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Sample records for recombinant adeno-associated virus-mediated

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

    PubMed Central

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

    2015-01-01

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

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

    PubMed

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

    2015-01-01

    Biopharming for the production of recombinant pharmaceutical proteins in the mammary gland of transgenic animals is an attractive but laborious alternative compared to mammalian cell fermentation. The disadvantage of the lengthy process of genetically modifying an entire animal could be circumvented with somatic transduction of only the mammary epithelium with recombinant, replication-defective viruses. While other viral vectors offer very limited scope for this approach, vectors based on adeno-associated virus (AAV) appear to be ideal candidates because AAV is helper-dependent, does not induce a strong immune response and has no association with disease. Here, we sought to test the suitability of recombinant AAV (rAAV) for biopharming. Using reporter genes, we showed that injected rAAV efficiently transduced mouse mammary cells. When rAAV encoding human myelin basic protein (hMBP) was injected into the mammary glands of mice and rabbits, this resulted in the expression of readily detectable protein levels of up to 0.5 g/L in the milk. Furthermore we demonstrated that production of hMBP persisted over extended periods and that protein expression could be renewed in a subsequent lactation by re-injection of rAAV into a previously injected mouse gland. PMID:26463440

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

    PubMed

    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

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

    PubMed

    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

  5. Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

    PubMed Central

    Bertran, J; Miller, J L; Yang, Y; Fenimore-Justman, A; Rueda, F; Vanin, E F; Nienhuis, A W

    1996-01-01

    Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration. PMID:8794313

  6. Recombinant adeno-associated virus-mediated global anterograde delivery of glial cell line-derived neurotrophic factor to the spinal cord: comparison of rubrospinal and corticospinal tracts in the rat.

    PubMed

    Foust, Kevin D; Flotte, Terence R; Reier, Paul J; Mandel, Ronald J

    2008-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of spinal lower motoneurons. Gene delivery is a promising strategy to deliver therapeutic molecules to these vulnerable cells. However, definition of an optimal route of delivery capable of accessing neurons over a considerable extent of the neuraxis represents a significant logistical problem. Intramuscular vector injections are not ideal as this approach would involve hundreds of injections to completely treat an ALS patient and also would be dependent on retrograde transport of the viral platform of choice. Alternatively, upper motoneurons could deliver trophic factors over considerable distances by anterograde transport after a relatively localized intracerebral injection. To test this approach, the present study was designed to compare the corticospinal (CST) and rubrospinal (RST) tracts for their ability to transport recombinant adeno-associated virus serotype 5 (rAAV5)-derived green fluorescent protein (GFP) or glial cell line-derived neurotrophic factor (GDNF) to the spinal cord. Unilateral injections of rAAV5-GFP into the red nucleus (RN) or motor cortex of normal rats produced GFP-positive fibers in the appropriate descending tracts extending to the lumbar spinal cord. For both tracts, GFP-positive axonal projections into the spinal gray matter were consistently observed. GDNF immunohistochemistry demonstrated that confirmed RN injections resulted in GDNF-positive fibers projecting into spinal gray matter as seen in the GFP group. In contrast, confirmed cortical rAAV5-GDNF injections resulted in less evident staining in spinal cord. Spinal cord GDNF levels were elevated at distances up to 72 mm from the injection sites, and confirmed that RST-related GDNF transport to spinal cord surpassed CST-associated delivery. PMID:18072858

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

    PubMed

    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

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

    PubMed

    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 (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and 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

  9. Adeno-associated Virus-mediated Rescue of Neonatal Lethality in Argininosuccinate Synthetase-deficient Mice

    PubMed Central

    Kok, Cindy Y; Cunningham, Sharon C; Carpenter, Kevin H; Dane, Allison P; Siew, Susan M; Logan, Grant J; Kuchel, Philip W; Alexander, Ian E

    2013-01-01

    Viral vectors based on adeno-associated virus (AAV) are showing exciting promise in gene therapy trials targeting the adult liver. A major challenge in extending this promise to the pediatric liver is the loss of episomal vector genomes that accompanies hepatocellular proliferation during liver growth. Hence maintenance of sufficient transgene expression will be critical for success in infants and children. We therefore set out to explore the therapeutic efficacy and durability of liver-targeted gene transfer in the challenging context of a neonatal lethal urea cycle defect, using the argininosuccinate synthetase deficient mouse. Lethal neonatal hyperammonemia was prevented by prenatal and early postnatal vector delivery; however, hyperammonemia subsequently recurred limiting survival to no more than 33 days despite vector readministration. Antivector antibodies acquired in milk from vector-exposed dams were subsequently shown to be blocking vector readministration, and were avoided by crossfostering vector-treated pups to vector-naive dams. In the absence of passively acquired antivector antibodies, vector redelivery proved efficacious with mice surviving to adulthood without recurrence of significant hyperammonemia. These data demonstrate the potential of AAV vectors in the developing liver, showing that vector readministration can be used to counter growth-associated loss of transgene expression provided the challenge of antivector humoral immunity is addressed. PMID:23817206

  10. Adeno-associated virus mediated gene transfer of Shepherdin inhibits gallbladder carcinoma growth in vitro and in vivo.

    PubMed

    Zhu, Aijun; Ren, Yu; Wang, Ning; Jin, Qiuyue; Zhang, Dongchang; Yang, Guangxiao; Wang, Quanying

    2015-11-01

    Gene therapy, a significantly crucial strategy for treatment of malignancies, has been gradually accepted in recent years. However, this therapeutic approach has being facing great challenges concerning problems which include complicated development of cancer with multiple gene control, effective target shortage, low efficiency of gene transferring and safety of the vector delivery system. Shepherdin, a novel peptidomimetic molecule designed from Lys-79 to Leu-87 of survivin, has been identified as a tumor suppressor with the function that can not only competitively interfere with the interaction between survivin and Hsp90 (heat shock protein-90) leading to the degradation of survivin to anti-tumor, but also competitively target the ATP-dependent binding pocket of Hsp90 resulting in the dysfunction of Hsp90 chaperone to cell apoptosis via a mitochondrial dependent or independent pathway. In the present study, we designed and constructed a recombinant Adeno-associated virus (rAAV) loading fusion gene NT4-TAT-6His-Shepherdin. The expression of Shepherdin in gallbladder carcinoma (GBC) cells was detected and its strong inhibitory effects against GBC growth were evaluated after AAV mediated gene transfer of Shepherdin into GBC cells and xenograft tumors. MTT assay and flow cytometric analysis demonstrated that rAAV containing Shepherdin gene could significantly inhibit the growth of GBC and this effect was closely associated with apoptosis. These results indicated that rAAV-NT4-TAT-6His-Shepherdin may be considered a novel therapeutic strategy in the gene therapy for gallbladder carcinoma. PMID:26143116

  11. [Adeno-associated virus mediated T-bet gene transfer into SGC-7901 cell to regulate IFN-gamma production].

    PubMed

    Qiu, Gufeng; Wang, Suoying; Wang, Shengjun; Shao, Qixiang; Ma, Jie; Yang, Ming; Xu, Xiaopeng; Mao, Chaoming; Su, Zhaoliang; Huang, Xinxiang; Xu, Huaxi

    2009-06-01

    In order to investigate the effect of T-bet on malignant cells, we selected SGC-7901, a kind of human gastric carcinoma cell line, and used gene clone technique and adeno-associated virus (AAV) packing technology, thus obtaining a recombinant rAAV-eGFP-T-bet and T-bet gene-transfected SGC-7901 cells. Then the function of T-bet gene-infected SGC-7901 cells was researched by detecting the levels of IFN-gamma and T-bet production. The results showed: (1) It was verified that rAAV-T-bet's packing was completed; (2) After SGC-7901 cells was transfected by rAAV-eGFP-T-bet, a green fluorescence was found in about 30%-40% SGC-7901s, and the gene of 1670 bp (T-bet) and 388 bp (IFN-gamma) were generated from SGC-7901s cells; (3) The proteins of IFN-gamma and T-bet secreted by SGC-7901 cells were also detected. These reveal that SGC-7901 cell is efficiently infected by rAAV encoding T-bet, which can induce transfected cells to secret IFN-gamma. It may be useful in the researches on cancer immune therapy of transfecting T-bet gene. PMID:19634682

  12. Rescue of skeletal muscles of gamma-sarcoglycan-deficient mice with adeno-associated virus-mediated gene transfer.

    PubMed

    Cordier, L; Hack, A A; Scott, M O; Barton-Davis, E R; Gao, G; Wilson, J M; McNally, E M; Sweeney, H L

    2000-02-01

    In humans, a subset of cases of Limb-girdle muscular dystrophy (LGMD) arise from mutations in the genes encoding one of the sarcoglycan (alpha, beta, gamma, or delta) subunits of the dystrophin-glycoprotein complex. While adeno-associated virus (AAV) is a potential gene therapy vector for these dystrophies, it is unclear if AAV can be used if a diseased muscle is undergoing rapid degeneration and necrosis. The skeletal muscles of mice lacking gamma-sarcoglycan (gsg-/- mice) differ from the animal models that have been evaluated to date in that the severity of the skeletal muscle pathology is much greater and more representative of that of humans with muscular dystrophy. Following direct muscle injection of a recombinant AAV [in which human gamma-sarcoglycan expression is driven by a truncated muscle creatine kinase (MCK) promoter/enhancer], we observed significant numbers of muscle fibers expressing gamma-sarcoglycan and an overall improvement of the histologic pattern of dystrophy. However, these results could be achieved only if injections into the muscle were prior to the development of significant fibrosis in the muscle. The results presented in this report show promise for AAV gene therapy for LGMD, but underscore the need for intervention early in the time course of the disease process. PMID:10933922

  13. Adeno-associated virus-mediated expression of β-hexosaminidase prevents neuronal loss in the Sandhoff mouse brain.

    PubMed

    Sargeant, Timothy J; Wang, Susan; Bradley, Josephine; Smith, Nicolas J C; Raha, Animesh A; McNair, Rosamund; Ziegler, Robin J; Cheng, Seng H; Cox, Timothy M; Cachón-González, Maria Begoña

    2011-11-15

    Sandhoff disease, a GM2 gangliosidosis caused by a deficiency in β-hexosaminidase, is characterized by progressive neurodegeneration. Although loss of neurons in association with lysosomal storage of glycosphingolipids occurs in patients with this disease, the molecular pathways that lead to the accompanying neurological defects are unclear. Using an authentic murine model of GM2 gangliosidosis, we examined the pattern of neuronal loss in the central nervous system and investigated the effects of gene transfer using recombinant adeno-associated viral vectors expressing β-hexosaminidase subunits (rAAV2/1-Hex). In 4-month-old Sandhoff mice with neurological deficits, cells staining positively for the apoptotic signature in the TUNEL reaction were found in the ventroposterior medial and ventroposterior lateral (VPM/VPL) nuclei of the thalamus. There was progressive loss of neuronal density in this region with age. Comparable loss of neuronal density was identified in the lateral vestibular nucleus of the brainstem and a small but statistically significant loss was present in the ventral spinal cord. Loss of neurons was not detected in other regions that were analysed. Administration of rAAV2/1-Hex into the brain of Sandhoff mice prevented the decline in neuronal density in the VPM/VPL. Preservation of neurons in the VPM/VPL was variable at the humane endpoint in treated animals, but correlated directly with increased lifespan. Loss of neurons was localized to only a few regions in the Sandhoff brain and was prevented by rAAV-mediated transfer of β-hexosaminidase gene function at considerable distances from the site of vector administration. PMID:21852247

  14. Adeno-Associated Virus-Mediated Correction of a Canine Model of Glycogen Storage Disease Type Ia

    PubMed Central

    Weinstein, David A.; Correia, Catherine E.; Conlon, Thomas; Specht, Andrew; Verstegen, John; Onclin-Verstegen, Karine; Campbell-Thompson, Martha; Dhaliwal, Gurmeet; Mirian, Layla; Cossette, Holly; Falk, Darin J.; Germain, Sean; Clement, Nathalie; Porvasnik, Stacy; Fiske, Laurie; Struck, Maggie; Ramirez, Harvey E.; Jordan, Juan; Andrutis, Karl; Chou, Janice Y.; Byrne, Barry J.

    2010-01-01

    Abstract Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-α. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver. PMID:20163245

  15. A next step in adeno-associated virus-mediated gene therapy for neurological diseases: regulation and targeting

    PubMed Central

    Chtarto, Abdelwahed; Bockstael, Olivier; Tshibangu, Terence; Dewitte, Olivier; Levivier, Marc; Tenenbaum, Liliane

    2013-01-01

    Recombinant adeno-associated virus (rAAV) vectors mediating long term transgene expression are excellent gene therapy tools for chronic neurological diseases. While rAAV2 was the first serotype tested in the clinics, more efficient vectors derived from the rh10 serotype are currently being evaluated and other serotypes are likely to be tested in the near future. In addition, aside from the currently used stereotaxy-guided intraparenchymal delivery, new techniques for global brain transduction (by intravenous or intra-cerebrospinal injections) are very promising. Various strategies for therapeutic gene delivery to the central nervous system have been explored in human clinical trials in the past decade. Canavan disease, a genetic disease caused by an enzymatic deficiency, was the first to be approved. Three gene transfer paradigms for Parkinson's disease have been explored: converting L-dopa into dopamine through AADC gene delivery in the putamen; synthesizing GABA through GAD gene delivery in the overactive subthalamic nucleus and providing neurotrophic support through neurturin gene delivery in the nigro-striatal pathway. These pioneer clinical trials demonstrated the safety and tolerability of rAAV delivery in the human brain at moderate doses. Therapeutic effects however, were modest, emphasizing the need for higher doses of the therapeutic transgene product which could be achieved using more efficient vectors or expression cassettes. This will require re-addressing pharmacological aspects, with attention to which cases require either localized and cell-type specific expression or efficient brain-wide transgene expression, and when it is necessary to modulate or terminate the administration of transgene product. The ongoing development of targeted and regulated rAAV vectors is described. PMID:23331189

  16. Correction of multiple striated muscles in murine Pompe disease through adeno-associated virus-mediated gene therapy.

    PubMed

    Sun, Baodong; Young, Sarah P; Li, Ping; Di, Chunhui; Brown, Talmage; Salva, Maja Z; Li, Songtao; Bird, Andrew; Yan, Zhen; Auten, Richard; Hauschka, Stephen D; Koeberl, Dwight D

    2008-08-01

    Glycogen storage disease type II (Pompe disease; MIM 232300) stems from the deficiency of acid alpha-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. An adeno-associated virus 2/8 (AAV2/8) vector containing the muscle creatine kinase (MCK) (CK1) reduced glycogen content by approximately 50% in the heart and quadriceps in GAA-knockout (GAA-KO) mice; furthermore, an AAV2/8 vector containing the hybrid alpha-myosin heavy chain enhancer-/MCK enhancer-promoter (MHCK7) cassette reduced glycogen content by >95% in heart and >75% in the diaphragm and quadriceps. Transduction with an AAV2/8 vector was higher in the quadriceps than in the gastrocnemius. An AAV2/9 vector containing the MHCK7 cassette corrected GAA deficiency in the distal hindlimb, and glycogen accumulations were substantially cleared by human GAA (hGAA) expression therein; however, the analogous AAV2/7 vector achieved much lower efficacy. Administration of the MHCK7-containing vectors significantly increased striated muscle function as assessed by increased Rotarod times at 18 weeks after injection, whereas the CK1-containing vector did not increase Rotarod performance. Importantly, type IIb myofibers in the extensor digitalis longus (EDL) were transduced, thereby correcting a myofiber type that is unresponsive to enzyme replacement therapy. In summary, AAV8 and AAV9-pseudotyped vectors containing the MHCK7 regulatory cassette achieved enhanced efficacy in Pompe disease mice. PMID:18560415

  17. Correction of Multiple Striated Muscles in Murine Pompe Disease Through Adeno-associated Virus-Mediated Gene Therapy

    PubMed Central

    Sun, Baodong; Young, Sarah P.; Li, Ping; Di, Chunhui; Brown, Talmage; Salva, Maia Z.; Li, Songtao; Bird, Andrew; Yan, Zhen; Auten, Richard; Hauschka, Stephen D.; Koeberl, Dwight D.

    2009-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) stems from the deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. We hypothesized that systemic administration of an adeno-associated virus (AAV) vector containing a muscle specific regulatory cassette could drive efficacious transgene expression in GAA-knockout (GAA-KO) mice. AAV2/8 vectors containing the muscle creatine kinase (CK1) or hybrid α-myosin heavy chain enhancer-/muscle creatine kinase enhancer-promoter (MHCK7) cassettes were compared. The CK1 reduced glycogen content by approximately 50% in the heart and quadriceps, in comparison to untreated GAA-KO mice, whereas the MHCK7 containing vector reduced glycogen content even further: >95% in heart and >75% in the diaphragm and quadriceps. Administration of the MHCK7-containing vector significantly increased striated muscle function as assessed by increased Rotarod times at 18 weeks post-injection, whereas the CK1-containing vector did not increase Rotarod performance. Transduction efficiency was evaluated with an AAV2/8 vector in which MHCK7 drives alkaline-phosphatase, revealing that many more myofibers were transduced in the quadriceps than in the gastrocnemius. An AAV2/9 vector containing the MHCK7 cassette corrected GAA deficiency in the skeletal muscles of the distal limb, including the gastrocnemius, extensor digitalis longus, and soleus; furthermore, glycogen accumulations were substantially cleared by hGAA expression therein. Importantly, type IIb myofibers in the extensor digitalis longus were transduced, thereby correcting a myofiber type that is unresponsive to enzyme replacement therapy. In summary, AAV8 and AAV9-pseudotyped vectors containing the MHCK7 regulatory cassette achieved enhanced efficacy in Pompe disease mice. PMID:18560415

  18. Recombinant adeno-associated virus targets passenger gene expression to cones in primate retina

    NASA Astrophysics Data System (ADS)

    Mancuso, Katherine; Hendrickson, Anita E.; Connor, Thomas B., Jr.; Mauck, Matthew C.; Kinsella, James J.; Hauswirth, William W.; Neitz, Jay; Neitz, Maureen

    2007-05-01

    Recombinant adeno-associated virus (rAAV) is a promising vector for gene therapy of photoreceptor-based diseases. Previous studies have demonstrated that rAAV serotypes 2 and 5 can transduce both rod and cone photoreceptors in rodents and dogs, and it can target rods, but not cones in primates. Here we report that using a human cone-specific enhancer and promoter to regulate expression of a green fluorescent protein (GFP) reporter gene in an rAAV-5 vector successfully targeted expression of the reporter gene to primate cones, and the time course of GFP expression was able to be monitored in a living animal using the RetCam II digital imaging system.

  19. Inexpensive, serotype-independent protocol for native and bioengineered recombinant adeno-associated virus purification

    PubMed Central

    Arden, Erik; Metzger, Joseph M.

    2016-01-01

    Recombinant adeno-associated virus (AAV) is a valuable and often used gene therapy vector. With increased demand for highly purified virus comes the need for a standardized purification procedure that is applicable across many serotypes and includes bioengineered viruses. Currently cesium chloride banding or affinity chromatography are the predominate forms of purification. These approaches expose the final purified virus to toxic contaminants or are highly capsid dependent and may require significant optimization to isolate purified AAV. These methods may also limit crude viral lysate processing volume resulting in a significant loss of viral titer. To circumvent these issues, we have developed an AAV purification protocol independent of toxic compounds, supernatant volume and capsid moiety. This purification method standardizes virus purification across native serotype and bioengineered mosaic capsids. PMID:27294171

  20. Manufacturing of recombinant adeno-associated viral vectors for clinical trials

    PubMed Central

    Clément, Nathalie; Grieger, Joshua C

    2016-01-01

    The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings. PMID:27014711

  1. Antitumor effect and biological pathways of a recombinant adeno-associated virus as a human renal cell carcinoma suppressor.

    PubMed

    Chen, Jie; Ruan, Xiyun; Wang, Shaomei; Zhang, Bin; Liu, Bo; Sun, Zeqiang; Liu, Qingyong

    2014-11-01

    The aims of this work are to study the antitumor effect of the adeno-associated virus on the xenografted tumors of chick embryo chorioallantoic membrane and predict potential genes and biological pathways which are associated with renal cell carcinoma. The adeno-associated virus NT4-TAT-6 × His-VHLbeta was constructed and identified. Then, chick embryos with xenografted tumor were divided into three groups and respectively inoculated with rAAV/NT4-TAT-6 × His-VHLbeta (group A), empty virus (group B), and phosphate-buffered saline (group C, the control subject). Antitumor effect in each group was investigated by means of immunofluorescence observation. Genes interacted with von Hippel-Lindau were screened by Search Tool for the Retrieval of Interacting Genes/Proteins database, while pathway analysis were performed based on Kyoto Encyclopedia of Genes and Genomes. The growth of xenografted tumors inoculated with recombinant adeno-associated virus was slower than the control subjects. The tumor volumes of group A showed significant difference compared with group B and group C (P < 0.05). Growth of xenografted tumors which administered with the recombinant adeno-associated virus was inhibited. Among the protein-protein interaction network, TCEB2, HIF1A, TCEB1, CUL2, RBX1, and PHF17 were hub genes which might be involved in the development of renal cell carcinoma. The most significant signaling pathway was renal cell carcinoma. In this paper, we constructed and identified the recombinant adeno-associated virus NT4-TAT-6 × His-VHLbeta and studied the antitumor effect of the adeno-associated virus on xenografted tumors of chicken embryo chorioallantoic membrane. In addition, genes in the protein-protein interaction network which are associated with renal cell carcinoma were revealed and the biological pathway of renal cell carcinoma was identified. Our results provide a gene-therapeutic agent for the treatment of human renal cell carcinoma. PMID:25091575

  2. Human Treg responses allow sustained recombinant adeno-associated virus–mediated transgene expression

    PubMed Central

    Mueller, Christian; Chulay, Jeffrey D.; Trapnell, Bruce C.; Humphries, Margaret; Carey, Brenna; Sandhaus, Robert A.; McElvaney, Noel G.; Messina, Louis; Tang, Qiushi; Rouhani, Farshid N.; Campbell-Thompson, Martha; Fu, Ann Dongtao; Yachnis, Anthony; Knop, David R.; Ye, Guo-jie; Brantly, Mark; Calcedo, Roberto; Somanathan, Suryanarayan; Richman, Lee P.; Vonderheide, Robert H.; Hulme, Maigan A.; Brusko, Todd M.; Wilson, James M.; Flotte, Terence R.

    2013-01-01

    Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1–AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy. PMID:24231351

  3. Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors.

    PubMed

    Burnham, Brenda; Nass, Shelley; Kong, Elton; Mattingly, MaryEllen; Woodcock, Denise; Song, Antonius; Wadsworth, Samuel; Cheng, Seng H; Scaria, Abraham; O'Riordan, Catherine R

    2015-12-01

    Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality. PMID:26414997

  4. Recombinant Adeno-Associated Virus Serotype 6 Efficiently Transduces Primary Human Melanocytes

    PubMed Central

    Verdon, Daniel; Chen, Jennifer; Taylor, John A.; Dunbar, P. Rod

    2013-01-01

    The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7–78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6–12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes. PMID:23646140

  5. Gene transfer in the liver using recombinant adeno-associated virus

    PubMed Central

    Ahmed, Seemin Seher; Li, Jia; Godwin, Jonathan; Gao, Guangping; Zhong, Li

    2013-01-01

    Liver-directed gene transfer and gene therapy are rapidly gaining attention primarily because the liver is centrally involved in a variety of metabolic functions that are affected in various inherited disorders. Recombinant adeno-associated virus (rAAV) is a popular gene delivery vehicle for gene therapy and intravenous delivery of some rAAV serotypes results in very efficient transduction of the liver. rAAV-mediated and liver-directed gene transfer can help in creating somatic transgenic animals or disease models and studying the function of various genes and miRNAs. The liver is the target tissue for gene therapy of many inborn metabolic diseases and may also be exploited as a “bio-factory” for the production of coagulation factors, insulin and growth hormones and other non-hepatic proteins. Hence efficient delivery of transgenes and small RNAs to the liver by rAAV vectors has been of long-standing interest to research scientists and clinicians alike. PMID:23686826

  6. Production of CFTR-null and CFTR-DeltaF508 heterozygous pigs by adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer.

    PubMed

    Rogers, Christopher S; Hao, Yanhong; Rokhlina, Tatiana; Samuel, Melissa; Stoltz, David A; Li, Yuhong; Petroff, Elena; Vermeer, Daniel W; Kabel, Amanda C; Yan, Ziying; Spate, Lee; Wax, David; Murphy, Clifton N; Rieke, August; Whitworth, Kristin; Linville, Michael L; Korte, Scott W; Engelhardt, John F; Welsh, Michael J; Prather, Randall S

    2008-04-01

    Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (DeltaF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice. PMID:18324337

  7. Adeno-Associated Virus Mediated Delivery of An Engineered Protein that Combines the Complement Inhibitory Properties of CD46, CD55 and CD59

    PubMed Central

    Leaderer, Derek; Cashman, Siobhan M.; Kumar-Singh, Rajendra

    2015-01-01

    Background A variety of disorders are associated with the activation of complement. CD46, CD55 and CD59 are the major membrane associated regulators of complement on human cells. Previously, we have found that independent expression of CD55, CD46 or CD59 through gene transfer protects murine tissues against human complement mediated attack. Herein we investigated the potential of combining the complement regulatory properties of CD46, CD55 and CD59 into single gene products expressed from an adeno-associated virus (AAV) vector in a soluble non-membrane anchored form. Methods Minigenes encoding the complement regulatory domains from CD46, CD55 and CD59 (SACT) or CD55 and CD59 (DTAC) were cloned into an AAV vector. The specific regulatory activity of each component of SACT and DTAC was measured in vitro. The recombinant AAV vectors were injected into the peritoneum of mice and the efficacy of the transgene products for being able to protect murine liver vasculature against human complement, specifically the membrane attack complex (MAC) was measured. Results SACT and DTAC exhibited properties similar to CD46, CD55 and CD59 or CD55 and CD59 respectively in vitro. AAV mediated delivery of SACT or DTAC protected murine liver vasculature from human MAC deposition by 63.2% and 56.7% respectively. Conclusions When delivered to mice in vivo via an AAV vector, SACT and DTAC are capable of limiting human complement mediated damage. SACT and DTAC merit further study as potential therapies for complement mediated disorders when delivered via a gene therapy approach. PMID:25917932

  8. Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

    PubMed Central

    Teramoto, S.; Bartlett, J. S.; McCarty, D.; Xiao, X.; Samulski, R. J.; Boucher, R. C.

