAs such as bta-miR-487b, miR-181b, and miR-421. The implications in health-related processes have been illustrated in the functional analysis. This work demonstrates the data-driven computational analysis is highly promising to study novel molecular characteristics of transportable microRNAs while bypassing the complex mechanistic details.

  8. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    Steven W Paugh

    2016-02-01

    Full Text Available MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16 for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

  9. MicroRNA-338 and microRNA-21 co-transfection for the treatment of rat sciatic nerve injury.

    Wang, Jianyong; Muheremu, Aikeremujiang; Zhang, Ming; Gong, Kai; Huang, Chuyi; Ji, Yuchen; Wei, Yujun; Ao, Qiang

    2016-06-01

    The objective of this study is to find if co-transfecting microRNA-338 and microRNA-21 into the neurons in the spinal cord can promote functional recovery after peripheral nerve injury in rats. Animals were divided into three groups: 20 animals in the GFP control vector group (group A), 20 animals in the GFP experimental vector group (group B) and ten animals in the normal control group. Right sciatic nerves of animals in groups A and B were transected and were bridged with collagen nerve conduits with 10 mm distance between the stumps. 3 µl GFP control vector or 3 µl lentiviral vectors encoding the sequence of microRNA-338 and microRNA-21 were injected in the conduit. 8 weeks after the surgery, the treatment effect was evaluated by functional analysis, electrophysiological analysis, immunohistochemical analysis as well as transmitting electronic microscope observations in all the rats. Animals treated with microRNA-338 and microRNA-21 showed significantly better recovery than GFP control group animals by means of functional analysis (Sciatic nerve index -47.7 ± 2.5 vs -59.4 ± 3.7), electrophysiological analysis (Conduction velocity 20.5 ± 2.8 vs 10.5 ± 1.4 m/s), ratio of wet weight of the gastrocnemius muscles (0.83 ± 0.03 vs 0.55 ± 0.06), axon diameter (5.0 ± 1.8 µm vs 4.0 ± 2.2), myelin sheath thickness (1.4 ± 0.43 vs 0.80 ± 0.31 µm) and G-ratio (0.80 ± 0.06 vs 0.75 ± 0.04). Lentiviral vectors encoding microRNA 338 and 21 might be explored in the future as potential therapeutic intervention to promote nerve regeneration. PMID:26909749

  10. Identification of Drosophila MicroRNA Targets

    Stark Alexander

    2003-01-01

    Full Text Available MicroRNAs (miRNAs are short RNA molecules that regulate gene expression by binding to target messenger RNAs and by controlling protein production or causing RNA cleavage. To date, functions have been assigned to only a few of the hundreds of identified miRNAs, in part because of the difficulty in identifying their targets. The short length of miRNAs and the fact that their complementarity to target sequences is imperfect mean that target identification in animal genomes is not possible by standard sequence comparison methods. Here we screen conserved 3' UTR sequences from the Drosophila melanogaster genome for potential miRNA targets. The screening procedure combines a sequence search with an evaluation of the predicted miRNA-target heteroduplex structures and energies. We show that this approach successfully identifies the five previously validated let-7, lin-4, and bantam targets from a large database and predict new targets for Drosophila miRNAs. Our target predictions reveal striking clusters of functionally related targets among the top predictions for specific miRNAs. These include Notch target genes for miR-7, proapoptotic genes for the miR-2 family, and enzymes from a metabolic pathway for miR-277. We experimentally verified three predicted targets each for miR-7 and the miR-2 family, doubling the number of validated targets for animal miRNAs. Statistical analysis indicates that the best single predicted target sites are at the border of significance; thus, target predictions should be considered as tentative until experimentally validated. We identify features shared by all validated targets that can be used to evaluate target predictions for animal miRNAs. Our initial evaluation and experimental validation of target predictions suggest functions for two miRNAs. For others, the screen suggests plausible functions, such as a role for miR-277 as a metabolic switch controlling amino acid catabolism. Cross-genome comparison proved essential

  11. Sequence fingerprints of microRNA conservation.

    Bing Shi

    Full Text Available It is known that the conservation of protein-coding genes is associated with their sequences both various species, such as animals and plants. However, the association between microRNA (miRNA conservation and their sequences in various species remains unexplored. Here we report the association of miRNA conservation with its sequence features, such as base content and cleavage sites, suggesting that miRNA sequences contain the fingerprints for miRNA conservation. More interestingly, different species show different and even opposite patterns between miRNA conservation and sequence features. For example, mammalian miRNAs show a positive/negative correlation between conservation and AU/GC content, whereas plant miRNAs show a negative/positive correlation between conservation and AU/GC content. Further analysis puts forward the hypothesis that the introns of protein-coding genes may be a main driving force for the origin and evolution of mammalian miRNAs. At the 5' end, conserved miRNAs have a preference for base U, while less-conserved miRNAs have a preference for a non-U base in mammals. This difference does not exist in insects and plants, in which both conserved miRNAs and less-conserved miRNAs have a preference for base U at the 5' end. We further revealed that the non-U preference at the 5' end of less-conserved mammalian miRNAs is associated with miRNA function diversity, which may have evolved from the pressure of a highly sophisticated environmental stimulus the mammals encountered during evolution. These results indicated that miRNA sequences contain the fingerprints for conservation, and these fingerprints vary according to species. More importantly, the results suggest that although species share common mechanisms by which miRNAs originate and evolve, mammals may develop a novel mechanism for miRNA origin and evolution. In addition, the fingerprint found in this study can be predictor of miRNA conservation, and the findings are helpful

  12. MicroRNA profiling in intraocular medulloepitheliomas.

    Deepak P Edward

    Full Text Available To study the differential expression of microRNA (miRNA profiles between intraocular medulloepithelioma (ME and normal control tissue (CT.Total RNA was extracted from formalin fixed paraffin embedded (FFPE intraocular ME (n=7 and from age matched ciliary body controls (n=8. The clinical history and phenotype was recorded. MiRNA profiles were determined using the Affymetrix GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation of significantly dysregulated miRNA was confirmed by quantitative real-time PCR. The web-based DNA Intelligent Analysis (DIANA-miRPath v2.0 was used to perform enrichment analysis of differentially expressed (DE miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG pathway.The pathologic evaluation revealed one benign (benign non-teratoid, n=1 and six malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4. A total of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly (P<0.05 in the tumor specimens. Many of these significantly dysregulated miRNAs were known to play various roles in carcinogenesis and tumor behavior. RT-PCR validated three significantly upregulated miRNAs and three significantly downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive human cartilage. Enriched pathway analysis suggested a significant association of upregulated miRNAs with 15 pathways involved in prion disease and several types of cancer. The pathways involving significantly downregulated miRNAs included the toll-like receptor (TLR (p<4.36E-16 and Nuclear Factor kappa B (NF-κB signaling pathways (p<9.00E-06.We report significantly dysregulated miRNAs in intraocular ME tumors, which exhibited abnormal profiles in other cancers as well such as retinoblastoma and glioblastoma. Pathway analysis

  13. MicroRNAs: The Missing Link in the Biology of Graft-Versus-Host Disease?

    Atarod, Sadaf; Dickinson, Anne Mary

    2013-01-01

    Graft-versus-host disease (GVHD) is still the major complication of allogeneic hematopoietic stem cell transplantation. Despite extensive studies in understanding the pathophysiology of GVHD, its pathogenesis remains unclear. Recently, important functions of microRNAs have been demonstrated in various autoimmune diseases and cancers such as psoriasis and lymphoma. This review highlights the need to investigate the role of microRNAs in GVHD and hypothesizes that microRNAs may be one of the mis...

  14. Role of microRNAs from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma

    Calvo, Katherine R.; Landgren, C. Ola; Roccaro, Aldo M.; Ghobrial, Irene M.

    2011-01-01

    microRNAs are increasingly recognized as significant players in oncogenesis and tumor biology through post-transcriptional gene regulation impacting broad pathways of proliferation, differentiation, apoptosis, metastasis and cell survival. Recent studies have found abnormal expression of microRNAs in multiple myeloma (MM). Currently, the precise role of these microRNAs in the biology of MM remains to be elucidated, although they are predicted to be involved in plasma cell proliferation, survi...

  15. Distinct microRNA alterations characterize high- and low-grade bladder cancer.

    Catto, J W F; Miah, S; Owen, H C; Bryant, H.; Myers, K.; Dudziec, E.; Larre, S.; Milo, M.; Rehman, I; Rosario, D. J.; Di Martino, E; Knowles, M. A.; Meuth, M; Harris, A.L.; Hamdy, F C

    2009-01-01

    Urothelial carcinoma of the bladder (UCC) is a common disease that arises by at least two different molecular pathways. The biology of UCC is incompletely understood, making the management of this disease difficult. Recent evidence implicates a regulatory role for microRNA in cancer. We hypothesized that altered microRNA expression contributes to UCC carcinogenesis. To test this hypothesis, we examined the expression of 322 microRNAs and their processing machinery in 78 normal and malignant u...

  16. MicroRNAs in Respiratory Disease. A Clinician’s Overview

    Brown, Derek; Rahman, Mohammad; Nana-Sinkam, S Patrick

    2014-01-01

    Since their initial discovery in the early 1990s, microRNAs have now become the focus of a multitude of lines of investigation ranging from basic biology to translational applications in the clinic. Previously believed to be of no biological relevance, microRNAs regulate processes fundamental to human health and disease. In diseases of the lung, microRNAs have been implicated in developmental programming, as drivers of disease, potential therapeutic targets, and clinical biomarkers; however, ...

  17. The Potential of MicroRNAs in Personalized Medicine against Cancers

    Anne Saumet; Anthony Mathelier; Charles-Henri Lecellier

    2014-01-01

    MicroRNAs orchestrate the expression of the genome and impact many, if not all, cellular processes. Their deregulation is thus often causative of human malignancies, including cancers. Numerous studies have implicated microRNAs in the different steps of tumorigenesis including initiation, progression, metastasis, and resistance to chemo/radiotherapies. Thus, microRNAs constitute appealing targets for novel anticancer therapeutic strategies aimed at restoring their expression or function. As m...

  18. MicroRNAs are potential therapeutic targets in fibrosing kidney disease: lessons from animal models

    Duffield, Jeremy S.; Grafals, Monica; Portilla, Didier

    2012-01-01

    Chronic disease of the kidneys has reached epidemic proportions in industrialized nations. New therapies are urgently sought. Using a combination of animal models of kidney disease and human biopsy samples, a pattern of dysregulated microRNA expression has emerged which is common to chronic diseases. A number of these dysregulated microRNA have recently been shown to have functional consequences for the disease process and therefore may be potential therapeutic targets. We highlight microRNA-...

  19. MicroRNAs are potential therapeutic targets in fibrosing kidney disease: lessons from animal models

    Duffield, Jeremy S; Grafals, Monica; Portilla, Didier

    2012-01-01

    Chronic disease of the kidneys has reached epidemic proportions in industrialized nations. New therapies are urgently sought. Using a combination of animal models of kidney disease and human biopsy samples, a pattern of dysregulated microRNA expression has emerged which is common to chronic diseases. A number of these dysregulated microRNA have recently been shown to have functional consequences for the disease process and therefore may be potential therapeutic targets. We highlight microRNA-21, the most comprehensively studied microRNA in the kidney so far. MicroRNA-21 is expressed widely in healthy kidney but studies from knockout mice indicate it is largely inert. Although microRNA-21 is upregulated in many cell compartments including leukocytes, epithelial cells and myofibroblasts, the inert microRNA-21 also appears to become activated, by unclear mechanisms. Mice lacking microRNA-21 are protected from kidney injury and fibrosis in several distinct models of kidney disease, and systemically administered oligonucleotides that specifically bind to the active site in microRNA-21, inhibiting its function, recapitulate the genetic deletion of microRNA-21, suggesting inhibitory oligonucleotides may have therapeutic potential. Recent studies of microRNA-21 targets in kidney indicate that it normally functions to silence metabolic pathways including fatty acid metabolism and pathways that prevent Reactive Oxygen Species generation in peroxisomes and mitochondria in epithelial cells and myofibroblasts. Targeting specific pathogenic microRNAs in a specific manner is feasible in vivo and may be a new therapeutic target in disease of the kidney PMID:25018773

  20. Intra-Platform Repeatability and Inter-Platform Comparability of MicroRNA Microarray Technology

    Sato, Fumiaki; Tsuchiya, Soken; Terasawa, Kazuya; Tsujimoto, Gozoh

    2009-01-01

    Over the last decade, DNA microarray technology has provided a great contribution to the life sciences. The MicroArray Quality Control (MAQC) project demonstrated the way to analyze the expression microarray. Recently, microarray technology has been utilized to analyze a comprehensive microRNA expression profiling. Currently, several platforms of microRNA microarray chips are commercially available. Thus, we compared repeatability and comparability of five different microRNA microarray platfo...

  1. MicroRNAs and lung cancer: Biology and applications in diagnosis and prognosis

    Mallick Reema; Patnaik Santosh; Yendamuri Sai

    2010-01-01

    MicroRNAs are tiny non-coding RNA molecules which play important roles in the epigenetic control of cellular processes by preventing the translation of proteins from messenger RNAs (mRNAs). A single microRNA can target different mRNAs, and an mRNA can be targeted by multiple microRNAs. Such complex interplays underlie many molecular pathways in cells, and specific roles for many microRNAs in physiological as well as pathological phenomena have been identified. Changes in expression of microRN...

  2. Properties and kinetics of microRNA regulation through canonical seed sites

    Chen, Jerry S.; Revilla, Arra C; Guerrero, Michael; Abygail M Gumbayan; Zeller, Robert W.

    2015-01-01

    MicroRNAs are a fundamental class of small RNAs involved in post-transcriptional gene regulation; however, the mechanism by which microRNAs regulate their gene targets in animals remains poorly understood. Practically, a mechanistic understanding of microRNA binding and regulation is crucial for the rational design of microRNA-based vectors for RNA interference. In this report, we focus on the largest known class of microRNA targets, the canonical seed targets, and explore the factors involve...

  3. MicroRNA expression profiling in neurogenesis of adipose tissue-derived stem cells

    Jung Ah Cho; Ho Park; Eun Hye Lim; Kyo Won Lee

    2011-04-01

    Adipose tissue-derived stem cells (ADSCs) are one population of adult stem cells that can self renew and differentiate into multiple lineages. Because of advantages in method and quantity of acquisition, ADSCs are gaining attention as an alternative source of bone marrow mesenchymal stem cells. In this study, we performed microRNA profiling of undifferentiated and of neurally-differentiated ADSCs to identify the responsible microRNAs in neurogenesis using this type of stem cell. MicroRNAs from four different donors were analysed by microarray. Compared to the undifferentiation control, we identified 39–101 microRNAs with more than two-fold higher expression and 3–9 microRNAs with two-fold lower expression. The identified microRNAs were further analysed in terms of gene ontology (GO) in relation with neurogenesis, based on their target mRNAs predicted by computational analysis. This study revealed the specific microRNAs involved in neurogenesis via microRNA microarray, and may provide the basic information for genetic induction of adult stem cell differentiation using microRNAs.

  4. miRBase: annotating high confidence microRNAs using deep sequencing data.

    Kozomara, Ana; Griffiths-Jones, Sam

    2014-01-01

    We describe an update of the miRBase database (http://www.mirbase.org/), the primary microRNA sequence repository. The latest miRBase release (v20, June 2013) contains 24 521 microRNA loci from 206 species, processed to produce 30 424 mature microRNA products. The rate of deposition of novel microRNAs and the number of researchers involved in their discovery continue to increase, driven largely by small RNA deep sequencing experiments. In the face of these increases, and a range of microRNA annotation methods and criteria, maintaining the quality of the microRNA sequence data set is a significant challenge. Here, we describe recent developments of the miRBase database to address this issue. In particular, we describe the collation and use of deep sequencing data sets to assign levels of confidence to miRBase entries. We now provide a high confidence subset of miRBase entries, based on the pattern of mapped reads. The high confidence microRNA data set is available alongside the complete microRNA collection at http://www.mirbase.org/. We also describe embedding microRNA-specific Wikipedia pages on the miRBase website to encourage the microRNA community to contribute and share textual and functional information. PMID:24275495

  5. Differential expression of microRNAs in dorsal root ganglia after sciatic nerve injury

    Anjie Lu; Zufa Huang; Chaoyue Zhang; Xianfang Zhang; Jiuhong Zhao; Haiying Zhang; Quanpeng Zhang; Song Wu; Xinan Yi

    2014-01-01

    This study investigated the possible involvement of microRNAs in the regulation of genes that participate in peripheral neural regeneration. A microRNA microarray analysis was conducted and 23 microRNAs were identiifed whose expression was signiifcantly changed in rat dorsal root ganglia after sciatic nerve transection. The expression of one of the downregulated microRNAs, microRNA-214, was validated using quantitative reverse transcriptase-PCR. MicroRNA-214 was predicted to target the 3′-untranslated region of Slit-Robo GTPase-activating protein 3. In situ hybridization veriifed that microRNA-214 was located in the cytoplasm of dorsal root ganglia primary neurons and was downregulated following sciatic nerve transection. Moreover, a com-bination of in situ hybridization and immunohistochemistry revealed that microRNA-214 and Slit-Robo GTPase-activating protein 3 were co-localized in dorsal root ganglion primary neu-rons. Western blot analysis suggested that Slit-Robo GTPase-activating protein 3 was upregulated in dorsal root ganglion neurons after sciatic nerve transection. These data demonstrate that mi-croRNA-214 is located and differentially expressed in dorsal root ganglion primary neurons and may participate in regulating the gene expression of Slit-Robo GTPase-activating protein 3 after sciatic nerve transection.

  6. MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

    Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA

  7. Vive la différence: biogenesis and evolution of microRNAs in plants and animals

    Axtell, Michael J.; Westholm, Jakub O.; Eric C Lai

    2011-01-01

    MicroRNAs are pervasive in both plants and animals, but many aspects of their biogenesis, function and evolution differ. We reveal how these differences contribute to characteristic features of microRNA evolution in the two kingdoms.

  8. MicroRNA 10a marks regulatory T cells

    Jeker, Lukas T; Zhou, Xuyu; Gershberg, Kseniya;

    2012-01-01

    MicroRNAs (miRNAs) are crucial for regulatory T cell (Treg) stability and function. We report that microRNA-10a (miR-10a) is expressed in Tregs but not in other T cells including individual thymocyte subsets. Expression profiling in inbred mouse strains demonstrated that non-obese diabetic (NOD...... vitro TGF-ß-induced, iTregs do not express miR-10a unless cultured in the presence of retinoic acid (RA) which has been associated with increased stability of iTreg, suggesting that miR-10a might play a role in stabilizing Treg. However, genetic ablation of miR-10a neither affected the number and...