    1998-01-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  9. Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: comparison with adenovirus vectors.

    PubMed

    Teramoto, S; Bartlett, J S; McCarty, D; Xiao, X; Samulski, R J; Boucher, R C

    1998-11-01

    Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should

  10. Recombinant Adeno-Associated Virus Utilizes Cell-Specific Infectious Entry Mechanisms

    PubMed Central

    Weinberg, Marc S.; Nicolson, Sarah; Bhatt, Aadra P.; McLendon, Michael; Li, Chengwen

    2014-01-01

    ABSTRACT Understanding the entry and trafficking mechanism(s) of recombinant adeno-associated virus (rAAV) into host cells can lead to evolution in capsid and vector design and delivery methods, resulting in enhanced transduction and therapeutic gene expression. Variability of findings regarding the early entry pathway of rAAV supports the possibility that rAAV, like other viruses, can utilize more than one infectious entry pathway. We tested whether inhibition of macropinocytosis impacted rAAV transduction of HeLa cells compared to hepatocellular carcinoma cell lines. We found that macropinocytosis inhibitor cytochalasin D blocked rAAV transduction of HeLa cells (>2-fold) but enhanced (10-fold) transduction in HepG2 and Huh7 lines. Similar results were obtained with another macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl) amiloride (EIPA). The augmented transduction was due to neither viral binding nor promoter activity, affected multiple rAAV serotypes (rAAV2, rAAV2-R585E, and rAAV8), and influenced single-stranded and self-complementary virions to comparable extents. Follow-up studies using CDC42 inhibitor ML141 and p21-activated kinase 1 (PAK1) siRNA knockdown also resulted in enhanced HepG2 transduction. Microscopy revealed that macropinocytosis inhibition correlated with expedited nuclear entry of the rAAV virions into HepG2 cells. Enhancement of hepatocellular rAAV transduction extended to the mouse liver in vivo (4-fold enhancement) but inversely blocked heart tissue transduction (13-fold). This evidence of host cell-specific rAAV entry pathways confers a potent means for controlling and enhancing vector delivery and could help unify the divergent accounts of rAAV cellular entry mechanisms. IMPORTANCE There is a recognized need for improved rAAV vector targeting strategies that result in delivery of fewer total particles, averting untoward toxicity and/or an immune response against the vector. A critical step in rAAV transduction is entry and early

  11. Impurity of recombinant adeno-associated virus type 2 affects the transduction characteristics following subretinal injection in the rat.

    PubMed

    Shen, Wei-Yong; Lai, Yvonne K Y; Lai, Chooi-May; Rakoczy, P Elizabeth

    2004-02-01

    We recently reported that different purification methods of recombinant adeno-associated virus type 2 (rAAV2) affect the transduction characteristics following subretinal injection. In this study, we examined the roles of contaminant proteins from the HEK-293 cells and helper adenovirus, inactivation of helper adenovirus and cell stress induced by DNA-damaging agents in rAAV-mediated retinal transduction. Our results showed that contaminating factors/proteins resulting from the helper E1 deleted adenovirus are possibly responsible for efficient RPE transduction. Future studies of these factors will undoubtedly lead to development of new therapeutic approaches to PR- and RPE-specific retinal diseases. PMID:14659960

  12. Protection against henipavirus infection by use of recombinant adeno-associated virus-vector vaccines.

    PubMed

    Ploquin, Aurélie; Szécsi, Judit; Mathieu, Cyrille; Guillaume, Vanessa; Barateau, Véronique; Ong, Kien Chai; Wong, Kum Thong; Cosset, François-Loïc; Horvat, Branka; Salvetti, Anna

    2013-02-01

    Nipah virus (NiV) and Hendra virus (HeV) are closely related, recently emerged paramyxoviruses that are capable of causing considerable morbidity and mortality in several mammalian species, including humans. Henipavirus-specific vaccines are still commercially unavailable, and development of novel antiviral strategies to prevent lethal infections due to henipaviruses is highly desirable. Here we describe the development of adeno-associated virus (AAV) vaccines expressing the NiV G protein. Characterization of these vaccines in mice demonstrated that a single intramuscular AAV injection was sufficient to induce a potent and long-lasting antibody response. Translational studies in hamsters further demonstrated that all vaccinated animals were protected against lethal challenge with NiV. In addition, this vaccine induced a cross-protective immune response that was able to protect 50% of the animals against a challenge by HeV. This study presents a new efficient vaccination strategy against henipaviruses and opens novel perspectives on the use of AAV vectors as vaccines against emergent diseases. PMID:23175762

  13. Preferential Targeting of Disseminated Liver Tumors Using a Recombinant Adeno-Associated Viral Vector

    PubMed Central

    Della Peruta, Marco; Badar, Adam; Rosales, Cecilia; Chokshi, Shilpa; Kia, Azadeh; Nathwani, Devhrut; Galante, Eva; Yan, Ran; Arstad, Erik; Davidoff, Andrew M.; Williams, Roger; Lythgoe, Mark F.

    2015-01-01

    Abstract A novel selectively targeting gene delivery approach has been developed for advanced hepatocellular carcinoma (HCC), a leading cause of cancer mortality whose prognosis remains poor. We combine the strong liver tropism of serotype-8 capsid-pseudotyped adeno-associated viral vectors (AAV8) with a liver-specific promoter (HLP) and microRNA-122a (miR-122a)-mediated posttranscriptional regulation. Systemic administration of our AAV8 construct resulted in preferential transduction of the liver and encouragingly of HCC at heterotopic sites, a finding that could be exploited to target disseminated disease. Tumor selectivity was enhanced by inclusion of miR-122a-binding sequences (ssAAV8-HLP-TK-122aT4) in the expression cassette, resulting in abrogation of transgene expression in normal murine liver but not in HCC. Systemic administration of our tumor-selective vector encoding herpes simplex virus-thymidine kinase (TK) suicide gene resulted in a sevenfold reduction in HCC growth in a syngeneic murine model without toxicity. In summary, we have developed a systemically deliverable gene transfer approach that enables high-level expression of therapeutic genes in HCC but not normal tissues, thus improving the prospects of safe and effective treatment for advanced HCC. PMID:25569358

  14. BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus hybrid vector.

    PubMed

    Yamamoto, H; Ishimura, M; Ochiai, M; Takada, H; Kusuhara, K; Nakatsu, Y; Tsuzuki, T; Mitani, K; Hara, T

    2016-02-01

    X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting vector (HD-Ad.AAV BTK vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK vector via homologous recombination in hematopoietic stem cells. PMID:26280081

  15. Adeno-Associated Virus-Mediated Overexpression of LARGE Rescues α-Dystroglycan Function in Dystrophic Mice with Mutations in the Fukutin-Related Protein

    PubMed Central

    Vannoy, Charles H.; Xu, Lei; Keramaris, Elizabeth; Lu, Pei; Xiao, Xiao

    2014-01-01

    Abstract Multiple genes (e.g., POMT1, POMT2, POMGnT1, ISPD, GTDC2, B3GALNT2, FKTN, FKRP, and LARGE) are known to be involved in the glycosylation pathway of α-dystroglycan (α-DG). Mutations of these genes result in muscular dystrophies with wide phenotypic variability. Abnormal glycosylation of α-DG with decreased extracellular ligand binding activity is a common biochemical feature of these genetic diseases. While it is known that LARGE overexpression can compensate for defects in a few aforementioned genes, it is unclear whether it can also rescue defects in FKRP function. We examined adeno-associated virus (AAV)-mediated LARGE or FKRP overexpression in two dystrophic mouse models with loss-of-function mutations: (1) Largemyd (LARGE gene) and (2) FKRPP448L (FKRP gene). The results agree with previous findings that overexpression of LARGE can ameliorate the dystrophic phenotypes of Largemyd mice. In addition, LARGE overexpression in the FKRPP448L mice effectively generated functional glycosylation (hyperglycosylation) of α-DG and improved dystrophic pathologies in treated muscles. Conversely, FKRP transgene overexpression failed to rescue the defect in glycosylation and improve the phenotypes of the Largemyd mice. Our findings suggest that AAV-mediated LARGE gene therapy may still be a viable therapeutic strategy for dystroglycanopathies with FKRP deficiency. PMID:24635668

  16. Producing Recombinant Adeno-Associated Virus in Foster Cells: Overcoming Production Limitations Using a Baculovirus–Insect Cell Expression Strategy

    PubMed Central

    Virag, Tamas; Cecchini, Sylvain

    2009-01-01

    Abstract Establishing pharmacological parameters, such as efficacy, routes of administration, and toxicity, for recombinant adeno-associated virus (rAAV) vectors is a prerequisite for gaining acceptance for clinical applications. In fact, even a therapeutic window, that is, the dose range between therapeutic efficacy and toxicity, has yet to be determined for rAAV in vivo. Multiphase clinical trials investigating the safety and efficacy of recombinant AAV-based therapeutics will require unprecedented vector production capacity to meet the needs of preclinical toxicology studies, and the progressive clinical protocol phases of safety/dose escalation (phase I), efficacy (phase II), and high-enrollment, multicenter evaluations (phase III). Methods of rAAV production capable of supporting such trials must be scalable, robust, and efficient. We have taken advantage of the ease of scalability of nonadherent cell culture techniques coupled with the inherent efficiency of viral infection to develop an rAAV production method based on recombinant baculovirus-mediated expression of AAV components in insect-derived suspension cells. PMID:19604040

  17. Thymosin Beta-4 Recombinant Adeno-associated Virus Enhances Human Nucleus Pulposus Cell Proliferation and Reduces Cell Apoptosis and Senescence

    PubMed Central

    Wang, Yuan-Yi; Zhu, Qing-San; Wang, Yi-Wei; Yin, Ruo-Feng

    2015-01-01

    Background: Thymosin beta-4 (TB-4) is considered key roles in tissue development, maintenance and pathological processes. The study aimed to prove TB-4 positive biological function on nucleus pulposus (NP) cell apoptosis and slowing the process of cell aging while increasing the cell proliferation. Methods: TB-4 recombinant adeno-associated virus (AAV) was constructed and induced to human NP cells. Cell of same group were cultured without gene modification as controlled group. Proliferation capacity and cell apoptosis were observed during 6 passages of the cells. Morphology and expression of the TB-4 gene were documented as parameter of cell activity during cell passage. Results: NP cells with TB-4 transfection has normal TB-4 expression and exocytosis. NP cells with TB-4 transfection performed significantly higher cell activity than that at the control group in each generation. TB-4 recombinant AAV-transfected human NP cells also show slower cell aging, lower cell apoptosis and higher cell proliferation than control group. Conclusions: TB-4 can prevent NP cell apoptosis, slow NP cell aging and promote NP cell proliferation. AAV transfection technique was able to highly and stably express TB-4 in human NP cells, which may provide a new pathway for innovation in the treatment of intervertebral disc degenerative diseases. PMID:26021512

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

    PubMed

    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

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

    PubMed

    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

  20. Recombinant adeno-associated virus vector: use for transgene expression and anterograde tract tracing in the CNS

    PubMed Central

    Chamberlin, Nancy L.; Du, Bin; de Lacalle, Sonsoles; Saper, Clifford B.

    2016-01-01

    We used a recombinant adeno-associated virus vector (AAV) to deliver a foreign gene, green fluorescent protein (GFP), into mature neurons in adult rat CNS in vivo. Microinjections of AAV as small as 50 nl transduced hundreds of neurons at the injection site. There was virtually no retrograde transport as fewer than one neuron per brain was found distant from the injection site that exhibited GFP immunoreactivity. The gene product, GFP, filled the entire neuronal cytoplasmic compartment; GFP immunoreactivity was robust in cell bodies, axons, and nerve terminals. There was no tissue damage at the injection sites or pathogenicity indicated by changes in astrocytic or microglial markers. There was no inflammatory response as judged by leukocytic invasion. Gene expression in transduced cells was robust and apparently permanent: there was no evidence of phenotypic reversion up to 12 weeks following infection. AAV is an excellent vector for introducing foreign genes into mature CNS neurons. Not only might it be an ideal vehicle for gene therapy, but also the GFP-containing AAV presents a new strategy for tracing long axonal pathways in the CNS, which is difficult with current tracers (PHAL, biotinylated dextrans). PMID:9630611

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

    PubMed Central

    Buclez, Pierre-Olivier; Dias Florencio, Gabriella; Relizani, Karima; Beley, Cyriaque; Garcia, Luis; Benchaouir, Rachid

    2016-01-01

    Recombinant adeno-associated viruses (rAAV) are largely used for gene transfer in research, preclinical developments, and clinical trials. Their broad in vivo biodistribution and long-term efficacy in postmitotic tissues make them good candidates for numerous gene transfer applications. Upstream processes able to produce large amounts of rAAV were developed, particularly those using baculovirus expression vector system. In parallel, downstream processes present a large panel of purification methods, often including multiple and time consuming steps. Here, we show that simple tangential flow filtration, coupled with an optimized iodixanol-based isopycnic density gradient, is sufficient to purify several liters of crude lysate produced by baculovirus expression vector system in only one working day, leading to high titers and good purity of rAAV products. Moreover, we show that the viral vectors retain their in vitro and in vivo functionalities. Our results demonstrate that simple, rapid, and relatively low-cost methods can easily be implemented for obtaining a high-quality grade of gene therapy products based on rAAV technology. PMID:27226971

  2. Noninvasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus.

    PubMed

    Wang, S; Olumolade, O O; Sun, T; Samiotaki, G; Konofagou, E E

    2015-01-01

    Recombinant adeno-associated virus (rAAV) has shown great promise as a potential cure for neurodegenerative diseases. The existence of the blood-brain barrier (BBB), however, hinders efficient delivery of the viral vectors. Direct infusion through craniotomy is the most commonly used approach to achieve rAAV delivery, which carries increased risks of infection and other complications. Here, we report a focused ultrasound (FUS)-facilitated noninvasive rAAV delivery paradigm that is capable of producing targeted and neuron-specific transductions. Oscillating ultrasound contrast agents (microbubbles), driven by FUS waves, temporarily 'unlock' the BBB, allowing the systemically administrated rAAVs to enter the brain parenchyma, while maintaining their bioactivity and selectivity. Taking the advantage of the neuron-specific promoter synapsin, rAAV gene expression was triggered almost exclusively (95%) in neurons of the targeted caudate-putamen region. Both behavioral assessment and histological examination revealed no significant long-term adverse effects (in the brain and several other critical organs) for this combined treatment paradigm. Results from this study demonstrated the feasibility and safety for the noninvasive, targeted rAAV delivery, which might have open a new avenue in gene therapy in both preclinical and clinical settings. PMID:25354683

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

    PubMed

    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

  4. Non-invasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus

    PubMed Central

    Wang, Shutao; Olumolade, Oluyemi O.; Sun, Tao; Samiotaki, Gesthimani; Konofagou, Elisa E.

    2015-01-01

    Recombinant adeno-associated virus (rAAV) has shown great promise as a potential cure for neurodegenerative diseases. The existence of the blood-brain barrier (BBB), however, hinders efficient delivery of the viral vectors. Direct infusion through craniotomy is the most commonly used approach to achieve rAAV delivery, which carries increased risks of infection and other complications. Here we report a focused ultrasound (FUS) facilitated, non-invasive rAAV delivery paradigm that is capable of producing targeted and neuron-specific transductions. Oscillating ultrasound contrast agents (i.e. microbubbles), driven by focused ultrasound waves, temporarily “unlocking” the BBB, allowing the systemically administrated rAAVs to enter the brain parenchyma, while maintaining their bioactivity and selectivity. Taking the advantage of the neuron-specific promoter-synapsin, rAAV gene expression was triggered almost exclusively (95%) in neurons of the targeted (i.e. caudate-putamen) region. Both behavioral assessment and histological examination revealed no significant long term adverse effects (in the brain and several other critical organs) for this combined treatment paradigm. Results from this study demonstrated the feasibility and safety for the non-invasive, targeted rAAV delivery technique, which might have provided a new arena for gene therapy in both pre-clinical and clinical settings. PMID:25354683

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

    SciTech Connect

    Zhao Weihong; Wu Jianqing ||; Zhong Li; Chen Linyuan; Weigel-Kelley, Kirsten A. |; Qing Keyun; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H. |; Srivastava, Arun |. E-mail: asrivastava@gtc.ufl.edu

    2006-09-30

    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 {approx}25-fold in WT MEFs, but only by {approx}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 {approx}23-fold in WT MEFs, but only {approx}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, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}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

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

    PubMed

    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. Frequency and Spectrum of Genomic Integration of Recombinant Adeno-Associated Virus Serotype 8 Vector in Neonatal Mouse Liver▿

    PubMed Central

    Inagaki, Katsuya; Piao, Chuncheng; Kotchey, Nicole M.; Wu, Xiaolin; Nakai, Hiroyuki

    2008-01-01

    Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 × 1011 vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a β-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least ∼0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was ∼0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 103 hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum. PMID:18614641

  8. Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles.

    PubMed Central

    Chiorini, J A; Yang, L; Liu, Y; Safer, B; Kotin, R M

    1997-01-01

    We have cloned and characterized the full-length genome of adeno-associated virus type 4 (AAV4). The genome of AAV4 is 4,767 nucleotides in length and contains an expanded p5 promoter region compared to AAV2 and AAV3. Within the inverted terminal repeat (ITR), several base changes were identified with respect to AAV2. However, these changes did not affect the ability of this region to fold into a hairpin structure. Within the ITR, the terminal resolution site and Rep binding sites were conserved; however, the Rep binding site was expanded from three GAGC repeats to four. The Rep gene product of AAV4 shows greater than 90% homology to the Rep products of serotypes 2 and 3, with none of the changes occurring in regions which had previously been shown to affect the known functions of Rep68 or Rep78. Most of the differences in the capsid proteins lie in regions which are thought to be on the exterior surface of the viral capsid. It is these unique regions which are most likely to be responsible for the lack of cross-reacting antibodies and the altered tissue tropism compared to AAV2. The results of our studies, performed with a recombinant version of AAV4 carrying a lacZ reporter gene, suggest that AAV4 can transduce human, monkey, and rat cells. Furthermore, comparison of transduction efficiencies in a number of cell lines, competition cotransduction experiments, and the effect of trypsin on transduction efficiency all suggest that the cellular receptor for AAV4 is distinct from that of AAV2. PMID:9261407

  9. Calcium-ion-modulated ceramic hydroxyapatite resin for the scalable purification of recombinant Adeno-Associated Virus serotype 9.

    PubMed

    Qu, Weihong; Wang, Mingxi; Wu, Yaqing; Lv, Yinghui; Wang, Qizhao; Xu, Ruian

    2015-05-15

    Column chromatography has been widely used as a scalable purification strategy for recombinant adeno-associated virus (rAAV) vectors. The rAAV1, 2, 4, 5, 6, 8 and 9 serotypes could be separated using affinity resins, ion exchange resins or other types of resins. Apatite resin has displayed outstanding performance in protein purification in the past 10 years, and ceramic hydroxyapatite (CHT) chromatography resin with a polyethylene glycol (PEG) modulation has recently been used for rAAV1 and rAAV9 vectors. This study reports the use of CHT chromatography modulated by calcium ions instead of PEG for rAAV9 purification. Calcium-ion-containing buffers effectively improve the inclusion of CHT as a capture resin, the resin-binding capacity and the yield. The optimum calcium ion concentration is 30ppm, and the optimum pH is 7.0. A frontal analysis indicated that the binding capacity of CHT at 2ml/min reaches 65.1mg total protein per ml of resin. A previously developed purification strategy consists of CHT followed by ANX anion exchange chromatography. The vector yield of this approach is approximately 70%, and a software analysis indicated a vector purity exceeding 98%. The residual host cell (HEK293) protein contents are 24.75±2.32ng and 67.21±2.10ng, and the Benzonase residue contents are 1.55±0.10pg and 1.95±0.16ng per 10(13) vector genome copies (G.C.) separated by CHT/ANX and CsCl. In addition, CHT/ANX yields 798.44±50.10pg of plasmid DNA and 2.17±0.11ng of HEK293 DNA, while CsCl purification yields 840.27±76.14pg of plasmid DNA and 2.43±0.19 of HEK293 DNA. The two methods produce vectors with similar in vitro and in vivo potencies. The results indicated that the CHT/ANX method is suitable for the scalable purification of the rAAV9 vector. PMID:25841202

  10. Adeno-associated virus mediated SOD gene therapy protects the retinal ganglion cells from chronic intraocular pressure elevation induced injury via attenuating oxidative stress and improving mitochondrial dysfunction in a rat model

    PubMed Central

    Jiang, Wenmin; Tang, Luosheng; Zeng, Jun; Chen, Baihua

    2016-01-01

    Purpose: This study aimed to determine whether chronic intraocular pressure (IOP) elevation induces retinal oxidative stress and alters mitochondrial morphology and function of retinal ganglion cells (RGC) and to explore the effects of AAV-SOD2 gene therapy on the RGC survival and mitochondrial dysfunction. Methods: Chronic experimental glaucoma was induced unilaterally in adult male Sprague-Dawley rats by laser burns at trabecular meshwork and episcleral veins 2 times with an interval of one week. One eye of each rat was intravitreally pretreated with recombinant adeno-associated virus expressing SOD2 (AAV-SOD2) or recombinant AAV expressing GFP (AAV-GFP) 21 days before glaucoma induction. RGCs counting, morphometric analysis of retina and optic nerve, and detection of activities of retinal SOD2 and catalase, MDA, mitochondrial morphology, mitochondrial dynamin protein OPA1 and DRP-1 expressions were conducted at 4, 8, 12 and 24 weeks. Results: Severe RGC loss, degeneration of optic nerve, reduced thickness of RGC layer and nerve fiber layer, significant decrease in total SOD and catalase activities, mitochondrial dysfunction and increased MDA were observed at 4, 8, 12 and 24 weeks after glaucoma. Pretreatment with AAV-SOD2 significantly reduced MDA and attenuated the damage to RGCs through a mitochondria-related pathway. Conclusion: AAV mediated pre-treatment with SOD2 is able to attenuate oxidative stress and improve mitochondrial dysfunction of RGC and optic nerve secondary to glaucoma. Thus, SOD2 may be used to prevent the retinal RGCs from glaucoma, which provides a promising strategy for glaucoma therapy. PMID:27158370

  11. Transduction of folate receptor cDNA into cervical carcinoma cells using recombinant adeno-associated virions delays cell proliferation in vitro and in vivo.

    PubMed Central

    Sun, X L; Murphy, B R; Li, Q J; Gullapalli, S; Mackins, J; Jayaram, H N; Srivastava, A; Antony, A C

    1995-01-01

    Although folate receptors (FRs) mediate folate uptake into cells, the independent role of FRs in cell proliferation remains unclear. We tested the hypothesis that transduction of FR cDNA in sense or antisense orientation using recombinant adeno-associated virus modulated FR expression and altered proliferation of cervical carcinoma cells (which constitutively overexpress FR genes). We determined that the integration of recombinant adeno-associated virions was not site specific. When compared with untransduced cells, sense and antisense FR cDNA-transduced cells exhibited an increase and decrease in FR mRNA and FR expression on the cell surface, respectively. However, when compared with antisense FR cDNA-transduced and untransduced cells, sense FR cDNA-transduced cells exhibited statistically significant (a) increased in total FRs, (b) smaller colonies, (c) lowered cell proliferation in vitro, and (d) less tumor volume with dramatic prolongation of tumor doubling times (225.6 h vs. 96 h) after transplantation into nude mice. Finally, (f) using single cell-derived transduced clones, an inverse relationship between cell proliferation and FR expression was established (r = 0.90, P < 0.001). Thus, transduction of sense/antisense FR cDNA into cervical carcinoma cells modulated expression of FRs and had an impact on cell proliferation in vitro and in vivo. Images PMID:7657824

  12. Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications.

    PubMed

    Strobel, Benjamin; Miller, Felix D; Rist, Wolfgang; Lamla, Thorsten

    2015-08-01

    Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the distribution of intact, bioactive AAVs and contaminants, respectively. Moreover, we tested four different polishing methods (ultrafiltration, size-exclusion chromatography, hollow-fiber tangential flow filtration, and polyethylene glycol precipitation) implemented after the iodixanol gradient for their ability to deplete iodixanol and protein contaminations. Last, we conducted a side-by-side comparison of the CsCl and iodixanol/ultrafiltration protocol. Our results demonstrate that iodixanol-purified AAV preparations show higher vector purity but harbor more (∼20%) empty particles as compared with CsCl-purified vectors (<1%). Using mass spectrometry, we analyzed prominent protein impurities in the AAV vector product, thereby identifying known and new, possibly AAV-interacting proteins as major contaminants. Thus, our study not only provides a helpful guide for the many laboratories entering the AAV field, but also builds a basis for further investigation of cellular processes involved in AAV vector assembly and trafficking. PMID:26222983

  13. Comparative Analysis of Cesium Chloride- and Iodixanol-Based Purification of Recombinant Adeno-Associated Viral Vectors for Preclinical Applications

    PubMed Central

    Strobel, Benjamin; Miller, Felix D.; Rist, Wolfgang; Lamla, Thorsten

    2015-01-01

    Cesium chloride (CsCl)- and iodixanol-based density gradients represent the core step in most protocols for serotype-independent adeno-associated virus (AAV) purification established to date. However, despite controversial reports about the purity and bioactivity of AAV vectors derived from each of these protocols, systematic comparisons of state-of-the-art variants of these methods are sparse. To define exact conditions for such a comparison, we first fractionated both gradients to analyze the distribution of intact, bioactive AAVs and contaminants, respectively. Moreover, we tested four different polishing methods (ultrafiltration, size-exclusion chromatography, hollow-fiber tangential flow filtration, and polyethylene glycol precipitation) implemented after the iodixanol gradient for their ability to deplete iodixanol and protein contaminations. Last, we conducted a side-by-side comparison of the CsCl and iodixanol/ultrafiltration protocol. Our results demonstrate that iodixanol-purified AAV preparations show higher vector purity but harbor more (∼20%) empty particles as compared with CsCl-purified vectors (<1%). Using mass spectrometry, we analyzed prominent protein impurities in the AAV vector product, thereby identifying known and new, possibly AAV-interacting proteins as major contaminants. Thus, our study not only provides a helpful guide for the many laboratories entering the AAV field, but also builds a basis for further investigation of cellular processes involved in AAV vector assembly and trafficking. PMID:26222983

  14. Adeno-Associated Virus Capsid Proteins May Play a Role in Transcription and Second-Strand Synthesis of Recombinant Genomes

    PubMed Central

    Salganik, Maxim; Aydemir, Fikret; Nam, Hyun-Joo; McKenna, Robert; Agbandje-McKenna, Mavis

    2014-01-01

    A group of four interacting amino acids in adeno-associated virus type 8 (AAV8) called the pH quartet has been shown to undergo a structural change when subjected to acidic pH comparable to that seen in endosomal compartments. We examined the phenotypes of mutants with mutations in these amino acids as well as several nearby residues in the background of AAV2. We found that three of the mutations in this region (Y704A, E562A, and E564A) produce normal titers of mature capsids but are extremely defective for transduction (>107-fold). The remaining mutants were also defective for transduction, but the defect in these mutants (E563A, E561A, H526A, and R389A) is not as severe (3- to 22-fold). Two other mutants (Y700A and Y730A) were found to be defective for virus assembly. One of the extremely defective mutants (Y704A) was found to enter the cell, traffic to the nucleus, and uncoat its DNA nearly as efficiently as the wild type. This suggested that some step after nuclear entry and uncoating was defective. To see if the extremely defective mutants were impaired in second-strand synthesis, the Y704A, E562A, and E564A mutants containing self-complementary DNA were compared with virus containing single-stranded genomes. Two of the mutants (Y704A and E564A) showed 1-log and 3-log improvements in infectivity, respectively, while the third mutant (E562A) showed no change. This suggested that inhibition of second-strand synthesis was responsible for some but not most of the defect in these mutants. Comparison of Y704A mRNA synthesis with that of the wild-type capsid showed that accumulation of steady-state mRNA in the Y704A mutant was reduced 450-fold, even though equal genome numbers were uncoated. Our experiments have identified a novel capsid function. They suggest that AAV capsids may play a role in the initiation of both second-strand synthesis and transcription of the input genome. PMID:24198419

  15. Characterization of Wild-Type Adeno-Associated Virus Type 2-Like Particles Generated during Recombinant Viral Vector Production and Strategies for Their Elimination

    PubMed Central

    Wang, Xu-Shan; Khuntirat, Benjawan; Qing, Keyun; Ponnazhagan, Selvarangan; Kube, Dagmar M.; Zhou, Shangzhen; Dwarki, Varavani J.; Srivastava, Arun

    1998-01-01

    The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence

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

    PubMed Central

    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

  17. Postentry Processing of Recombinant Adeno-Associated Virus Type 1 and Transduction of the Ferret Lung Are Altered by a Factor in Airway Secretions

    PubMed Central

    Yan, Ziying; Sun, Xingshen; Evans, Idil A.; Tyler, Scott R.; Song, Yi; Liu, Xiaoming; Sui, Hongshu

    2013-01-01

    Abstract We recently created a cystic fibrosis ferret model that acquires neonatal lung infection. To develop lung gene therapies for this model, we evaluated recombinant adeno-associated virus (rAAV)-mediated gene transfer to the neonatal ferret lung. Unlike in vitro ferret airway epithelial (FAE) cells, in vivo infection of the ferret lung with rAAV1 required proteasome inhibitors to achieve efficient airway transduction. We hypothesized that differences in transduction between these two systems were because of an in vivo secreted factor that alter the transduction biology of rAAV1. Indeed, treatment of rAAV1 with ferret airway secretory fluid (ASF) strongly inhibited rAAV1, but not rAAV2, transduction of primary FAE and HeLa cells. Properties of the ASF inhibitory factor included a strong affinity for the AAV1 capsid, heat-stability, negative charge, and sensitivity to endoproteinase Glu-C. ASF-treated rAAV1 dramatically inhibited apical transduction of FAE ALI cultures (512-fold), while only reducing viral entry by 55-fold, suggesting that postentry processing of virus was influenced by the inhibitor factor. Proteasome inhibitors rescued transduction in the presence of ASF (∼1600-fold) without effecting virus internalization, while proteasome inhibitors only enhanced transduction 45-fold in the absence of ASF. These findings demonstrate that a factor in lung secretions can influence intracellular processing of rAAV1 in a proteasome-dependent fashion. PMID:23948055

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

    PubMed

    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

  19. Preclinical safety evaluation of recombinant adeno-associated virus 2 vector encoding human tumor necrosis factor receptor-immunoglobulin Fc fusion gene.

    PubMed

    Zhou, Xiaobing; Shen, Lianzhong; Liu, Li; Wang, Chao; Qi, Weihong; Zhao, Aizhi; Wu, Xiaobing; Li, Bo

    2016-03-01

    Recombinant adeno-associated virus (rAAV) 2 vector gene therapy offers promise for the healing of Rheumatoid arthritis. To support the clinical development of the candidate gene therapeutic product in China, a comprehensive preclinical safety assessment of rAAV2 encoding human TNF receptor-immunoglobulin Fc fusion gene (rAAV2/human TNFR:Fc), were conducted in 3 species of experimental animals. No abnormal findings were observed in mice following single intravenous administration with test article. Compared with the control group, no differences in mean body weight, food consumption in rats and monkeys following the repeated intraarticular administration with rAAV2/human TNFR:Fc. There were also no significant adverse effects due to treatment noted by clinical chemistry, hematology and pathology assessments. After intraarticular administration with rAAV2/human TNFR:Fc, the vector DNA initially distributed to spleen, lymph nodes, and joint synovium. The vector DNA cleared rapidly as it could be detected mainly at the site of injection by 91 d post-administration (182 d for monkey). Taken together, localized delivery of rAAV2/human TNFR:Fc showed no significant toxicity in mice, rats, and monkeys, which support the planned clinical evaluation of this product. PMID:26837862

  20. Recombinant adeno-associated virus-delivered anginex inhibits angiogenesis and growth of HUVECs by regulating the Akt, JNK and NF-κB signaling pathways.

    PubMed

    Ma, Ke; Wang, Chuying; Geng, Qianqian; Fan, Yangwei; Ning, Jing; Yang, Haixia; Dong, Xuyuan; Dong, Danfeng; Guo, Yuyan; Wei, Xin; Li, Enxiao; Wu, Yinying

    2016-06-01

    Anginex is an artificial synthetic small molecule β-sheet-forming peptide shown to have anti-angiogenesis and antitumor effects in various solid tumors. However, its molecular mechanism remains largely unclear and efficient delivery methods for anginex remains to be developed. We report on the development of recombinant adeno-associated virus (rAAV2)-delivered anginex and the underlying mechanism of anti-angiogenesis and antitumor effects of anginex. We have successfully developed the rAAV2 vector to efficiently express anginex (rAAV2‑anginex). Transduction of rAAV2-anginex significantly induced apoptosis, and inhibited the proliferation, migration, invasion and tube formation of human umbilical vein endothelial cells in vitro. Western blot analysis revealed that rAAV2‑anginex inhibited the phosphorylation of Akt, while inducing the phosphorylation of JNK and activation of the NF-κB signaling pathway. In an in vivo CAM assay and xenograft model of SKOV3, rAAV2-anginex significantly reduced microvessel density (MVD) and vascular endothelial growth factor 165 (VEGF165), as demonstrated by immunohistochemistry analysis. Importantly, rAAV2-anginex inhibited tumor growth in an ovarian cancer SKOV3 cell nude mouse xenograft model. Our results suggest that rAAV2-anginex may inhibit tumor angiogenesis and growth through regulating Akt, JNK and NF-κB signaling pathways. PMID:27035232

  1. Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery.

    PubMed

    Weber, Michel; Rabinowitz, Joseph; Provost, Nathalie; Conrath, Hervé; Folliot, Sébastien; Briot, Delphine; Chérel, Yan; Chenuaud, Pierre; Samulski, Jude; Moullier, Philippe; Rolling, Fabienne

    2003-06-01

    We previously described chimeric recombinant adeno-associated virus (rAAV) vectors 2/4 and 2/5 as the most efficient vectors in rat retina. We now characterize these two vectors carrying the CMV.gfp genome following subretinal injection in the Wistar rat, beagle dog, and cynomolgus macaque. Both serotypes displayed stable GFP expression for the duration of the experiment (6 months) in all three animal models. Similar to the AAV-2 serotype, AAV-2/5 transduced both RPE and photoreceptor cells, with higher level of transduction in photoreceptors, whereas rAAV-2/4 transduction was unambiguously restricted to RPE cells. This unique specificity found conserved among all three species makes AAV-2/4-derived vectors attractive for retinal diseases originating in RPE such as Leber congenital amaurosis (RPE65) or retinitis pigmentosa due to a mutated mertk gene. To provide further important preclinical data, vector shedding was monitored by PCR in various biological fluids for 2 months post-rAAV administration. Following rAAV-2/4 and -5 subretinal delivery in dogs (n = 6) and in nonhuman primates (n = 2), vector genome was found in lacrymal and nasal fluids for up to 3-4 days and in the serum for up to 15-20 days. Overall, these findings will have a practical impact on the development of future gene therapy trials of retinal diseases. PMID:12788651

  2. Immobilization of FLAG-Tagged Recombinant Adeno-Associated Virus 2 onto Tissue Engineering Scaffolds for the Improvement of Transgene Delivery in Cell Transplants

    PubMed Central

    Shi, Wen-Jie; Bai, Xue-Jia; Jia, Shu-Qin; Zhang, Chen-Guang; Ding, Wei

    2015-01-01

    The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures. PMID:26035716

  3. Short-lived recombinant adeno-associated virus transgene expression in dystrophic muscle is associated with oxidative damage to transgene mRNA

    PubMed Central

    Dupont, Jean-Baptiste; Tournaire, Benoit; Georger, Christophe; Marolleau, Béatrice; Jeanson-Leh, Laurence; Ledevin, Mireille; Lindenbaum, Pierre; Lecomte, Emilie; Cogné, Benjamin; Dubreil, Laurence; Larcher, Thibaut; Gjata, Bernard; Van Wittenberghe, Laetitia; Le Guiner, Caroline; Penaud-Budloo, Magalie; Snyder, Richard O; Moullier, Philippe; Léger, Adrien

    2015-01-01

    Preclinical gene therapy strategies using recombinant adeno-associated virus (AAV) vectors in animal models of Duchenne muscular dystrophy have shown dramatic phenotype improvements, but long-lasting efficacy remains questionable. It is believed that in dystrophic muscles, transgene persistence is hampered, notably by the progressive loss of therapeutic vector genomes resulting from muscle fibers degeneration. Intracellular metabolic perturbations resulting from dystrophin deficiency could also be additional factors impacting on rAAV genomes and transgene mRNA molecular fate. In this study, we showed that rAAV genome loss is not the only cause of reduced transgene mRNA level and we assessed the contribution of transcriptional and post-transcriptional factors. We ruled out the implication of transgene silencing by epigenetic mechanisms and demonstrated that rAAV inhibition occurred mostly at the post-transcriptional level. Since Duchenne muscular dystrophy (DMD) physiopathology involves an elevated oxidative stress, we hypothesized that in dystrophic muscles, transgene mRNA could be damaged by oxidative stress. In the mouse and dog dystrophic models, we found that rAAV-derived mRNA oxidation was increased. Interestingly, when a high expression level of a therapeutic transgene is achieved, oxidation is less pronounced. These findings provide new insights into rAAV transductions in dystrophic muscles, which ultimately may help in the design of more effective clinical trials. PMID:26029721

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

    PubMed

    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 × 10(13) v.g./ml (the total titer was 4.17 × 10(13) v.g.) from the 4 × 10(9) HEK293 cells. This novel chromatography-based method will facilitate scale-up of manufacturing for clinical applications in gene therapy. PMID:26913289

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

    PubMed Central

    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

  6. An Adenovirus Type 5 Mutant with the Preterminal Protein Gene Deleted Efficiently Provides Helper Functions for the Production of Recombinant Adeno-Associated Virus

    PubMed Central

    Maxwell, Ian H.; Maxwell, Francoise; Schaack, Jerome

    1998-01-01

    Production of recombinant adeno-associated virus (rAAV) requires helper functions that have routinely been provided by infection of the producer cells with adenovirus. Complete removal and/or inactivation of progeny adenovirus, present in such rAAV preparations, presents significant difficulty. Here, we report that an adenovirus type 5 (Ad5) mutant with the preterminal protein (pTP) gene deleted can provide helper function for the growth of rAAV. At high multiplicity, Ad5dl308ΔpTP was as efficient as the phenotypically wild-type Ad5dl309 in permitting growth of rAAV. Use of Ad5dl308ΔpTP, which is incapable of replication in the absence of complementation for pTP, as a helper avoids the need to remove contaminating adenovirus infectious activity by heat inactivation or by purification. Comparison of the transducing ability of rAAV generated with either Ad5dl308ΔpTP or Ad5dl309 as a helper demonstrated that the heat inactivation protocol generally used does not remove all of the helper Ad5dl309 function. PMID:9733887

  7. The effect of recombinant adeno-associated virus-adiponectin (rAAV2/1-Acrp30) on glycolipid dysmetabolism and liver morphology in diabetic rats.

    PubMed

    Long, Wen; Hui Ju, Zhong; Fan, Zhang; Jing, Wang; Qiong, Li

    2014-09-15

    Adiponectin is an adipocytokine derived from adipocytes with insulin resistance-improving and anti-inflammatory activities. The level of Adiponectin is decreased in obesity, insulin resistance and Type 2 diabetes mellitus. The administration of recombinant adiponectin has been shown to improve hyperglycemia and insulin resistance in diabetic mice. Therefore, we investigated the effects of recombinant adeno-associated virus-adiponectin (rAAV2/1-Acrp30) on the glycolipid profile and liver morphology in streptozotocin-induced diabetic rats. Animals were fed a high-fat/high-glucose diet for 4weeks and diabetes induced by intraperitoneal administration of streptozotocin. The animals were divided randomly into four groups: diabetes control group, rAAV2/1-Acrp30 treatment group, vacuity virus group, and normal control group. Compared with diabetic rats and those in the vacuity virus group, animals treated with rAAV2/1-Acrp30 exhibited significantly lower values for glycaemic and lipidic profiles, and significantly higher levels of HDL. Although APN expression increased in the liver tissue, serum levels were not significantly increased. However, the rAAV2/1-Acrp30 treated animals showed amelioration of hepatic disease, accompanied by marked reduction in the expression of NF-κBp65 and IκBα. The results suggest that rAAV2/1-Acrp30 ameliorates glycolipid dysmetabolism and hepatic disease in streptozotocin-induced diabetic rats. These observations indicate that the function of rAAV2/1-Acrp30 is mediated by downregulated expression of NF-κBp65 and IκBα. PMID:25019654

  8. Enhancement of Recombinant Adeno-Associated Virus Type 2-Mediated Transgene Expression in a Lung Epithelial Cell Line by Inhibition of the Epidermal Growth Factor Receptor

    PubMed Central

    Smith, Andrew D.; Collaco, Roy F.; Trempe, James P.

    2003-01-01

    Recombinant adeno-associated viruses (rAAVs) have attracted considerable interest as gene delivery systems because they show long-term expression in vivo and transduce numerous cell types. Limitations to successful gene transduction from rAAVs have prompted investigations of a variety of treatments to enhance transgene expression from rAAV vectors. Tyrphostin-1, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, dramatically enhances rAAV transgene expression. Elegant studies have demonstrated that a single-strand D-sequence-binding protein (ssDBP) is phosphorylated by EGFR and binds to the D sequence element in the AAV terminal repeat (TR). Binding of the Tyr-phosphorylated ssDBP prevents conversion of single-stranded vector DNA to a double-strand conformation. We observed dramatic increases in transgene expression in lung epithelial cells (IB3) with tyrphostin treatment. Gel shift analysis of ssDBP revealed that its DNA binding characteristics were unchanged after tyrphostin treatment or adenovirus infection. Tyrphostin stimulated rAAV transgene expression to a greater extent than adenovirus coinfection. Southern hybridizations revealed that the vector DNA remained in the single-strand conformation in tyrphostin-treated cells but double-stranded replicative form monomer DNA was most abundant in adenovirus-infected cells. Northern analyses revealed that tyrphostin treatment enhanced mRNA accumulation more than in adenovirus-infected cultures even though replicative form DNA was undetectable. Analysis of the JNK, ERK, and p38K mitogen-activated protein kinase pathways revealed that tyrphostin treatment stimulated the activity of JNK and p38K. Our data suggest that tyrphostin-induced alteration of stress response pathways results in dramatic enhancement of transcription on linear vector DNA templates in the IB3 cell line. These results expand the downstream targets of the EGFR in regulating rAAV transduction. PMID:12743297

  9. Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-CB-hRS1, a Recombinant Adeno-Associated Virus Vector Expressing Retinoschisin

    PubMed Central

    Ye, Guo-Jie; Budzynski, Ewa; Sonnentag, Peter; Miller, Paul E.; Sharma, Alok K.; Ver Hoeve, James N.; Howard, Kellie; Knop, David R.; Neuringer, Martha; McGill, Trevor; Stoddard, Jonathan; Chulay, Jeffrey D.

    2015-01-01

    Applied Genetic Technologies Corporation is developing rAAV2tYF-CB-hRS1, a recombinant adeno-associated virus (rAAV) vector for treatment of X-linked retinoschisis (XLRS), an inherited retinal disease characterized by splitting (schisis) of retinal layers causing poor vision. We report here results of a study evaluating the safety and biodistribution of rAAV2tYF-CB-hRS1 in normal cynomolgus macaques. Three groups of male animals (n = 6 per group) received an intravitreal injection in one eye of either vehicle, or rAAV2tYF-CB-hRS1 at one of two dose levels (4 × 1010 or 4 × 1011 vg/eye). Half the animals were sacrificed after 14 days and the others after 91 or 115 days. The intravitreal injection procedure was well tolerated in all groups. Serial ophthalmic examinations demonstrated a dose-related anterior and posterior segment inflammatory response that improved over time. There were no test article-related effects on intraocular pressure, electroretinography, visual evoked potential, hematology, coagulation, clinical chemistry, or gross necropsy observations. Histopathological examination demonstrated minimal or moderate mononuclear infiltrates in 6 of 12 vector-injected eyes. Immunohistochemical staining showed RS1 labeling of the ganglion cell layer at the foveal slope in vector-injected eyes at both dose levels. Serum anti-AAV antibodies were detected in 4 of 6 vector-injected animals at the day 15 sacrifice and all vector-injected animals at later time points. No animals developed antibodies to RS1. Biodistribution studies demonstrated high levels of vector DNA in the injected eye but minimal or no vector DNA in any other tissue. These results support the use of rAAV2tYF-CB-hRS1 in clinical studies in patients with XLRS. PMID:26390090

  10. Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB signaling pathway

    PubMed Central

    SUN, Zhan; MA, Yi-Tong; CHEN, Bang-Dang; LIU, Fen

    2014-01-01

    Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the underlying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease. PMID:25593580

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

    PubMed

    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

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

    PubMed Central

    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. Additionally, the transgene was expressed in some brain areas up to the frontal cortex and the olfactory bulb. The rAAV was distributed predominantly in the spinal cord, where its genome copy was over ten times that of the peripheral organs. Compared with intravenous injection, another method for rAAV delivery to the broad CNS, the intrathecal injection reduced the dosage of rAAV required to achieve similar or higher levels of transgene expression in the CNS by ∼100 fold. Finally, the transduced areas were colocalized with the perivascular spaces of Virchow-Robin, from which the rAAV spreads further into the CNS parenchyma, thus suggesting that rAAV penetrated the CNS parenchyma through this pathway. Taken together, we have defined a fast and efficient method to deliver widespread transgene expression in mature spinal cord in mice. This method can be applied to stably overexpress or silence gene expression in the spinal cord to investigate gene functions in mammalian CNS. Additionally, this method can be applied to validate therapeutic targets for spinal cord diseases. PMID:26050084

  13. Characterization of Cognitive Deficits in Rats Overexpressing Human Alpha-Synuclein in the Ventral Tegmental Area and Medial Septum Using Recombinant Adeno-Associated Viral Vectors

    PubMed Central

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

    2013-01-01

    Intraneuronal inclusions containing alpha-synuclein (a-syn) constitute one of the pathological hallmarks of Parkinson's disease (PD) and are accompanied by severe neurodegeneration of A9 dopaminergic neurons located in the substantia nigra. Although to a lesser extent, A10 dopaminergic neurons are also affected. Neurodegeneration of other neuronal populations, such as the cholinergic, serotonergic and noradrenergic cell groups, has also been documented in PD patients. Studies in human post-mortem PD brains and in rodent models suggest that deficits in cholinergic and dopaminergic systems may be associated with the cognitive impairment seen in this disease. Here, we investigated the consequences of targeted overexpression of a-syn in the mesocorticolimbic dopaminergic and septohippocampal cholinergic pathways. Rats were injected with recombinant adeno-associated viral vectors encoding for either human wild-type a-syn or green fluorescent protein (GFP) in the ventral tegmental area and the medial septum/vertical limb of the diagonal band of Broca, two regions rich in dopaminergic and cholinergic neurons, respectively. Histopathological analysis showed widespread insoluble a-syn positive inclusions in all major projections areas of the targeted nuclei, including the hippocampus, neocortex, nucleus accumbens and anteromedial striatum. In addition, the rats overexpressing human a-syn displayed an abnormal locomotor response to apomorphine injection and exhibited spatial learning and memory deficits in the Morris water maze task, in the absence of obvious spontaneous locomotor impairment. As losses in dopaminergic and cholinergic immunoreactivity in both the GFP and a-syn expressing animals were mild-to-moderate and did not differ from each other, the behavioral impairments seen in the a-syn overexpressing animals appear to be determined by the long term persisting neuropathology in the surviving neurons rather than by neurodegeneration. PMID:23705016

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

    PubMed

    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

  15. Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells.

    PubMed Central

    Miller, J L; Donahue, R E; Sellers, S E; Samulski, R J; Young, N S; Nienhuis, A W

    1994-01-01

    Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content. Images PMID:7524085

  16. An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

    PubMed Central

    Schreiber, Claire A.; Sakuma, Toshie; Izumiya, Yoshihiro; Holditch, Sara J.; Hickey, Raymond D.; Bressin, Robert K.; Basu, Upamanyu; Koide, Kazunori; Asokan, Aravind; Ikeda, Yasuhiro

    2015-01-01

    Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors. PMID:26244496

  17. A Single Injection of Recombinant Adeno-Associated Virus into the Lumbar Cistern Delivers Transgene Expression Throughout the Whole Spinal Cord.

    PubMed

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

    2016-07-01

    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 60 to 90 % of the cells in the spinal cord. The transgene was expressed in all cell types, including neurons, glia, ependymal cells, and endothelial cells. Additionally, the transgene was expressed in some brain areas up to the frontal cortex and the olfactory bulb. The rAAV was distributed predominantly in the spinal cord, where its genome copy was over ten times that of the peripheral organs. Compared with intravenous injection, another method for rAAV delivery to the broad central nervous system (CNS), the intrathecal injection reduced the dosage of rAAV required to achieve similar or higher levels of transgene expression in the CNS by ~100-fold. Finally, the transduced areas were co-localized with the perivascular spaces of Virchow-Robin, from which the rAAV spreads further into the CNS parenchyma, thus suggesting that rAAV penetrated the CNS parenchyma through this pathway. Taken together, we have defined a fast and efficient method to deliver widespread transgene expression in mature spinal cord in mice. This method can be applied to stably overexpress or silence gene expression in the spinal cord to investigate gene functions in mammalian CNS. Additionally, this method can be applied to validate therapeutic targets for spinal cord diseases. PMID:26050084

  18. Heat-shock Treatment-mediated Increase in Transduction by Recombinant Adeno-associated Virus 2 Vectors Is Independent of the Cellular Heat-shock Protein 90*

    PubMed Central

    Zhong, Li; Qing, Keyun; Si, Yue; Chen, Linyuan; Tan, Mengqun; Srivastava, Arun

    2007-01-01

    Recombinant adeno-associated virus 2 (AAV) vectors transduction efficiency varies greatly in different cell types. We have described that a cellular protein, FKBP52, in its phosphorylated form interacts with the D-sequence in the viral inverted terminal repeat, inhibits viral second strand DNA synthesis, and limits transgene expression. Here we investigated the role of cellular heat-shock protein 90 (HSP90) in AAV transduction because FKBP52 forms a complex with HSP90, and because heat-shock treatment augments AAV transduction efficiency. Heat-shock treatment of HeLa cells resulted in tyrosine dephosphorylation of FKBP52, led to stabilization of the FKBP52-HSP90 complex, and resulted in ∼6-fold increase in AAV transduction. However, when HeLa cells were pre-treated with tyrphostin 23, a specific inhibitor of cellular epidermal growth factor receptor tyrosine kinase, which phosphorylates FKBP52 at tyrosine residues, heat-shock treatment resulted in a further 18-fold increase in AAV transduction. HSP90 was shown to be a part of the FKBP52-AAV D-sequence complex, but HSP90 by itself did not bind to the D-sequence. Geldanamycin treatment, which disrupts the HSP90-FKBP52 complex, resulted in >22-fold increase in AAV transduction in heat-shock-treated cells compared with heat shock alone. Deliberate overexpression of the human HSP90 gene resulted in a significant decrease in AAV-mediated transduction in tyrphostin 23-treated cells, whereas down-modulation of HSP90 levels led to a decrease in HSP90-FKBP52-AAV D-sequence complex formation, resulting in a significant increase in AAV transduction following pre-treatment with tyrphostin 23. These studies suggest that the observed increase in AAV transduction efficiency following heat-shock treatment is unlikely to be mediated by HSP90 alone and that increased levels of HSP90, in the absence of heat shock, facilitate binding of FKBP52 to the AAV D-sequence, thereby leading to inhibition of AAV-mediated transgene expression

  19. Recombinant adeno-associated virus type 2 replication and packaging is entirely supported by a herpes simplex virus type 1 amplicon expressing Rep and Cap.