  9. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    Paugh, Steven W; Coss, David R; Bao, Ju; Laudermilk, Lucas T; Grace, Christy R; Ferreira, Antonio M; Waddell, M Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F; Panetta, John C; Wilkinson, Mark R; Pui, Ching-Hon; Naeve, Clayton W; Uberbacher, Edward C; Bonten, Erik J; Evans, William E

    2016-02-01

    MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, ptriplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

  10. Therapeutic evaluation of microRNA-15a and microRNA-16 in ovarian cancer

    Dhar Dwivedi, Shailendra Kumar; Mustafi, Soumyajit Banerjee; Mangala, Lingegowda S.; Jiang, Dahai; Pradeep, Sunila; Rodriguez-Aguayo, Cristian; Ling, Hui; Ivan, Cristina; Mukherjee, Priyabrata; Calin, George A.; Lopez-Berestein, Gabriel; Sood, Anil K.; Bhattacharya, Resham

    2016-01-01

    Treatment of chemo-resistant ovarian cancer (OvCa) remains clinically challenging and there is a pressing need to identify novel therapeutic strategies. Here we report that multiple mechanisms that promote OvCa progression and chemo-resistance could be inhibited by ectopic expression of miR-15a and miR-16. Significant correlations between low expression of miR-16, high expression of BMI1 and shortened overall survival (OS) were noted in high grade serous (HGS) OvCa patients upon analysis of The Cancer Genome Atlas (TCGA). Targeting BMI1, in vitro with either microRNA reduced clonal growth of OvCa cells. Additionally, epithelial to mesenchymal transition (EMT) as well as expression of the cisplatin transporter ATP7B were inhibited by miR-15a and miR-16 resulting in decreased degradation of the extra-cellular matrix and enhanced sensitization of OvCa cells to cisplatin. Nanoliposomal delivery of the miR-15a and miR-16 combination, in a pre-clinical chemo-resistant orthotopic mouse model of OvCa, demonstrated striking reduction in tumor burden compared to cisplatin alone. Thus, with the advent of miR replacement therapy some of which are in Phase 2 clinical trials, miR-15a and miR-16 represent novel ammunition in the anti-OvCa arsenal. PMID:26918603

  11. Efficient Silencing of Endogenous MicroRNAs Using Artificial MicroRNAs in Arabidopsis thaliana

    Andrew L. Eamens; Claire Agius; Neil A. Smith; Peter M. Waterhouse; Ming-Bo Wang

    2011-01-01

    We report here that the expression of endogenous microRNAs (miRNAs) can be efficiently silenced in Arabidopsis thaliana (Arabidopsis) using artificial miRNA (amiRNA) technology. We demonstrate that an amiRNA designed to target a mature miRNA directs silencing against all miRNA family members, whereas an amiRNA designed to target the stem-loop region of a miRNA precursor transcript directs silencing against only the individual family member targeted.Furthermore, our results indicate that amiRNAs targeting both the mature miRNA and stem-loop sequence direct RNA silencing through cleavage of the miRNA precursor transcript, which presumably occurs in the nucleus of a plant cell during the initial stages of miRNA biogenesis. This suggests that small RNA (sRNA)-guided RNA cleavage in plants occurs not only in the cytoplasm, but also in the nucleus. Many plant miRNA gene families have been identified via sequencing and bioinformatic analysis, but, to date, only a small tranche of these have been functionally characterized due to a lack of effective forward or reverse genetic tools. Our findings therefore provide a new and powerful reverse-genetic tool for the analysis of miRNA function in plants.

  12. Pathological microRNAs in acute cardiovascular diseases and microRNA therapeutics

    Syed Salman Ali; Chandra Kala; Mohd Abid; Nabeel Ahmad; Uma Shankar Sharma; Najam Ali Khan

    2016-01-01

    Cardiovascular diseases are one of the leading causes of morbidity and mortality. In recent researches, it is demonstrated that microRNAs (miRNAs) are expressed exten-sively in cardiovascular system and regulate gene expression in various cardiovascular diseases. Here, we are giving overview on number of miRNAs involved in patho-physiology of various cardiovascular diseases, and diagnostic and therapeutic potentials of miRNAs in these diseases. MiRNAs are a group of small non-coding mRNAs with approximately 18–22 nucleotides in length that regulate gene expression post tran-scriptionally. MiRNAs are regulated in various cardiovascular diseases like hyperten-sion, congestive heart failure, congenital heart defects, coronary artery disease and stroke. Some of these miRNAs also act as potential biomarker of these cardiovascular diseases. Inhibition of these miRNAs via different approaches like chemically modified antisense oligonucleotide, antagomirs, and locked nucleic acids serves as effective approaches for inactivating pathological miRNAs. Clinical trials are being conducted on therapeutic and diagnostic potentials of miRNAs. However, extensive researches are required to explore the therapeutic and diagnostic values of miRNAs as successful as classical approaches.

  13. Rapid generation of microRNA sponges for microRNA inhibition.

    Joost Kluiver

    Full Text Available MicroRNA (miRNA sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

  14. Androgen-Responsive MicroRNAs in Mouse Sertoli Cells

    Subbarayalu Panneerdoss; Yao-Fu Chang; Kalyan C Buddavarapu; Hung-I Harry Chen; Gunapala Shetty; Huizhen Wang; Yidong Chen; T Rajendra Kumar; Rao, Manjeet K.

    2012-01-01

    Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs – small, non-coding RNAs – are one class of androgen-regulated trans-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the ...

  15. Practical Aspects of microRNA Target Prediction

    Witkos, T.M; Koscianska, E; Krzyzosiak, W J

    2011-01-01

    microRNAs (miRNAs) are endogenous non-coding RNAs that control gene expression at the posttranscriptional level. These small regulatory molecules play a key role in the majority of biological processes and their expression is also tightly regulated. Both the deregulation of genes controlled by miRNAs and the altered miRNA expression have been linked to many disorders, including cancer, cardiovascular, metabolic and neurodegenerative diseases. Therefore, it is of particular interest to reliabl...

  16. MicroRNAs enrichment in GWAS of complex human phenotypes

    2015-01-01

    Background The genotype information carried by Genome-wide association studies (GWAS) seems to have the potential to explain more of the ‘missing heritability’ of complex human phenotypes, given improved statistical approaches. Several lines of evidence support the involvement of microRNA (miRNA) and other non-coding RNA in complex human traits and diseases. We employed a novel, genetic annotation-informed enrichment method for GWAS that captures more polygenic effects than standard GWAS anal...

  17. MicroRNA Target Recognition and Regulatory Functions

    Bartel, David P.

    2009-01-01

    MicroRNAs (miRNAs) are endogenous ~23-nt RNAs that can play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

  18. Computational methods for the identification of microRNA targets

    Zhou, Xiaofeng

    2010-01-01

    Yang Dai1, Xiaofeng Zhou21Department of Bioengineering, Department of Computer Science, College of Engineering, 2Center for Molecular Biology of Oral Diseases, College of Dentistry, and Graduate College, UIC Cancer Center, University of Illinois at Chicago, Chicago, IL, USAAbstract: MicroRNAs are pivotal regulators of development and cellular homeostasis. They act as post-transcriptional regulators, which control the stability and translation efficiency of their target mRNAs. The prediction o...

  19. The role of microRNAs in bladder cancer

    ENOKIDA, HIDEKI; YOSHINO, HIROFUMI; Matsushita, Ryosuke; Nakagawa, Masayuki

    2016-01-01

    Bladder cancer (BC) is the fifth most common cancer worldwide and is associated with significant morbidity and mortality. The prognosis of muscle invasive BC is poor, and recurrence is common after radical surgery or chemotherapy. Therefore, new diagnostic methods and treatment modalities are critical. MicroRNAs (miRNAs), a class of small noncoding RNAs, regulate the expression of protein-coding genes by repressing translation or cleaving RNA transcripts in a sequence-specific manner. miRNAs ...

  20. MicroRNAs as potential therapeutic targets in kidney disease

    Gomez, Ivan G.; Grafals, Monica; Portilla, Didier; Duffield, Jeremy S.

    2013-01-01

    One cornerstone of Chronic Kidney Disease (CKD) is fibrosis, as kidneys are susceptible due to their high vascularity and predisposition to ischemia. Presently, only therapies targeting the angiotensin receptor are used in clinical practice to retard the progression of CKD. Thus, there is a pressing need for new therapies designed to treat the damaged kidney. Several independent laboratories have identified a number of microRNAs that are dysregulated in human and animal models of CKD. We will...

  1. microRNAs: Implications for air pollution research

    Jardim, Melanie J., E-mail: melaniejardim@gmail.com [Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, Chapel Hill, NC (United States)

    2011-12-01

    The purpose of this review is to provide an update of the current understanding on the role of microRNAs in mediating genetic responses to air pollutants and to contemplate on how these responses ultimately control susceptibility to ambient air pollution. Morbidity and mortality attributable to air pollution continues to be a growing public health concern worldwide. Despite several studies on the health effects of ambient air pollution, underlying molecular mechanisms of susceptibility and disease remain elusive. In the last several years, special attention has been given to the role of epigenetics in mediating, not only genetic and physiological responses to certain environmental insults, but also in regulating underlying susceptibility to environmental stressors. Epigenetic mechanisms control the expression of gene products, both basally and as a response to a perturbation, without affecting the sequence of DNA itself. These mechanisms include structural regulation of the chromatin structure, such as DNA methylation and histone modifications, and post-transcriptional gene regulation, such as microRNA mediated repression of gene expression. microRNAs are small noncoding RNAs that have been quickly established as key regulators of gene expression. As such, miRNAs have been found to control several cellular processes including apoptosis, proliferation and differentiation. More recently, research has emerged suggesting that changes in the expression of some miRNAs may be critical for mediating biological, and ultimately physiological, responses to air pollutants. Although the study of microRNAs, and epigenetics as a whole, has come quite far in the field of cancer, the understanding of how these mechanisms regulate gene-environment interactions to environmental exposures in everyday life is unclear. This article does not necessarily reflect the views and policies of the US EPA.

  2. Functional screens for cancer-modulating microRNAs

    Poell, J.B.

    2012-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that control various cellular processes by inhibiting messenger RNAs (mRNAs) with partially complementary target sites. Many molecular pathways that are commonly perturbed in cancer, are subject to miRNA regulation. Introducing or reconstituting specific miRNAs in cancer cells may disrupt cancer progression, and ameliorate disease outcome. To identify cancer-modulating miRNAs, I created a lentiviral expression library containing the majority of all...

  3. Lowly Expressed Human MicroRNA Genes Evolve Rapidly

    Liang, Han; Li, Wen-Hsiung

    2009-01-01

    To study the evolution of human microRNAs (miRNAs), we examined nucleotide variation in humans, sequence divergence between species, and genomic clustering patterns for miRNAs with different expression levels. We found that expression level is a major indicator of the rate of evolution and that ∼30% of currently annotated human miRNA genes are almost free of selective pressure.

  4. microRNAs may sharpen spatial expression patterns

    Levine, Erel; McHale, Peter; Levine, Herbert

    2007-01-01

    The precise layout of gene expression patterns is a crucial step in development. Formation of a sharp boundary between high and low expression domains requires a genetic mechanism which is both sensitive and robust to fluctuations, a demand that may not be easily achieved by morphogens alone. Recently it has been demonstrated that small RNAs (and, in particular, microRNAs) play many roles in embryonic development. While some RNAs are essential for embryogenesis, others are limited to fine-tun...

  5. MicroRNA as therapeutic targets for treatment of depression

    Hansen KF; Obrietan K

    2013-01-01

    Katelin F Hansen, Karl Obrietan Department of Neuroscience, Ohio State University, Columbus, OH, USA Abstract: Depression is a potentially life-threatening mental disorder affecting approximately 300 million people worldwide. Despite much effort, the molecular underpinnings of clinical depression remain poorly defined, and current treatments carry limited therapeutic efficacy and potentially burdensome side effects. Recently, small noncoding RNA molecules known as microRNA (miRNA) have gaine...

  6. MicroRNAs in multiple myeloma and related bone disease

    Rossi, Marco; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2015-01-01

    MicroRNAs (miRNAs) are short non coding RNAs aberrantly expressed in solid and hematopoietic malignancies where they play a pivotal function as post-transcriptional regulators of gene expression. Recent reports have unveiled a central role of miRNAs in multiple myeloma onset and progression and preclinical findings are progressively disclosing their potential therapeutic value as drugs or targets. In this review, we provide the basic insights of miRNA biology and function, showing how these m...

  7. MicroRNA in carcinogenesis & cancer diagnostics: A new paradigm

    Ahmad, Javed; Seyed E Hasnain; Siddiqui, Maqsood A; Ahamed, Maqusood; Musarrat, Javed; Al-Khedhairy, Abdulaziz A.

    2013-01-01

    MicroRNAs (miRNAs) are small 22-25 nucleotides long non-coding RNAs, that are conserved during evolution, and control gene expression in metazoan animals, plants, viruses, and bacteria primarily at post-transcriptional and transcriptional levels. MiRNAs ultimately regulate target gene expression by degrading the corresponding mRNA and/or inhibiting their translation. Currently, the critical functions of miRNAs have been established in regulating immune system, cell proliferation, differentiat...

  8. Retrovirus infected cells contain viral microRNAs

    Klase Zachary A; Sampey Gavin C; Kashanchi Fatah

    2013-01-01

    Abstract The encoding of microRNAs in retroviral genomes has remained a controversial hypothesis despite significant supporting evidence in recent years. A recent publication demonstrating the production of functional miRNAs from the retrovirus bovine leukemia virus adds further credence to the fact that retroviruses do indeed encode their own miRNAs. Here we comment on the importance of this paper to the field, as well as examine the other known examples of miRNAs encoded by RNA viruses.

  9. Portable surface plasmon resonance biosensor to detect micrornas

    Šípová, Hana; Zhang, S. J.; Galas, D.; Wang, K.; Homola, Jiří

    Vol. XConference on Optical Chemical Sensors and Biosensors. Praha : Institute of Photonics and Electronics AS CR, v.v.i, 2010 - (Homola, J.). s. 208-208 ISBN 978-80-86269-20-7. [EUROPT(R)ODE X – X.Conference on Optical Chemical Sensors and Biosensors. 28.03.2010-31.3.2010, Praha] Institutional research plan: CEZ:AV0Z20670512 Keywords : surface plasmon resonance * microRNA * cancer diagnostics Subject RIV: JB - Sensors, Measurment, Regulation

  10. Helicobacter pylori and microRNAs: Relation with innate immunity and progression of preneoplastic conditions.

    Libânio, Diogo; Dinis-Ribeiro, Mário; Pimentel-Nunes, Pedro

    2015-10-10

    The accepted paradigm for intestinal-type gastric cancer pathogenesis is a multistep progression from chronic gastritis induced by Helicobacter pylori (H. pylori) to gastric atrophy, intestinal metaplasia, dysplasia and ultimately gastric cancer. The genetic and molecular mechanisms underlying disease progression are still not completely understood as only a fraction of colonized individuals ever develop neoplasia suggesting that bacterial, host and environmental factors are involved. MicroRNAs are noncoding RNAs that may influence H. pylori-related pathology through the regulation of the transcription and expression of various genes, playing an important role in inflammation, cell proliferation, apoptosis and differentiation. Indeed, H. pylori have been shown to modify microRNA expression in the gastric mucosa and microRNAs are involved in the immune host response to the bacteria and in the regulation of the inflammatory response. MicroRNAs have a key role in the regulation of inflammatory pathways and H. pylori may influence inflammation-mediated gastric carcinogenesis possibly through DNA methylation and epigenetic silencing of tumor suppressor microRNAs. Furthermore, microRNAs influenced by H. pylori also have been found to be involved in cell cycle regulation, apoptosis and epithelial-mesenchymal transition. Altogether, microRNAs seem to have an important role in the progression from gastritis to preneoplastic conditions and neoplastic lesions and since each microRNA can control the expression of hundreds to thousands of genes, knowledge of microRNAs target genes and their functions are of paramount importance. In this article we present a comprehensive review about the role of microRNAs in H. pylori gastric carcinogenesis, identifying the microRNAs downregulated and upregulated in the infection and clarifying their biological role in the link between immune host response, inflammation, DNA methylation and gastric carcinogenesis. PMID:26468448

  11. MicroRNA dysregulation in Spinal Cord Injury: causes, consequences and therapeutics

    Manuel eNieto-Díaz

    2014-02-01

    Full Text Available Trauma to the spinal cord causes permanent disability to more than 180,000 people every year worldwide. The initial mechanical damage triggers a complex set of secondary events involving the neural, vascular, and immune systems that largely determine the functional outcome of the spinal cord injury (SCI. Cellular and biochemical mechanisms responsible for this secondary injury largely depend on activation and inactivation of specific gene programs. Recent studies indicate that microRNAs function as gene expression switches in key processes of the SCI. Microarray data from rodent contusion models reveal that SCI induces changes in the global microRNA expression patterns. Variations in microRNA abundance largely result from alterations in the expression of the cells at the damaged spinal cord. However, microRNA expression levels after SCI are also influenced by the infiltration of immune cells to the injury site and the death and migration of specific neural cells after injury. Evidences on the role of microRNAs in the SCI pathophysiology have come from different sources. Bioinformatic analysis of microarray data has been used to identify specific variations in microRNA expression underlying transcriptional changes in target genes, which are involved in key processes in the SCI. Direct evidences on the role of microRNAs in SCI are scarcer, although recent studies have identified several microRNAs (miR-21, miR/486, miR-20 involved in key mechanisms of the SCI such as cell death or astrogliosis, among others. From a clinical perspective, different evidences make clear that microRNAs can be potent therapeutic tools to manipulate cell state and molecular processes in order to enhance functional recovery. The present article reviews the actual knowledge on how injury affects microRNA expression and the meaning of these changes in the SCI pathophysiology, to finally explore the clinical potential of microRNAs in the SCI.