    PubMed Central

    Conway, J E; Zolotukhin, S; Muzyczka, N; Hayward, G S; Byrne, B J

    1997-01-01

    Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have recently been shown to have great utility as gene transfer agents both in vitro and in vivo. One of the problems associated with the use of rAAV vectors has been the difficulty of large-scale vector production. Low-efficiency plasmid transfection of the rAAV vector and complementing AAV type 2 (AAV-2) functions (rep and cap) followed by superinfection with adenovirus has been the standard approach to rAAV production. The objectives of this study were to demonstrate the ability of a recombinant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 Rep and Cap to support replication and packaging of rAAV vectors. HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supplying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively. Replication of the amplicon stocks is not inhibited by the presence of AAV-2 Rep proteins, which highlights important differences between HSV-1 and adenovirus replication and the mechanism of providing helper function for productive AAV infection. Coinfection of rAAV and HSV-RC/KOS resulted in the replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were transfected into cells followed by HSV-RC/KOS infection and when two rAAV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27. Production of infectious rAAV by rescue from two rAAV proviral cell lines has also been achieved with HSV-RC/KOS and HSV-RC/d27. The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by supplying rep and cap by transfection followed by adenovirus superinfection. Importantly, no detectable wild-type AAV-2 is generated with this approach. These results demonstrate

  20. Recombinant adeno-associated virus expressing human papillomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer.

    PubMed

    Liu, D W; Tsao, Y P; Kung, J T; Ding, Y A; Sytwu, H K; Xiao, X; Chen, S L

    2000-03-01

    In this study, we explore a potential vaccine for human papillomavirus (HPV)-induced tumors, using heat shock protein as an adjuvant, a peptide vaccine for safety, and adeno-associated virus (AAV) as a gene delivery vector. The tumor vaccine was devised by constructing a chimeric gene which contained HPV type 16 E7 cytotoxic T-lymphocyte (CTL) epitope DNA (M. C. Feltkamp, H. L. Smits, M. P. Vierboom, R. P. Minnaar, B. M. de Jongh, J. W. Drijfhout, J. ter Schegget, C. J. Melief, and W. M. Kast, Eur. J. Immunol. 23:2242-2249, 1993) fused with the heat shock protein gene as a tumor vaccine delivered via AAV. Our results demonstrate that this vaccine can eliminate tumor cells in syngeneic animals and induce CD4- and CD8-dependent CTL activity in vitro. Moreover, studies with knockout mice with distinct T-cell deficiencies confirm that CTL-induced tumor protection is CD4 and CD8 dependent. Taken together, the evidence indicates that this chimeric gene delivered by AAV has potential as a cervical cancer vaccine. PMID:10684306

  1. Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17-36 epitopes.

    PubMed

    Jagu, Subhashini; Karanam, Balusubramanyam; Wang, Joshua W; Zayed, Hatem; Weghofer, Margit; Brendle, Sarah A; Balogh, Karla K; Tossi, Kerstin Pino; Roden, Richard B S; Christensen, Neil D

    2015-10-13

    Vaccination with the minor capsid protein L2, notably the 17-36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17-36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum±MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17-36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant. PMID:26382603

  2. Durable immunity to oncogenic human papillomaviruses elicited by adjuvanted recombinant Adeno-associated virus-like particle immunogen displaying L2 17–36 epitopes

    PubMed Central

    Jagu, Subhashini; Karanam, Balusubramanyam; Wang, Joshua W.; Zayed, Hatem; Weghofer, Margit; Brendle, Sarah A.; Balogh, Karla K.; Tossi, Kerstin Pino; Roden, Richard B.S.; Christensen, Neil D.

    2016-01-01

    Vaccination with the minor capsid protein L2, notably the 17–36 neutralizing epitope, induces broadly protective antibodies, although the neutralizing titers attained in serum are substantially lower than for the licensed L1 VLP vaccines. Here we examine the impact of other less reactogenic adjuvants upon the induction of durable neutralizing serum antibody responses and protective immunity after vaccination with HPV16 and HPV31 L2 amino acids 17–36 inserted at positions 587 and 453 of VP3, respectively, for surface display on Adeno-Associated Virus 2-like particles [AAVLP (HPV16/31L2)]. Mice were vaccinated three times subcutaneously with AAVLP (HPV16/31L2) at two week intervals at several doses either alone or formulated with alum, alum and MPL, RIBI adjuvant or Cervarix. The use of adjuvant with AAVLP (HPV16/31L2) was necessary in mice for the induction of L2-specific neutralizing antibody and protection against vaginal challenge with HPV16. While use of alum was sufficient to elicit durable protection (>3 months after the final immunization), antibody titers were increased by addition of MPL and RIBI adjuvants. To determine the breadth of immunity, rabbits were immunized three times with AAVLP (HPV16/31L2) either alone, formulated with alum ± MPL, or RIBI adjuvants, and after serum collection, the animals were concurrently challenged with HPV16/31/35/39/45/58/59 quasivirions or cottontail rabbit papillomavirus (CRPV) at 6 or 12 months post-immunization. Strong protection against all HPV types was observed at both 6 and 12 months post-immunization, including robust protection in rabbits receiving the vaccine without adjuvant. In summary, vaccination with AAVLP presenting HPV L2 17–36 epitopes at two sites on their surface induced cross-neutralizing serum antibody, immunity against HPV16 in the genital tract, and long-term protection against skin challenge with the 7 most common oncogenic HPV types when using a clinically relevant adjuvant. PMID:26382603

  3. Enhanced Transgene Expression from Recombinant Single-Stranded D-Sequence-Substituted Adeno-Associated Virus Vectors in Human Cell Lines In Vitro and in Murine Hepatocytes In Vivo

    PubMed Central

    Wang, Yuan; Lu, Yuan; Wang, Lina; Jayandharan, Giridhara R.; Aslanidi, George V.; Li, Baozheng; Cheng, Binbin; Ma, Wenqin; Lentz, Thomas; Ling, Changquan; Xiao, Xiao; Samulski, R. Jude; Muzyczka, Nicholas

    2014-01-01

    ABSTRACT We have previously reported that the removal of a 20-nucleotide sequence, termed the D sequence, from both ends of the inverted terminal repeats (ITRs) in the adeno-associated virus serotype 2 (AAV2) genome significantly impairs rescue, replication, and encapsidation of the viral genomes (X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Mol Biol 250:573–580, 1995; X. S. Wang, S. Ponnazhagan, and A. Srivastava, J Virol 70:1668–1677, 1996). Here we describe that replacement of only one D sequence in either ITR restores each of these functions, but DNA strands of only single polarity are encapsidated in mature progeny virions. Since most commonly used recombinant AAV vectors contain a single-stranded DNA (ssDNA), which is transcriptionally inactive, efficient transgene expression from AAV vectors is dependent upon viral second-strand DNA synthesis. We have also identified a transcription suppressor sequence in one of the D sequences, which shares homology with the binding site for the cellular NF-κB-repressing factor (NRF). The removal of this D sequence from, and replacement with a sequence containing putative binding sites for transcription factors in, single-stranded AAV (ssAAV) vectors significantly augments transgene expression both in human cell lines in vitro and in murine hepatocytes in vivo. The development of these genome-modified ssAAV vectors has implications not only for the basic biology of AAV but also for the optimal use of these vectors in human gene therapy. IMPORTANCE The results of the studies described here not only have provided novel insights into some of the critical steps in the life cycle of a human virus, the adeno-associated virus (AAV), that causes no known disease but have also led to the development of novel recombinant AAV vectors which are more efficient in allowing increased levels of gene expression. Thus, these studies have significant implications for the potential use of these novel AAV vectors in human gene therapy

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

    PubMed

    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

    2008-04-01

    Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene-deficient model in the domestic ferret using recombinant adeno-associated virus-mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338

  5. Comparative analysis of cytotoxic T lymphocyte response induced by dendritic cells pulsed with recombinant adeno-associated virus carrying α-fetoprotein gene or cancer cell lysate.

    PubMed

    Zhou, Jun; Ma, Ping; Li, Jun; Song, Wei

    2015-04-01

    Hepatocellular carcinoma (HCC) is one of the most common and difficult to treat types of cancer worldwide. Antigen‑targeted immunotherapy has the potential to be a novel and effective adjuvant for use in HCC. In the present study, recombinant adeno‑associated virus carrying the α‑fetoprotein gene (rAAV/AFP) and cancer cell lysates were used to pulse antigen‑presenting dendritic cells (DCs) in order to stimulate a cytotoxic T lymphocyte (CTL) response against HCC. rAAV/AFP‑pulsed and cancer cell lysate‑pulsed DCs resulted in a mature DC phenotype with high expression of major histocompatibility complex (MHC) class I, MHC class II, CD80, CD83 and CD86 molecules. However, rAAV/AFP‑pulsed DCs exhibited superiority over cancer cell lysate‑pulsed DCs in terms of stimulating proliferation of T cells, activating T cells to secret interferon‑γ (IFN‑γ) and inducing an AFP‑specific MHC class I‑restricted CTL response. The current data suggest that pulsing of DCs using rAAV/AFP is more effective than the cancer cell lysate‑pulsing technique, and that this technique may be used for the development of immunotherapy in AFP‑positive HCC. PMID:25484119

  6. Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

    PubMed Central

    Weimer, Anja; Madry, Henning; Venkatesan, Jagadeesh K; Schmitt, Gertrud; Frisch, Janina; Wezel, Anna; Jung, Jochen; Kohn, Dieter; Terwilliger, Ernest F; Trippel, Stephen B; Cucchiarini, Magali

    2012-01-01

    Administration of therapeutic genes to human osteoarthritic (OA) cartilage is a potential approach to generate effective, durable treatments against this slow, progressive disorder. Here, we tested the ability of recombinant adeno-associated virus (rAAV)-mediated overexpression of human insulinlike growth factor (hIGF)-I to reproduce an original surface in human OA cartilage in light of the pleiotropic activities of the factor. We examined the proliferative, survival and anabolic effects of the rAAV-hIGF-I treatment in primary human normal and OA chondrocytes in vitro and in explant cultures in situ compared with control (reporter) vector delivery. Efficient, prolonged IGF-I secretion via rAAV stimulated the biological activities of OA chondrocytes in all the systems evaluated over extended periods of time, especially in situ, where it allowed for the long-term reconstruction of OA cartilage (at least for 90 d). Remarkably, production of high, stable amounts of IGF-I in OA cartilage using rAAV advantageously modulated the expression of central effectors of the IGF-I axis by downregulating IGF-I inhibitors (IGF binding protein [IGFBP]-3 and IGFBP4) while up-regulating key potentiators (IGFBP5, the IGF-I receptor and downstream mitogen-activated protein kinase/extracellular signal–regulated kinase 1/2 [MAPK/ERK-1/2] and phosphatidylinisitol-3/Akt [PI3K/Akt] signal transduction pathways), probably explaining the enhanced responsiveness of OA cartilage to IGF-I treatment. These findings show the benefits of directly providing an IGF-I sequence to articular cartilage via rAAV for the future treatment of human osteoarthritis. PMID:22160392

  7. [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)].

    PubMed

    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

  8. Adeno-associated viral vectors for clinical gene transfer studies.

    PubMed

    Snyder, Richard O; Francis, Joyce

    2005-06-01

    Recombinant adeno-associated viral (rAAV) vectors can mediate the safe and long-term correction of genetic diseases in animal models following a single administration. These pre-clinical studies are the basis of human trials that have shown rAAV vector persistence and safety in humans following delivery to lung, sinus, skeletal muscle, brain and liver. Transient disease correction has also been demonstrated in humans treated for hemophilia B and cystic fibrosis using AAV2 vectors. The physiochemical properties of rAAV vector virions are amenable to industry accepted manufacturing methodologies, long-term storage and direct in vivo administration. Recombinant adeno-associated virus vectors are manufactured in compliance with current Good Manufacturing Practices (cGMPs) as outlined in the Code of Federal Regulations (21CFR). To meet these requirements, manufacturing controls and quality systems are established, including 1) adequate facilities and equipment, 2) personnel who have relevant education or experience and are trained for specific assigned duties, 3) raw materials that are qualified for use and 4) a process (including production, purification, formulation, filling, storage and shipping) that is controlled, aseptic, reliable and consistent. Quality systems including Quality Control (QC) and Quality Assurance (QA) are also implemented. These manufacturing procedures and quality systems are designed so the product meets its release specifications to ensure that patients receive a safe, pure, potent and stable investigational drug. PMID:15975008

  9. Adeno-associated virus: fit to serve.

    PubMed

    Zinn, Eric; Vandenberghe, Luk H

    2014-10-01

    Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue tropism, and limited viral host response has made it an attractive vector system for gene therapy. The viral protein capsid, the primary interface with the host, is the main determinant for these phenotypes, is highly variable, and is most subject to pressures during replication. Here, we explore the evolutionary path of AAV and other parvoviruses in respect to these phenotypes, as well as directed evolution and engineering strategies that have exploited the lessons learned from natural selection in order to address remaining limitations of AAV as a therapeutic gene transfer platform. PMID:25128609

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

    ERIC Educational Resources Information Center

    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…

  11. 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.

    PubMed

    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

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

    PubMed

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

    2004-06-01

    To better understand the relationship between primate adeno-associated viruses (AAVs) and those of other mammals, we have cloned and sequenced the genome of an AAV found as a contaminant in two isolates of bovine adenovirus that was reported to be serologically distinct from primate AAVs. The bovine AAV (BAAV) genome has 4,693 bp, and its organization is similar to that of other AAV isolates. The left-hand open reading frame (ORF) and both inverted terminal repeats (ITRs) have the highest homology with the rep ORF and ITRs of AAV serotype 5 (AAV-5) (89 and 96%, respectively). However, the right-hand ORF was only 55% identical to the AAV-5 capsid ORF; it had the highest homology with the capsid ORF of AAV-4 (76%). By comparing the BAAV cap sequence with a model of an AAV-4 capsid, we mapped the regions of BAAV VP1 that are divergent from AAV-4. These regions are located on the outside of the capsid and are partially located in exposed loops. BAAV was not neutralized by antisera raised against recombinant AAV-2, AAV-4, or AAV-5, and it demonstrated a unique cell tropism profile in four human cancer cell lines, suggesting that BAAV might have transduction activity distinct from that of other isolates. A murine model of salivary gland gene transfer was used to evaluate the in vivo performance of recombinant BAAV. Recombinant BAAV-mediated gene transfer was 11 times more efficient than that with AAV-2. Overall, these data suggest that vectors based on BAAV could be useful for gene transfer applications. PMID:15163744

  13. The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

    PubMed Central

    Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

    2004-01-01

    Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

  14. Adeno-Associated Viral Vectors for Mapping, Monitoring, and Manipulating Neural Circuits

    PubMed Central

    Betley, J. Nicholas

    2011-01-01

    Abstract Understanding the structure and function of neural circuits is central is neuroscience research. To address the associated questions, new genetically encoded tools have been developed for mapping, monitoring, and manipulating neurons. Essential to implementation of these tools is their selective delivery to defined neuronal populations in the brain. This has been facilitated by recent improvements in cell type–specific transgene expression using recombinant adeno-associated viral vectors. Here, we highlight these developments and discuss areas for improvement that could further expand capabilities for neural circuit analysis. PMID:21319997

  15. Effects of Adeno-Associated Virus DNA Hairpin Structure on Recombination‡

    PubMed Central

    Choi, Vivian W.; Samulski, R. Jude; McCarty, Douglas M.

    2005-01-01

    Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence. PMID:15890919

  16. Adeno-associated virus for cystic fibrosis gene therapy.

    PubMed

    Martini, S V; Rocco, P R M; Morales, M M

    2011-11-01

    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. PMID:21952739

  17. Glycan binding avidity determines the systemic fate of adeno-associated virus type 9.

    PubMed

    Shen, Shen; Bryant, Kelli D; Sun, Junjiang; Brown, Sarah M; Troupes, Andrew; Pulicherla, Nagesh; Asokan, Aravind

    2012-10-01

    Glycans are key determinants of host range and transmissibility in several pathogens. In the case of adeno-associated viruses (AAV), different carbohydrates serve as cellular receptors in vitro; however, their contributions in vivo are less clear. A particularly interesting example is adeno-associated virus serotype 9 (AAV9), which displays systemic tropism in mice despite low endogenous levels of its primary receptor (galactose) in murine tissues. To understand this further, we studied the effect of modulating glycan binding avidity on the systemic fate of AAV9 in mice. Intravenous administration of recombinant sialidase increased tissue levels of terminally galactosylated glycans in several murine tissues. These conditions altered the systemic tropism of AAV9 into a hepatotropic phenotype, characterized by markedly increased sequestration within the liver sinusoidal endothelium and Kupffer cells. In contrast, an AAV9 mutant with decreased glycan binding avidity displayed a liver-detargeted phenotype. Altering glycan binding avidity also profoundly affected AAV9 persistence in blood circulation. Our results support the notion that high glycan receptor binding avidity appears to impart increased liver tropism, while decreased avidity favors systemic spread of AAV vectors. These findings may not only help predict species-specific differences in tropism for AAV9 on the basis of tissue glycosylation profiles, but also provide a general approach to tailor AAV vectors for systemic or hepatic gene transfer by reengineering capsid-glycan interactions. PMID:22787229

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

    PubMed

    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

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

    PubMed Central

    An, Heeyoung; Cho, Doo-Wan; Lee, Seung Eun; Yang, Young-Su

    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 brain. One month after injection, monkeys were sacrificed, and then the presence of viral infection by expression of reporter fluorescence proteins was examined. Tissues were sectioned and stained with NeuN and GFAP antibodies for identifying neuronal cells or astrocytes, respectively, and viral reporter GFP-expressing cells were counted. We found that while lentivirus infected mostly astrocytes, AAV infected neurons at a higher rate than astrocytes. Moreover, astrocytes showed reactiveness when cells were infected by virus, likely due to virus-mediated neuroinflammation. The Sholl analysis was done to compare the hypertrophy of infected and uninfected astrocytes by virus. The lentivirus infected astrocytes showed negligible hypertrophy whereas AAV infected astrocytes showed significant changes in morphology, compared to uninfected astrocytes. In the brain of cynomolgus monkey, lentivirus shows tropism for astrocytes over neurons without much reactivity in astrocytes, whereas AAV shows tropism for neurons over glial cells with a significant reactivity in astrocytes. We conclude that AAV is best-suited for gene delivery to neurons, whereas lentivirus is the best choice for gene delivery to astrocytes in the brain of cynomolgus monkeys. PMID:26924933

  20. Biophysical and Ultrastructural Characterization of Adeno-Associated Virus Capsid Uncoating and Genome Release

    PubMed Central

    Horowitz, Eric D.; Rahman, K. Shefaet; Bower, Brian D.; Dismuke, David J.; Falvo, Michael R.; Griffith, Jack D.

    2013-01-01

    We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release. PMID:23269804

  1. Adeno-associated Virus as a Mammalian DNA Vector

    PubMed Central

    SALGANIK, MAX; HIRSCH, MATTHEW L.; SAMULSKI, RICHARD JUDE

    2015-01-01

    In the nearly five decades since its accidental discovery, adeno-associated virus (AAV) has emerged as a highly versatile vector system for both research and clinical applications. A broad range of natural serotypes, as well as an increasing number of capsid variants, has combined to produce a repertoire of vectors with different tissue tropisms, immunogenic profiles and transduction efficiencies. The story of AAV is one of continued progress and surprising discoveries in a viral system that, at first glance, is deceptively simple. This apparent simplicity has enabled the advancement of AAV into the clinic, where despite some challenges it has provided hope for patients and a promising new tool for physicians. Although a great deal of work remains to be done, both in studying the basic biology of AAV and in optimizing its clinical application, AAV vectors are currently the safest and most efficient platform for gene transfer in mammalian cells. PMID:26350320

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

    PubMed

    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

  3. Chemical Modulation of Endocytic Sorting Augments Adeno-associated Viral Transduction.

    PubMed

    Berry, Garrett E; Asokan, Aravind

    2016-01-01

    Intracellular trafficking of viruses can be influenced by a variety of inter-connected cellular sorting and degradation pathways involving endo-lysosomal vesicles, the ubiquitin-proteasome system, and autophagy-based or endoplasmic reticulum-associated machinery. In the case of recombinant adeno-associated viruses (AAV), proteasome inhibitors are known to prevent degradation of ubiquitinated AAV capsids, thereby leading to increased nuclear accumulation and transduction. However, the impact of other cellular degradation pathways on AAV trafficking is not well understood. In the current study, we screened a panel of small molecules focused on modulating different cellular degradation pathways and identified eeyarestatin I (EerI) as a novel reagent that enhances AAV transduction. EerI improved AAV transduction by an order of magnitude regardless of vector dose, genome architecture, cell type, or serotype. This effect was preceded by sequestration of AAV within enlarged vesicles that were dispersed throughout the cytoplasm. Specifically, EerI treatment redirected AAV particles toward large vesicles positive for late endosomal (Rab7) and lysosomal (LAMP1) markers. Notably, MG132 and EerI (proteasomal and endoplasmic reticulum-associated degradation inhibitors, respectively) appear to enhance AAV transduction by increasing the intracellular accumulation of viral particles in a mutually exclusive fashion. Taken together, our results expand on potential strategies to redirect recombinant AAV vectors toward more productive trafficking pathways by deregulating cellular degradation mechanisms. PMID:26527686

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

    PubMed

    High, Katherine A; Anguela, Xavier M

    2016-04-15

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

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

    PubMed

    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

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

    PubMed

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

    2016-02-01

    Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr(-/-) (also known as Au040320(-/-) and Kiaa0319l(-/-)) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection. PMID:26814968

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

    SciTech Connect

    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.

  8. Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia.