  12. MicroRNA expression profiles in human cancer cells after ionizing radiation

    MicroRNAs are regulators of central cellular processes and are implicated in the pathogenesis and prognosis of human cancers. MicroRNAs also modulate responses to anti-cancer therapy. In the context of radiation oncology microRNAs were found to modulate cell death and proliferation after irradiation. However, changes in microRNA expression profiles in response to irradiation have not been comprehensively analyzed so far. The present study's intend is to present a broad screen of changes in microRNA expression following irradiation of different malignant cell lines. 1100 microRNAs (Sanger miRBase release version 14.0) were analyzed in six malignant cell lines following irradiation with clinically relevant doses of 2.0 Gy. MicroRNA levels 6 hours after irradiation were compared to microRNA levels in non-irradiated cells using the 'Geniom Biochip MPEA homo sapiens'. Hierarchical clustering analysis revealed a pattern, which significantly (p = 0.014) discerned irradiated from non-irradiated cells. The expression levels of a number of microRNAs known to be involved in the regulation of cellular processes like apoptosis, proliferation, invasion, local immune response and radioresistance (e. g. miR-1285, miR-24-1, miR-151-5p, let-7i) displayed 2 - 3-fold changes after irradiation. Moreover, several microRNAs previously not known to be radiation-responsive were discovered. Ionizing radiation induced significant changes in microRNA expression profiles in 3 glioma and 3 squamous cell carcinoma cell lines. The functional relevance of these changes is not addressed but should by analyzed by future work especially focusing on clinically relevant endpoints like radiation induced cell death, proliferation, migration and metastasis

  13. Accurate microRNA target prediction correlates with protein repression levels

    Simossis Victor A

    2009-09-01

    Full Text Available Abstract Background MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease. Results DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction. Conclusion Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT

  14. Retrieving relevant experiments: The case of microRNA microarrays.

    Açıcı, Koray; Terzi, Yunus Kasım; Oğul, Hasan

    2015-08-01

    Content-based retrieval of biological experiments in large public repositories is a recent challenge in computational biology and bioinformatics. The task is, in general, to search in a database using a query-by-example without any experimental meta-data annotation. Here, we consider a more specific problem that seeks a solution for retrieving relevant microRNA experiments from microarray repositories. A computational framework is proposed with this objective. The framework adapts a normal-uniform mixture model for identifying differentially expressed microRNAs in microarray profiling experiments. A rank-based thresholding scheme is offered to binarize real-valued experiment fingerprints based on differential expression. An effective similarity metric is introduced to compare categorical fingerprints, which in turn infers the relevance between two experiments. Two different views of experimental relevance are evaluated, one for disease association and another for embryonic germ layer, to discern the retrieval ability of the proposed model. To the best of our knowledge, the experiment retrieval task is investigated for the first time in the context of microRNA microarrays. PMID:26116091

  15. MicroRNA Regulation of Epithelial to Mesenchymal Transition

    Mohammed L. Abba

    2016-01-01

    Full Text Available Epithelial to mesenchymal transition (EMT is a central regulatory program that is similar in many aspects to several steps of embryonic morphogenesis. In addition to its physiological role in tissue repair and wound healing, EMT contributes to chemo resistance, metastatic dissemination and fibrosis, amongst others. Classically, the morphological change from epithelial to mesenchymal phenotype is characterized by the appearance or loss of a group of proteins which have come to be recognized as markers of the EMT process. As with all proteins, these molecules are controlled at the transcriptional and translational level by transcription factors and microRNAs, respectively. A group of developmental transcription factors form the backbone of the EMT cascade and a large body of evidence shows that microRNAs are heavily involved in the successful coordination of mesenchymal transformation and vice versa, either by suppressing the expression of different groups of transcription factors, or otherwise acting as their functional mediators in orchestrating EMT. This article dissects the contribution of microRNAs to EMT and analyzes the molecular basis for their roles in this cellular process. Here, we emphasize their interaction with core transcription factors like the zinc finger enhancer (E-box binding homeobox (ZEB, Snail and Twist families as well as some pluripotency transcription factors.

  16. Silencing microRNA-155 ameliorates experimental autoimmune encephalomyelitis.

    Murugaiyan, Gopal; Beynon, Vanessa; Mittal, Akanksha; Joller, Nicole; Weiner, Howard L

    2011-09-01

    IFN-γ-producing Th1 and IL-17-producing Th17 cells are the key participants in various autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Although both of these T cell subsets are known to be regulated by specific transcription factors and cytokines, the role of microRNAs that control these two inflammatory T cell subsets and whether targeting microRNAs can have therapeutic effects are not known. In this study, we show that microRNA-155 (Mir-155) expression is elevated in CD4(+) T cells during EAE, and Mir-155(-/-) mice had a delayed course and reduced severity of disease and less inflammation in the CNS. The attenuation of EAE in Mir-155(-/-) mice was associated with a decrease in Th1 and Th17 responses in the CNS and peripheral lymphoid organs. The T cell-intrinsic function of Mir-155(-/-) was demonstrated by the resistance of Mir-155(-/-) CD4(+) T cell-repleted Rag-1(-/-) mice to EAE. Finally, we found that anti-Mir-155 treatment reduced clinical severity of EAE when given before and after the appearance of clinical symptoms. These findings demonstrate that Mir-155 confers susceptibility to EAE by affecting inflammatory T cell responses and identify Mir-155 as a new target for therapeutic intervention in multiple sclerosis. PMID:21788439

  17. MIRE:A GRAPHICAL R PACKAGE FOR MICRORNA RELATED ANALYSIS

    Xing-qi Yan; Kang Tu; Lu Xie; Yi-xue Li; Bin Yin; Yan-hua Gong; Jian-gang Yuan; Bo-qin Qiang; Xiao-zhong Peng

    2008-01-01

    Objective To provide a set of useful analysis tools for the researchers to explore the microRNA data.Methods The R language was used for generating the Graphical Users Interface and implementing most functions.Some Practical Extraction and Report Language (Perl) scripts were used for parsing source fries.Results We developed a graphical R package named miRE,which was designated for the analysis of microRNA functions,genomic organization,etc.This package provided effective and convenient tools for molecular biologists to deal with routine analyses in microRNA-related research.With its help,the users would be able to build a desktopcentered microRNA research environment quite easily and effectively,miRE is freely available at http://www.biosino.org/~kanghu/WorkPresentation/miRE/miRE.html.A detailed user manual and tutorials with example code and image are also available.Conclusion miRE is a tool providing an open-source,user-friendly,integrated interface for microRNA-related analysis.With its help,researchers can perform microRNA-related analysis more efficiently.

  18. MicroRNAs in Hepatobiliary and Pancreatic Cancers

    Yoshimasa eSaito

    2011-09-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs that function as endogenous silencers of numerous target genes. Hundreds of human miRNAs have been identified in the human genome. miRNAs are expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation. Aberrant expression of miRNAs may also contribute to the development and progression of human hepatobiliary and pancreatic cancers. Recent studies have shown that some miRNAs play roles as tumor suppressors or oncogenes in hepatobiliary and pancreatic cancers. miR-122, let-7 family, and miR-101 are downregulated in hepatocellular carcinoma (HCC, suggesting that it is a potential tumor suppressor of HCC. miR-221 and miR-222 are up-regulated in HCC and may act as oncogenic miRNAs in hepatocarcinogenesis. miRNA expression profiling may be a powerful clinical tool for diagnosis and regulation of miRNA expression could be a novel therapeutic strategy for hepatobiliary and pancreatic cancers. In this review, we summarize current knowledge about the roles of important tumor suppressor microRNAs and oncogenic microRNAs in hepatobiliary and pancreatic cancers.

  19. MicroRNA characterize genetic diversity and drug resistance in pediatric acute lymphoblastic leukemia

    D. Schotte (Diana); R.X. de Menezes (Renee); F. Akbari Moqadam (Farhad); L.M. Khankahdani (Leila Mohammadi); E.A.M. Lange-Turenhout (Ellen); C. Chen (Caifu); R. Pieters (Rob); M.L. den Boer (Monique)

    2011-01-01

    textabstractBackground MicroRNA regulate the activity of protein-coding genes including those involved in hematopoietic cancers. The aim of the current study was to explore which microRNA are unique for seven different subtypes of pediatric acute lymphoblastic leukemia. Design and Methods Expression

  20. MicroRNA expression profiling in relation to the genetic heterogeneity of acute myeloid leukemia

    M. Jongen-Lavrencic (Mojca); S.M. Sun; M.K. Dijkstra; P.J.M. Valk (Peter); B. Löwenberg (Bob)

    2008-01-01

    textabstractAcute myeloid leukemia (AML) is a highly diverse disease characterized by various cytogenetic and molecular abnormalities. MicroRNAs are small noncoding RNAs that show variable expression during myeloid differentiation. MicroRNA expression in marrow blasts in 215 cases of newly diagnosed

  1. ULTRASOUND AND MICROBUBBLE-INDUCED LOCAL DELIVERY OF MICRORNA-BASED THERAPEUTICS

    Kwekkeboom, Rick F. J.; Lei, Zhiyong; Bogaards, Sylvia J. P.; Aiazian, Eric; Kamp, Otto; Paulus, Walter J.; Sluijter, Joost P. G.; Musters, Rene J. P.

    2015-01-01

    MicroRNAs are involved in many pathologic processes and are a promising target for therapeutic intervention. However, successful, localized delivery of microRNA-based therapeutics is lacking. In this study, cationic ultrasound-responsive microbubbles (MBs) were used to deliver microRNA blockers and

  2. Approccio genomico e funzionale allo studio delle alterazioni dei microRNA nelle MPN.

    L. Tozzi

    2012-01-01

    Alterazioni dell'espressione dei microRNA sono state descritte in numerosi tumori sia solidi che ematologici. In questo studio sono stati utilizzati dati funzionali e dati derivanti dalle più recenti tecnologie di analisi genomica high-throughput per identificare subset di microRNA alterati nelle cellule neoplastiche e caratterizzare il loro possibile ruolo.

  3. MicroRNA silencing and the development of novel therapies for liver disease

    Szabo, Gyongyi; Sarnow, Peter; Bala, Shashi

    2012-01-01

    In recent years microRNAs have emerged as crucial small non-coding RNA molecules with diverse roles in various diseases including diseases of the liver. In this review, we highlight the latest advances in the field of microRNA biology and their potential as emerging therapeutic targets in liver disease.

  4. Guidelines for the functional annotation of microRNAs using the Gene Ontology

    D'Eustachio, Peter; Smith, Jennifer R.; Zampetaki, Anna

    2016-01-01

    MicroRNA regulation of developmental and cellular processes is a relatively new field of study, and the available research data have not been organized to enable its inclusion in pathway and network analysis tools. The association of gene products with terms from the Gene Ontology is an effective method to analyze functional data, but until recently there has been no substantial effort dedicated to applying Gene Ontology terms to microRNAs. Consequently, when performing functional analysis of microRNA data sets, researchers have had to rely instead on the functional annotations associated with the genes encoding microRNA targets. In consultation with experts in the field of microRNA research, we have created comprehensive recommendations for the Gene Ontology curation of microRNAs. This curation manual will enable provision of a high-quality, reliable set of functional annotations for the advancement of microRNA research. Here we describe the key aspects of the work, including development of the Gene Ontology to represent this data, standards for describing the data, and guidelines to support curators making these annotations. The full microRNA curation guidelines are available on the GO Consortium wiki (http://wiki.geneontology.org/index.php/MicroRNA_GO_annotation_manual). PMID:26917558

  5. A MicroRNA Repertoire for Functional Genome Research in rainbow trout (Oncorhynchus mykiss)

    MicroRNAs are small, highly conserved, non-coding RNAs that regulate gene expression of target mRNAs through cleavage or translational inhibition. MicroRNAs are most often identified through computational prediction from genome sequences. The rainbow trout genome sequence is not available yet, which...

  6. Circulating microRNA expression profiles associated with systemic lupus erythematosus

    Carlsen, Anting Liu; Schetter, Aaron J; Nielsen, Christoffer;

    2013-01-01

    OBJECTIVE: To evaluate the specificity of expression patterns of cell-free, circulating microRNAs in systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma and 45 different specific mature microRNAs were determined using quantitative reverse transcription polymerase chain...

  7. Immunopathologic Processes in Sympathetic Ophthalmia as Signified by MicroRNA Profiling

    Kaneko, Yutaka; Wu, Guey Shuang; Saraswathy, Sindhu; Vasconcelos-Santos, Daniel V.; Rao, Narsing A.

    2012-01-01

    Recent discovery of microRNAs and their negative gene regulation have provided a new understanding in the pathogenesis of inflammatory diseases. This study demonstrated microRNA expression profiling and their likely role in sympathetic ophthalmia, using formalin-fixed, paraffin-embedded samples.

  8. Histone deacetylase 1 enhances microRNA processing via deacetylation of DGCR8

    Wada, Taeko; Kikuchi, Jiro; Furukawa, Yusuke

    2012-01-01

    HDACs are known to control gene expression via promoter histone deacetylation. Furukawa and colleagues now show that, in addition to this role, HDAC1 enhances microRNA production by acetylation of DGCR8, one of the components of the Microprocessor complex that is involved in the early steps of microRNA biogenesis.

  9. LNA-modified oligonucleotides mediate specific inhibition of microRNA function

    Ørom, Ulf Andersson; Kauppinen, Sakari; Lund, Anders H

    2006-01-01

    microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been...

  10. Guidelines for the functional annotation of microRNAs using the Gene Ontology.

    Huntley, Rachael P; Sitnikov, Dmitry; Orlic-Milacic, Marija; Balakrishnan, Rama; D'Eustachio, Peter; Gillespie, Marc E; Howe, Doug; Kalea, Anastasia Z; Maegdefessel, Lars; Osumi-Sutherland, David; Petri, Victoria; Smith, Jennifer R; Van Auken, Kimberly; Wood, Valerie; Zampetaki, Anna; Mayr, Manuel; Lovering, Ruth C

    2016-05-01

    MicroRNA regulation of developmental and cellular processes is a relatively new field of study, and the available research data have not been organized to enable its inclusion in pathway and network analysis tools. The association of gene products with terms from the Gene Ontology is an effective method to analyze functional data, but until recently there has been no substantial effort dedicated to applying Gene Ontology terms to microRNAs. Consequently, when performing functional analysis of microRNA data sets, researchers have had to rely instead on the functional annotations associated with the genes encoding microRNA targets. In consultation with experts in the field of microRNA research, we have created comprehensive recommendations for the Gene Ontology curation of microRNAs. This curation manual will enable provision of a high-quality, reliable set of functional annotations for the advancement of microRNA research. Here we describe the key aspects of the work, including development of the Gene Ontology to represent this data, standards for describing the data, and guidelines to support curators making these annotations. The full microRNA curation guidelines are available on the GO Consortium wiki (http://wiki.geneontology.org/index.php/MicroRNA_GO_annotation_manual). PMID:26917558

  11. MicroRNAs and their targets from Arabidopsis to rice:half conserved and half diverged

    Ligeng Ma

    2010-01-01

    @@ MicroRNAs are 21-to 24-nucleotide long, endogenous non-coding RNAs in eukaryotes (Hannon, 2002). Mature microRNAs generated by Dicer are incorporated into an RNA-induced silencing complex (RISC), resulting in gene silencing via the cleavage of a target mRNA or the repression of target mRNA translation (Qi et al, 2005).

  12. MicroRNAs, HIV and HCV: a complex relation towards pathology.

    Piedade, Diogo; Azevedo-Pereira, José Miguel

    2016-05-01

    MicroRNAs are small non-coding RNAs that modulate protein production by post-transcriptional gene regulation. They impose gene expression control by interfering with mRNA translation and stability in cell cytoplasm through a mechanism involving specific binding to mRNA based on base pair complementarity. Because of their intracellular replication cycle it is no surprise that viruses evolved in a way that allows them to use microRNAs to infect, replicate and persist in host cells. Several ways of interference between virus and host-cell microRNA machinery have been described. Most of the time, viruses drastically alter host-cell microRNA expression or synthesize their own microRNA to facilitate infection and pathogenesis. HIV and HCV are two prominent examples of this complex interplay revealing how fine-tuning of microRNA expression is crucial for controlling key host pathways that allow viral infection and replication, immune escape and persistence. In this review we delve into the mechanisms underlying cellular and viral-encoded microRNA functions in the context of HIV and HCV infections. We focus on which microRNAs are differently expressed and deregulated upon viral infection and how these alterations dictate the fate of virus and cell. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27059433

  13. MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival

    Pezzella Francesco

    2011-05-01

    Full Text Available Abstract Background MicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0 of purified tumor (CD138+ cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS and 9 controls. Results Unsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129 were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40% suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59% of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14 and t(11;14 and del(13q. Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS in MM, ten of which were significant by univariate (logrank survival analysis. Conclusions In summary, this work has identified aberrantly expressed microRNAs associated with the

  14. MicroRNA immunobiology: when microRNA chemists meet immunologists

    Youhai H Chen

    2011-01-01

    Although interdisciplinary research has been heralded as the engine of basic discovery for decades,many in the immunology community have been taken back by the recent marriage between microRNA (miRNA) and immunology.MicroRNAs were first discovered by Ambros and colleagues in 1993.1 They are small untranslated RNAs,highly conserved between different eukaryotic species.2-5 They are encoded by specific genes in the genome,which are controlled at the transcriptional level in a manner similar to protein-encoding genes.2 Following the synthesis of the primary miRNA by RNA polymerase Ⅱ or Ⅲ,nuclear processing by the enzyme Drosha produces a primary miRNA transcript which can be shuttled into the cytoplasm 2 Final production of the mature miRNA species requires further cytoplasmic processing by an RNase Ⅲ enzyme called Dicer,producing a 19- to 24-base pair product,capable of being incorporated into the RNA-induced silencing complex.The RNA-induced silencing complex,in turn,is able to use the 'seed sequence' of the miRNA to recognize complementary mRNA transcripts for degradation or translational silencing.To date,more than 800 human miRNAs have been identified,regulating an estimated 50% of all human genes.Each miRNA appears to regulate the expression of tens to hundreds of genes,thereby functioning as 'master-switches' that regulate and coordinate multiple cellular pathways in important processes such as embryonic development and oncogenesis,as well as cellular growth and proliferation.

  15. MicroRNA as a Novel Modulator in Head and Neck Squamous Carcinoma

    Li-Hsin Chen

    2010-01-01

    Full Text Available MicroRNAs have emerged as important regulators of cell proliferation, development, cancer formation, stress responses, cell death, and other physiological conditions in the past decade. On the other hand, head and neck cancer is one of the top ten most common cancers worldwide. Recent advances in microRNAs have revealed their prominent role in regulating gene expression and provided new aspects of applications in diagnosis, prognosis, and therapeutic strategies in head and neck squamous carcinoma. In the present paper, we focus on microRNAs showing significant differences between normal and tumor cells or between cells with differential ability of metastasis. We also emphasize specific microRNAs that could modulate tumor cell properties, such as apoptosis, metastasis, and proliferation. These microRNAs possess the potential to be applied on clinical therapy in the future.

  16. Identification of reference genes for relative quantification of circulating microRNAs in bovine serum.

    In-Seon Bae

    Full Text Available Circulating microRNAs in body fluids have been implicated as promising biomarkers for physiopathology disorders. Currently, the expression levels of circulating microRNAs are estimated by reverse transcription quantitative real-time polymerase chain reaction. Use of appropriate reference microRNAs for normalization is critical for accurate microRNA expression analysis. However, no study has systematically investigated reference genes for evaluating circulating microRNA expression in cattle. In this study, we describe the identification and characterization of appropriate reference microRNAs for use in the normalization of circulating microRNA levels in bovine serum. We evaluated the expression stability of ten candidate reference genes in bovine serum by using reverse transcription quantitative real-time polymerase chain reaction. Data were analyzed using geNorm, NormFinder, and BestKeeper statistical algorithms. The results consistently showed that a combination of miR-93 and miR-127 provided the most stably expressed reference. The suitability of these microRNAs was validated, and even when compared among different genders or breeds, the combination of miR-93 and miR-127 was ranked as the most stable microRNA reference. Therefore, we conclude that this combination is the optimal endogenous reference for reverse transcription quantitative real-time polymerase chain reaction-based detection of microRNAs in bovine serum. The data presented in this study are crucial to successful biomarker discovery and validation for the diagnosis of physiopathological conditions in cattle.

  17. Roles of avian herpesvirus microRNAs in infection, latency, and oncogenesis.

    Morgan, Robin W; Burnside, Joan

    2011-01-01

    MicroRNAs have been reported for the avian herpesviruses Marek's disease virus 1 (MDV1; oncogenic), Marek's disease virus 2 (MDV2; non-oncogenic), herpesvirus of turkeys (HVT), and infectious laryngotracheitis virus (ILTV). No obvious phylogenetic relationships exist among the avian herpesvirus microRNAs, but the general genomic locations of microRNA clusters are conserved, with microRNAs being located in the repeat regions of the genomes. In some cases, microRNAs are antisense to open reading frames. Among MDV1 field isolates with different virulence properties, microRNAs are highly conserved, and variations that have been observed lie in putative promoter regions. One cluster of MDV1 microRNAs lies upstream of the meq gene, and this cluster is more highly expressed in tumors caused by an extremely virulent MDV1 isolate compared to tumors caused by a less virulent isolate. Several of the avian herpesvirus microRNAs are orthologs of microRNAs in other species. For example, mdv1-miR-M4 shares a seed sequence with gga-miR-155 (also shared with Kaposi sarcoma herpesvirus (KSHV) kshv-miR-K12), mdv2-miR-M21 shares a seed with miR-29b, and hvt-miR-H14 shares a seed sequence with miR-221. Functional analyses of avian herpesvirus microRNAs include a variety of in vitro assays to demonstrate potential function as well as the use of mutants that can exploit the ability to assess phenotypes experimentally in the natural host. This article is part of a Special Issue entitled:MicroRNA's in viral gene regulation. PMID:21683170

  18. Identification of differentially expressed microRNAs in human male breast cancer

    Schipper Elisa

    2010-03-01

    Full Text Available Abstract Background The discovery of small non-coding RNAs and the subsequent analysis of microRNA expression patterns in human cancer specimens have provided completely new insights into cancer biology. Genetic and epigenetic data indicate oncogenic or tumor suppressor function of these pleiotropic regulators. Therefore, many studies analyzed the expression and function of microRNA in human breast cancer, the most frequent malignancy in females. However, nothing is known so far about microRNA expression in male breast cancer, accounting for approximately 1% of all breast cancer cases. Methods The expression of 319 microRNAs was analyzed in 9 primary human male breast tumors and in epithelial cells from 15 male gynecomastia specimens using fluorescence-labeled bead technology. For identification of differentially expressed microRNAs data were analyzed by cluster analysis and selected statistical methods. Expression levels were validated for the most up- or down-regulated microRNAs in this training cohort using real-time PCR methodology as well as in an independent test cohort comprising 12 cases of human male breast cancer. Results Unsupervised cluster analysis separated very well male breast cancer samples and control specimens according to their microRNA expression pattern indicating cancer-specific alterations of microRNA expression in human male breast cancer. miR-21, miR519d, miR-183, miR-197, and miR-493-5p were identified as most prominently up-regulated, miR-145 and miR-497 as most prominently down-regulated in male breast cancer. Conclusions Male breast cancer displays several differentially expressed microRNAs. Not all of them are shared with breast cancer biopsies from female patients indicating male breast cancer specific alterations of microRNA expression.

  19. Increased microRNA-155 and decreased microRNA-146a may promote ocular inflammation and proliferation in Graves’ ophthalmopathy

    Li, Kaijun; Du, Yi; Jiang, Ben-Li; He, Jian-feng

    2014-01-01

    Graves’ ophthalmopathy is an inflammatory autoimmune disease of the orbit, characterized by inflammation and proliferation of the orbital tissue caused by CD4+T cells and orbital fibroblasts. Despite recent substantial findings regarding its cellular and molecular foundations, the pathogenesis of Graves’ ophthalmopathy remains unclear. Accumulating data suggest that microRNAs play important roles in the pathophysiology of autoimmunity and proliferation. Specifically, microRNA-155 (miR-155) ca...

  20. Prognostic microRNAs in cancer tissue from patients operated for pancreatic cancer--five microRNAs in a prognostic index

    Schultz, Nicolai A; Andersen, Klaus; Roslind, Anne;

    2012-01-01

    The aim of the present study was to identify a panel of microRNAs (miRNAs) that can predict overall survival (OS) in non micro-dissected cancer tissues from patients operated for pancreatic cancer (PC).......The aim of the present study was to identify a panel of microRNAs (miRNAs) that can predict overall survival (OS) in non micro-dissected cancer tissues from patients operated for pancreatic cancer (PC)....

  1. MicroRNAs of Gallid and Meleagrid herpesviruses show generally conserved genomic locations and are virus-specific.

    Waidner, Lisa A; Morgan, Robin W; Anderson, Amy S; Bernberg, Erin L; Kamboj, Sachin; Garcia, Maricarmen; Riblet, Silva M; Ouyang, Ming; Isaacs, Grace K; Markis, Milos; Meyers, Blake C; Green, Pamela J; Burnside, Joan

    2009-05-25

    Many herpesviruses, including Marek's disease viruses (MDV1 and MDV2), encode microRNAs. In this study, we report microRNAs of two related herpesviruses, infectious laryngotracheitis virus (ILTV) and herpesvirus of turkeys (HVT), as well as additional MDV2 microRNAs. The genome locations, but not microRNA sequences, are conserved among all four of these avian herpesviruses. Most are clustered in the repeats flanking the unique long region (I/TR(L)), except in ILTV which lacks these repeats. Two abundant ILTV microRNAs are antisense to the immediate early gene ICP4. A homologue of host microRNA, gga-miR-221, was found among the HVT microRNAs. Additionally, a cluster of HVT microRNAs was found in a region containing two locally duplicated segments, resulting in paralogous HVT microRNAs with 96-100% identity. The prevalence of microRNAs in the genomic repeat regions as well as in local repeats suggests the importance of genetic plasticity in herpesviruses for microRNA evolution and preservation of function. PMID:19328516

  2. MicroRNA profiling of pericardial fluid samples from patients with heart failure.

    Suvi M Kuosmanen

    Full Text Available Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate biomarkers due to their relative stability and easy quantification from clinical samples. Pericardial fluid contains hormones secreted by the heart and is known to reflect the cardiac function. In this study, we sought to investigate whether pericardial fluid contains microRNAs and if so, whether they could be used to distinguish between different cardiovascular pathologies and disease stages.Pericardial fluid was collected from heart failure patients during open-heart surgery. MicroRNA profiles of altogether 51 patients were measured by quantitative real-time PCR (qPCR using Exiqon human panels I and II. On the average, 256 microRNAs were detected per sample, and 70 microRNAs out of 742 profiled microRNAs were detected in every sample. The five most abundant microRNAs in pericardial fluid were miR-21-5p, miR-451a, miR-125b-5p, let-7b-5p and miR-16-5p. No specific signatures for cardiovascular pathologies or clinically assessed heart failure stages could be detected from the profiles and, overall, microRNA profiles of the samples were found to be very similar despite the heterogeneity in the study population.Measured microRNA profiles did not separate the samples according to the clinical features of the patients. However, several previously identified heart failure marker microRNAs were detected. The pericardial fluid microRNA profile appeared to be a result of an active and selective secretory process indicating that microRNAs may act as paracrine signalling

  3. Detection of microRNAs in archival cytology urine smears.

    Francesca Simonato

    Full Text Available MicroRNAs' dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC. Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205 in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples (mean 1.27±1.43 µg, range 0.06-4.60 µg. Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears (slope= -3.4084; R-squared=0.99; efficiency=1.94. miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers.

  4. Identification of microRNAs in the coral Stylophora pistillata.

    Liew, Yi Jin

    2014-03-21

    Coral reefs are major contributors to marine biodiversity. However, they are in rapid decline due to global environmental changes such as rising sea surface temperatures, ocean acidification, and pollution. Genomic and transcriptomic analyses have broadened our understanding of coral biology, but a study of the microRNA (miRNA) repertoire of corals is missing. miRNAs constitute a class of small non-coding RNAs of ∼22 nt in size that play crucial roles in development, metabolism, and stress response in plants and animals alike. In this study, we examined the coral Stylophora pistillata for the presence of miRNAs and the corresponding core protein machinery required for their processing and function. Based on small RNA sequencing, we present evidence for 31 bona fide microRNAs, 5 of which (miR-100, miR-2022, miR-2023, miR-2030, and miR-2036) are conserved in other metazoans. Homologues of Argonaute, Piwi, Dicer, Drosha, Pasha, and HEN1 were identified in the transcriptome of S. pistillata based on strong sequence conservation with known RNAi proteins, with additional support derived from phylogenetic trees. Examination of putative miRNA gene targets indicates potential roles in development, metabolism, immunity, and biomineralisation for several of the microRNAs. Here, we present first evidence of a functional RNAi machinery and five conserved miRNAs in S. pistillata, implying that miRNAs play a role in organismal biology of scleractinian corals. Analysis of predicted miRNA target genes in S. pistillata suggests potential roles of miRNAs in symbiosis and coral calcification. Given the importance of miRNAs in regulating gene expression in other metazoans, further expression analyses of small non-coding RNAs in transcriptional studies of corals should be informative about miRNA-affected processes and pathways.

  5. Identification of microRNAs in the coral Stylophora pistillata.

    Yi Jin Liew

    Full Text Available Coral reefs are major contributors to marine biodiversity. However, they are in rapid decline due to global environmental changes such as rising sea surface temperatures, ocean acidification, and pollution. Genomic and transcriptomic analyses have broadened our understanding of coral biology, but a study of the microRNA (miRNA repertoire of corals is missing. miRNAs constitute a class of small non-coding RNAs of ∼22 nt in size that play crucial roles in development, metabolism, and stress response in plants and animals alike. In this study, we examined the coral Stylophora pistillata for the presence of miRNAs and the corresponding core protein machinery required for their processing and function. Based on small RNA sequencing, we present evidence for 31 bona fide microRNAs, 5 of which (miR-100, miR-2022, miR-2023, miR-2030, and miR-2036 are conserved in other metazoans. Homologues of Argonaute, Piwi, Dicer, Drosha, Pasha, and HEN1 were identified in the transcriptome of S. pistillata based on strong sequence conservation with known RNAi proteins, with additional support derived from phylogenetic trees. Examination of putative miRNA gene targets indicates potential roles in development, metabolism, immunity, and biomineralisation for several of the microRNAs. Here, we present first evidence of a functional RNAi machinery and five conserved miRNAs in S. pistillata, implying that miRNAs play a role in organismal biology of scleractinian corals. Analysis of predicted miRNA target genes in S. pistillata suggests potential roles of miRNAs in symbiosis and coral calcification. Given the importance of miRNAs in regulating gene expression in other metazoans, further expression analyses of small non-coding RNAs in transcriptional studies of corals should be informative about miRNA-affected processes and pathways.

  6. Contribution of MicroRNAs to autoimmune diseases.

    Garo, Lucien P; Murugaiyan, Gopal

    2016-05-01

    MicroRNAs are a class of evolutionarily conserved, short non-coding RNAs that post-transcriptionally modulate the expression of multiple target genes. They are implicated in almost every biological process, including pathways involved in immune homeostasis, such as immune cell development, central and peripheral tolerance, and T helper cell differentiation. Alterations in miRNA expression and function can lead to major dysfunction of the immune system and mediate susceptibility to autoimmune disease. Here, we discuss the role of miRNAs in the maintenance of immune tolerance to self-antigens and the gain or loss of miRNA functions on tissue inflammation and autoimmunity. PMID:26943802

  7. Micromanaging Vascular Biology: Tiny MicroRNAs Play Big Band

    Sen, Chandan K.; Gordillo, Gayle M.; Khanna, Savita; Roy, Sashwati

    2009-01-01

    Micro-RNAs (miRNAs) are estimated to regulate 30% of the human genome primarily through translational repression. In 2005–2008, the first series of observations establishing the key significance of miRNAs in the regulation of vascular biology came from experimental studies involved in arresting miRNA biogenesis to deplete the miRNA pools of vascular tissues and cells. Dicer-dependent biogenesis of miRNA is required for blood vessel development during embryogenesis and wound healing. miRNAs re...

  8. MicroRNAs in Experimental Models of Movement Disorders

    Soon-Tae Lee; Manho Kim

    2011-01-01

    MicroRNAs (miRNAs) are small RNAs comprised of 20–25 nucleotides that regulates gene expression by inducing translational repression or degradation of target mRNA. The importance of miRNAs as a mediator of disease pathogenesis and therapeutic targets is rapidly emerging in neuroscience, as well as oncology, immunology, and cardiovascular diseases. In Parkinson’s disease and related disorders, multiple studies have identified the implications of specific miRNAs and the polymorphisms of miRNA t...

  9. MicroRNAs in cancers and neurodegenerative disorders

    Saito, Yoshimasa; Saito, Hidetsugu

    2012-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs which function as endogenous silencers of various target genes. miRNAs are expressed in a tissue-specific manner and playing important roles in cell proliferation, apoptosis, and differentiation during mammalian development. Links between miRNAs and the initiation and progression of human diseases including cancer are becoming increasingly apparent. Recent studies have revealed that some miRNAs such as miR-9, miR-29 family, and miR-34 family are di...

  10. Intratumoral Heterogeneity of MicroRNA Expression in Rectal Cancer

    Anne Haahr Mellergaard Eriksen; Rikke Fredslund Andersen; Boye Schnack Nielsen; Flemming Brandt Sørensen; Ane Lindegaard Appelt; Anders Jakobsen; Torben Frøstrup Hansen

    2016-01-01

    Introduction An increasing number of studies have investigated microRNAs (miRNAs) as potential markers of diagnosis, treatment and prognosis. So far, agreement between studies has been minimal, which may in part be explained by intratumoral heterogeneity of miRNA expression. The aim of the present study was to assess the heterogeneity of a panel of selected miRNAs in rectal cancer, using two different technical approaches. Materials and Methods The expression of the investigated miRNAs was an...

  11. MicroRNA-21 regulates stemness in cancer cells

    Kang, Hong-Yo

    2013-01-01

    MicroRNA-21 (miR-21) functions have been linked to cancer progression and chemo- or radiotherapy resistance. While an increasing number of studies have reported a potential role of miR-21 expression in promoting growth of a small population of stem/progenitor cells, knowledge on its role as a regulator of stemness in cancers remains limited. In a previous issue of Stem Cell Research &Therapy, Chung and colleagues provide evidence that miR-21 is highly expressed in stem/progenitor populations ...

  12. MicroRNA in autoimmunity and autoimmune diseases

    Pauley, Kaleb M.; Cha, Seunghee; Chan, Edward K. L.

    2009-01-01

    MicroRNAs (miRNAs) are small conserved non-coding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3′ untranslated region (UTR) of specific messenger RNAs (mRNAs) for degradation or translational repression. miRNA-mediated gene regulation is critical for normal cellular functions such as the cell cycle, differentiation, and apoptosis, and as much as one-third of human mRNAs may be miRNA targets. Emerging evidence has demonstrated that miRNAs play a vital rol...

  13. MicroRNAs meet calcium: joint venture in ER proteostasis.

    Finger, Fabian; Hoppe, Thorsten

    2014-11-01

    The endoplasmic reticulum (ER) is a cellular compartment that has a key function in protein translation and folding. Maintaining its integrity is of fundamental importance for organism's physiology and viability. The dynamic regulation of intraluminal ER Ca(2+) concentration directly influences the activity of ER-resident chaperones and stress response pathways that balance protein load and folding capacity. We review the emerging evidence that microRNAs play important roles in adjusting these processes to frequently changing intracellular and environmental conditions to modify ER Ca(2+) handling and storage and maintain ER homeostasis. PMID:25372053

  14. Cloning and expression of new microRNAs from zebrafish

    Kloosterman, Wigard P.; Steiner, Florian A.; Berezikov, Eugene; de Bruijn, Ewart; Van de Belt, Jose; Verheul, Mark; Cuppen, Edwin; Ronald H A Plasterk

    2006-01-01

    MicroRNAs (miRNAs) play an important role in development and regulate the expression of many animal genes by post-transcriptional gene silencing. Here we describe the cloning and expression of new miRNAs from zebrafish. By high-throughput sequencing of small-RNA cDNA libraries from 5-day-old zebrafish larvae and adult zebrafish brain we found 139 known miRNAs and 66 new miRNAs. For 65 known miRNAs and for 11 new miRNAs we also cloned the miRNA star sequence. We analyzed the temporal and spati...

  15. Global characterization of microRNAs in Trichomonas gallinae

    Xu, Min-Jun; Qiu, Shen-Ben; Nisbet, Alasdair J.; Fu, Jing-Hua; Shao, Chang-Chun; Zhu, Xing-Quan

    2014-01-01

    Background Trichomonas gallinae is a protozoan parasite causing trichomonosis in many species of domestic poultry and birds world-wide. microRNAs (miRNAs) are a class of small non-coding RNAs that play key roles in gene regulation. However, no miRNAs have been characterized from T. gallinae. Methods Here, we investigated the global miRNA profile of this parasite by high throughput sequencing technology, bioinformatics platform analysis and quantitative RT-PCR. Results Three miRNA candidates, ...

  16. Control of metastatic progression by microRNA regulatory networks.

    Pencheva, Nora; Tavazoie, Sohail F

    2013-06-01

    Aberrant microRNA (miRNA) expression is a defining feature of human malignancy. Specific miRNAs have been identified as promoters or suppressors of metastatic progression. miRNAs control metastasis through divergent or convergent regulation of metastatic gene pathways. Some miRNA regulatory networks govern cell-autonomous cancer phenotypes, whereas others modulate the cell-extrinsic composition of the metastatic microenvironment. The use of small RNAs as probes into the molecular and cellular underpinnings of metastasis holds promise for the identification of candidate genes for potential therapeutic intervention. PMID:23728460

  17. Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

    Galli, Vanessa; Guzman, Frank; de Oliveira, Luiz F. V.; Loss-Morais, Guilherme; Körbes, Ana P.; Silva, Sérgio D. A.; Margis-Pinheiro, Márcia M. A. N.; Margis, Rogério

    2014-01-01

    MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identifica...