    PubMed

    Li, C; Narkbunnam, N; Samulski, R J; Asokan, A; Hu, G; Jacobson, L J; Manco-Johnson, M J; Monahan, P E

    2012-03-01

    Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector and has recently been used in patients with hemophilia. One limitation of AAV application is that most humans have experienced wild-type AAV serotype 2 exposure, which frequently generates neutralizing antibodies (NAbs) that may inhibit rAAV2 vector transduction. Employing alternative serotypes of rAAV vectors may circumvent this problem. We investigated the development of NAbs in early childhood by examining sera gathered prospectively from 62 children with hemophilia A, participating in a multi-institutional hemophilia clinical trial (the Joint Outcome Study). Clinical applications in hemophilia therapy have been suggested for serotypes AAV2, AAV5 and AAV8, therefore NAbs against these serotypes were serially assayed over a median follow-up of 4 years. NAbs prevalence increased during early childhood for all serotypes. NAbs against AAV2 (43.5%) were observed more frequently and at higher titers compared with both AAV5 (25.8%) and AAV8 (22.6%). NAbs against AAV5 or AAV8 were rarely observed in the absence of co-prevalent and higher titer AAV2 NAbs, suggesting that NAbs to AAV5 and AAV8 were detected following AAV2 exposure due to partial cross-reactivity of AAV2-directed NAbs. The results may guide rational design of clinical trials using alternative AAV serotypes and suggest that younger patients who are given AAV gene therapy will benefit from the lower prevalence of NAbs. PMID:21697954

  9. Inducible long-term gene expression in brain with adeno-associated virus gene transfer.

    PubMed

    Haberman, R P; McCown, T J; Samulski, R J

    1998-12-01

    Recombinant adeno-associated virus (rAAV) vectors hold promise for treating a number of neurological disorders due to the ability to deliver long-term gene expression without toxicity or immune response. Critical to these endeavors will be controlled expression of the therapeutic gene in target cells. We have constructed and tested a dual cassette rAAV vector carrying a reporter gene under the control of the tetracycline-responsive system and the tetracycline transactivator. Transduction in vitro resulted in stable expression from the vector that can be suppressed 20-fold by tetracycline treatment. In vivo experiments, carried out to 6 weeks, demonstrated that vector-transduced expression is sustained until doxycycline administration upon which reporter gene expression is reduced. Moreover, the suppression of vector-driven expression can be reversed by removal of the drug. These studies demonstrate long-term regulated gene expression from rAAV vectors. This system will provide a valuable approach for controlling vector gene expression both in vitro and in vivo. PMID:10023439

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

    PubMed Central

    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

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

    PubMed

    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

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

    PubMed Central

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

    2008-01-01

    Somatic cell gene targeting combined with nuclear transfer cloning presents tremendous potential for the creation of new, large-animal models of human diseases. Mouse disease models often fail to reproduce human phenotypes, underscoring the need for the generation and study of alternative disease models. Mice deficient for CFTR have been poor models for cystic fibrosis (CF), lacking many aspects of human CF lung disease. In this study, we describe the production of a CFTR gene–deficient model in the domestic ferret using recombinant adeno-associated virus–mediated gene targeting in fibroblasts, followed by nuclear transfer cloning. As part of this approach, we developed a somatic cell rejuvenation protocol using serial nuclear transfer to produce live CFTR-deficient clones from senescent gene-targeted fibroblasts. We transferred 472 reconstructed embryos into 11 recipient jills and obtained 8 healthy male ferret clones heterozygous for a disruption in exon 10 of the CFTR gene. To our knowledge, this study represents the first description of genetically engineered ferrets and describes an approach that may be of substantial utility in modeling not only CF, but also other genetic diseases. PMID:18324338

  13. Adeno-associated virus (AAV) gene delivery in stem cell therapy.

    PubMed

    Brown, Nolan J; Hirsch, Matthew L

    2015-11-01

    The past 30 years have witnessed the development of cell and gene therapies for the treatment of diverse human diseases. Each of these approaches has inherent advantages and disadvantages; however, the two methods align in that, essentially, they are both methods of foreign DNA delivery to complement, eradicate, or supplement nucleotide sequences important for human health. As discussed herein, the combination of these therapies (gene therapy in stem cells), particularly in an ex vivo context, offers powerful genetic engineering which is applicable to the treatment of both genetic and acquired maladies ranging from blood diseases to the treatment of HIV infection. Of the existing gene therapy approaches, including non-viral and viral vectors, those based on adeno-associated virus (AAV) are currently at the forefront as they have been safely used in hundreds of clinical trials and have demonstrated remarkable success in treating blindness and hemophilia B. However, AAV vectors used in combination with cell-based therapies have not transitioned to the clinic. Instead, adenoviral, retroviral, and lentiviral vectors are the preferred choice for the modification of stem cells prior to patient infusion. This review provides a general background of AAV gene therapy and cell therapies, and highlights reports demonstrating apparently conflicting data of productive transduction and vector-induced toxicity using recombinant AAV in stem and stem-like cells. PMID:26645905

  14. Super-resolution imaging of nuclear import of adeno-associated virus in live cells.

    PubMed

    Kelich, Joseph M; Ma, Jiong; Dong, Biao; Wang, Qizhao; Chin, Mario; Magura, Connor M; Xiao, Weidong; Yang, Weidong

    2015-01-01

    Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9-12 nm and 2-20 ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy. PMID:26665132

  15. Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.

    PubMed

    Xu, R; Janson, C G; Mastakov, M; Lawlor, P; Young, D; Mouravlev, A; Fitzsimons, H; Choi, K L; Ma, H; Dragunow, M; Leone, P; Chen, Q; Dicker, B; During, M J

    2001-09-01

    This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette. PMID:11571569

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

    PubMed Central

    Nahid, M Abu; Gao, Guangping

    2014-01-01

    Introduction Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8+ T-cell response against AAV capsid in humans. Areas covered In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application. Expert opinion Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application. PMID:24386892

  17. Super-resolution imaging of nuclear import of adeno-associated virus in live cells

    PubMed Central

    Kelich, Joseph M; Ma, Jiong; Dong, Biao; Wang, Qizhao; Chin, Mario; Magura, Connor M; Xiao, Weidong; Yang, Weidong

    2015-01-01

    Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9–12 nm and 2–20 ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy. PMID:26665132

  18. Adeno-Associated Viral-Mediated Catalase Expression Suppresses Optic Neuritis in Experimental Allergic Encephalomyelitis

    NASA Astrophysics Data System (ADS)

    Guy, John; Qi, Xiaoping; Hauswirth, William W.

    1998-11-01

    Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

  19. In vivo model of adeno-associated virus vector persistence and rescue.

    PubMed Central

    Afione, S A; Conrad, C K; Kearns, W G; Chunduru, S; Adams, R; Reynolds, T C; Guggino, W B; Cutting, G R; Carter, B J; Flotte, T R

    1996-01-01

    Gene therapy vectors based on human DNA viruses could be mobilized or rescued from individuals who are subsequently infected with the corresponding wild-type (wt) helper viruses. This phenomenon has been effectively modeled in vitro with both adenovirus (Ad) and adeno-associated virus (AAV) vectors but has not previously been studied in vivo. In the current study, we have developed an in vivo model to study the interactions of a recombinant AAV vector (AAV-CFTR) with wt AAV type 2 (AAV2) and a host range mutant Ad (Ad2HR405) for which monkey cells are permissive (D.E.Brough, S.A.Rice, S.Sell, and D.F.Klessig, J. Virol. 55:206-212, 1985). AAV-CFTR was administered to the respiratory epithelium of the nose or lung of rhesus macaques. Primary cells were harvested from the infusion site at time points up to 3 months after vector administration to confirm vector DNA persistence. Vector DNA was present in episomal form and could be rescued in vitro only by addition of wt AAV2 and Ad. In in vivo rescue studies, vector was administered before or after wt-AAV2 and Ad2HR405 infection, and the shedding of AAV-CFTR was examined. Ad2HR405 and wt-AAV2 infections were established in the nose with concomitant administration. wt-AAV2 replication occurred in the lung when virus was administered directly at a high titer to the lower respiratory tract. AAV-CFTR vector rescue was also observed in the latter setting. Although these studies were performed with small numbers of animals within each group, it appears that AAV-CFTR DNA persists in the primate respiratory tract and that this model may be useful for studies of recombinant AAV vector rescue. PMID:8627804

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

    PubMed

    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

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

    PubMed Central

    Wang, Lina; Yin, Zifei; Wang, Yuan; Lu, Yuan; Zhang, Daniel; Srivastava, Arun; Ling, Changquan

    2015-01-01

    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

  2. Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy.

    PubMed

    Hagen, Sven; Baumann, Tobias; Wagner, Hanna J; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M; Müller, Kristian M

    2014-01-01

    The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

  3. Integrin alphaVbeta5 is not involved in adeno-associated virus type 2 (AAV2) infection.

    PubMed

    Qiu, J; Brown, K E

    1999-11-25

    alphaVbeta5 integrin was recently proposed as a coreceptor for adeno-associated virus type 2 (AAV2) infection (Summerford et al., 1999, Nat. Med. 5, 78-82), based mainly on the direct binding of AAV2 to denatured beta5 by virus overlay assay. In studies using purified natural or recombinant human integrin alphaVbeta5 we were unable to demonstrate AAV2 binding, either by virus overlay or by liquid binding assay. Furthermore, neither purified integrin alphaVbeta5, nor RGD peptides, nor functional blocking monoclonal antibody blocked rAAV2 transduction. These data strongly suggest that integrin alphaVbeta5 is not involved in AAV2 infection. PMID:10562505

  4. Lipofection of Purified Adeno-Associated Virus Rep68 Protein: toward a Chromosome-Targeting Nonviral Particle

    PubMed Central

    Lamartina, Stefania; Roscilli, Giuseppe; Rinaudo, Daniela; Delmastro, Paola; Toniatti, Carlo

    1998-01-01

    Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV. PMID:9696870

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed Central

    Gruenert, Anja K.; Czugala, Marta; Mueller, Chris; Schmeer, Marco; Schleef, Martin; Kruse, Friedrich E.; Fuchsluger, Thomas A.

    2016-01-01

    Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic. PMID:27023329

  8. My Life with Adeno-Associated Virus: A Long Time Spent Studying a Short Genome

    PubMed Central

    2013-01-01

    My 45 years of studying the molecular biology of adeno-associated virus are recounted. Additional activities as a mentor, department chair, and medical school administrator are described, as are my activities in the public sphere, which involved national issues related to science policy and medical education. PMID:23781880

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

    PubMed

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

    2014-01-01

    Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures. PMID:24758837

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

    PubMed Central

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

    2014-01-01

    Antibody combination therapeutics (ACTs) are polyvalent biopharmaceuticals that are uniquely suited for the control of complex diseases, including antibiotic resistant infectious diseases, autoimmune disorders and cancers. However, ACTs also represent a distinct manufacturing challenge because the independent manufacture and subsequent mixing of monoclonal antibodies quickly becomes cost prohibitive as more complex mixtures are envisioned. We have developed a virus-free recombinant protein expression platform based on adeno-associated viral (AAV) elements that is capable of rapid and consistent production of complex antibody mixtures in a single batch format. Using both multiplexed immunoassays and cation exchange (CIEX) chromatography, cell culture supernatants generated using our system were assessed for stability of expression and ratios of the component antibodies over time. Cultures expressing combinations of three to ten antibodies maintained consistent expression levels and stable ratios of component antibodies for at least 60 days. Cultures showed remarkable reproducibility following cell banking, and AAV-based cultures showed higher stability and productivity than non-AAV based cultures. Therefore, this non-viral AAV-based expression platform represents a predictable, reproducible, quick and cost effective method to manufacture or quickly produce for preclinical testing recombinant antibody combination therapies and other recombinant protein mixtures. PMID:24758837

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

    PubMed Central

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

    1989-01-01

    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. Images PMID:2536816

  12. Tissue-Specific Expression of Herpes Simplex Virus Thymidine Kinase Gene Delivered by Adeno-Associated Virus Inhibits the Growth of Human Hepatocellular Carcinoma in Athymic Mice

    NASA Astrophysics Data System (ADS)

    Su, Hua; Lu, Ronghua; Chang, Judy C.; Kan, Yuet Wai

    1997-12-01

    About 70% of hepatocellular carcinomas are known to express α -fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α -fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

  13. Syntaxin 5-Dependent Retrograde Transport to the trans-Golgi Network Is Required for Adeno-Associated Virus Transduction

    PubMed Central

    Nonnenmacher, Mathieu E.; Cintrat, Jean-Christophe; Gillet, Daniel

    2014-01-01

    ABSTRACT Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment. IMPORTANCE Gene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV

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

    SciTech Connect

    Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael; Moullier, Philippe; Salvetti, Anna . E-mail: anna.salvetti@univ-nantes.fr

    2005-05-10

    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.

  15. Biochemical Correction of Very Long–chain Acyl-CoA Dehydrogenase Deficiency Following Adeno-associated Virus Gene Therapy

    PubMed Central

    Merritt, J. Lawrence; Nguyen, Tien; Daniels, Jan; Matern, Dietrich; Schowalter, David B.

    2009-01-01

    We report the development of a gene replacement strategy for very long–chain acyl-CoA dehydrogenase (VLCAD) deficiency. VLCAD is a mitochondrial enzyme involved in fatty acid β-oxidation, a key step in energy production during times of fasting or stress. Deficiency of VLCAD classically presents as hepatic dysfunction, hypoglycemia, cardiomyopathy, rhabdomyolysis, and/or sudden death. While dietary therapy for VLCAD deficiency has proven beneficial in preventing some symptoms, a risk of metabolic catastrophic decompensation remains throughout life during times of increased energy demand. We designed a recombinant adeno-associated virus (AAV) expressing the human VLCAD gene (AAV8-hVLCAD). To demonstrate its in vivo activity, AAV8-hVLCAD was administered via the tail vein to VLCAD-knockout mice. A reduction in accumulated serum long-chain acylcarnitines and increased fasting tolerance judged on blood glucose concentrations were observed as of 11 days postinjections through >100 days. Western analysis of liver, skeletal muscle, and heart extracts using PEP1 anti-hVLCAD antibody revealed short-term hVLCAD expression in the liver and muscle and longer-term expression in heart. This demonstrates the ability of human VLCAD to correct the biochemical phenotype of VLCAD-deficient mice. PMID:19156135

  16. Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies.

    PubMed

    Clément, Nathalie; Knop, David R; Byrne, Barry J

    2009-08-01

    The ability of recombinant adeno-associated viral (rAAV) vectors to exhibit minimal immunogenicity and little to no toxicity or inflammation while eliciting robust, multiyear gene expression in vivo are only a few of the salient features that make them ideally suited for many gene therapy applications. A major hurdle for the use of rAAV in sizeable research and clinical applications is the lack of efficient and versatile large-scale production systems. Continued progression toward flexible, scalable production techniques is a prerequisite to support human clinical evaluation of these novel biotherapeutics. This review examines the current state of large-scale production methods that employ the herpes simplex virus type 1 (HSV) platform to produce rAAV vectors for gene delivery. Improvements have substantially advanced the HSV/AAV hybrid method for large-scale rAAV manufacture, facilitating the generation of highly potent, clinical-grade purity rAAV vector stocks. At least one human clinical trial employing rAAV generated via rHSV helper-assisted replication is poised to commence, highlighting the advances and relevance of this production method. PMID:19569968

  17. Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer.

    PubMed

    Walters, R W; Yi, S M; Keshavjee, S; Brown, K E; Welsh, M J; Chiorini, J A; Zabner, J

    2001-06-01

    Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors. PMID:11262413

  18. The impact of minimally oversized adeno-associated viral vectors encoding human factor VIII on vector potency in vivo

    PubMed Central

    Kyostio-Moore, Sirkka; Berthelette, Patricia; Piraino, Susan; Sookdeo, Cathleen; Nambiar, Bindu; Jackson, Robert; Burnham, Brenda; O’Riordan, Catherine R; Cheng, Seng H; Armentano, Donna

    2016-01-01

    Recombinant adeno-associated viral (rAAV) vectors containing oversized genomes provide transgene expression despite low efficiency packaging of complete genomes. Here, we characterized the properties of oversized rAAV2/8 vectors (up to 5.4 kb) encoding human factor VIII (FVIII) under the transcriptional control of three liver promoters. All vectors provided sustained production of active FVIII in mice for 7 months and contained comparable levels of vector genomes and complete expression cassettes in liver. Therefore, for the 5.4 kb genome size range, a strong expression cassette was more important for FVIII production than the vector genome size. To evaluate the potency of slightly oversized vectors, a 5.1 kb AAVrh8R/FVIII vector was compared to a 4.6 kb (wild-type size) vector with an identical expression cassette (but containing a smaller C1-domain deleted FVIII) for 3 months in mice. The 5.1 kb vector had twofold to threefold lower levels of plasma FVIII protein and liver vector genomes than that obtained with the 4.6 kb vector. Vector genomes for both vectors persisted equally and existed primarily as high molecular weight concatemeric circular forms in liver. Taken together, these results indicate that the slightly oversized vectors containing heterogeneously packaged vector genomes generated a functional transgene product but exhibited a twofold to threefold lower in vivo potency. PMID:26958574

  19. Identification of adeno-associated viral vectors suitable for intestinal gene delivery and modulation of experimental colitis.

    PubMed

    Polyak, Steven; Mach, Annette; Porvasnik, Stacy; Dixon, Lisa; Conlon, Thomas; Erger, Kirsten E; Acosta, Andres; Wright, Amy J; Campbell-Thompson, Martha; Zolotukhin, Irene; Wasserfall, Clive; Mah, Cathryn

    2012-02-01

    Effective gene transfer with sustained gene expression is an important adjunct to the study of intestinal inflammation and future therapy in inflammatory bowel disease. Recombinant adeno-associated virus (AAV) vectors are ideal for gene transfer and long-term transgene expression. The purpose of our study was to identify optimal AAV pseudotypes for transduction of the epithelium in the small intestine and colon, which could be used for studies in experimental colitis. The tropism and transduction efficiencies of AAV pseudotypes 1-10 were examined in murine small intestine and colon 8 wk after administration by real-time PCR and immunohistochemistry. The clinical and histopathological effects of IL-10-mediated intestinal transduction delivered by AAVrh10 were examined in the murine IL-10⁻/⁻ enterocolitis model. Serum IL-10 levels and IL-10 expression were followed by ELISA and real-time PCR, respectively. AAV pseudotypes 4, 7, 8, 9, and 10 demonstrated optimal intestinal transduction. Transgene expression was sustained 8 wk after administration and was frequently observed in enteroendocrine cells. Long-term IL-10 gene expression and serum IL-10 levels were observed following AAV transduction in an IL-10-/- model of enterocolitis. Animals treated with AAVrh10-IL-10 had lower disease activity index scores, higher colon weight-to-length ratios, and lower microscopic inflammation scores. This study identifies novel AAV pseudotypes with small intestine and colon tropism and sustained transgene expression capable of modulating mucosal inflammation in a murine model of enterocolitis. PMID:22114116

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

    PubMed Central

    Dias Florencio, Gabriella; Precigout, Guillaume; Beley, Cyriaque; Buclez, Pierre-Olivier; Garcia, Luis; Benchaouir, Rachid

    2015-01-01

    Recombinant adeno-associated viruses (rAAV) are promising candidates for gene therapy approaches. The last two decades were particularly fruitful in terms of processes applied in the production and purification of this type of gene transfer vectors. This rapid technological evolution led to better yields and higher levels of vector purity. Recently, some reports showed that rAAV produced by transient tri-transfection method in adherent human embryonic kidney 293 cells can be harvested directly from supernatant, leading to easier and faster purification compared to classical virus extraction from cell pellets. Here, we compare these approaches with new vector recovery method using small quantity of detergent at the initial clarification step to treat the whole transfected cell culture. Coupled with tangential flow filtration and iodixanol-based isopycnic density gradient, this new method significantly increases rAAV yields and conserves high vector purity. Moreover, this approach leads to the reduction of the total process duration. Finally, the vectors maintain their functionality, showing unexpected higher in vitro and in vivo transduction efficacies. This new development in rAAV downstream process once more demonstrates the great capacity of these vectors to easily accommodate to large panel of methods, able to furthermore ameliorate their safety, functionality, and scalability. PMID:26207258

  1. Adeno-Associated Virus-Based Gene Therapy for CNS Diseases

    PubMed Central

    Hocquemiller, Michaël; Giersch, Laura; Audrain, Mickael; Parker, Samantha; Cartier, Nathalie

    2016-01-01

    Gene therapy is at the cusp of a revolution for treating a large spectrum of CNS disorders by providing a durable therapeutic protein via a single administration. Adeno-associated virus (AAV)-mediated gene transfer is of particular interest as a therapeutic tool because of its safety profile and efficiency in transducing a wide range of cell types. The purpose of this review is to describe the most notable advancements in preclinical and clinical research on AAV-based CNS gene therapy and to discuss prospects for future development based on a new generation of vectors and delivery. PMID:27267688

  2. [Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

    PubMed

    Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

    2012-01-01

    Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials. PMID:23544311

  3. Adeno-Associated Viral Vector-Mediated Transgene Expression Is Independent of DNA Methylation in Primate Liver and Skeletal Muscle

    PubMed Central

    Léger, Adrien; Le Guiner, Caroline; Nickerson, Michael L.; McGee Im, Kate; Ferry, Nicolas; Moullier, Philippe; Snyder, Richard O.; Penaud-Budloo, Magalie

    2011-01-01

    Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model. PMID:21687632

  4. The state of the art of adeno-associated virus-based vectors in gene therapy

    PubMed Central

    Coura, Renata dos Santos; Nardi, Nance Beyer

    2007-01-01

    The adeno-associated virus (AAV) has rapidly gained popularity in gene therapy since the establishment of the first AAV2 infectious clone, in 1982, due to some of their distinguishing characteristics such as lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression. Notably over the past decade, this virus has attracted considerable interest as a gene therapy vector, and about 85% of the currently available 2,041 PubMed references on adeno-associated viruses have been published during this time. The exponential progress of AAV-based vectors has been made possible by the advances in the knowledge of the virology and biology of this virus, which allows great improvement in AAV vectors construction and a better comprehension of their operation. Moreover, with the recent discovery of novel AAV serotypes, there is virtually one preferred serotype for nearly every organ or tissue to target. Thus, AAV-based vectors have been successfully overcoming the main gene therapy challenges such as transgene maintenance, safety and host immune response, and meeting the desirable vector system features of high level of safety combined with clinical efficacy and versatility in terms of potential applications. Consequently, AAV is increasingly becoming the vector of choice for a wide range of gene therapy approaches. This report will highlight the state of the art of AAV-based vectors studies and the advances on the use of AAV vectors for several gene therapy approaches. PMID:17939872

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

    PubMed

    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

  6. Use of Adeno-Associated and Herpes Simplex Viral Vectors for In Vivo Neuronal Expression in Mice

    PubMed Central

    Penrod, Rachel D.; Wells, Audrey M.; Carlezon, William A.; Cowan, Christopher W.

    2015-01-01

    Adeno-associated viruses and the herpes simplex virus are the two most widely used vectors for the in vivo expression of exogenous genes. Advances in the development of these vectors have enabled remarkable temporal and spatial control of gene expression. This unit provides methods for storing, delivering, and verifying expression of adeno-associated and herpes simplex viruses in the adult mouse brain. It also describes important considerations for experiments using in vivo expression of these viral vectors, including serotype and promoter selection, as well as timing of expression. Additional protocols are provided that describe methods for preliminary experiments to determine the appropriate conditions for in vivo delivery. PMID:26426386

  7. Efficient Transduction of Corneal Stroma by Adeno-Associated Viral Serotype Vectors for Implications in Gene Therapy of Corneal Diseases.

    PubMed

    Lu, Yi; Ai, Jianzhong; Gessler, Dominic; Su, Qin; Tran, Karen; Zheng, Qiang; Xu, Xun; Gao, Guangping

    2016-08-01

    Corneal disease is one of the leading causes of blindness worldwide. Gene therapy is an attractive therapeutic strategy for corneal diseases, but currently underdeveloped. Recombinant adeno-associated viral (rAAV) vectors have emerged as a highly promising gene therapy platform. This study aims to identify rAAV vectors that can efficiently transduce corneal stroma for potential applications in studying pathophysiology of corneal diseases and therapeutic development. We characterized 14 rAAV serotypes expressing enhanced green fluorescent protein (EGFP), for cell specificity and transduction efficiency after either intrastromal injection or topical administration in mouse corneas in vivo. Our results show that intrastromal injections of rAAVrh.8, rAAVrh.10, rAAVrh.39, and rAAVrh.43 efficiently transduce mouse corneal stroma in vivo, and that topical administrations of rAAVrh.10 and rAAVrh.39 subsequent to epithelial scraping generate detectable transgene expression. In vivo imaging analysis revealed that transgene expression became detectable by 1 week postadministration, peaked at 2 weeks, and lasted for the duration of the study (i.e., 4 weeks). Both rAAVrh.10 and rAAVrh.39 transduced more than 50% of keratocytes, the major cell type in the corneal stroma, by intrastromal injection and 30% by topical administration. Histopathology indicated that rAAV transduction of cornea caused no morphological adverse effects. Overall, our findings suggest that some rAAV serotype vectors can efficiently transduce corneal stroma in vivo, constituting a potentially powerful and safe gene delivery platform for gene therapy of corneal diseases. PMID:27001051

  8. Avian Adeno-Associated Virus Vector Efficiently Transduces Neurons in the Embryonic and Post-Embryonic Chicken Brain

    PubMed Central

    Matsui, Ryosuke; Tanabe, Yasuto; Watanabe, Dai

    2012-01-01

    The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits. PMID:23144948

  9. Inhibition and promotion of tumor growth with adeno-associated virus carcinoembryonic antigen vaccine and Toll-like receptor agonists.

    PubMed

    Triozzi, P L; Aldrich, W; Ponnazhagan, S

    2011-12-01

    Carcinoembryonic antigen (CEA) is a cancer vaccines' target. Several features of recombinant adeno-associated virus (rAAV) are attractive for vaccine applications. Combining other viral vector vaccines with Toll-like receptor (TLR) agonists enhances antitumor immunity. Wild-type and CEA transgenic (Tg) mice were immunized with rAAV-expressing CEA, the TLR9 agonist, oligodinucleotide (ODN)1826 and the TLR7 agonist, imiquimod. Mice were challenged with MC38 colon tumor cells and MC38 cells expressing CEA. rAAV-CEA immunization combined with ODN1826 or imiquimod enhanced CEA-specific T-helper 1 immunity and protected against tumor challenge in wild-type but not in CEA-Tg mice. In contrast, immunization with rAAV-CEA in CEA-Tg mice could abrogate the antitumor effects of ODN1826 and promote tumor growth. Compared to wild-type, CEA-Tg mice were characterized by a greater myeloid suppressor cell and T-helper 2 response to TLR agonists and to syngeneic tumors. Depleting PDCA1(+) plasmacytoid dendritic cells and Gr1(+) myeloid cells increased anti-CEA immune responses in CEA-Tg mice to rAAV-CEA-ODN1826 immunization, whereas depleting CD25(+) T cells did not. There are differences in the response of wild-type and CEA-Tg mice to rAAV-CEA, TLR agonists and syngeneic tumor. In CEA-Tg mice, tumor growth can be promoted with rAAV-CEA and TLR agonists. Dendritic and myeloid cells play a regulatory role. PMID:21869824

  10. Laser-evoked synaptic transmission in cultured hippocampal neurons expressing Channelrhodopsin-2 delivered by adeno-associated virus

    PubMed Central

    Wang, Jennifer; Hasan, Mazahir T.; Seung, H. Sebastian

    2009-01-01

    We present a method for studying synaptic transmission in mass cultures of dissociated hippocampal neurons based on patch clamp recording combined with laser stimulation of neurons expressing Channelrhodopsin-2 (ChR2). Our goal was to use the high spatial resolution of laser illumination to come as close as possible to the ideal of identifying monosynaptically coupled pairs of neurons, which is conventionally done using microisland rather than mass cultures. Using recombinant adeno-associated virus (rAAV) to deliver the ChR2 gene, we focused on the time period between 14 and 20 days in vitro, during which expression levels are high, and spontaneous bursting activity has not yet started. Stimulation by wide-field illumination is sufficient to make the majority of ChR2-expressing neurons spike. Stimulation with a laser spot at least 10 μm in diameter also produces action potentials, but in a reduced fraction of neurons. We studied synaptic transmission by voltage-clamping a neuron with low expression of ChR2 and scanning a 40 μm laser spot at surrounding locations. Responses were observed to stimulation at a subset of locations in the culture, indicating spatial localization of stimulation. Pharmacological means were used to identify responses that were synaptic. Many responses were of smaller amplitude than those typically found in microisland cultures. We were unable to find an entirely reliable criterion for distinguishing between monosynaptic and polysynaptic responses. However, we propose that postsynaptic currents with small amplitudes, simple shapes, and latencies not much greater than 8 msec are reasonable candidates for monosynaptic interactions. PMID:19560489

  11. Targeting adeno-associated virus and adenoviral gene therapy for hepatocellular carcinoma.

    PubMed

    Wang, Yi-Gang; Huang, Pan-Pan; Zhang, Rong; Ma, Bu-Yun; Zhou, Xiu-Mei; Sun, Yan-Fang

    2016-01-01

    Human hepatocellular carcinoma (HCC) heavily endangers human heath worldwide. HCC is one of most frequent cancers in China because patients with liver disease, such as chronic hepatitis, have the highest cancer susceptibility. Traditional therapeutic approaches have limited efficacy in advanced liver cancer, and novel strategies are urgently needed to improve the limited treatment options for HCC. This review summarizes the basic knowledge, current advances, and future challenges and prospects of adeno-associated virus (AAV) and adenoviruses as vectors for gene therapy of HCC. This paper also reviews the clinical trials of gene therapy using adenovirus vectors, immunotherapy, toxicity and immunological barriers for AAV and adenoviruses, and proposes several alternative strategies to overcome the therapeutic barriers to using AAV and adenoviruses as vectors. PMID:26755879

  12. Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

    SciTech Connect

    Malkinson, Mertyn . E-mail: malkins@agri.huji.ac.il; Winocour, Ernest . E-mail: ernest.winocour@weizmann.ac.il

    2005-06-05

    The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM. AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.