  18. MicroRNAs and their role in viral infection

    MU Junjie; YAO Xue; CHEN Qimin; GENG Yunqi; QIAO Wentao

    2007-01-01

    Recently,a class of about 22 nucleotides (nt)small RNA has been discovered in many eukaryotes,termed microRNAs (miRNAs),which have a variety of functions.Many recent findings have demonstrated that viruses can also encode their own miRNAs.Meanwhile,other findings reveal a relationship between host miRNA and viral infection.These findings suggest a tight relationship between host and viral infection via miRNA pathway.This article introduces the miRNAs encoded by viruses and reviews the advances of the interaction of the mammalian host miRNAs and viral infection.

  19. PDGF induced microRNA alterations in cancer cells

    Shao, Minghai; Rossi, Simona; Chelladurai, Bhadrani; Shimizu, Masayoshi; Ntukogu, Obiageli; Ivan, Mircea; Calin, George A.; Matei, Daniela

    2011-01-01

    Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust c...

  20. Comprehensive Analysis of Rhesus Lymphocryptovirus MicroRNA Expression▿ †

    Riley, Kasandra J.-L.; Rabinowitz, Gabrielle S.; Steitz, Joan A.

    2010-01-01

    Rhesus lymphocryptovirus (rLCV) and Epstein-Barr virus (EBV) are closely related gammaherpesviruses that infect and cause disease in rhesus monkeys and humans, respectively. Thus, rLCV is an important model system for EBV pathogenesis. Both rLCV and EBV express microRNAs (miRNAs), several conserved in sequence and genomic location. We have applied deep sequencing technology to obtain an inventory of rLCV miRNA expression in latently rLCV-infected monkey B cells. Our data confirm the presence ...

  1. MicroRNA in cell differentiation and development

    SHI Yi; JIN YouXin

    2009-01-01

    The regulation of gene expression by microRNAs (miRNAs) Is a recently discovered pattern of gene regulation in animals and plants. MiRNAs have been implicated in various aspects of animal develop-ment and cell differentiation, such as early embryonic development, neuronal development, muscle development, and lymphocyte development, by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators in animal development and are po-tential causes of human diseases. Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

  2. miRviewer: a multispecies microRNA homologous viewer

    Kiezun Adam; Artzi Shay; Modai Shira; Volk Naama; Isakov Ofer; Shomron Noam

    2012-01-01

    Abstract Background MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression via binding to the 3' ends of mRNAs. MiRNAs have been associated with many cellular events ascertaining their central role in gene regulation. In order to better understand miRNAs of interest it is of utmost importance to learn about the genomic conservation of these genes. Findings The miRviewer web-server, presented here, encompasses all known miRNAs of currently fully annotated animal genomes in ...

  3. The role of microRNAs in human liver cancers.

    Braconi, Chiara; Henry, Jon C; Kogure, Takayuki; Schmittgen, Thomas; Patel, Tushar

    2011-12-01

    Hepatocellular carcinoma (HCC) is a primary malignancy of the liver of global importance. Recent studies of the expression and role of microRNA (miRNA) in HCC are providing new insights into disease pathogenesis. In addition, therapeutic efforts targeting specific miRNAs are being evaluated in animal models of HCC. The potential of miRNAs as biomarkers of disease or prognostic markers is being explored. Herein, we review studies of miRNA expression in human HCC, and discuss recent advances in knowledge about the involvement and role of selected miRNAs in disease pathogenesis, as biomarkers, or as therapeutic targets for HCC. PMID:22082761

  4. MicroRNA-145 regulates human corneal epithelial differentiation.

    Sharon Ka-Wai Lee

    Full Text Available BACKGROUND: Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC epithelia containing corneal epithelial progenitor cells (CEPCs and early transit amplifying cells, and their role in corneal epithelium. METHODOLOGY/PRINCIPAL FINDINGS: Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test. This was validated by quantitative polymerase chain reaction (qPCR. Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance. As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8 expression in both human corneal epithelial cells

  5. Identification and Function of MicroRNAs Encoded by Herpesviruses

    Zhi-qing Bai; Xiu-fen Lei; Lin-ding Wang; Shou-jiang Gao

    2008-01-01

    MicroRNAs (miRNAs) play important roles in eukaryotes,plants and some viruses.It is increasingly clear that miRNAs-encoded by viruses can affect the viral life cycle and host physiology.Viral miRNAs could repress the innate and adaptive host immunity,modulate cellular signaling pathways,and regulate the expression of cellular and viral genes.These functions facilitate viral acute and persistent infections,and have profound effects on the host cell survival and disease progression.Here,we discuss the miRNAs encoded by herpesviruses,and their regulatory roles involved in virus-host interactions.

  6. Artificial micrornas and their applications in plant molecular biology

    Pérez-Quintero Álvaro Luis; López Camilo

    2012-01-01

    Artificial microRNAs (amiRNAs) are modified endogenous microRNA precursors in which the miRNA:miRNA* duplex is replaced with sequences designed to silence any desired gene. amiRNAs are used as part of new genetic transformation techniques in eukaryotes and have proven to be effective and to excel over other RNA-mediated gene silencing methods in both specificity and stability. amiRNAs can be designed to silence single or multiple genes, it is also possible to construct dimeric amiR...

  7. MicroRNA miR-125b causes leukemia

    Bousquet, Marina; Harris, Marian H.; Zhou, Beiyan; Lodish, Harvey F.

    2010-01-01

    MicroRNA miR-125b has been implicated in several kinds of leukemia. The chromosomal translocation t(2;11)(p21;q23) found in patients with myelodysplasia and acute myeloid leukemia leads to an overexpression of miR-125b of up to 90-fold normal. Moreover, miR-125b is also up-regulated in patients with B-cell acute lymphoblastic leukemia carrying the t(11;14)(q24;q32) translocation. To decipher the presumed oncogenic mechanism of miR-125b, we used transplantation experiments in mice. All mice tr...

  8. Identification of 20 microRNAs from Oryza sativa

    Wang, Jia-Fu; Zhou, Hui; Chen, Yue-Qin; Luo, Qing-Jun; Qu, Liang-Hu

    2004-01-01

    MicroRNAs (miRNAs) are present in both plant and animal kingdoms and represents a growing family of non-coding RNAs. These tiny RNAs act as small guides and direct negative regulations usually in the process of development through sequence complementarity to target mRNAs. Although a large number of miRNAs have been identified from various animals, so far plant miRNA studies have focused mainly on Arabidopsis. Here we describe the identification of 20 miRNAs from a rice cDNA library. All the m...

  9. MicroRNAs and the genetic network in aging

    Inukai, Sachi; Slack, Frank

    2013-01-01

    MicroRNAs (miRNAs) comprise a class of small RNAs important for the post-transcriptional regulation of numerous biological processes. Their combinatorial mode of function, in which an individual miRNA can target many genes and multiple miRNAs share targets, makes them especially suited for regulating processes and pathways at the “network” level. In particular, miRNAs have recently been implicated in aging which is a complex process known to involve multiple pathways. Findings from genome-wid...

  10. Effect of MicroRNA Modulation on Bioartificial Muscle Function

    Rhim, Caroline; Cheng, Cindy S.; Kraus, William E.; Truskey, George A.

    2010-01-01

    Cellular therapies have recently employed the use of small RNA molecules, particularly microRNAs (miRNAs), to regulate various cellular processes that may be altered in disease states. In this study, we examined the effect of transient muscle-specific miRNA inhibition on the function of three-dimensional skeletal muscle cultures, or bioartificial muscles (BAMs). Skeletal myoblast differentiation in vitro is enhanced by inhibiting a proliferation-promoting miRNA (miR-133) expressed in muscle t...

  11. Role of microRNAs in Arbovirus/Vector Interactions

    Sassan Asgari

    2014-01-01

    The role of microRNAs (miRNAs) as small non-coding RNAs in regulation of gene expression has been recognized. They appear to be involved in regulation of a wide range of cellular pathways that affect several biological processes such as development, the immune system, survival, metabolism and host-pathogen interactions. Arthropod-borne viruses impose great economic and health risks around the world. Recent advances in miRNA biology have shed some light on the role of these small RNAs in vecto...

  12. Brain expressed microRNAs implicated in schizophrenia etiology

    Hansen, Thomas; Olsen, Line; Lindow, Morten;

    2007-01-01

    Protein encoding genes have long been the major targets for research in schizophrenia genetics. However, with the identification of regulatory microRNAs (miRNAs) as important in brain development and function, miRNAs genes have emerged as candidates for schizophrenia-associated genetic factors....... Indeed, the growing understanding of the regulatory properties and pleiotropic effects that miRNA have on molecular and cellular mechanisms, suggests that alterations in the interactions between miRNAs and their mRNA targets may contribute to phenotypic variation....

  13. The hibernating South American marsupial, Dromiciops gliroides, displays torpor-sensitive microRNA expression patterns

    Hadj-Moussa, Hanane; Moggridge, Jason A.; Luu, Bryan E.; Quintero-Galvis, Julian F.; Gaitán-Espitia, Juan Diego; Nespolo, Roberto F.; Storey, Kenneth B.

    2016-01-01

    When faced with adverse environmental conditions, the marsupial Dromiciops gliroides uses either daily or seasonal torpor to support survival and is the only known hibernating mammal in South America. As the sole living representative of the ancient Order Microbiotheria, this species can provide crucial information about the evolutionary origins and biochemical mechanisms of hibernation. Hibernation is a complex energy-saving strategy that involves changes in gene expression that are elicited in part by microRNAs. To better elucidate the role of microRNAs in orchestrating hypometabolism, a modified stem-loop technique and quantitative PCR were used to characterize the relative expression levels of 85 microRNAs in liver and skeletal muscle of control and torpid D. gliroides. Thirty-nine microRNAs were differentially regulated during torpor; of these, 35 were downregulated in liver and 11 were differentially expressed in skeletal muscle. Bioinformatic analysis predicted that the downregulated liver microRNAs were associated with activation of MAPK, PI3K-Akt and mTOR pathways, suggesting their importance in facilitating marsupial torpor. In skeletal muscle, hibernation-responsive microRNAs were predicted to regulate focal adhesion, ErbB, and mTOR pathways, indicating a promotion of muscle maintenance mechanisms. These tissue-specific responses suggest that microRNAs regulate key molecular pathways that facilitate hibernation, thermoregulation, and prevention of muscle disuse atrophy. PMID:27090740

  14. New roles for ‘old’ microRNAs in nervous system function and disease

    Marion eHartl

    2013-12-01

    Full Text Available Since their discovery, microRNAs became prominent candidates providing missing links on how to explain the developmental and phenotypical variation within one species or among different species. In addition, microRNAs were implicated in diseases such as neurodegeneration and cancer. More recently, the regulation of animal behavior was shown to be influenced by microRNAs. In spite of their numerous functions, only a few microRNAs were discovered by using classic genetic approaches. Due to the very mild or redundant phenotypes of most microRNAs or their genomic location within introns of other genes many regulatory microRNAs were missed. In this review, we focus on three microRNAs first identified in a forward genetic screen in invertebrates for their essential function in animal development, namely bantam, let-7 and miR-279. All three are essential for survival, are not located in introns of other genes, and are highly conserved among species. We highlight their important functions in the nervous system and discuss their emerging roles, especially during nervous system disease and behavior.

  15. The Regulatory Roles of MicroRNAs in Bone Remodeling and Perspectives as Biomarkers in Osteoporosis

    Mengge Sun

    2016-01-01

    Full Text Available MicroRNAs are involved in many cellular and molecular activities and played important roles in many biological and pathological processes, such as tissue formation, cancer development, diabetes, neurodegenerative diseases, and cardiovascular diseases. Recently, it has been reported that microRNAs can modulate the differentiation and activities of osteoblasts and osteoclasts, the key cells that are involved in bone remodeling process. Meanwhile, the results from our and other research groups showed that the expression profiles of microRNAs in the serum and bone tissues are significantly different in postmenopausal women with or without fractures compared to the control. Therefore, it can be postulated that microRNAs might play important roles in bone remodeling and that they are very likely to be involved in the pathological process of postmenopausal osteoporosis. In this review, we will present the updated research on the regulatory roles of microRNAs in osteoblasts and osteoclasts and the expression profiles of microRNAs in osteoporosis and osteoporotic fracture patients. The perspective of serum microRNAs as novel biomarkers in bone loss disorders such as osteoporosis has also been discussed.

  16. Prioritization of disease microRNAs through a human phenome-microRNAome network

    Liu Yunlong

    2010-05-01

    Full Text Available Abstract Background The identification of disease-related microRNAs is vital for understanding the pathogenesis of diseases at the molecular level, and is critical for designing specific molecular tools for diagnosis, treatment and prevention. Experimental identification of disease-related microRNAs poses considerable difficulties. Computational analysis of microRNA-disease associations is an important complementary means for prioritizing microRNAs for further experimental examination. Results Herein, we devised a computational model to infer potential microRNA-disease associations by prioritizing the entire human microRNAome for diseases of interest. We tested the model on 270 known experimentally verified microRNA-disease associations and achieved an area under the ROC curve of 75.80%. Moreover, we demonstrated that the model is applicable to diseases with which no known microRNAs are associated. The microRNAome-wide prioritization of microRNAs for 1,599 disease phenotypes is publicly released to facilitate future identification of disease-related microRNAs. Conclusions We presented a network-based approach that can infer potential microRNA-disease associations and drive testable hypotheses for the experimental efforts to identify the roles of microRNAs in human diseases.

  17. The hibernating South American marsupial, Dromiciops gliroides, displays torpor-sensitive microRNA expression patterns.

    Hadj-Moussa, Hanane; Moggridge, Jason A; Luu, Bryan E; Quintero-Galvis, Julian F; Gaitán-Espitia, Juan Diego; Nespolo, Roberto F; Storey, Kenneth B

    2016-01-01

    When faced with adverse environmental conditions, the marsupial Dromiciops gliroides uses either daily or seasonal torpor to support survival and is the only known hibernating mammal in South America. As the sole living representative of the ancient Order Microbiotheria, this species can provide crucial information about the evolutionary origins and biochemical mechanisms of hibernation. Hibernation is a complex energy-saving strategy that involves changes in gene expression that are elicited in part by microRNAs. To better elucidate the role of microRNAs in orchestrating hypometabolism, a modified stem-loop technique and quantitative PCR were used to characterize the relative expression levels of 85 microRNAs in liver and skeletal muscle of control and torpid D. gliroides. Thirty-nine microRNAs were differentially regulated during torpor; of these, 35 were downregulated in liver and 11 were differentially expressed in skeletal muscle. Bioinformatic analysis predicted that the downregulated liver microRNAs were associated with activation of MAPK, PI3K-Akt and mTOR pathways, suggesting their importance in facilitating marsupial torpor. In skeletal muscle, hibernation-responsive microRNAs were predicted to regulate focal adhesion, ErbB, and mTOR pathways, indicating a promotion of muscle maintenance mechanisms. These tissue-specific responses suggest that microRNAs regulate key molecular pathways that facilitate hibernation, thermoregulation, and prevention of muscle disuse atrophy. PMID:27090740

  18. Intronic microRNAs support their host genes by mediating synergistic and antagonistic regulatory effects

    Krumsiek Jan

    2010-04-01

    Full Text Available Abstract Background MicroRNA-mediated control of gene expression via translational inhibition has substantial impact on cellular regulatory mechanisms. About 37% of mammalian microRNAs appear to be located within introns of protein coding genes, linking their expression to the promoter-driven regulation of the host gene. In our study we investigate this linkage towards a relationship beyond transcriptional co-regulation. Results Using measures based on both annotation and experimental data, we show that intronic microRNAs tend to support their host genes by regulation of target gene expression with significantly correlated expression patterns. We used expression data of three differentiating cell types and compared gene expression profiles of host and target genes. Many microRNA target genes show expression patterns significantly correlated with the expressions of the microRNA host genes. By calculating functional similarities between host and predicted microRNA target genes based on GO annotations, we confirm that many microRNAs link host and target gene activity in an either synergistic or antagonistic manner. Conclusions These two regulatory effects may result from fine tuning of target gene expression functionally related to the host or knock-down of remaining opponent target gene expression. This finding allows to extend the common practice of mapping large scale gene expression data to protein associated genes with functionality of co-expressed intronic microRNAs.

  19. RNA degradation compromises the reliability of microRNA expression profiling

    Muckenthaler Martina U

    2009-12-01

    Full Text Available Abstract Background MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles. Results Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP or microRNA-specific real-time quantitative PCR (miQPCR. Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays. Conclusion MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven.

  20. Circulating microRNA-200 Family as Diagnostic Marker in Hepatocellular Carcinoma.

    Sameer A Dhayat

    Full Text Available In this clinical study, we aimed to evaluate the role of circulating microRNA-200 family as a non-invasive tool to identify patients with cirrhosis-associated hepatocellular carcinoma (HCC.Prognosis of HCC remains poor with increasing incidence worldwide, mainly related to liver cirrhosis. So far, no reliable molecular targets exist for early detection of HCC at surgically manageable stages. Recently, we identified members of the microRNA-200 family as potential diagnostic markers of cirrhosis-associated HCC in patient tissue samples. Their value as circulating biomarkers for HCC remained undefined.Blood samples and clinicopathological data of consecutive patients with liver diseases were collected prospectively. Expression of the microRNA-200 family was investigated by qRT-PCR in blood serum samples of 22 HCC patients with and without cirrhosis. Serum samples of patients with non-cancerous chronic liver cirrhosis (n = 22 and of healthy volunteers (n = 15 served as controls.MicroRNA-141 and microRNA-200a were significantly downregulated in blood serum of patients with HCC compared to liver cirrhosis (p<0.007 and healthy controls (p<0.002. MicroRNA-141 and microRNA-200a could well discriminate patients with cirrhosis-associated HCC from healthy volunteers with area under the receiver-operating characteristic curve (AUC values of 0.85 and 0.82, respectively. Additionally, both microRNAs could differentiate between HCC and non-cancerous liver cirrhosis with a fair accuracy.Circulating microRNA-200 family members are significantly deregulated in patients with HCC and liver cirrhosis. Further studies are necessary to confirm the diagnostic value of the microRNA-200 family as accurate serum marker for cirrhosis-associated HCC.