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

    PubMed Central

    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

  14. Adeno-associated virus expresses transgenes in hair follicles and epidermis.

    PubMed

    Hengge, U R; Mirmohammadsadegh, A

    2000-09-01

    Adeno-associated virus (AAV) vectors are nonpathogenic, integrating DNA vectors capable of transducing dividing and nondividing cells with the potential of long-term expression. Evaluating this interesting vector system in the skin for the first time, we found that an AAV vector containing the lacZ gene (AAVlacZ) led to the expression of beta-galactosidase for more than 6 weeks following in vivo injection. Interestingly, expression was present not only in dividing and postmitotic epidermal keratinocytes but also in hair follicle epithelial cells and eccrine sweat glands. However, expression upon readministration was limited. Functional studies in swine using human erythropoietin were hampered by immunogenicity. Thus, AAV seems to be the only vector to date that efficiently targets hair follicle epithelial cells. It may also be useful when longer term expression in keratinocytes than that achievable by direct injection of plasmid DNA is desired. PMID:10985948

  15. Adeno-associated virus vectors: potential applications for cancer gene therapy

    PubMed Central

    Li, Chengwen; Bowles, Dawn E; van Dyke, Terry; Samulski, Richard Jude

    2006-01-01

    Augmenting cancer treatment by protein and gene delivery continues to gain momentum based on success in animal models. The primary hurdle of fully exploiting the arsenal of molecular targets and therapeutic transgenes continues to be efficient delivery. Vectors based on adeno-associated virus (AAV) are of particular interest as they are capable of inducing transgene expression in a broad range of tissues for a relatively long time without stimulation of a cell-mediated immune response. Perhaps the most important attribute of AAV vectors is their safety profile in phase I clinical trials ranging from CF to Parkinson’s disease. The utility of AAV vectors as a gene delivery agent in cancer therapy is showing promise in preclinical studies. In this review, we will focus on the basic biology of AAV as well as recent progress in the use of this vector in cancer gene therapy. PMID:15962012

  16. Targeting adeno-associated virus and adenoviral gene therapy for hepatocellular carcinoma

    PubMed Central

    Wang, Yi-Gang; Huang, Pan-Pan; Zhang, Rong; Ma, Bu-Yun; Zhou, Xiu-Mei; Sun, Yan-Fang

    2016-01-01

    Human hepatocellular carcinoma (HCC) heavily endangers human heath worldwide. HCC is one of most frequent cancers in China because patients with liver disease, such as chronic hepatitis, have the highest cancer susceptibility. Traditional therapeutic approaches have limited efficacy in advanced liver cancer, and novel strategies are urgently needed to improve the limited treatment options for HCC. This review summarizes the basic knowledge, current advances, and future challenges and prospects of adeno-associated virus (AAV) and adenoviruses as vectors for gene therapy of HCC. This paper also reviews the clinical trials of gene therapy using adenovirus vectors, immunotherapy, toxicity and immunological barriers for AAV and adenoviruses, and proposes several alternative strategies to overcome the therapeutic barriers to using AAV and adenoviruses as vectors. PMID:26755879

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

    PubMed Central

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

    2015-01-01

    Biodistribution analysis is a key step in the evaluation of adeno-associated virus (AAV) capsid variants, whether natural isolates or produced by rational design or directed evolution. Indeed, when screening candidate vectors, accurate knowledge about which tissues are infected and how efficiently is essential. We describe the design, validation, and application of a new vector, pTR-UF50-BC, encoding a bioluminescent protein, a fluorescent protein and a DNA barcode, which can be used to visualize localization of transduction at the organism, organ, tissue, or cellular levels. In addition, by linking capsid variants to different barcoded versions of the vector and amplifying the barcode region from various tissue samples using barcoded primers, biodistribution of viral genomes can be analyzed with high accuracy and efficiency. PMID:26793739

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

    PubMed Central

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

    2008-01-01

    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. PMID:18997346

  19. Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

    PubMed Central

    Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

    2009-01-01

    Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

  20. Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface

    PubMed Central

    Bardelli, Martino; Zárate-Pérez, Francisco; Agúndez, Leticia; Linden, R. Michael

    2016-01-01

    ABSTRACT The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA+ domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle. IMPORTANCE The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a

  1. Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

    PubMed Central

    Sen, Dwaipayan; Gadkari, Rupali A; Sudha, Govindarajan; Gabriel, Nishanth; Kumar, Yesupatham Sathish; Selot, Ruchita; Samuel, Rekha; Rajalingam, Sumathi; Ramya, V.; Nair, Sukesh C.; Srinivasan, Narayanaswamy; Srivastava, Alok

    2013-01-01

    Abstract Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host–cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T→Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S→A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (∼9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8

  2. OneBac: Platform for Scalable and High-Titer Production of Adeno-Associated Virus Serotype 1–12 Vectors for Gene Therapy

    PubMed Central

    Mietzsch, Mario; Grasse, Sabrina; Zurawski, Catherine; Weger, Stefan; Bennett, Antonette; Agbandje-McKenna, Mavis; Muzyczka, Nicholas; Zolotukhin, Sergei

    2014-01-01

    Abstract Scalable and genetically stable recombinant adeno-associated virus (rAAV) production systems combined with facile adaptability for an extended repertoire of AAV serotypes are required to keep pace with the rapidly increasing clinical demand. For scalable high-titer production of the full range of rAAV serotypes 1–12, we developed OneBac, consisting of stable insect Sf9 cell lines harboring silent copies of AAV1–12 rep and cap genes induced upon infection with a single baculovirus that also carries the rAAV genome. rAAV burst sizes reach up to 5×105 benzonase-resistant, highly infectious genomic particles per cell, exceeding typical yields of current rAAV production systems. In contrast to recombinant rep/cap baculovirus strains currently employed for large-scale rAAV production, the Sf9rep/cap cell lines are genetically stable, leading to undiminished rAAV burst sizes over serial passages. Thus, OneBac combines full AAV serotype options with the capacity for stable scale-up production, the current bottleneck for the transition of AAV from gene therapy trials to routine clinical treatment. PMID:24299301

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

    SciTech Connect

    McCraw, Dustin M.; O'Donnell, Jason K.; Taylor, Kenneth A.; Stagg, Scott M.; Chapman, Michael S.

    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.

  4. Adeno-associated virus type 2 binding study on model heparan sulfate surface

    NASA Astrophysics Data System (ADS)

    Negishi, Atsuko; Liu, Jian; McCarty, Douglas; Samulski, Jude; Superfine, Richard

    2003-11-01

    Understanding the mechanisms involved in virus infections is useful in its application in areas such as gene therapy, drug development and delivery, and biosensors. In collaboration with UNC Gene Therapy Center and School of Pharmacy, we are specifically looking at the interaction between human parvovirus adeno-associated virus type 2 (AAV2), a potential viral vector, and heparan sulfate proteoglycan (HSPG), a known cell surface receptor for AAV2. Recent development in glycobiology has shown that some protein-polysaccharide binding is sugar sequence dependent. Heparan sulfate (HS) is a polysaccharide chain of sulfated iduronic/glucuronic and sulfate glucosamine residues and can be differentiated into sequence specific structures by enzymes. These enzymatic modifications, known as heparan sulfate sulfotransferase modified modifications, have been shown to change the biological nature of heparan sulfate such as specific binding to proteins and viruses. For understanding HS-assisted viral infection mechanisms, we are interested in investigating the binding affinity and stability of AAV to different HS structures. We have developed a model heparan sulfate surface in which AAV adsorption studies are done and analyzed using the atomic force microscope (AFM). In addition, a miniArray assay has been created to facilitate to this study. Adsorption studies are done in 4 white LED wells with approximately 3 mm2 reaction areas which minimize sample use and waste.

  5. Overcoming the Cystic Fibrosis Sputum Barrier to Leading Adeno-associated Virus Gene Therapy Vectors

    PubMed Central

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

    2014-01-01

    Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes. PMID:24869933

  6. Gene transfer of arginine kinase to skeletal muscle using adeno-associated virus

    PubMed Central

    Forbes, Sean C.; Bish, Lawrence T.; Ye, Fan; Spinazzola, Janelle; Baligand, Celine; Plant, Daniel; Vandenborne, Krista; Barton, Elisabeth R.; Sweeney, H. Lee; Walter, Glenn A.

    2014-01-01

    In this study we tested the feasibility of non-invasively measuring phosphoarginine (PArg) after gene delivery of arginine kinase (AK) using an adeno-associated virus (AAV) to murine hindlimbs. This was achieved by evaluating the time course, regional distribution, and metabolic flux of PArg using 31 phosphorus magnetic resonance spectroscopy (31P-MRS). AK gene was injected into the gastrocnemius of the left hindlimb of C57Bl10 mice (age 5wk, male) using self-complementary AAV, type 2/8 with desmin promoter. Non-localized 31P-MRS data were acquired over nine months after injection using 11.1-T and 17.6-T Bruker Avance spectrometers. In addition, 31P 2-D chemical shift imaging and saturation transfer experiments were performed to examine the spatial distribution and metabolic flux of PArg, respectively. PArg was evident in each injected mouse hindlimb after gene delivery, increased until 28 weeks, and remained elevated for at least nine months (p<.05). Furthermore, PArg was primarily localized to the injected posterior hindimb region with the metabolite being in exchange with ATP. Overall, the results show the viability of AAV gene transfer of AK gene to skeletal muscle, and provide support of PArg as a reporter that can be utilized to non-invasively monitor the transduction of genes for therapeutic interventions. PMID:24572791

  7. Gene therapy for choroideremia using an adeno-associated viral (AAV) vector.

    PubMed

    Barnard, Alun R; Groppe, Markus; MacLaren, Robert E

    2015-03-01

    Choroideremia is an outer retinal degeneration with a characteristic clinical appearance that was first described in the nineteenth century. The disorder begins with reduction of night vision and gradually progresses to blindness by middle age. The appearance of the fundus in sufferers is recognizable by the characteristic pale color caused by the loss of the outer retina, retinal-pigmented epithelium, and choroidal vessels, leading to exposure of the underlying sclera. Choroideremia shows X-linked recessive inheritance and the choroideremia gene (CHM) was one of the first to be identified by positional cloning in 1990. Subsequent identification and characterization of the CHM gene, which encodes Rab escort protein 1 (REP1), has led to better comprehension of the disease and enabled advances in genetic diagnosis. Despite several decades of work to understand the exact pathogenesis, no established treatments currently exist to stop or even slow the progression of retinal degeneration in choroideremia. Encouragingly, several specific molecular and clinical features make choroideremia an ideal candidate for treatment with gene therapy. This work describes the considerations and challenges in the development of a new clinical trial using adeno-associated virus (AAV) encoding the CHM gene. PMID:25359548

  8. Site-specific integration of adeno-associated virus involves partial duplication of the target locus

    PubMed Central

    Henckaerts, Els; Dutheil, Nathalie; Zeltner, Nadja; Kattman, Steven; Kohlbrenner, Erik; Ward, Peter; Clément, Nathalie; Rebollo, Patricia; Kennedy, Marion; Keller, Gordon M.; Linden, R. Michael

    2009-01-01

    A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences. PMID:19372372

  9. The SUMOylation Pathway Restricts Gene Transduction by Adeno-Associated Viruses

    PubMed Central

    Henrich, Katharina; Chen, Qingxin; Beneke, Jürgen; Matula, Petr; Rohr, Karl; Kaderali, Lars; Beil, Nina; Erfle, Holger; Kleinschmidt, Jürgen A.; Müller, Martin

    2015-01-01

    Adeno-associated viruses are members of the genus dependoviruses of the parvoviridae family. AAV vectors are considered promising vectors for gene therapy and genetic vaccination as they can be easily produced, are highly stable and non-pathogenic. Nevertheless, transduction of cells in vitro and in vivo by AAV in the absence of a helper virus is comparatively inefficient requiring high multiplicity of infection. Several bottlenecks for AAV transduction have previously been described, including release from endosomes, nuclear transport and conversion of the single stranded DNA into a double stranded molecule. We hypothesized that the bottlenecks in AAV transduction are, in part, due to the presence of host cell restriction factors acting directly or indirectly on the AAV-mediated gene transduction. In order to identify such factors we performed a whole genome siRNA screen which identified a number of putative genes interfering with AAV gene transduction. A number of factors, yielding the highest scores, were identified as members of the SUMOylation pathway. We identified Ubc9, the E2 conjugating enzyme as well as Sae1 and Sae2, enzymes responsible for activating E1, as factors involved in restricting AAV. The restriction effect, mediated by these factors, was validated and reproduced independently. Our data indicate that SUMOylation targets entry of AAV capsids and not downstream processes of uncoating, including DNA single strand conversion or DNA damage signaling. We suggest that transiently targeting SUMOylation will enhance application of AAV in vitro and in vivo. PMID:26625259

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

    PubMed Central

    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

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

    PubMed Central

    Liu, Yarong; Kim, Young Joo; Ji, Man; Fang, Jinxu; Siriwon, Natnaree; Zhang, Li I; Wang, Pin

    2014-01-01

    Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy. PMID:26015948

  12. Prevalence and genetic diversity of adeno-associated viruses in bats from China.

    PubMed

    Li, Yan; Ge, Xingyi; Hon, Chung-Chau; Zhang, Huajun; Zhou, Peng; Zhang, Yunzhi; Wu, Yi; Wang, Lin-Fa; Shi, Zhengli

    2010-10-01

    Bats are increasingly being recognized as important natural reservoirs of different viruses. Adeno-associated viruses (AAVs) are widely distributed in primates and their distribution in bats is unknown. In this study, a total of 370 faecal swab samples from 19 bat species were collected from various provinces of China and examined for the presence of AAVs. The mean prevalence rate was 22.4% (83 positives out of 370 samples), ranging from 10 to 38.9% among different bat species. The genome sequence spanning the entire rep-cap ORFs was determined from one chosen AAV-positive sample (designated BtAAV-YNM). Phylogenetic analysis of the entire rep-cap ORF coding sequences suggested that BtAAV-YNM is relatively distant to known primate AAVs, but phylogenetically closer to porcine AAV strain Po3. Further analysis of the partial cap ORF sequences of bat AAV samples (n=49) revealed a remarkably large genetic diversity, with an average pairwise nucleotide identity of only 84.3%. Co-presence of multiple distinctive genotypes of bat AAV within an individual sample was also observed. These results demonstrated that diverse AAVs might be widely distributed in bat populations. PMID:20573859

  13. Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.

    PubMed

    Walker, S L; Wonderling, R S; Owens, R A

    1997-09-01

    The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity. PMID:9261429

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

    PubMed Central

    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

  15. Efficacy of an adeno-associated virus 8-pseudotyped vector in glycogen storage disease type II.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Franco, Luis M; Young, Sarah P; Schneider, Ayn; Bird, Andrew; Amalfitano, Andrea; Chen, Y-T; Koeberl, Dwight D

    2005-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease) causes death in infancy from cardiorespiratory failure. The underlying deficiency of acid alpha-glucosidase (GAA; acid maltase) can be corrected by liver-targeted gene therapy in GSD-II, if secretion of GAA is accompanied by receptor-mediated uptake in cardiac and skeletal muscle. An adeno-associated virus (AAV) vector encoding human (h) GAA was pseudotyped as AAV8 (AAV2/8) and injected intravenously into immunodeficient GSD-II mice. High levels of hGAA were maintained in plasma for 24 weeks following AAV2/8 vector administration. A marked increase in vector copy number in the liver was demonstrated for the AAV2/8 vector compared to the analogous AAV2/2 vector. GAA deficiency in the heart and skeletal muscle was corrected with the AAV2/8 vector in male GSD-II mice, consistent with receptor-mediated uptake of hGAA. Male GSD-II mice demonstrated complete correction of glycogen storage in heart and diaphragm with the AAV2/8 vector, while female GSD-II mice had correction only in the heart. A biomarker for GSD-II was reduced in both sexes following AAV2/8 vector administration. Therefore, GAA production with an AAV2/8 vector in a depot organ, the liver, generated evidence for efficacious gene therapy in a mouse model for GSD-II. PMID:15585406

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

    PubMed

    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

  17. Ultrasound Targeted Microbubble Destruction Stimulates Cellular Endocytosis in Facilitation of Adeno-Associated Virus Delivery

    PubMed Central

    Jin, Li-Fang; Li, Fan; Wang, Hui-Ping; Wei, Fang; Qin, Peng; Du, Lian-Fang

    2013-01-01

    The generally accepted mechanism for ultrasound targeted microbubble destruction (UTMD) to enhance drug and gene delivery is through sonoporation. However, passive uptake of adeno-associated virus (AAV) into cells following sonoporation does not adequately explain observations of enhanced transduction by UTMD. This study investigated alternative mechanisms of UTMD enhancement in AAV delivery. UTMD significantly enhanced transduction efficiency of AAV in a dose-dependent manner. UTMD stimulated a persistent uptake of AAV into the cytoplasm and nucleus. This phenomenon occurred over several hours, suggesting that some viral particles are endocytosed by cells rather than exclusively passing through pores created by sonoporation. Additionally, UTMD enhanced clathrin expression and accumulation at the plasma membrane suggesting greater clathrin-mediated endocytosis following UTMD. Transmission electron microscopy (TEM) revealed that UTMD stimulated formation of clathrin-coated pits (CPs) and uncoated pits (nCPs). Furthermore, inhibition of clathrin-mediated endocytosis partially blocked the enhancement of AAV uptake following UTMD. The results of this study implicate endocytosis as a mechanism that contributes to UTMD-enhanced AAV delivery. PMID:23652832

  18. Gene transfer of arginine kinase to skeletal muscle using adeno-associated virus.

    PubMed

    Forbes, S C; Bish, L T; Ye, F; Spinazzola, J; Baligand, C; Plant, D; Vandenborne, K; Barton, E R; Sweeney, H L; Walter, G A

    2014-04-01

    In this study, we tested the feasibility of non-invasively measuring phosphoarginine (PArg) after gene delivery of arginine kinase (AK) using an adeno-associated virus (AAV) to murine hindlimbs. This was achieved by evaluating the time course, regional distribution and metabolic flux of PArg using (31)phosphorus magnetic resonance spectroscopy ((31)P-MRS). AK gene was injected into the gastrocnemius of the left hindlimb of C57Bl10 mice (age 5 weeks, male) using self-complementary AAV, type 2/8 with desmin promoter. Non-localized (31)P-MRS data were acquired over 9 months after injection using 11.1-T and 17.6-T Bruker Avance spectrometers. In addition, (31)P two-dimensional chemical shift imaging and saturation transfer experiments were performed to examine the spatial distribution and metabolic flux of PArg, respectively. PArg was evident in each injected mouse hindlimb after gene delivery, increased until 28 weeks, and remained elevated for at least 9 months (P<0.05). Furthermore, PArg was primarily localized to the injected posterior hindimb region and the metabolite was in exchange with ATP. Overall, the results show the viability of AAV gene transfer of AK gene to skeletal muscle, and provide support of PArg as a reporter that can be used to non-invasively monitor the transduction of genes for therapeutic interventions. PMID:24572791

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    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

  2. A Precise Chemical Strategy To Alter the Receptor Specificity of the Adeno-Associated Virus.

    PubMed

    Kelemen, Rachel E; Mukherjee, Raja; Cao, Xiaofu; Erickson, Sarah B; Zheng, Yunan; Chatterjee, Abhishek

    2016-08-26

    The ability to target the adeno-associated virus (AAV) to specific types of cells, by altering the cell-surface receptor it binds, is desirable to generate safe and efficient therapeutic vectors. Chemical attachment of receptor-targeting agents onto the AAV capsid holds potential to alter its tropism, but is limited by the lack of site specificity of available conjugation strategies. The development of an AAV production platform is reported that enables incorporation of unnatural amino acids (UAAs) into specific sites on the virus capsid. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. Retargeting ability was site-dependent, underscoring the importance of achieving site-selective capsid modification. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity. PMID:27483453

  3. Analysis of mutations in adeno-associated virus Rep protein in vivo and in vitro.

    PubMed Central

    McCarty, D M; Ni, T H; Muzyczka, N

    1992-01-01

    The adeno-associated virus (AAV) Rep protein is required for both viral DNA replication and transactivation of the AAV promoters. Here we report the effects of mutations in the rep gene on transcription and replication in vivo and terminal repeat binding and terminal resolution site (trs) endonuclease activities in vitro. In all, we examined 10 in-frame deletions and 14 amino acid substitution mutations at eight positions. The point mutations were targeted to regions that are highly conserved among the parvovirus nonstructural proteins and include the extended ATPase domain of the AAV Rep protein. The mutations identify at least two noncontiguous regions of Rep which are essential for terminal repeat binding (amino acids 134 to 242 and amino acids 415 to 490). Mutations in either region render the protein inactive for both DNA replication and transactivation. In addition, mutations within a putative ATPase region also cause defects in replication and transactivation in vivo as well as in the ATP-dependent trs endonuclease activity in vitro. These results suggest that Rep transactivates via a novel mechanism which may require both DNA binding and an enzymatic activity, namely, ATPase or DNA helicase activity. Images PMID:1318396

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

    SciTech Connect

    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.

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

    SciTech Connect

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

    2012-09-17

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

  6. Oligomeric Properties of Adeno-Associated Virus Rep68 Reflect Its Multifunctionality

    PubMed Central

    Zarate-Perez, Francisco; Mansilla-Soto, Jorge; Bardelli, Martino; Burgner, John W.; Villamil-Jarauta, Maria; Kekilli, Demet; Samso, Monserrat

    2013-01-01

    The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA+ domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle. PMID:23152528

  7. Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase

    SciTech Connect

    Li Bin; Duysen, Ellen G.; Poluektova, Larisa Y.; Murrin, L. Charles . E-mail: cmurrin@unmc.edu; Lockridge, Oksana . E-mail: olockrid@unmc.edu

    2006-07-15

    Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months in plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC 3.1.1.8) in motor endplates.

  8. Electron Microscopy Analysis of a Disaccharide Analog complex Reveals Receptor Interactions of Adeno-Associated Virus

    PubMed Central

    Xie, Qing; Spilman, Michael; Meyer, Nancy L.; Lerch, Thomas F.; Stagg, Scott M.; Chapman, Michael S.