  1. PTEN and PDCD4 are Bona Fide Targets of microRNA-21 in Human Cholangiocarcinoma

    Chang-zheng Liu; Wei Liu; Yi Zheng; Jin-mei Su; Jing-jing Li; Lan Yu; Xiao-dong He; Song-sen Chen

    2012-01-01

    Objective To investigate the expression profile of microRNA-21 in human cholangiocarcinoma tissues and to validate its bona fide targets in human cholangiocarcinoma cells.Methods The expression profile ofmicroRNA-21 in human cholangiocarcinoma tissues and cholangiocarcinoma cell line,QBC939,was evaluated by using real-time PCR analysis.The bona fide targets of microRNA-21 were analyzed and confirmed by dual luciferase reporter gene assay and western blot,respectively.The expressional correlation of microRNA-21 and its targets was probed in human cholangiocarcinoma tissues by using real-time PCR,locked nucleic acid in situ hybridization (LNA-ISH),and immunohistochemistry analysis.Results Real-time PCR analysis revealed that microRNA-21 expression depicted a significant up-regulation in human cholangiocarcinoma tissues about 5.6-fold as compared to the matched normal bile duct tissues (P<0.05).The dual luciferase reporter gene assay revealed endogenous microRNA-21 in cholangiocarcinoma cell line,QBC939,inhibited the luciferase reporter activities of wild-type PTEN (P<0.01) and PDCD4 (P<0.05) and had no this effect on mutated PTEN and PDCD4.Moreover,loss of microRNA-21 function led to a significant increase of PTEN and PDCD4 protein levels in QBC939 cells.Elevated microRNA-21 levels were accompanied by marked reductions of PTEN and PDCD4 expression in the same cholangiocarcinoma tissue.Conclusion microRNA-21 expression is up-regulated in human cholangiocarcinoma and PTEN,PDCD4 are direct effectors of microRNA-21.

  2. Functional Implications of Human-Specific Changes in Great Ape microRNAs

    García-Ramallo, Eva; Torruella-Loran, Ignasi; Fernández-Bellon, Hugo; Abelló, Teresa; Kondova, Ivanela; Bontrop, Ronald; Hvilsom, Christina; Navarro, Arcadi; Marquès-Bonet, Tomàs; Espinosa-Parrilla, Yolanda

    2016-01-01

    microRNAs are crucial post-transcriptional regulators of gene expression involved in a wide range of biological processes. Although microRNAs are highly conserved among species, the functional implications of existing lineage-specific changes and their role in determining differences between humans and other great apes have not been specifically addressed. We analyzed the recent evolutionary history of 1,595 human microRNAs by looking at their intra- and inter-species variation in great apes using high-coverage sequenced genomes of 82 individuals including gorillas, orangutans, bonobos, chimpanzees and humans. We explored the strength of purifying selection among microRNA regions and found that the seed and mature regions are under similar and stronger constraint than the precursor region. We further constructed a comprehensive catalogue of microRNA species-specific nucleotide substitutions among great apes and, for the first time, investigated the biological relevance that human-specific changes in microRNAs may have had in great ape evolution. Expression and functional analyses of four microRNAs (miR-299-3p, miR-503-3p, miR-508-3p and miR-541-3p) revealed that lineage-specific nucleotide substitutions and changes in the length of these microRNAs alter their expression as well as the repertoires of target genes and regulatory networks. We suggest that the studied molecular changes could have modified crucial microRNA functions shaping phenotypes that, ultimately, became human-specific. Our work provides a frame to study the impact that regulatory changes may have in the recent evolution of our species. PMID:27105073

  3. MicroRNAs in the pathophysiology and treatment of status epilepticus

    David Henshall

    2013-11-01

    Full Text Available MicroRNAs are an important class of non-coding RNA which function as post-transcriptional regulators of gene expression in cells, repressing and fine-tuning protein output. Prolonged seizures (status epilepticus, SE can cause damage to brain regions such as the hippocampus and result in cognitive deficits and the pathogenesis of epilepsy. Emerging work in animal models has found that SE produces select changes to microRNAs within the brain. Similar changes in over 20 microRNAs have been found in the hippocampus in two or more studies, suggesting conserved microRNA responses after SE. The microRNA changes that accompany SE are predicted to impact levels of multiple proteins involved in neuronal morphology and function, gliosis, neuroinflammation and cell death. MicroRNA expression also displays select changes in the blood after SE, supporting blood genomic profiling as potential molecular biomarkers of seizure-damage or epileptogenesis. Intracerebral delivery of chemically-modified antisense oligonucleotides (antagomirs has been shown to have potent, specific and long-lasting effects on brain levels of microRNAs. Targeting miR-34a, miR-132 and miR-184 has been reported to alter seizure-induced neuronal death, whereas targeting miR-134 was neuroprotective, reduced seizure severity during status epilepticus and reduced the later emergence of recurrent spontaneous seizures. These studies support roles for microRNAs in the pathophysiology of status epilepticus and microRNAs may represent novel therapeutic targets to reduce brain injury and epileptogenesis.

  4. Elevated levels of circulating microRNA-200 family members correlate with serous epithelial ovarian cancer

    Kan Casina WS

    2012-12-01

    Full Text Available Abstract Background There is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC. MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers. Methods To identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28 and age-matched healthy donors (n = 28. Serum microRNA levels were assessed by quantitative RT-PCR following preamplification. Results microRNA (miR-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48 were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P  Conclusions We identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

  5. The Role of microRNAs in the Pathogenesis of Herpesvirus Infection

    Diogo Piedade

    2016-06-01

    Full Text Available MicroRNAs (miRNAs are small non-coding RNAs important in gene regulation. They are able to regulate mRNA translation through base-pair complementarity. Cellular miRNAs have been involved in the regulation of nearly all cellular pathways, and their deregulation has been associated with several diseases such as cancer. Given the importance of microRNAs to cell homeostasis, it is no surprise that viruses have evolved to take advantage of this cellular pathway. Viruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. Moreover, viral inhibition of key proteins from the microRNA pathway and important changes in cellular microRNA pool have been reported upon viral infection. In addition, viruses have developed multiple mechanisms to avoid being targeted by cellular microRNAs. This complex interaction between host and viruses to control the microRNA pathway usually favors viral infection and persistence by either reducing immune detection, avoiding apoptosis, promoting cell growth, or promoting lytic or latent infection. One of the best examples of this virus-host-microRNA interplay emanates from members of the Herperviridae family, namely the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2, human cytomegalovirus (HCMV, human herpesvirus 8 (HHV-8, and the Epstein–Barr virus (EBV. In this review, we will focus on the general functions of microRNAs and the interactions between herpesviruses, human hosts, and microRNAs and will delve into the related mechanisms that contribute to infection and pathogenesis.

  6. Bioinformatics analysis of HPV associated host microRNA functions and identification of viral microRNA

    Qian, Kui

    2013-01-01

    Human papillomaviruses (HPVs) form a large family among double stranded DNA (dsDNA) viruses, some types of which are the major causes of cervical cancer. HPV 16 is widely distributed and the most common high-risk HPV type and approximately half of the cervical cancers are associated with HPV type 16. Of the three HPV 16 encoded oncogenes, the function of E5 in regulating viral replication and pathogenesis is less well understood than E6 and E7. The microRNAs (miRNAs) are important small nonco...

  7. Analysis of microarray-identified genes and microRNAs associated with drug resistance in ovarian cancer.

    Zou, Jing; Yin, Fuqiang; Wang, Qi; Zhang, Wei; Li, Li

    2015-01-01

    The aim of this study was to identify potential microRNAs and genes associated with drug resistance in ovarian cancer through web-available microarrays. The drug resistant-related microRNA microarray dataset GS54665 and mRNA dataset GSE33482, GSE28646, and GSE15372 were downloaded from the Gene Expression Omnibus database. Dysregulated microRNAs/genes were screened with GEO2R and were further identified in SKOV3 (SKOV3/DDP) and A2780 (A2780/DDP) cells by real-time quantitative PCR (qRT-PCR), and then their associations with drug resistance was analyzed by comprehensive bioinformatic analyses. Nine microRNAs (microRNA-199a-5p, microRNA-199a-3p, microRNA-199b-3p, microRNA-215, microRNA-335, microRNA-18b, microRNA-363, microRNA-645 and microRNA-141) and 38 genes were identified to be differentially expressed in drug-resistant ovarian cancer cells, with seven genes (NHSL1, EPHA3, USP51, ZSCAN4, EPHA7, SNCA and PI15) exhibited exactly the same expression trends in all three microarrays. Biological process annotation and pathway enrichment analysis of the 9 microRNAs and 38 genes identified several drug resistant-related signaling pathways, and the microRNA-mRNA interaction revealed the existence of a targeted regulatory relationship between the 9 microRNAs and most of the 38 genes. The expression of 9 microRNAs and the 7 genes by qRT-PCR in SKOV3/DDP and A2780/DDP cells indicating a consistent expression profile with the microarrays. Among those, the expression of EPHA7 and PI15 were negatively correlated with that of microRNA-141, and they were also identified as potential targets of this microRNA via microRNA-mRNA interaction. We thus concluded that microRNA-141, EPHA7, and PI15 might jointly participate in the regulation of drug resistance in ovarian cancer and serve as potential targets in targeted therapies. PMID:26261572

  8. Methods for the Identification of PTEN-Targeting MicroRNAs.

    Tuccoli, Andrea; Vitiello, Marianna; Marranci, Andrea; Russo, Francesco; Poliseno, Laura

    2016-01-01

    The identification of PTEN-targeting microRNAs usually starts from an in silico bioinformatic prediction and then requires a careful experimental validation that exploits both heterologous and endogenous systems. Here we describe the methods used to carry on these analyses and experiments, examining pitfalls and alternatives for each step. Moreover, we give an overview of the latest high-throughput microRNA target identification techniques which offer a more comprehensive view of the microRNAs that can bind a fundamental tumor suppressor such as PTEN. PMID:27033074

  9. PARma: identification of microRNA target sites in AGO-PAR-CLIP data.

    Erhard, Florian; Dölken, Lars; Jaskiewicz, Lukasz; Zimmer, Ralf

    2013-01-01

    PARma is a complete data analysis software for AGO-PAR-CLIP experiments to identify target sites of microRNAs as well as the microRNA binding to these sites. It integrates specific characteristics of the experiments into a generative model. The model and a novel pattern discovery tool are iteratively applied to data to estimate seed activity probabilities, cluster confidence scores and to assign the most probable microRNA. Based on differential PAR-CLIP analysis and comparison to RIP-Chip data, we show that PARma is more accurate than existing approaches. PARma is available from http://www.bio.ifi.lmu.de/PARma. PMID:23895117

  10. mESAdb: microRNA Expression and Sequence Analysis Database

    Kaya, Koray D.; Karakülah, Gökhan; Yakıcıer, Cengiz M.; Acar, Aybar C.; Konu, Özlen

    2010-01-01

    microRNA expression and sequence analysis database (http://konulab.fen.bilkent.edu.tr/mirna/) (mESAdb) is a regularly updated database for the multivariate analysis of sequences and expression of microRNAs from multiple taxa. mESAdb is modular and has a user interface implemented in PHP and JavaScript and coupled with statistical analysis and visualization packages written for the R language. The database primarily comprises mature microRNA sequences and their target data, along with selected...

  11. Cepred: predicting the co-expression patterns of the human intronic microRNAs with their host genes.

    Dong Wang

    Full Text Available Identifying the tissues in which a microRNA is expressed could enhance the understanding of the functions, the biological processes, and the diseases associated with that microRNA. However, the mechanisms of microRNA biogenesis and expression remain largely unclear and the identification of the tissues in which a microRNA is expressed is limited. Here, we present a machine learning based approach to predict whether an intronic microRNA show high co-expression with its host gene, by doing so, we could infer the tissues in which a microRNA is high expressed through the expression profile of its host gene. Our approach is able to achieve an accuracy of 79% in the leave-one-out cross validation and 95% on an independent testing dataset. We further estimated our method through comparing the predicted tissue specific microRNAs and the tissue specific microRNAs identified by biological experiments. This study presented a valuable tool to predict the co-expression patterns between human intronic microRNAs and their host genes, which would also help to understand the microRNA expression and regulation mechanisms. Finally, this framework can be easily extended to other species.

  12. MicroRNAs in Breastmilk and the Lactating Breast: Potential Immunoprotectors and Developmental Regulators for the Infant and the Mother

    Mohammed Alsaweed

    2015-10-01

    Full Text Available Human milk (HM is the optimal source of nutrition, protection and developmental programming for infants. It is species-specific and consists of various bioactive components, including microRNAs, small non-coding RNAs regulating gene expression at the post-transcriptional level. microRNAs are both intra- and extra-cellular and are present in body fluids of humans and animals. Of these body fluids, HM appears to be one of the richest sources of microRNA, which are highly conserved in its different fractions, with milk cells containing more microRNAs than milk lipids, followed by skim milk. Potential effects of exogenous food-derived microRNAs on gene expression have been demonstrated, together with the stability of milk-derived microRNAs in the gastrointestinal tract. Taken together, these strongly support the notion that milk microRNAs enter the systemic circulation of the HM fed infant and exert tissue-specific immunoprotective and developmental functions. This has initiated intensive research on the origin, fate and functional significance of milk microRNAs. Importantly, recent studies have provided evidence of endogenous synthesis of HM microRNA within the human lactating mammary epithelium. These findings will now form the basis for investigations of the role of microRNA in the epigenetic control of normal and aberrant mammary development, and particularly lactation performance.

  13. Targeting the microRNA-21/AP1 axis by 5-fluorouracil and pirarubicin in human hepatocellular carcinoma

    He, Xiaodong; Li, Jingjing; Guo, Weidong; Liu, Wei; Yu, Jia; Song, Wei; Dong, Lei; Wang, Fang; Yu, Shuangni; Zheng, Yi; Chen, Songsen; Kong, Yan; Liu, Changzheng

    2015-01-01

    MicroRNAs function as oncomiRs and tumor suppressors in diverse cancers. However, the utility of specific microRNAs in predicting the clinical benefit of chemotherapy has not been well-established. Here, we investigated the correlation between microRNA-21 expression and hepatic arterial infusion chemotherapy with 5-fluorouracil and pirarubicin (HAIC) for hepatocellular carcinoma (HCC). We found that HCC patients with low microRNA-21 levels in tumors tended to have a longer time to recurrence and disease-free survival. We demonstrated that microRNA-21 suppression in combination with 5-fluorouracil and pirarubicin treatment inhibited tumor growth in subcutaneous xenograft mice models. Mechanistically, the AP-1 and microRNA-21-mediated axis was verified to be a therapeutic target of cytotoxic drugs and deregulation of this axis led to an enhanced cell growth in HCC. Taken together, our findings demonstrate that microRNA-21 is a chemotherapy responsive microRNA and can serve as a prognostic biomarker for HCC patients undergoing HAIC. Targeting microRNA-21 enhances the effect of chemotherapeutic drugs, thereby suggesting that microRNA-21 suppression in combination with HAIC may be a novel approach for HCC treatment. PMID:25544773

  14. MicroRNA Polymorphisms in Cancer: A Literature Analysis

    Pipan, Veronika; Zorc, Minja; Kunej, Tanja, E-mail: tanja.kunej@bf.uni-lj.si [Department of Animal Science, Biotechnical Faculty, University of Ljubljana, Groblje 3, SI-1230 Domzale (Slovenia)

    2015-09-09

    Single nucleotide polymorphisms (SNPs) located in microRNA (miRNA) genes (miR-SNPs) have attracted increasing attention in recent years due to their involvement in the development of various types of cancer. Therefore, a systematic review on this topic was needed. From 55 scientific publications we collected 20 SNPs, which are located within 18 miRNA encoding genes and have been associated with 16 types of cancer. Among 20 miRNA gene polymorphisms 13 are located within the premature miRNA region, five within mature, and two within mature seed miRNA region. We graphically visualized a network of miRNA-cancer associations which revealed miRNA genes and cancer types with the highest number of connections. Our study showed that, despite a large number of variations currently known to be located within miRNA genes in humans, most of them have not yet been tested for association with cancer. MicroRNA SNPs collected in this study represent only 0.43% of known miRNA gene variations (20/4687). Results of the present study will be useful to researchers investigating the clinical use of miRNAs, such as the roles of miRNAs as diagnostic markers and therapeutic targets.

  15. Regulation of Pancreatic microRNA-7 Expression

    Sharon Kredo-Russo

    2012-01-01

    Full Text Available Genome-encoded microRNAs (miRNAs provide a posttranscriptional regulatory layer, which is important for pancreas development. Differentiation of endocrine cells is controlled by a network of pancreatic transcription factors including Ngn3 and NeuroD/Beta2. However, how specific miRNAs are intertwined into this transcriptional network is not well understood. Here, we characterize the regulation of microRNA-7 (miR-7 by endocrine-specific transcription factors. Our data reveal that three independent miR-7 genes are coexpressed in the pancreas. We have identified conserved blocks upstream of pre-miR-7a-2 and pre-miR-7b and demonstrated by functional assays that they possess promoter activity, which is increased by the expression of NeuroD/Beta2. These data suggest that the endocrine specificity of miR-7 expression is governed by transcriptional mechanisms and involves members of the pancreatic endocrine network of transcription factors.

  16. MicroRNA Polymorphisms in Cancer: A Literature Analysis

    Single nucleotide polymorphisms (SNPs) located in microRNA (miRNA) genes (miR-SNPs) have attracted increasing attention in recent years due to their involvement in the development of various types of cancer. Therefore, a systematic review on this topic was needed. From 55 scientific publications we collected 20 SNPs, which are located within 18 miRNA encoding genes and have been associated with 16 types of cancer. Among 20 miRNA gene polymorphisms 13 are located within the premature miRNA region, five within mature, and two within mature seed miRNA region. We graphically visualized a network of miRNA-cancer associations which revealed miRNA genes and cancer types with the highest number of connections. Our study showed that, despite a large number of variations currently known to be located within miRNA genes in humans, most of them have not yet been tested for association with cancer. MicroRNA SNPs collected in this study represent only 0.43% of known miRNA gene variations (20/4687). Results of the present study will be useful to researchers investigating the clinical use of miRNAs, such as the roles of miRNAs as diagnostic markers and therapeutic targets

  17. MicroRNAs in Honey Bee Caste Determination.

    Ashby, Regan; Forêt, Sylvain; Searle, Iain; Maleszka, Ryszard

    2016-01-01

    The cellular mechanisms employed by some organisms to produce contrasting morphological and reproductive phenotypes from the same genome remains one of the key unresolved issues in biology. Honeybees (Apis mellifera) use differential feeding and a haplodiploid sex determination system to generate three distinct organismal outcomes from the same genome. Here we investigate the honeybee female and male caste-specific microRNA and transcriptomic molecular signatures during a critical time of larval development. Both previously undetected and novel miRNAs have been discovered, expanding the inventory of these genomic regulators in invertebrates. We show significant differences in the microRNA and transcriptional profiles of diploid females relative to haploid drone males as well as between reproductively distinct females (queens and workers). Queens and drones show gene enrichment in physio-metabolic pathways, whereas workers show enrichment in processes associated with neuronal development, cell signalling and caste biased structural differences. Interestingly, predicted miRNA targets are primarily associated with non-physio-metabolic genes, especially neuronal targets, suggesting a mechanistic disjunction from DNA methylation that regulates physio-metabolic processes. Accordingly, miRNA targets are under-represented in methylated genes. Our data show how a common set of genetic elements are differentially harnessed by an organism, which may provide the remarkable level of developmental flexibility required. PMID:26739502

  18. Cellular Reprogramming Using Defined Factors and MicroRNAs

    Takanori Eguchi

    2016-01-01

    Full Text Available Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1, Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs and dental pulp stem cells (DPSCs are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs. This review reports histories, topics, and idea of cellular reprogramming.