    2013-01-01

    Mechanistic studies of macromolecular complexes often feature x-ray structures of complexes with bound ligands. The attachment of Adeno-Associated Virus (AAV) to cell surface glycosaminoglycans (GAGs) is an example that has not proven amenable to crystallography, because the binding of GAG analogs disrupts lattice contacts. The interactions of AAV with GAGs are of interest in mediating the cell specificity of AAV-based gene therapy vectors. Previous electron microscopy led to differing conclusions on the exact binding site and the existence of large ligand-induced conformational changes in the virus. Conformational changes are expected during cell entry, but it has remained unclear whether the electron microscopy provided evidence of their induction by GAG-binding. Taking advantage of automated data collection, careful processing and new methods of structure refinement, the structure of AAV-DJ complexed with sucrose octasulfate is determined by electron microscopy difference map analysis to 4.8 Å resolution. At this higher resolution, individual sulfate groups are discernible, providing a stereochemical validation of map interpretation, and highlighting interactions with two surface arginines that have been implicated in genetic studies. Conformational changes induced by the SOS are modest and limited to the loop most directly interacting with the ligand. While the resolution attainable will depend on sample order and other factors, there are an increasing number of macromolecular complexes that can be studied by cryo-electron microscopy at resolutions beyond 5 Å, for which the approaches used here could be used to characterize the binding of inhibitors and other small molecule effectors when crystallography is not tractable. PMID:24036405

  9. Molecular detection of adeno-associated virus in cases of spontaneous and intentional human abortion.

    PubMed

    Pereira, Christiane Curi; de Freitas, Luciana Bueno; de Vargas, Paulo Roberto Merçon; de Azevedo, Maria Luiza Borges; do Nascimento, Jussara Pereira; Spano, Liliana Cruz

    2010-10-01

    Pregnancy failure is a common event and often of unknown cause. Some viruses are thought to cause abortions including the adeno-associated viruses (AAV), viruses which are regarded as being without any definitive association to any human disease. This study investigated AAV infection in 81 human abortions, both spontaneous and intentional that occurred up to the 23rd week of gestation. Nucleic acid of AAV-2, 3, and 5 types from 118 decidual and chorionic tissues, collected from the patients in this study, was amplified by nested-PCR. In situ hybridization (ISH) was developed with a digoxigenin-labeled AAV probe in paraffin embedded tissues from the AAV positive cases. AAV was observed in 28.4% (23/81) of the cases, of which, 78.3% (18/23) were in the decidua and 21.7% (5/23) in the extravillous trophoblast, the chorionic plate, or chorionic villi fragments. AAV-2, the only type detected, occurred in 32.3% (22/68) and in 7.7% (1/13) of the spontaneous and intentional abortions, respectively. ISH revealed AAV in the decidua, chorionic tissue or chorionic plate and extravillous trophoblast. The detection of only AAV-2 type indicates that it is the most frequent in the population studied and/or shows tissue tropism. The presence of AAV in decidual or trophoblastic cells in cases of abortion, as observed by ISH, implies that the virus could jeopardize the pregnancy. The significant predominance in spontaneous cases suggests possibly a causal association between AAV and abortion. PMID:20827766

  10. Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9

    PubMed Central

    DiMattia, Michael A.; Nam, Hyun-Joo; Van Vliet, Kim; Mitchell, Matthew; Bennett, Antonette; Gurda, Brittney L.; McKenna, Robert; Olson, Norman H.; Sinkovits, Robert S.; Potter, Mark; Byrne, Barry J.; Aslanidi, George; Zolotukhin, Sergei; Muzyczka, Nicholas; Baker, Timothy S.

    2012-01-01

    Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, β-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less “pointed” than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype. PMID:22496238

  11. Adeno-Associated Virus 2-Mediated Hepatocellular Carcinoma is Very Rare in Korean Patients

    PubMed Central

    Park, Kyoung-Jin; Lee, Jongan; Park, June-Hee; Joh, Jae-Won; Kwon, Choon Hyuck David

    2016-01-01

    Background The incidence and etiology of hepatocellular carcinoma (HCC) vary widely according to race and geographic regions. The insertional mutagenesis of adeno-associated virus 2 (AAV2) has recently been considered a new viral etiology of HCC. The aim of this study was to investigate the frequency and clinical characteristics of AAV2 in Korean patients with HCC. Methods A total of 289 unrelated Korean patients with HCC, including 159 Hepatitis-B-related cases, 16 Hepatitis-C-related cases, and 114 viral serology-negative cases, who underwent surgery at the Samsung Medical Center in Korea from 2009 to 2014 were enrolled in this study. The presence of AAV2 in fresh-frozen tumor tissues was investigated by DNA PCR and Sanger sequencing. The clinical and pathological characteristics of AAV2-associated HCC in these patients were compared with previous findings in French patients. Results The AAV2 detection rate in Korean patients (2/289) was very low compared with that in French patients (11/193). Similar to the French patients, the Korean patients with AAV2-related HCC showed no signs of liver cirrhosis. The Korean patients were younger than the French patients with the same AAV2-associated HCC; the ages at diagnosis of the two Korean patients were 47 and 39 yr, while the median age of the 11 French patients was 55 yr (range 43-90 yr). Conclusions AAV2-associated HCC was very rare in Korean patients with HCC. Despite a limited number of cases, this study is the first to report the clinical characteristics of Korean patients with AAV2-associated HCC. These findings suggest epidemiologic differences in viral hepatocarcinogenesis between Korean and European patients. PMID:27374713

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

    PubMed

    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

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

    PubMed

    Schuster, Daniel J; Belur, Lalitha R; Riedl, Maureen S; Schnell, Stephen A; Podetz-Pedersen, Kelly M; Kitto, Kelley F; McIvor, R Scott; Vulchanova, Lucy; Fairbanks, Carolyn A

    2014-01-01

    We report the pattern of transgene expression across brain regions after intrathecal delivery of adeno-associated virus serotype 5 (AAV5). Labeling in hindbrain appeared to be primarily neuronal, and was detected in sensory nuclei of medulla, pontine nuclei, and all layers of cerebellar cortex. Expression in midbrain was minimal, and generally limited to isolated neurons and astrocytes in the cerebral peduncles. GFP immunoreactivity (-ir) in thalamus was most prominent in medial geniculate 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 neurons and non-neuronal cells. 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. PMID:25147505

  14. Production of CFTR-null and CFTR-ΔF508 heterozygous pigs by adeno-associated virus–mediated gene targeting and somatic cell nuclear transfer

    PubMed Central

    Rogers, Christopher S.; Hao, Yanhong; Rokhlina, Tatiana; Samuel, Melissa; Stoltz, David A.; Li, Yuhong; Petroff, Elena; Vermeer, Daniel W.; Kabel, Amanda C.; Yan, Ziying; Spate, Lee; Wax, David; Murphy, Clifton N.; Rieke, August; Whitworth, Kristin; Linville, Michael L.; Korte, Scott W.; Engelhardt, John F.; Welsh, Michael J.; Prather, Randall S.

    2008-01-01

    Progress toward understanding the pathogenesis of cystic fibrosis (CF) and developing effective therapies has been hampered by lack of a relevant animal model. CF mice fail to develop the lung and pancreatic disease that cause most of the morbidity and mortality in patients with CF. Pigs may be better animals than mice in which to model human genetic diseases because their anatomy, biochemistry, physiology, size, and genetics are more similar to those of humans. However, to date, gene-targeted mammalian models of human genetic disease have not been reported for any species other than mice. Here we describe the first steps toward the generation of a pig model of CF. We used recombinant adeno-associated virus (rAAV) vectors to deliver genetic constructs targeting the CF transmembrane conductance receptor (CFTR) gene to pig fetal fibroblasts. We generated cells with the CFTR gene either disrupted or containing the most common CF-associated mutation (ΔF508). These cells were used as nuclear donors for somatic cell nuclear transfer to porcine oocytes. We thereby generated heterozygote male piglets with each mutation. These pigs should be of value in producing new models of CF. In addition, because gene-modified mice often fail to replicate human diseases, this approach could be used to generate models of other human genetic diseases in species other than mice. PMID:18324337

  15. 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.

    PubMed

    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

  16. Directed Evolution of a Novel Adeno-associated Virus (AAV) Vector That Crosses the Seizure-compromised Blood–Brain Barrier (BBB)

    PubMed Central

    Gray, Steven J; Blake, Bonita L; Criswell, Hugh E; Nicolson, Sarah C; Samulski, R Jude; McCown, Thomas J

    2009-01-01

    DNA shuffling and directed evolution were employed to develop a novel adeno-associated virus (AAV) vector capable of crossing the seizure-compromised blood–brain barrier (BBB) and transducing cells in the brain. Capsid DNA from AAV serotypes 1–6, 8, and 9 were shuffled and recombined to create a library of chimeric AAVs. One day after kainic acid–induced limbic seizure activity in rats, the virus library was infused intravenously (i.v.), and 3 days later, neuron-rich cells were mechanically dissociated from seizure-sensitive brain sites, collected and viral DNA extracted. After three cycles of selection, green fluorescent protein (GFP)–packaged clones were administered directly into brain or i.v. 1 day after kainic acid–induced seizures. Several clones that were effective after intracranial administration did not transduce brain cells after the i.v. administration. However, two clones (32 and 83) transduced the cells after direct brain infusion and after i.v. administration transduced the cells that were localized to the piriform cortex and ventral hippocampus, areas exhibiting a seizure-compromised BBB. No transduction occurred in areas devoid of BBB compromise. Only one parental serotype (AAV8) exhibited a similar expression profile, but the biodistribution of 32 and 83 diverged dramatically from this parental serotype. Thus, novel AAV vectors have been created that can selectively cross the seizure-compromised BBB and transduce cells. PMID:20040913

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

    PubMed Central

    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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

    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 < 0.05) and AAV8 (p < 0.01) neutralizing antibodies (NAbs) compared with historical controls. Those with positive NAb titers were typically older than 18 years (p < 0.05 all serotypes) or had received solid organ transplantation (p < 0.01 AAV8, AAV9). The mut(0) patients who had not been transplanted (n = 24)-that is, the subset with the greatest need for improved treatments-represented the seronegative majority, with 21 out of 24 patients lacking Abs against all AAV capsids tested. The unexpected lack of NAbs against AAV in this patient population has encouraging implications for systemic gene delivery as a treatment for mut MMA. PMID:26790480

  20. Synthetic scaffold coating with adeno-associated virus encoding BMP2 to promote endogenous bone repair.

    PubMed

    Dupont, Kenneth M; Boerckel, Joel D; Stevens, Hazel Y; Diab, Tamim; Kolambkar, Yash M; Takahata, Masahiko; Schwarz, Edward M; Guldberg, Robert E

    2012-03-01

    Biomaterial scaffolds functionalized to stimulate endogenous repair mechanisms via the incorporation of osteogenic cues offer a potential alternative to bone grafting for the treatment of large bone defects. We first quantified the ability of a self-complementary adeno-associated viral vector encoding bone morphogenetic protein 2 (scAAV2.5-BMP2) to enhance human stem cell osteogenic differentiation in vitro. In two-dimensional culture, scAAV2.5-BMP2-transduced human mesenchymal stem cells (hMSCs) displayed significant increases in BMP2 production and alkaline phosphatase activity compared with controls. hMSCs and human amniotic-fluid-derived stem cells (hAFS cells) seeded on scAAV2.5-BMP2-coated three-dimensional porous polymer Poly(ε-caprolactone) (PCL) scaffolds also displayed significant increases in BMP2 production compared with controls during 12 weeks of culture, although only hMSC-seeded scaffolds displayed significantly increased mineral formation. PCL scaffolds coated with scAAV2.5-BMP2 were implanted into critically sized immunocompromised rat femoral defects, both with or without pre-seeding of hMSCs, representing ex vivo and in vivo gene therapy treatments, respectively. After 12 weeks, defects treated with acellular scAAV2.5-BMP2-coated scaffolds displayed increased bony bridging and had significantly higher bone ingrowth and mechanical properties compared with controls, whereas defects treated with scAAV2.5-BMP2 scaffolds pre-seeded with hMSCs failed to display significant differences relative to controls. When pooled, defect treatment with scAAV2.5-BMP2-coated scaffolds, both with or without inclusion of pre-seeded hMSCs, led to significant increases in defect mineral formation at all time points and increased mechanical properties compared with controls. This study thus presents a novel acellular bone-graft-free endogenous repair therapy for orthotopic tissue-engineered bone regeneration. PMID:21695398

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

    SciTech Connect

    Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

    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.

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

    SciTech Connect

    DiMattia, Michael; Govindasamy, Lakshmanan; Levy, Hazel C.; Gurda-Whitaker, Brittney; Kalina, Amy; Kohlbrenner, Erik; Chiorini, John A.; McKenna, Robert; Muzyczka, Nicholas; Zolotukhin, Sergei; Agbandje-McKenna, Mavis

    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.

  3. Herpes Simplex Virus Type 1/Adeno-Associated Virus rep+ Hybrid Amplicon Vector Improves the Stability of Transgene Expression in Human Cells by Site-Specific Integration

    PubMed Central

    Wang, Y.; Camp, S. M.; Niwano, M.; Shen, X.; Bakowska, J. C.; Breakefield, X. O.; Allen, P. D.

    2002-01-01

    Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep− HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep+ HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep− vector 3′ AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep+, but not the rep−, hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep+ hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by “deconcatenating” the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells. PMID:12072515

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

    PubMed

    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

  5. A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

    PubMed

    Xiao, X; Xiao, W; Li, J; Samulski, R J

    1997-02-01

    Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both r

  6. Adeno-Associated Virus Type 5 Utilizes Alternative Translation Initiation To Encode a Small Rep40-Like Protein▿

    PubMed Central

    Farris, K. David; Pintel, David J.

    2010-01-01

    Alternative splicing of adeno-associated virus type 2 (AAV2) P19-generated pre-mRNAs generates the small Rep proteins Rep52 and Rep40, which differ in their carboxyl termini. Both proteins are required for optimal packaging of AAV2 genomes. AAV5 Rep-encoding P19-generated transcripts are primarily polyadenylated within the central intron and not efficiently spliced; however, surprisingly, AAV5 was found to generate high levels of a Rep40-like protein. The AAV5 Rep40-like protein was generated by internal initiation and has the same C terminus as Rep52. Although precluded from using alternative splicing to generate multiple Rep isoforms, AAV5 ensures the production of a Rep40-like protein by utilizing a novel internal translation initiation event. PMID:19889770

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

    PubMed Central

    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

  8. Determination of anti-adeno-associated virus vector neutralizing antibody titer with an in vitro reporter system.

    PubMed

    Meliani, Amine; Leborgne, Christian; Triffault, Sabrina; Jeanson-Leh, Laurence; Veron, Philippe; Mingozzi, Federico

    2015-04-01

    Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors. PMID:25819687

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

    SciTech Connect

    Lerch, Thomas F.; Chapman, Michael S.

    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.

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

    SciTech Connect

    Lerch, Thomas F.; Chapman, Michael S.

    2012-02-05

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

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

    PubMed

    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

  12. Adeno-Associated Virus at 50: A Golden Anniversary of Discovery, Research, and Gene Therapy Success—A Personal Perspective

    PubMed Central

    Hastie, Eric

    2015-01-01

    Abstract Fifty years after the discovery of adeno-associated virus (AAV) and more than 30 years after the first gene transfer experiment was conducted, dozens of gene therapy clinical trials are in progress, one vector is approved for use in Europe, and breakthroughs in virus modification and disease modeling are paving the way for a revolution in the treatment of rare diseases, cancer, as well as HIV. This review will provide a historical perspective on the progression of AAV for gene therapy from discovery to the clinic, focusing on contributions from the Samulski lab regarding basic science and cloning of AAV, optimized large-scale production of vectors, preclinical large animal studies and safety data, vector modifications for improved efficacy, and successful clinical applications. PMID:25807962

  13. Modification of some biological properties of HeLa cells containing adeno-associated virus DNA integrated into chromosome 17.

    PubMed Central

    Walz, C; Schlehofer, J R

    1992-01-01

    Parvoviruses are known to interfere with cellular transformation and carcinogenesis. Since infecting adeno-associated virus (AAV) frequently integrates its DNA into the cellular genome, we analyzed whether this integration influences the transformed phenotype of the human tumor cell line HeLa. Analysis of three independent HeLa cell clones with integrated AAV DNA (HA-3x, HA-16, and HA-28) revealed the following phenotypic changes of these cells: (i) reduced growth rate, (ii) increased serum requirement, (iii) reduced capacity for colony formation in soft agar, (iv) reduced cloning efficiency on plastic, (v) elevated sensitivity to genotoxic agents (N-methyl-N'-nitro-N-nitrosoguanidine, 7,12-dimethylbenz[a]anthracene, human tumor necrosis factor alpha, UV irradiation [256 nm], and heat [42 degrees C]), and (vi) reduced sensitivity to the cytolytic effect of parvovirus H-1. Reduced growth rate and enhanced sensitivity to gamma irradiation were also observed in vivo when tumors from AAV DNA-containing HeLa cells were transplanted into nude mice. This alteration of the biological properties of HeLa cells was independent of the number of AAV genomes integrated, the physical structure of integrated AAV DNA, and the transcription of AAV genes. Integration of AAV DNA was found to occur preferentially on the long arm of chromosome 17 in the three HeLa cell clones analyzed. These findings demonstrate that genomic integration of AAV DNA can alter the biological properties of human tumor cells. Images PMID:1313913

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

    PubMed Central

    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

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

    PubMed Central

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

    2013-01-01

    Abstract 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 Largemyd 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

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

    PubMed

    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

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

    PubMed Central

    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

  18. Long-Term Sex-Biased Correction of Circulating Propionic Acidemia Disease Markers by Adeno-Associated Virus Vectors

    PubMed Central

    Guenzel, Adam J.; Collard, Renata; Kraus, Jan P.; Matern, Dietrich

    2015-01-01

    Abstract Propionic academia (PA) occurs because of mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase, a pivotal enzyme in the breakdown of certain amino acids and odd-chain fatty acids. There is no cure for PA, but dietary protein restriction and liver transplantation can attenuate its symptoms. We show here that a single intravenous injection of adeno-associated virus 2/8 (AAV8) or AAVrh10 expressing PCCA into PA hypomorphic mice decreased systemic propionylcarnitine and methyl citrate for up to 1.5 years. However, long-term phenotypic correction was always better in male mice. AAV-mediated PCCA expression was similar in most tissues in males and females at early time points and differed only in the liver. Over 1.5 years, luciferase and PCCA expression remained elevated in cardiac tissue for both sexes. In contrast, transgene expression in the liver and skeletal muscles of female, but not male, mice waned—suggesting that these tissues were major sinks for systemic phenotypic correction. These data indicate that single systemic intravenous therapy by AAV vectors can mediate long-term phenotype correction for PA. However, tissue-specific loss of expression in females reduces efficacy when compared with males. Whether similar sex-biased AAV effects occur in human gene therapy remains to be determined. PMID:25654275

  19. Intracerebroventricular delivery of self-complementary adeno-associated virus serotype 9 to the adult rat brain.

    PubMed

    Donsante, A; McEachin, Z; Riley, J; Leung, C H; Kanz, L; O'Connor, D M; Boulis, N M

    2016-05-01

    Gene therapy for the central nervous system is poised to become a powerful treatment for numerous neurological disorders. Adeno-associated viral vectors based on serotype 9 (AAV9) have proven themselves to be strong candidates for delivering gene-based therapies throughout the brain and spinal cord when administered intravenously, intrathecally, intracisternally, and intracerebroventricularly (i.c.v.). Previous studies of i.c.v.-delivered self-complimentary AAV9 have been performed in neonatal mice with delivery of a single dose. However, before clinical trials can be considered, more information is required about the dose-response relationship for transduction efficiency in adult animals. In the current study, three doses of self-complementary AAV9 were administered to adult rats. High levels of transduction were observed in the hippocampus, cerebellum and cerebral cortex, and transduction increased with increasing dosage. Both neurons and astrocytes were transduced. There was no evidence of astrocytosis at the doses tested. Preliminary results from pigs receiving i.c.v. self-complementary AAV9 are also presented. The results of this study will serve to inform dosing studies in large animal models before clinical testing. PMID:26824881

  20. In vivo adeno-associated viral vector-mediated genetic engineering of white and brown adipose tissue in adult mice.

    PubMed

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-12-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes. PMID:24043756

  1. Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation

    SciTech Connect

    Trempe, J.P.; Carter, B.J.

    1988-01-01

    The authors studied the effects of the adeno-associated virus (AAV) rep gene on the control of gene expression from the AAV p/sub 40/ promoter in 293 cells in the absence of an adenovirus coinfection. AAV vectors containing the chloramphenicol acetyltransferase (cat) gene were used to measure the levels of cat expression and steady-state mRNA from p/sub 40/. When the rep gene was present in cis or in trans, cat expression from p/sub 40/ was decreased 3- to 10-fold, but there was a 2- to 10-fold increase in the level of p/sub 40/ mRNA. Conversely, cat expression increased and the p/sub 40/ mRNA level decreased in the absence of the rep gene. Both wild-type and carboxyl-terminal truncated Rep proteins were capable of eliciting both effects. These data suggest two roles for the pleiotropic AAV rep gene: as a translational inhibitor and as a positive regulator of p/sub 40/ mRNA levels. They also provide additional evidence for a cis-acting negative regulatory region which decreases RNA from the AAV p/sub 5/ promoter in a fashion independent of rep.

  2. Novel adeno-associated viral vector delivering the utrophin gene regulator jazz counteracts dystrophic pathology in mdx mice.

    PubMed

    Strimpakos, Georgios; Corbi, Nicoletta; Pisani, Cinzia; Di Certo, Maria Grazia; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Gabanella, Francesca; Monaco, Lucia; Mattei, Elisabetta; Passananti, Claudio

    2014-09-01

    Over-expression of the dystrophin-related gene utrophin represents a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. We developed the artificial zinc finger transcription factor "Jazz" that up-regulates both the human and mouse utrophin promoter. We observed a significant recovery of muscle strength in dystrophic Jazz-transgenic mdx mice. Here we demonstrate the efficacy of an experimental gene therapy based on the systemic delivery of Jazz gene in mdx mice by adeno-associated virus (AAV). AAV serotype 8 was chosen on the basis of its high affinity for skeletal muscle. Muscle-specific expression of the therapeutic Jazz gene was enhanced by adding the muscle α-actin promoter to the AAV vector (mAAV). Injection of mAAV8-Jazz viral preparations into mdx mice resulted in muscle-specific Jazz expression coupled with up-regulation of the utrophin gene. We show a significant recovery from the dystrophic phenotype in mAAV8-Jazz-treated mdx mice. Histological and physiological analysis revealed a reduction of fiber necrosis and inflammatory cell infiltration associated with functional recovery in muscle contractile force. The combination of ZF-ATF technology with the AAV delivery can open a new avenue to obtain a therapeutic strategy for treatment of DMD. PMID:24469912

  3. ENHANCED GENE DELIVERY IN PORCINE VASCULATURE TISSUE FOLLOWING INCORPORATION OF ADENO-ASSOCIATED VIRUS NANOPARTICLES INTO POROUS SILICON MICROPARTICLES

    PubMed Central

    McConnell, Kellie I.; Rhudy, Jessica; Yokoi, Kenji; Gu, Jianhua; Mack, Aaron; Suh, Junghae; La Francesca, Saverio; Sakamoto, Jason; Serda, Rita E.

    2014-01-01

    There is an unmet clinical need to increase lung transplant successes, patient satisfaction and to improve mortality rates. We offer the development of a nanovector-based solution that will reduce the incidence of lung ischemic reperfusion injury (IRI) leading to graft organ failure through the successful ex vivo treatment of the lung prior to transplantation. The innovation is in the integrated application of our novel porous silicon (pSi) microparticles carrying adeno-associated virus (AAV) nanoparticles, and the use of our ex vivo lung perfusion/ventilation system for the modulation of pro-inflammatory cytokines initiated by ischemic pulmonary conditions prior to organ transplant that often lead to complications. Gene delivery of anti-inflammatory agents to combat the inflammatory cascade may be a promising approach to prevent IRI following lung transplantation. The rationale for the device is that the microparticle will deliver a large payload of virus to cells and serve to protect the AAV from immune recognition. The microparticle-nanoparticle hybrid device was tested both in vitro on cell monolayers and ex vivo using either porcine venous tissue or a pig lung transplantation model, which recapitulates pulmonary IRI that occurs clinically post-transplantation. Remarkably, loading AAV vectors into pSi microparticles increases gene delivery to otherwise non-permissive endothelial cells. PMID:25180449

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

    PubMed Central

    Wang, Lili; Calcedo, Roberto; Bell, Peter; Lin, Jianping; Grant, Rebecca L; Siegel, Don L

    2011-01-01

    Abstract 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) were injected intravenously with AAV8 vector expressing green fluorescent protein. Pre-existing antibody titers in excess of 1:10 substantially diminished hepatocyte transduction that, in the absence of NAbs, was highly efficient. Vector-specific NAb diminished liver deposition of genomes and unexpectedly increased genome distribution to the spleen. The majority of animals showed high-level and stable sequestration of vector capsid protein by follicular dendritic cells of splenic germinal centers. These studies illustrate how natural immunity to a virus that is related to a vector can impact the efficacy and potential safety of in vivo gene therapy. We propose to use the in vitro transduction inhibition assay to evaluate research subjects before gene therapy and to preclude from systemic AAV8 trials those that have titers in excess of 1:10. PMID:21476868

  5. Hydrostatic Isolated Limb Perfusion with Adeno-associated Virus Vectors Enhances Correction of Skeletal Muscle in Pompe Disease

    PubMed Central

    Sun, Baodong; Li, Songtao; Bird, Andrew; Koeberl, Dwight D.

    2010-01-01

    Glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) stems from the inherited deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. We hypothesized that hydrostatic isolated limb perfusion (ILP) administration of an adeno-associated virus (AAV) vector containing a muscle specific promoter could achieve relatively higher transgene expression in the hindlimb muscles of GAA-knockout (GAA-KO) mice, in comparison with intravenous (IV) administration. ILP adminstration of AAV2/8 vectors encoding alkaline phosphatase or human GAA transduced skeletal muscles of the hindlimb widely, despite the relatively low number of vector particles administered (1×1011), and IV administration of an equivalent vector dose failed to transduce skeletal muscle detectably. Similarly, ILP administration of fewer vector particles of the AAV2/9 vector encoding human GAA (3×1010) transduced skeletal muscles of the hindlimb widely and significantly reduced glycogen content to, in comparison with IV administration. The only advantage for IV administration was moderately high level transduction of cardiac muscle, which demonstrated compellingly that ILP administration sequestered vector particles within the perfused limb. Reduction of glycogen storage in the extensor digitorum longus demonstrated the potential advantage of ILP-mediated delivery of AAV vectors in Pompe disease, because type II myofibers are resistant to enzyme replacement therapy. Thus, ILP will enhance AAV transduction of multiple skeletal muscles while reducing the required dosages in terms of vector particle numbers. PMID:20686508

  6. Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer

    PubMed Central

    Kwon, Inchan

    2007-01-01

    Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure–function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. “Shielding” polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating ‘designer’ gene delivery vectors with enhanced properties. PMID:17763830

  7. Liver-Directed Adeno-Associated Virus Serotype 8 Gene Transfer Rescues a Lethal Murine Model of Citrullinemia Type 1

    PubMed Central

    Chandler, Randy J.; Tarasenko, Tatiana N.; Cusmano-Ozog, Kristina; Sun, Qin; Sutton, V. Reid; Venditti, Charles P.; McGuire, Peter J.