  19. Cellular Reprogramming Using Defined Factors and MicroRNAs.

    Eguchi, Takanori; Kuboki, Takuo

    2016-01-01

    Development of human bodies, organs, and tissues contains numerous steps of cellular differentiation including an initial zygote, embryonic stem (ES) cells, three germ layers, and multiple expertized lineages of cells. Induced pluripotent stem (iPS) cells have been recently developed using defined reprogramming factors such as Nanog, Klf5, Oct3/4 (Pou5f1), Sox2, and Myc. This outstanding innovation is largely changing life science and medicine. Methods of direct reprogramming of cells into myocytes, neurons, chondrocytes, and osteoblasts have been further developed using modified combination of factors such as N-myc, L-myc, Sox9, and microRNAs in defined cell/tissue culture conditions. Mesenchymal stem cells (MSCs) and dental pulp stem cells (DPSCs) are also emerging multipotent stem cells with particular microRNA expression signatures. It was shown that miRNA-720 had a role in cellular reprogramming through targeting the pluripotency factor Nanog and induction of DNA methyltransferases (DNMTs). This review reports histories, topics, and idea of cellular reprogramming. PMID:27382371

  20. Muscle-specific microRNAs in skeletal muscle development.

    Horak, Martin; Novak, Jan; Bienertova-Vasku, Julie

    2016-02-01

    Proper muscle function constitutes a precondition for good heath and an active lifestyle during an individual's lifespan and any deviations from normal skeletal muscle development and its functions may lead to numerous health conditions including e.g. myopathies and increased mortality. It is thus not surprising that there is an increasing need for understanding skeletal muscle developmental processes and the associated molecular pathways, especially as such information could find further uses in therapy. The understanding of complex skeletal muscle developmental networks was broadened with the discovery of microRNA (miRNA) molecules. MicroRNAs are evolutionary conserved small non-coding RNAs capable of negatively regulating gene expression on a post-transcriptional level by means of miRNA-mRNA interaction. Several miRNAs expressed exclusively in muscle have been labeled myomiRs. MyomiRs represent an integral part of skeletal muscle development, i.e. playing a significant role during skeletal muscle proliferation, differentiation and regeneration. The purpose of this review is to provide a summary of current knowledge regarding the involvement of myomiRs in the individual phases of myogenesis and other aspects of skeletal muscle biology, along with an up-to-date list of myomiR target genes and their functions in skeletal muscle and miRNA-related therapeutic approaches and future prospects. PMID:26708096

  1. Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development.

    Choi, Yoori; Hwang, Do Won; Kim, Mee Young; Kim, Joo Yeon; Sun, Woong; Lee, Dong Soo

    2016-01-01

    MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs. PMID:27462205

  2. Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development

    Choi, Yoori; Hwang, Do won; Kim, Mee Young; Kim, Joo Yeon; Sun, Woong; Lee, Dong Soo

    2016-01-01

    MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs. PMID:27462205

  3. Expression and survival prediction of microRNA-155 in hepatocellular carcinoma after liver transplantation

    韩中博

    2013-01-01

    Objective To explore the expression of microRNA-155in hepatocellular carcinoma(HCC)and its contribution to recurrence and prognosis of HCC after liver transplantation(LT).Methods The expression levels

  4. Expression and survival prediction of microRNA-155 in hepatocellular carcinoma after liver transplantation

    韩中博

    2013-01-01

    Objective To explore the expression of microRNA-155in hepatocellular carcinoma(HCC)and its contribution to recurrence and prognosis of HCC after liver transplantation(LT).Methods The expression levels of

  5. Polychlorinated biphenyl exposure alters the expression profile of microRNAs associated with vascular diseases.

    Wahlang, Banrida; Petriello, Michael C; Perkins, Jordan T; Shen, Shu; Hennig, Bernhard

    2016-09-01

    Exposure to persistent organic pollutants, including polychlorinated biphenyls (PCBs) is correlated with multiple vascular complications including endothelial cell dysfunction and atherosclerosis. PCB-induced activation of the vasculature subsequently leads to oxidative stress and induction of pro-inflammatory cytokines and adhesion proteins. Gene expression of these cytokines/proteins is known to be regulated by small, endogenous oligonucleotides known as microRNAs that interact with messenger RNA. MicroRNAs are an acknowledged component of the epigenome, but the role of environmentally-driven epigenetic changes such as toxicant-induced changes in microRNA profiles is currently understudied. The objective of this study was to determine the effects of PCB exposure on microRNA expression profile in primary human endothelial cells using the commercial PCB mixture Aroclor 1260. Samples were analyzed using Affymetrix GeneChip® miRNA 4.0 arrays for high throughput detection and selected microRNA gene expression was validated (RT-PCR). Microarray analysis identified 557 out of 6658 microRNAs that were changed with PCB exposure (p<0.05). In-silico analysis using MetaCore database identified 21 of these microRNAs to be associated with vascular diseases. Further validation showed that Aroclor 1260 increased miR-21, miR-31, miR-126, miR-221 and miR-222 expression levels. Upregulated miR-21 has been reported in cardiac injury while miR-126 and miR-31 modulate inflammation. Our results demonstrated evidence of altered microRNA expression with PCB exposure, thus providing novel insights into mechanisms of PCB toxicity. PMID:27288564

  6. MicroRNA Profiling of Pericardial Fluid Samples from Patients with Heart Failure

    Kuosmanen, Suvi M.; Hartikainen, Juha; Hippeläinen, Mikko; Kokki, Hannu; Levonen, Anna-Liisa; Tavi, Pasi

    2015-01-01

    Aims Multicellular organisms maintain vital functions through intercellular communication. Release of extracellular vesicles that carry signals to even distant target organs is one way of accomplishing this communication. MicroRNAs can also be secreted from the cells in exosomes and act as paracrine signalling molecules. In addition, microRNAs have been implicated in the pathogenesis of a large number of diseases, including cardiovascular diseases, and are considered as promising candidate bi...

  7. Comparative Characterization of Cardiac Development Specific microRNAs: Fetal Regulators for Future.

    Yashika Rustagi

    Full Text Available MicroRNAs (miRNAs are small, conserved RNAs known to regulate several biological processes by influencing gene expression in eukaryotes. The implication of miRNAs as another player of regulatory layers during heart development and diseases has recently been explored. However, there is no study which elucidates the profiling of miRNAs during development of heart till date. Very limited miRNAs have been reported to date in cardiac context. In addition, integration of large scale experimental data with computational and comparative approaches remains an unsolved challenge.The present study was designed to identify the microRNAs implicated in heart development using next generation sequencing, bioinformatics and experimental approaches. We sequenced six small RNA libraries prepared from different developmental stages of the heart using chicken as a model system to produce millions of short sequence reads. We detected 353 known and 703 novel miRNAs involved in heart development. Out of total 1056 microRNAs identified, 32.7% of total dataset of known microRNAs displayed differential expression whereas seven well studied microRNAs namely let-7, miR-140, miR-181, miR-30, miR-205, miR-103 and miR-22 were found to be conserved throughout the heart development. The 3'UTR sequences of genes were screened from Gallus gallus genome for potential microRNA targets. The target mRNAs were appeared to be enriched with genes related to cell cycle, apoptosis, signaling pathways, extracellular remodeling, metabolism, chromatin remodeling and transcriptional regulators. Our study presents the first comprehensive overview of microRNA profiling during heart development and prediction of possible cardiac specific targets and has a big potential in future to develop microRNA based therapeutics against cardiac pathologies where fetal gene re-expression is witnessed in adult heart.

  8. RNA sequencing reveals a depletion of collagen targeting microRNAs in Dupuytren’s disease

    Riester, Scott M; Arsoy, Diren; Camilleri, Emily T.; Dudakovic, Amel; Paradise, Christopher R.; Evans, Jared M.; Torres-Mora, Jorge; Rizzo, Marco; Kloen, Peter; Julio, Marianna Kruithof-de; van Wijnen, Andre J; Kakar, Sanjeev

    2015-01-01

    Background Dupuytren’s disease is an inherited disorder in which patients develop fibrotic contractures of the hand. Current treatment strategies include surgical excision or enzymatic digestion of fibrotic tissue. MicroRNAs, which are key posttranscriptional regulators of genes expression, have been shown to play an important regulatory role in disorders of fibrosis. Therefore in this investigation, we apply high throughput next generation RNA sequencing strategies to characterize microRNA e...

  9. Differential Expression of MicroRNAs in Response to Drought Stress in Maize

    LI Jing-sheng; FU Feng-ling; AN Ming; ZHOU Shu-feng; SHE Yue-hui; LI Wan-chen

    2013-01-01

    Drought is one of the major abiotic stresses that limit maize productivity. Apart from the principal transcriptional regulation, post-transcriptional regulation mediated by microRNAs appears to be the prevalent response of plants to abiotic stress. In this study, the differential expression of microRNAs in the previously evaluated drought-tolerant inbred lines R09 under drought stress was detected by microarray hybridization. The target genes of the differentially-expressed microRNAs were predicted by bioinformatics software WMD3 for plant target gene prediction. The possible regulation of the differentially-expressed microRNAs as well as their target genes in maize response to drought stress was analysed according to Gene Ontology. Sixty-eight microRNAs in 29 microRNA families were detected to be differentially expressed in the seedling of the drought-tolerant inbred line R09, accounting for 5.97% of the total number of the probes. The expression profiles were different between the two time points of the drought stress. The functions of the genes targeted by the differentially-expressed microRNAs involve multiple physiological and biochemical pathways of response to abiotic stress, such as transcription regulation, metabolism, signal transduction, hormone stimulation, and transmembrane transport. Under drought stress, the differential expression of microRNAs regulates the expression of their target genes, resulting in multiple responses of physiological and biochemical pathways relative to drought tolerance of maize. miR156, miR159 and miR319 families may play more important roles. The different members of the same family may play similar regulation effects in most cases.

  10. MicroRNAs in colorectal cancer as markers and targets: Recent advances

    Ye, Jing-jia; Cao, Jiang

    2014-01-01

    MicroRNAs are evolutionarily conserved small non-coding RNA molecules encoded by eukaryotic genomic DNA, and function in post-transcriptional regulation of gene expression via base-pairing with complementary sequences in target mRNAs, resulting in translational repression or degradation of target mRNAs. They represent one of the major types of epigenetic modification and play important roles in all aspects of cellular activities. Altered expression of microRNAs has been found in various human...

  11. Genetic variation in microRNA networks: the implications for cancer research

    Ryan, Bríd M.; Ana I Robles; Curtis C Harris

    2010-01-01

    Many studies have highlighted the role that microRNAs have in physiological processes and how their deregulation can lead to cancer. More recently it has been proposed that the presence of single nucleotide polymorphisms in microRNA genes, their processing machinery and target binding sites affects cancer risk, treatment efficacy and patient prognosis. In reviewing this new field of cancer biology, we describe the methodological approaches of these studies and make recommendations for which s...

  12. Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery

    Lv, Lin-Li; Cao, Yuhan; Liu, Dan; Xu, Min; Liu, Hong; Tang, Ri-Ning; Ma, Kun-Ling; Liu, Bi-Cheng

    2013-01-01

    Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes an...

  13. Expression Profile of microRNAs Regulating Proliferation and Differentiation in Mouse Adult Cardiac Stem Cells

    Brás-Rosário, Luis; Matsuda, Alex; Pinheiro, Ana Isabel; Gardner, Rui; Lopes, Telma; Amaral, Andreia; Gama-Carvalho, Margarida

    2013-01-01

    The identification of cardiac cells with stem cell properties changed the paradigm of the heart as a post mitotic organ. These cells proliferate and differentiate into cardiomyocytes, endothelial and vascular smooth muscle cells, providing for cardiac cell homeostasis and regeneration. microRNAs are master switches controlling proliferation and differentiation, in particular regulating stem cell biology and cardiac development. Modulation of microRNAs -regulated gene expression networks holds...

  14. microRNAs Associated with Drought Response in the Bioenergy Crop Sugarcane (Saccharum spp.)

    Ferreira, Thaís Helena; Gentile, Agustina; Vilela, Romel Duarte; Costa, Gustavo Gilson Lacerda; Dias, Lara Isys; Endres, Laurício; Menossi, Marcelo

    2012-01-01

    Sugarcane (Saccharum spp.) is one of the most important crops in the world. Drought stress is a major abiotic stress factor that significantly reduces sugarcane yields. However the gene network that mediates plant responses to water stress remains largely unknown in several crop species. Although several microRNAs that mediate post-transcriptional regulation during water stress have been described in other species, the role of the sugarcane microRNAs during drought stress has not been studied...

  15. An Optimized microRNA Backbone for Effective Single-Copy RNAi

    Christof Fellmann; Thomas Hoffmann; Vaishali Sridhar; Barbara Hopfgartner; Matthias Muhar; Mareike Roth; Dan Yu Lai; Inês A.M. Barbosa; Jung Shick Kwon; Yuanzhe Guan; Nishi Sinha; Johannes Zuber

    2013-01-01

    Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such “shRNAmirs” often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a system...

  16. Prognostic Role of Common MicroRNA Polymorphisms in Cancers: Evidence from a Meta-Analysis

    Xia, Lingzi; Ren, Yangwu; Fang, Xue; Yin, Zhihua; Li, Xuelian; Wu, Wei; Guan, Peng; Baosen ZHOU

    2014-01-01

    Background The morbidity and mortality of cancer increase remarkably every year. It's a heavy burden for family and society. The detection of prognostic biomarkers can help to improve the theraputic effect and prolong the lifetime of patients. microRNAs have an influential role in cancer prognosis. The results of articles discussing the relationship between microRNA polymorphisms and cancer prognosis are inconsistent. Methods We conduct a meta-analysis of 19 publications concerning the associ...

  17. MicroRNA 146 (Mir146) Modulates Spermatogonial Differentiation by Retinoic Acid in Mice1

    Huszar, Jessica M.; Payne, Christopher J.

    2012-01-01

    Impaired biogenesis of microRNAs disrupts spermatogenesis and leads to infertility in male mice. Spermatogonial differentiation is a key step in spermatogenesis, yet the mechanisms that control this event remain poorly defined. In this study, we discovered microRNA 146 (Mir146) to be highly regulated during spermatogonial differentiation, a process dependent on retinoic acid (RA) signaling. Mir146 transcript levels were diminished nearly 180-fold in differentiating spermatogonia when compared...

  18. Sensitive detection of melanoma metastasis using circulating microRNA expression profiles.

    Shiiyama, Rie; Fukushima, Satoshi; Jinnin, Masatoshi; Yamashita, Junji; Miyashita, Azusa; Nakahara, Satoshi; Kogi, Ai; Aoi, Jun; Masuguchi, Shinichi; Inoue, Yuji; Ihn, Hironobu

    2013-10-01

    Numerous studies have indicated that the serum levels of microRNAs are useful for the diagnosis or evaluation of activity in human diseases. However, determining the level of only one of the nearly 2000 microRNAs identified so far may be less significant. Accordingly, we examined the possibility that the expression pattern of multiple microRNAs in each patient may be a more reliable disease marker for melanoma, especially metastatic disease, focusing on the interaction among microRNAs. Six microRNAs (miR-9, miR-145, miR-150, miR-155, miR-203, and miR-205) were evaluated using real-time PCR in 11 patients with metastatic melanoma and in 16 patients without melanoma. The expression of the six microRNAs was significantly different between the patients with metastasis and those without it. MiR-9 and miR-205 and miR-203 and miR-205 showed significant correlations, and the combination of miR-9, miR-145, miR-150, miR-155, and miR-205 was more sensitive than when each miR was used individually to distinguish the patients with metastasis from those without it. This is the first report demonstrating the expression profiles of multiple microRNAs in melanoma patients. Clarifying the involvement of the microRNA network in the pathogenesis of melanoma may contribute to the development of new diagnostic tools and to advancing the understanding of this disease. PMID:23863473

  19. MicroRNA Expression Analyses in Preoperative Pancreatic Juice Samples of Pancreatic Ductal Adenocarcinoma

    Yoshihiko Sadakari; Takao Ohtsuka; Kenoki Ohuchida; Kosuke Tsutsumi; Shunichi Takahata; Masafumi Nakamura; Kazuhiro Mizumoto; Masao Tanaka

    1999-01-01

    Context Cytological assessment of pancreatic juice is commonly used to diagnose pancreatic ductal adenocarcinoma; however, the sensitivity of cytological assessment has been reported to be low. MicroRNAs are small RNAs regulating various cellular processes and have recently been identified as possible markers of malignant diseases including pancreatic ductal adenocarcinoma. Objective The purposes of this study were to prove the existence of microRNAs in pancreatic juice and to determine wheth...

  20. Performance Comparison of Digital microRNA Profiling Technologies Applied on Human Breast Cancer Cell Lines

    Knutsen, Erik; Fiskaa, Tonje; Ursvik, Anita; Jørgensen, Tor Erik; Perander, maria; Lund, Eiliv; Seternes, Ole Morten; Johansen, Steinar

    2013-01-01

    MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybr...

  1. Search for MicroRNAs Expressed by Intracellular Bacterial Pathogens in Infected Mammalian Cells

    Furuse, Yuki; Finethy, Ryan; Saka, Hector A.; Xet-Mull, Ana M.; Sisk, Dana M.; Smith, Kristen L. Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H.; Tobin, David M.; Cullen, Bryan R.

    2014-01-01

    MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin. PMID:25184567

  2. Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells.

    Yuki Furuse

    Full Text Available MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼ 23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin.

  3. Identification and Pathway Analysis of microRNAs with No Previous Involvement in Breast Cancer

    Rebollar-Vega, Rosa; Quintanar-Jurado, Valeria; Maffuz-Aziz, Antonio; Jimenez-Sanchez, Gerardo; Bautista-Piña, Veronica; Arellano-Llamas, Rocio; Hidalgo-Miranda, Alfredo

    2012-01-01

    microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value = 0.05, Fold Change = 2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The expression of 14 microRNAs was replicated in an independent set of 55 tumors. Bioinformatic analysis of mRNA targets of the altered miRNAs, identified oncogenes like ERBB2, YY1, several MAP kinases, and known tumor-suppressors like FOXA1 and SMAD4. Pathway analysis identified that some biological process which are important in breast carcinogenesis are affected by the altered microRNA expression, including signaling through MAP kinases and TP53 pathways, as well as biological processes like cell death and communication, focal adhesion and ERBB2-ERBB3 signaling. Our data identified the altered expression of several microRNAs whose aberrant expression might have an important impact on cancer-related cellular pathways and whose role in breast cancer has not been previously described. PMID:22438871

  4. Circulating microRNAs are associated with docetaxel chemotherapy outcome in castration-resistant prostate cancer

    Lin, H-M.; Castillo, L.; Mahon, K L; Chiam, K; Lee, B Y; Nguyen, Q.; Boyer, M. J.; Stockler, M R; Pavlakis, N; Marx, G.; Mallesara, G; Gurney, H; Clark, S J; Swarbrick, A; Daly, R. J.

    2014-01-01

    Background: Docetaxel is the first-line chemotherapy for castration-resistant prostate cancer (CRPC). However, response rates are ∼50% and determined quite late in the treatment schedule, thus non-responders are subjected to unnecessary toxicity. The potential of circulating microRNAs as early biomarkers of docetaxel response in CRPC patients was investigated in this study. Methods: Global microRNA profiling was performed on docetaxel-resistant and sensitive cell lines to identify candidate c...

  5. Emerging roles of chicken and viral microRNAs in avian disease

    Burnside Joan; Morgan Robin

    2011-01-01

    Abstract Background MicroRNAs are short RNAs (~22 nt) expressed by plants, animals and viruses that regulate gene expression post-transcriptionally, and their importance is highlighted by distinct patterns of expression in many physiological processes, including development, hematopoeisis, stress resistance, and disease. Our group has characterized the microRNAs encoded by the avian herpesviruses; namely, oncogenic Marek’s disease (MD) virus (MDV1), non-oncogenic MDV (MDV2) herpesvirus of tu...

  6. Diagnostic Potential of Circulating MicroRNA-21 in Hepatocellular Carcinoma.

    Waleed Alnoanmany

    2015-08-01

    Full Text Available Abstract Background amp AimsSeveral studies have reported the significance of circulating microRNA as a biochemical marker of cancer. However there are no reports on the significance of circulating microRNA in hepatocellular carcinoma. The aim of this study was to evaluate the significance of plasma microRNA-21 level as a biochemical marker for hepatocellular carcinoma.Materials amp Methods To identify the causal role of MicroRNA-21 in hepatocarcinogenesis we used a human model in which 30 diagnosed HCC cases of different stages 20 HCV positive cases and 20 healthy controls were tested for circulating microRNA-21 using whole blood samples taken from mentioned individuals at National Liver Institute Menoufiya University. MicroRNA extraction Amplification RT-PCR was done for all samples with other various biochemical analysis. Results Real-time RT-PCR analysis demonstrated upregulation of oncogenic miR-21 at different stages of hepatocarcinogenesis. On the other hand there were no significant miRNA-21 changes neither in HCV nor Control groups. ROC study showed that the best cutoff value for miR-21 was 3.93 Fold expression and the sensitivity was 93 while the specificity was 90. Compared to the cutoff value for AFP which was 91.7 ngmL and the sensitivity was 75.2 while the specificity was 92.3 .Conclusion Circulating MiRNA-21 level is more sensitive than AFP and highly specific as a biological marker for HCC also it is proved to be beneficial in early diagnosis of HCC.Targeting of microRNA-21 is sufficient to limit tumor cell proliferation and invasion in a manner that is likely to involve associated changes in multiple targets suggesting that suppression of microRNA-21 may be an approach for the treatment of hepatocellular carcinoma.

  7. Understanding alcoholism through microRNA signatures in brains of human alcoholics

    R. Dayne eMayfield

    2012-04-01

    Full Text Available Advances in the fields of genomics and genetics in the last decade have identified a large number of genes that can potentially influence alcohol-drinking behavior in humans as well as animal models. Consequently, the task of identifying efficient molecular targets that could be used to develop effective therapeutics against the disease has become increasingly daunting. One of the reasons for this is the fact that each of the many alcohol-responsive genes only contributes a small effect to the overall mechanism and disease phenotype, as is characteristic of complex traits. Current research trends are hence shifting towards the analysis of gene networks rather than emphasizing individual genes. The discovery of microRNAs and their mechanisms of action on regulation of transcript level and protein translation have made evident the utility of these small non-coding RNA molecules that act as central coordinators of multiple cross-communicating cellular pathways. Cells exploit the fact that a single microRNA can target hundreds of mRNA transcripts and that a single mRNA transcript can be simultaneously targeted by distinct microRNAs, to ensure fine-tuned and/or redundant control over a large number of cellular functions. By the same token, we can use these properties of microRNAs to develop novel, targeted strategies to combat complex disorders. In this review, we will focus on recent discoveries of microRNA signatures in brain of human alcoholics supporting the hypothesis that changes in gene expression and regulation by microRNAs are responsible for long-term neuroadaptations occurring during development of alcoholism. We also discuss insights into the potential modulation of epigenetic regulators by a subset of microRNAs. Taken together, microRNA activity may be controlling many of the cellular mechanisms already known to be involved in the development of alcoholism, and suggests potential targets for the development of novel therapeutic

  8. On combining microRNA analysis with DNA profiling in a single stream process

    van der Meer, Dieudonne J.; Williams, Graham

    2015-01-01

    MicroRNAs have a potential to be ideal forensic markers due to their small size (~22nt), high abundance per cell, and sensitive and specific PCR-based detection. Thousands of microRNAs are present in biological material and they are rich in information due to their tightly regulated and cell type specific expression. Their advantageous properties increase the chances of successful analysis from challenged crime scene samples. In addition, it has been demonstrated previously that informative m...

  9. MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

    Irma Virant-Klun; Anders Ståhlberg; Mikael Kubista; Thomas Skutella

    2016-01-01

    MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human ...

  10. Smoking-related microRNAs and mRNAs in human peripheral blood mononuclear cells.

    Su, Ming-Wei; Yu, Sung-Liang; Lin, Wen-Chang; Tsai, Ching-Hui; Chen, Po-Hua; Lee, Yungling Leo

    2016-08-15

    Teenager smoking is of great importance in public health. Functional roles of microRNAs have been documented in smoke-induced gene expression changes, but comprehensive mechanisms of microRNA-mRNA regulation and benefits remained poorly understood. We conducted the Teenager Smoking Reduction Trial (TSRT) to investigate the causal association between active smoking reduction and whole-genome microRNA and mRNA expression changes in human peripheral blood mononuclear cells (PBMC). A total of 12 teenagers with a substantial reduction in smoke quantity and a decrease in urine cotinine/creatinine ratio were enrolled in genomic analyses. In Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA), differentially expressed genes altered by smoke reduction were mainly associated with glucocorticoid receptor signaling pathway. The integrative analysis of microRNA and mRNA found eleven differentially expressed microRNAs negatively correlated with predicted target genes. CD83 molecule regulated by miR-4498 in human PBMC, was critical for the canonical pathway of communication between innate and adaptive immune cells. Our data demonstrated that microRNAs could regulate immune responses in human PBMC after habitual smokers quit smoking and support the potential translational value of microRNAs in regulating disease-relevant gene expression caused by tobacco smoke. PMID:27321975

  11. Circulating microRNAs as potential biomarkers for diagnosis of congenital heart defects

    Xie, Wan-qin; Zhou, Lin; Chen, Yong; Ni, Bin

    2016-01-01

    BACKGROUND: MicroRNAs are small non-coding RNAs of approximately 22 nucleotides in length, and play important regulatory roles in normal heart development and the pathogenesis of heart diseases. Recently, a few prospective studies have implicated the diagnostic role of microRNAs in congenital heart defects (CHD). DATA RESOURCES: This review retrieved the research articles in PubMed focusing on the altered microRNAs in cardiac tissue or serum of patients with CHD versus healthy normal controls, as well as the studies exploring circulating microRNAs as potential biomarkers for (fetal) CHD. RESULTS: Most of the studies of interest were conducted in recent years, implicating that the topic in this review is a newly emerging field and is drawing much attention. Moreover, a number of differentially expressed microRNAs between CHD specimens and normal controls have been reported. CONCLUSION: Circulating microRNAs may serve as potential biomarkers for diagnosis of CHD in the future, with more efforts paving the road to the aim. PMID:27313801

  12. A novel serum microRNA panel to discriminate benign from malignant ovarian disease.

    Langhe, Ream

    2015-01-28

    Ovarian cancer is the seventh most common cancer in women and the most frequent cause of gynaecological malignancy-related mortality in women. Currently, no standardized reliable screening test exists. MicroRNA profiling has allowed the identification of signatures associated with diagnosis, prognosis and response to treatment of human tumours. The aim of this study was to determine if a microRNA signature could distinguish between malignant and benign ovarian disease. A training set of 5 serous ovarian carcinomas and 5 benign serous cystadenomas were selected for the initial experiments. The validation set included 20 serous ovarian carcinomas and 20 benign serous cystadenomas. The serum\\/plasma focus microRNA Exiqon panel was used for the training set. For the validation set a pick and mix Exiqon panel, which focuses on microRNAs of interest was used. A panel of 4 microRNAs (let-7i-5p, miR-122, miR-152-5p and miR-25-3p) was significantly down regulated in cancer patients. These microRNAs target WNT signalling, AKT\\/mTOR and TLR-4\\/MyD88, which have previously been found to play a role in ovarian carcinogenesis and chemoresistance. let-7i-5p, miR-122, miR-152-5p and miR-25-3p could act as diagnostic biomarkers in ovarian cancer.

  13. MicroRNAs: From Female Fertility, Germ Cells, and Stem Cells to Cancer in Humans

    Irma Virant-Klun

    2016-01-01

    Full Text Available MicroRNAs are a family of naturally occurring small noncoding RNA molecules that play an important regulatory role in gene expression. They are suggested to regulate a large proportion of protein encoding genes by mediating the translational suppression and posttranscriptional control of gene expression. Recent findings show that microRNAs are emerging as important regulators of cellular differentiation and dedifferentiation, and are deeply involved in developmental processes including human preimplantation development. They keep a balance between pluripotency and differentiation in the embryo and embryonic stem cells. Moreover, it became evident that dysregulation of microRNA expression may play a fundamental role in progression and dissemination of different cancers including ovarian cancer. The interest is still increased by the discovery of exosomes, that is, cell-derived vesicles, which can carry different proteins but also microRNAs between different cells and are involved in cell-to-cell communication. MicroRNAs, together with exosomes, have a great potential to be used for prognosis, therapy, and biomarkers of different diseases including infertility. The aim of this review paper is to summarize the existent knowledge on microRNAs related to female fertility and cancer: from primordial germ cells and ovarian function, germinal stem cells, oocytes, and embryos to embryonic stem cells.

  14. Identification of Conserved and Novel MicroRNAs during Tail Regeneration in the Mexican Axolotl

    Micah D. Gearhart

    2015-09-01

    Full Text Available The Mexican axolotl salamander (Ambystoma mexicanum is one member of a select group of vertebrate animals that have retained the amazing ability to regenerate multiple body parts. In addition to being an important model system for regeneration, the axolotl has also contributed extensively to studies of basic development. While many genes known to play key roles during development have now been implicated in various forms of regeneration, much of the regulatory apparatus controlling the underlying molecular circuitry remains unknown. In recent years, microRNAs have been identified as key regulators of gene expression during development, in many diseases and also, increasingly, in regeneration. Here, we have used deep sequencing combined with qRT-PCR to undertake a comprehensive identification of microRNAs involved in regulating regeneration in the axolotl. Specifically, among the microRNAs that we have found to be expressed in axolotl tissues, we have identified 4564 microRNA families known to be widely conserved among vertebrates, as well as 59,811 reads of putative novel microRNAs. These findings support the hypothesis that microRNAs play key roles in managing the precise spatial and temporal patterns of gene expression that ensures the correct regeneration of missing tissues.

  15. Synaptic enrichment of microRNAs in adult mouse forebrain is related to structural features of their precursors

    Smalheiser Neil R

    2008-01-01

    Abstract Within mouse forebrain, a subset of microRNAs are significantly enriched in synaptoneurosomes (a synaptic fraction containing pinched-off dendritic spines) and a subset are significantly depleted relative to total forebrain homogenate. Here I show that, as a group, the pre-miR hairpin precursors of synaptically enriched microRNAs exhibit significantly different structural features than those that are non-enriched or depleted. Precursors of synaptically enriched microRNAs tend to hav...

  16. Regulation of MicroRNAs, and the Correlations of MicroRNAs and Their Targeted Genes by Zinc Oxide Nanoparticles in Ovarian Granulosa Cells.

    Zhao, Yong; Li, Lan; Min, Ling-Jiang; Zhu, Lian-Qin; Sun, Qing-Yuan; Zhang, Hong-Fu; Liu, Xin-Qi; Zhang, Wei-Dong; Ge, Wei; Wang, Jun-Jie; Liu, Jing-Cai; Hao, Zhi-Hui

    2016-01-01

    Zinc oxide (ZnO) nanoparticles (NPs) have been applied in numerous industrial products and personal care products like sunscreens and cosmetics. The released ZnO NPs from consumer and household products into the environment might pose potential health issues for animals and humans. In this study the expression of microRNAs and the correlations of microRNAs and their targeted genes in ZnO NPs treated chicken ovarian granulosa cells were investigated. ZnSO4 was used as the sole Zn2+ provider to differentiate the effects of NPs from Zn2+. It was found that ZnO-NP-5 μg/ml specifically regulated the expression of microRNAs involved in embryonic development although ZnO-NP-5 μg/ml and ZnSO4-10 μg/ml treatments produced the same intracellular Zn concentrations and resulted in similar cell growth inhibition. And ZnO-NP-5 μg/ml also specifically regulated the correlations of microRNAs and their targeted genes. This is the first investigation that intact NPs in ZnO-NP-5 μg/ml treatment specifically regulated the expression of microRNAs, and the correlations of microRNAs and their targeted genes compared to that by Zn2+. This expands our knowledge for biological effects of ZnO NPs and at the same time it raises the health concerns that ZnO NPs might adversely affect our biological systems, even the reproductive systems through regulation of specific signaling pathways. PMID:27196542

  17. Regulation of MicroRNAs, and the Correlations of MicroRNAs and Their Targeted Genes by Zinc Oxide Nanoparticles in Ovarian Granulosa Cells

    Zhao, Yong; Li, Lan; Min, Ling-Jiang; Zhu, Lian-Qin; Sun, Qing-Yuan; Zhang, Hong-Fu; Liu, Xin-Qi; Zhang, Wei-Dong; Ge, Wei; Wang, Jun-Jie; Liu, Jing-Cai

    2016-01-01

    Zinc oxide (ZnO) nanoparticles (NPs) have been applied in numerous industrial products and personal care products like sunscreens and cosmetics. The released ZnO NPs from consumer and household products into the environment might pose potential health issues for animals and humans. In this study the expression of microRNAs and the correlations of microRNAs and their targeted genes in ZnO NPs treated chicken ovarian granulosa cells were investigated. ZnSO4 was used as the sole Zn2+ provider to differentiate the effects of NPs from Zn2+. It was found that ZnO-NP-5 μg/ml specifically regulated the expression of microRNAs involved in embryonic development although ZnO-NP-5 μg/ml and ZnSO4-10 μg/ml treatments produced the same intracellular Zn concentrations and resulted in similar cell growth inhibition. And ZnO-NP-5 μg/ml also specifically regulated the correlations of microRNAs and their targeted genes. This is the first investigation that intact NPs in ZnO-NP-5 μg/ml treatment specifically regulated the expression of microRNAs, and the correlations of microRNAs and their targeted genes compared to that by Zn2+. This expands our knowledge for biological effects of ZnO NPs and at the same time it raises the health concerns that ZnO NPs might adversely affect our biological systems, even the reproductive systems through regulation of specific signaling pathways. PMID:27196542

  18. Regulation of MicroRNAs, and the Correlations of MicroRNAs and Their Targeted Genes by Zinc Oxide Nanoparticles in Ovarian Granulosa Cells.

    Yong Zhao

    Full Text Available Zinc oxide (ZnO nanoparticles (NPs have been applied in numerous industrial products and personal care products like sunscreens and cosmetics. The released ZnO NPs from consumer and household products into the environment might pose potential health issues for animals and humans. In this study the expression of microRNAs and the correlations of microRNAs and their targeted genes in ZnO NPs treated chicken ovarian granulosa cells were investigated. ZnSO4 was used as the sole Zn2+ provider to differentiate the effects of NPs from Zn2+. It was found that ZnO-NP-5 μg/ml specifically regulated the expression of microRNAs involved in embryonic development although ZnO-NP-5 μg/ml and ZnSO4-10 μg/ml treatments produced the same intracellular Zn concentrations and resulted in similar cell growth inhibition. And ZnO-NP-5 μg/ml also specifically regulated the correlations of microRNAs and their targeted genes. This is the first investigation that intact NPs in ZnO-NP-5 μg/ml treatment specifically regulated the expression of microRNAs, and the correlations of microRNAs and their targeted genes compared to that by Zn2+. This expands our knowledge for biological effects of ZnO NPs and at the same time it raises the health concerns that ZnO NPs might adversely affect our biological systems, even the reproductive systems through regulation of specific signaling pathways.

  19. Ontology-oriented retrieval of putative microRNAs in Vitis vinifera via GrapeMiRNA: a web database of de novo predicted grape microRNAs

    Fontana Paolo

    2009-06-01

    Full Text Available Abstract Background Two complete genome sequences are available for Vitis vinifera Pinot noir. Based on the sequence and gene predictions produced by the IASMA, we performed an in silico detection of putative microRNA genes and of their targets, and collected the most reliable microRNA predictions in a web database. The application is available at http://www.itb.cnr.it/ptp/grapemirna/. Description The program FindMiRNA was used to detect putative microRNA genes in the grape genome. A very high number of predictions was retrieved, calling for validation. Nine parameters were calculated and, based on the grape microRNAs dataset available at miRBase, thresholds were defined and applied to FindMiRNA predictions having targets in gene exons. In the resulting subset, predictions were ranked according to precursor positions and sequence similarity, and to target identity. To further validate FindMiRNA predictions, comparisons to the Arabidopsis genome, to the grape Genoscope genome, and to the grape EST collection were performed. Results were stored in a MySQL database and a web interface was prepared to query the database and retrieve predictions of interest. Conclusion The GrapeMiRNA database encompasses 5,778 microRNA predictions spanning the whole grape genome. Predictions are integrated with information that can be of use in selection procedures. Tools added in the web interface also allow to inspect predictions according to gene ontology classes and metabolic pathways of targets. The GrapeMiRNA database can be of help in selecting candidate microRNA genes to be validated.

  20. Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

    Wallace, Jared; Hu, Ruozhen; Mosbruger, Timothy L.; Dahlem, Timothy J.; Stephens, W. Zac; Rao, Dinesh S.; Round, June L.; O’Connell, Ryan M.

    2016-01-01

    Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that m...