    2013-01-01

    Citrullinemia type 1 (CTLN1) is an autosomal recessive disorder of metabolism caused by a deficiency of argininosuccinate synthetase. Despite optimal management, CTLN1 patients still suffer from lethal metabolic instability and experience life threatening episodes of acute hyperammonemia. A murine model of CTLN1 (fold/fold) that displays lethality within the first 21 days of life was used to determine the efficacy of adeno-associated viral (AAV) gene transfer as a potential therapy. An AAV serotype 8 (AAV8) vector was engineered to express the human ASS1 cDNA under the control of a liver-specific promoter (thyroxine binding globulin, TBG), AAV8-TBG-hASS1, and delivered to 7–10 day old mice via intraperitoneal injection. Greater than 95% of the mice were rescued from lethality and survival was extended beyond 100 days after receiving a single dose of vector. AAV8-TBG-hASS1 treatment resulted in liver specific expression of hASS1, increased ASS1 enzyme activity, reduction in plasma ammonia and citrulline concentrations, and significant phenotypic improvement of the fold/fold growth and skin phenotypes. These experiments highlight a gene transfer approach using AAV8 vector for liver targeted gene therapy that could serve as a treatment for CTLN1. PMID:24131980

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

    PubMed Central

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

    2016-01-01

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

  9. Adeno-associated virus vector-mediated minidystrophin gene therapy improves dystrophic muscle contractile function in mdx mice.

    PubMed

    Watchko, Jon; O'Day, Terry; Wang, Bing; Zhou, Liqiao; Tang, Ying; Li, Juan; Xiao, Xiao

    2002-08-10

    Duchenne muscular dystrophy (DMD) is the most common disabling and lethal genetic muscle disorder, afflicting 1 of every 3500 males. Patients with DMD experience progressive muscle degeneration and weakness and succumb to respiratory or cardiac failure by their early twenties. No treatment is currently available for DMD. Mutations in the dystrophin gene result in lack of a functional dystrophin protein in striated muscle, which induces instability in the muscle cell membrane leading to persistent muscle injury after contraction. We have previously created novel minidystrophin genes and demonstrated that adeno-associated virus (AAV)-mediated intramuscular delivery of the minigenes effectively ameliorated mdx dystrophic histopathology and led to normal cell membrane integrity for more than 1 year. In this paper, we investigated whether AAV-minidystrophin could also improve mdx muscle contractile function. Two-month-old adult male mdx mice, with established muscular dystrophy, were given a single-dose injection of an AAV-minidystrophin vector in the tibialis anterior (TA) muscle of one leg, with the untreated contralateral leg used as a control. The treated TA muscle showed both (1) a significant increase in isometric force generation and (2) a significant increase in resistance to lengthening activation-induced muscle force decrements. We conclude that AAV-minidystrophin gene treatment is effective in improving mdx muscle contractile function. PMID:12215266

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

    PubMed

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

    2015-03-01

    Propionic academia (PA) occurs because of mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase, a pivotal enzyme in the breakdown of certain amino acids and odd-chain fatty acids. There is no cure for PA, but dietary protein restriction and liver transplantation can attenuate its symptoms. We show here that a single intravenous injection of adeno-associated virus 2/8 (AAV8) or AAVrh10 expressing PCCA into PA hypomorphic mice decreased systemic propionylcarnitine and methyl citrate for up to 1.5 years. However, long-term phenotypic correction was always better in male mice. AAV-mediated PCCA expression was similar in most tissues in males and females at early time points and differed only in the liver. Over 1.5 years, luciferase and PCCA expression remained elevated in cardiac tissue for both sexes. In contrast, transgene expression in the liver and skeletal muscles of female, but not male, mice waned—suggesting that these tissues were major sinks for systemic phenotypic correction. These data indicate that single systemic intravenous therapy by AAV vectors can mediate long-term phenotype correction for PA. However, tissue-specific loss of expression in females reduces efficacy when compared with males. Whether similar sex-biased AAV effects occur in human gene therapy remains to be determined. PMID:25654275

  11. Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants

    PubMed Central

    Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

    2014-01-01

    Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

  12. Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants.

    PubMed

    Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

    2014-11-01

    Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of "virtual family shuffling" to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

  13. Treatment of congenital neurotransmitter deficiencies by intracerebral ventricular injection of an adeno-associated virus serotype 9 vector.

    PubMed

    Lee, Ni-Chung; Chien, Yin-Hsiu; Hu, Min-Hsiu; Liu, Wen-Shin; Chen, Pin-Wen; Wang, Wei-Hua; Tzen, Kai-Yuan; Byrne, Barry J; Hwu, Wuh-Liang

    2014-03-01

    Dopamine and serotonin are produced by distinct groups of neurons in the brain, and gene therapies other than direct injection have not been attempted to correct congenital deficiencies in such neurotransmitters. In this study, we performed gene therapy to treat knock-in mice with dopamine and serotonin deficiencies caused by a mutation in the aromatic L-amino acid decarboxylase (AADC) gene (Ddc(KI) mice). Intracerebral ventricular injection of neonatal mice with an adeno-associated virus (AAV) serotype 9 (AAV9) vector expressing the human AADC gene (AAV9-hAADC) resulted in widespread AADC expression in the brain. Without treatment, 4-week-old Ddc(KI) mice exhibited whole-brain homogenate dopamine and serotonin levels of 25% and 15% of normal, respectively. After gene therapy, the levels rose to 100% and 40% of normal, respectively. The gene therapy improved the growth rate and survival of Ddc(KI) mice and normalized their hindlimb clasping and cardiovascular dysfunctions. The behavioral abnormalities of the Ddc(KI) mice were partially corrected, and the treated Ddc(KI) mice were slightly more active than normal mice. No immune reactions resulted from the treatment. Therefore, a congenital neurotransmitter deficiency can be treated safely through inducing widespread expression of the deficient gene in neonatal mice. PMID:24251946

  14. A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia.

    PubMed

    Ostedgaard, Lynda S; Rokhlina, Tatiana; Karp, Philip H; Lashmit, Philip; Afione, Sandra; Schmidt, Michael; Zabner, Joseph; Stinski, Mark F; Chiorini, Jay A; Welsh, Michael J

    2005-02-22

    Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds approximately 4,900-5,000 bp. To partially circumvent this size constraint, we previously developed a CF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTRDeltaR). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate/early (CMVie) enhancer/promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTRDeltaR transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl(-) transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer. PMID:15703296

  15. A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia

    PubMed Central

    Ostedgaard, Lynda S.; Rokhlina, Tatiana; Karp, Philip H.; Lashmit, Philip; Afione, Sandra; Schmidt, Michael; Zabner, Joseph; Stinski, Mark F.; Chiorini, Jay A.; Welsh, Michael J.

    2005-01-01

    Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds ≈4,900–5,000 bp. To partially circumvent this size constraint, we previously developed a CF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTRΔR). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate/early (CMVie) enhancer/promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTRΔR transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl– transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer. PMID:15703296

  16. Rep-mediated nicking of the adeno-associated virus origin requires two biochemical activities, DNA helicase activity and transesterification.

    PubMed

    Brister, J R; Muzyczka, N

    1999-11-01

    The single-stranded adeno-associated virus (AAV) genome is flanked by terminal hairpinned origins of DNA replication (terminal repeats [TRs]) that are nicked at the terminal resolution site (trs) by the AAV Rep protein in an ATP-dependent, site-specific manner. Here we determine the minimal trs sequence necessary for Rep cleavage, 3'-CCGGT/TG-5', and show that this 7-base core sequence is required only on the nicked strand. We also identify a potential stem-loop structure at the trs. Interestingly, Rep nicking on a TR substrate that fixes this trs stem-loop in the extruded form no longer requires ATP. This suggests that ATP-dependent Rep helicase activity is necessary to unwind the duplex trs and extrude the stem-loop structure, prior to the ATP-independent Rep transesterification reaction. The extrusion of origin stem-loop structures prior to nicking appears to be a general mechanism shared by plant and animal viruses and bacterial plasmids. In the case of AAV, this mechanism of TR nicking would provide a possible regulatory function. PMID:10516041

  17. Impact of Preexisting Vector Immunity on the Efficacy of Adeno-Associated Virus-Based HIV-1 Gag Vaccines

    PubMed Central

    Lin, Jianping; Calcedo, Roberto; Vandenberghe, Luk H.; Figueredo, Joanita M.

    2008-01-01

    Abstract Vectors based on primate-derived adeno-associated virus (AAV) are being considered in the development of genetic vaccines against a number of diseases including infection with HIV-1. Preexisting immunity to the vaccine carrier as a result of natural infections could potentially compromise vaccine efficacy. This study evaluates the impact of neutralizing antibodies against AAV capsids on the ability of HIV-1 Gag-expressing vectors to elicit transgene-specific T and B cell responses. Mice were passively transferred with pooled human immunoglobulin at various doses to simulate human antivector humoral immunity. Vectors based on serotype 2, which were evaluated in the clinic, were compared with those created from the novel monkey isolates AAV7 and AAV8. Inhibition of AAV2-directed Gag responses occurred at doses of human immunoglobulin 10- to 20-fold less than was required to inhibit immunogenicity of AAV7 and AAV8 vectors. Cynomolgus macaques were screened for preexisting immunity to AAV7 and AAV8 and sera from individual animals were passively transferred into mice that were analyzed for AAV vaccine efficacy. There was a correlation between the level of preexisting capsid neutralizing titers and diminution of vaccine efficacy; sera from a number of animals with no detectable neutralizing antibodies showed partial vaccine inhibition, suggesting that the in vitro assay is less sensitive than the in vivo passive transfer assay for detecting neutralizing antibodies to AAV. PMID:18549307

  18. Inner Ear Gene Transfection in Neonatal Mice Using Adeno-Associated Viral Vector: A Comparison of Two Approaches

    PubMed Central

    Xia, Li; Yin, Shankai; Wang, Jian

    2012-01-01

    Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant. PMID:22912830

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

    PubMed

    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

  20. Adeno-associated virus-2 and its primary cellular receptor-Cryo-EM structure of a heparin complex

    SciTech Connect

    O'Donnell, Jason; Taylor, Kenneth A.; Chapman, Michael S.

    2009-03-15

    Adeno-associated virus serotype 2 (AAV-2) is a leading candidate vector for gene therapy. Cell entry starts with attachment to a primary receptor, Heparan Sulfate Proteoglycan (HSPG) before binding to a co-receptor. Here, cryo-electron microscopy provides direct visualization of the virus-HSPG interactions. Single particle analysis was performed on AAV-2 complexed with a 17 kDa heparin fragment at 8.3 A resolution. Heparin density covers the shoulder of spikes surrounding viral 3-fold symmetry axes. Previously implicated, positively charged residues R{sub 448/585}, R{sub 451/588} and R{sub 350/487} from another subunit cluster at the center of the heparin footprint. The footprint is much more extensive than apparent through mutagenesis, including R{sub 347/484}, K{sub 395/532} and K{sub 390/527} that are more conserved, but whose roles have been controversial. It also includes much of a region proposed as a co-receptor site, because prior studies had not revealed heparin interactions. Heparin density bridges over the viral 3-fold axes, indicating multi-valent attachment to symmetry-related binding sites.

  1. Adeno-associated virus 9 mediated FKRP gene therapy restores functional glycosylation of α-dystroglycan and improves muscle functions.

    PubMed

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

    2013-10-01

    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 a proline to leucine missense mutation (P448L). Our results showed that intraperitoneal administration of AAV9-FKRP resulted in systemic FKRP expression in all striated muscles examined with the highest levels in cardiac muscle. Consistent with our previous observations, FKRP protein is localized in the Golgi apparatus in myofibers. Expression of FKRP consequently restored functional glycosylation of α-DG in the skeletal and cardiac muscles. Significant improvement in dystrophic pathology, serum creatine kinase levels and muscle function was observed. Only limited FKRP transgene expression was detected in kidney and liver with no detectable toxicity. Our results provided evidence for the utility of AAV-mediated gene replacement therapy for FKRP-related muscular dystrophies. PMID:23817215

  2. In Vivo Adeno-Associated Viral Vector–Mediated Genetic Engineering of White and Brown Adipose Tissue in Adult Mice

    PubMed Central

    Jimenez, Veronica; Muñoz, Sergio; Casana, Estefania; Mallol, Cristina; Elias, Ivet; Jambrina, Claudia; Ribera, Albert; Ferre, Tura; Franckhauser, Sylvie; Bosch, Fatima

    2013-01-01

    Adipose tissue is pivotal in the regulation of energy homeostasis through the balance of energy storage and expenditure and as an endocrine organ. An inadequate mass and/or alterations in the metabolic and endocrine functions of adipose tissue underlie the development of obesity, insulin resistance, and type 2 diabetes. To fully understand the metabolic and molecular mechanism(s) involved in adipose dysfunction, in vivo genetic modification of adipocytes holds great potential. Here, we demonstrate that adeno-associated viral (AAV) vectors, especially serotypes 8 and 9, mediated efficient transduction of white (WAT) and brown adipose tissue (BAT) in adult lean and obese diabetic mice. The use of short versions of the adipocyte protein 2 or uncoupling protein-1 promoters or micro-RNA target sequences enabled highly specific, long-term AAV-mediated transgene expression in white or brown adipocytes. As proof of concept, delivery of AAV vectors encoding for hexokinase or vascular endothelial growth factor to WAT or BAT resulted in increased glucose uptake or increased vessel density in targeted depots. This method of gene transfer also enabled the secretion of stable high levels of the alkaline phosphatase marker protein into the bloodstream by transduced WAT. Therefore, AAV-mediated genetic engineering of adipose tissue represents a useful tool for the study of adipose pathophysiology and, likely, for the future development of new therapeutic strategies for obesity and diabetes. PMID:24043756

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

    SciTech Connect

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

    PubMed

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

    2016-01-01

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

  5. Highly Divergent Integration Profile of Adeno-Associated Virus Serotype 5 Revealed by High-Throughput Sequencing

    PubMed Central

    Janovitz, Tyler; Oliveira, Thiago; Sadelain, Michel

    2014-01-01

    ABSTRACT Adeno-associated virus serotype 5 (AAV-5) is a human parvovirus that infects a high percentage of the population. It is the most divergent AAV, the DNA sequence cleaved by the viral endonuclease is distinct from all other described serotypes and, uniquely, AAV-5 does not cross-complement the replication of other serotypes. In contrast to the well-characterized integration of AAV-2, no published studies have investigated the genomic integration of AAV-5. In this study, we analyzed more than 660,000 AAV-5 integration junctions using high-throughput integrant capture sequencing of infected human cells. The integration activity of AAV-5 was 99.7% distinct from AAV-2 and favored intronic sequences. Genome-wide integration was highly correlated with viral replication protein binding and endonuclease sites, and a 39-bp consensus integration motif was revealed that included these features. Algorithmic scanning identified 126 AAV-5 hot spots, the largest of which encompassed 3.3% of all integration events. The unique aspects of AAV-5 integration may provide novel tools for biotechnology and gene therapy. IMPORTANCE Viral integration into the host genome is an important aspect of virus host cell biology. Genomic integration studies of the small single-stranded AAVs have largely focused on site preferential integration of AAV-2, which depends on the viral replication protein (Rep). We have now established the first genome wide integration profile of the highly divergent AAV-5 serotype. Using integrant capture sequencing, more than 600,000 AAV-5 integration junctions in human cells were analyzed. AAV-5 integration hot spots were 99.7% distinct from AAV-2. Integration favored intronic sequences, occurred on all chromosomes, and integration hot spot distribution was correlated with human genomic GAGC repeats and transcriptional activity. These features support expansion of AAV-5 based vectors for gene transfer considerations. PMID:24335317

  6. TrkB gene therapy by adeno-associated virus enhances recovery after cervical spinal cord injury.

    PubMed

    Martínez-Gálvez, Gabriel; Zambrano, Juan M; Diaz Soto, Juan C; Zhan, Wen-Zhi; Gransee, Heather M; Sieck, Gary C; Mantilla, Carlos B

    2016-02-01

    Unilateral cervical spinal cord hemisection at C2 (C2SH) interrupts descending bulbospinal inputs to phrenic motoneurons, paralyzing the diaphragm muscle. Recovery after C2SH is enhanced by brain derived neurotrophic factor (BDNF) signaling via the tropomyosin-related kinase subtype B (TrkB) receptor in phrenic motoneurons. The role for gene therapy using adeno-associated virus (AAV)-mediated delivery of TrkB to phrenic motoneurons is not known. The present study determined the therapeutic efficacy of intrapleural delivery of AAV7 encoding for full-length TrkB (AAV-TrkB) to phrenic motoneurons 3 days post-C2SH. Diaphragm EMG was recorded chronically in male rats (n=26) up to 21 days post-C2SH. Absent ipsilateral diaphragm EMG activity was verified 3 days post-C2SH. A greater proportion of animals displayed recovery of ipsilateral diaphragm EMG activity during eupnea by 14 and 21 days post-SH after AAV-TrkB (10/15) compared to AAV-GFP treatment (2/11; p=0.031). Diaphragm EMG amplitude increased over time post-C2SH (p<0.001), and by 14 days post-C2SH, AAV-TrkB treated animals displaying recovery achieved 48% of the pre-injury values compared to 27% in AAV-GFP treated animals. Phrenic motoneuron mRNA expression of glutamatergic AMPA and NMDA receptors revealed a significant, positive correlation (r(2)=0.82), with increased motoneuron NMDA expression evident in animals treated with AAV-TrkB and that displayed recovery after C2SH. Overall, gene therapy using intrapleural delivery of AAV-TrkB to phrenic motoneurons is sufficient to promote recovery of diaphragm activity, adding a novel potential intervention that can be administered after upper cervical spinal cord injury to improve impaired respiratory function. PMID:26607912

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

    PubMed

    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

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

    PubMed

    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

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

    PubMed

    Luo, Xiaoyan; Hall, Gentzon; Li, Songtao; Bird, Andrew; Lavin, Peter J; Winn, Michelle P; Kemper, Alex R; Brown, Talmage T; Koeberl, Dwight D

    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. PMID:21730973

  10. Early, sustained efficacy of adeno-associated virus vector-mediated gene therapy in glycogen storage disease type Ia.

    PubMed

    Koeberl, D D; Sun, B D; Damodaran, T V; Brown, T; Millington, D S; Benjamin, D K; Bird, A; Schneider, A; Hillman, S; Jackson, M; Beaty, R M; Chen, Y T

    2006-09-01

    The deficiency of glucose-6-phosphatase (G6Pase) underlies life-threatening hypoglycemia and growth retardation in glycogen storage disease type Ia (GSD-Ia). An adeno-associated virus (AAV) vector encoding G6Pase was pseudotyped as AAV8 and administered to 2-week-old GSD-Ia mice (n = 9). Median survival was prolonged to 7 months following vector administration, in contrast to untreated GSD-Ia mice that survived for only 2 weeks. Although GSD-Ia mice were initially growth-retarded, treated mice increased fourfold in weight to normal size. Blood glucose was partially corrected by 2 weeks following treatment, whereas blood cholesterol normalized. Glucose-6-phosphatase activity was partially corrected to 25% of the normal level at 7 months of age in treated mice, and blood glucose during fasting remained lower in treated, affected mice than in normal mice. Glycogen storage was partially corrected in the liver by 2 weeks following treatment, but reaccumulated to pre-treatment levels by 7 months old (m.o.). Vector genome DNA decreased between 3 days and 3 weeks in the liver following vector administration, mainly through the loss of single-stranded genomes; however, double-stranded vector genomes were more stable. Although CD8+ lymphocytic infiltrates were present in the liver, partial biochemical correction was sustained at 7 m.o. The development of efficacious AAV vector-mediated gene therapy could significantly reduce the impact of long-term complications in GSD-Ia, including hypoglycemia, hyperlipidemia and growth failure. PMID:16672983

  11. Hepatorenal Correction in Murine Glycogen Storage Disease Type I With a Double-stranded Adeno-associated Virus Vector

    PubMed Central

    Luo, Xiaoyan; Hall, Gentzon; Li, Songtao; Bird, Andrew; Lavin, Peter J; Winn, Michelle P; Kemper, Alex R; Brown, Talmage T; Koeberl, Dwight D

    2011-01-01

    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. PMID:21730973

  12. Hydrostatic isolated limb perfusion with adeno-associated virus vectors enhances correction of skeletal muscle in Pompe disease.

    PubMed

    Sun, B; Li, S; Bird, A; Koeberl, D D

    2010-12-01

    Glycogen storage disease type II (Pompe disease; MIM 232300) stems from the inherited deficiency of acid-α-glucosidase (GAA; acid maltase; EC 3.2.1.20), which primarily involves cardiac and skeletal muscles. We hypothesized that hydrostatic isolated limb perfusion (ILP) administration of an adeno-associated virus (AAV) vector containing a muscle-specific promoter could achieve relatively higher transgene expression in the hindlimb muscles of GAA-knockout (GAA-KO) mice, in comparison with intravenous (IV) administration. ILP administration of AAV2/8 vectors encoding alkaline phosphatase or human GAA-transduced skeletal muscles of the hindlimb widely, despite the relatively low number of vector particles administered (1 × 10¹¹), and IV administration of an equivalent vector dose failed to transduce skeletal muscle detectably. Similarly, ILP administration of fewer vector particles of the AAV2/9 vector encoding human GAA (3 × 10¹⁰) transduced skeletal muscles of the hindlimb widely and significantly reduced glycogen content to, in comparison with IV administration. The only advantage for IV administration was moderately high-level transduction of cardiac muscle, which demonstrated compellingly that ILP administration sequestered vector particles within the perfused limb. Reduction of glycogen storage in the extensor digitorum longus demonstrated the potential advantage of ILP-mediated delivery of AAV vectors in Pompe disease, because type II myofibers are resistant to enzyme replacement therapy. Thus, ILP will enhance AAV transduction of multiple skeletal muscles while reducing the required dosages in terms of vector particle numbers. PMID:20686508

  13. Effects of Adeno-Associated Virus Serotype and Tissue-Specific Expression on Circulating Biomarkers of Propionic Acidemia

    PubMed Central

    Guenzel, Adam J.; Hillestad, Matthew L.; Matern, Dietrich

    2014-01-01

    Abstract Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca−/−(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease. PMID:25046265

  14. Effects of adeno-associated virus serotype and tissue-specific expression on circulating biomarkers of propionic acidemia.

    PubMed

    Guenzel, Adam J; Hillestad, Matthew L; Matern, Dietrich; Barry, Michael A

    2014-09-01

    Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca(-/-)(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease. PMID:25046265

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

    PubMed

    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

  16. Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse

    PubMed Central

    Schuster, Daniel J.; Dykstra, Jaclyn A.; Riedl, Maureen S.; Kitto, Kelley F.; Belur, Lalitha R.; McIvor, R. Scott; Elde, Robert P.; Fairbanks, Carolyn A.; Vulchanova, Lucy

    2014-01-01

    Adeno-associated virus serotype 9 (AAV9)-mediated gene transfer has been reported in central nervous system (CNS) and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP) across the mouse CNS and selected peripheral tissues after intrathecal (i.t.) or intravenous (i.v.) delivery of equivalent doses of single-stranded AAV9 vector. After i.t. delivery, GFP immunoreactivity (-ir) was observed in spinal neurons, primary afferent fibers and corresponding primary sensory neurons at all spinal levels. Robust transduction was seen in small and large dorsal root ganglion (DRG) neurons as well as trigeminal and vagal primary afferent neurons. Transduction efficiency in sensory ganglia was substantially lower in i.v. treated mice. In brain, i.v. delivery yielded GFP-immunoreactivity (-ir) primarily in spinal trigeminal tract, pituitary, and scattered isolated neurons and astrocytes. In contrast, after i.t. delivery, GFP-ir was widespread throughout CNS, with greater intensity and more abundant neuropil-like staining at 6 weeks compared to 3 weeks. Brain regions with prominent GFP-ir included cranial nerve nuclei, ventral pons, cerebellar cortex, hippocampus, pituitary, choroid plexus, and selected nuclei of midbrain, thalamus and hypothalamus. In cortex, GFP-ir was associated with blood vessels, and was seen in both neurons and astrocytes. In the periphery, GFP-ir in colon and ileum was present in the enteric nervous system in both i.v. and i.t. treated mice. Liver and adrenal cortex, but not adrenal medulla, also showed abundant GFP-ir after both routes of delivery. In summary, i.t. delivery yielded higher transduction efficiency in sensory neurons and the CNS. The observation of comparable gene transfer to peripheral tissues using the two routes indicates that a component of i.t. delivered vector is redistributed from the subarachnoid space to the systemic circulation. PMID:24959122

  17. Biodistribution of adeno-associated virus serotype 9 (AAV9) vector after intrathecal and intravenous delivery in mouse.

    PubMed

    Schuster, Daniel J; Dykstra, Jaclyn A; Riedl, Maureen S; Kitto, Kelley F; Belur, Lalitha R; McIvor, R Scott; Elde, Robert P; Fairbanks, Carolyn A; Vulchanova, Lucy

    2014-01-01

    Adeno-associated virus serotype 9 (AAV9)-mediated gene transfer has been reported in central nervous system (CNS) and peripheral tissues. The current study compared the pattern of expression of Green Fluorescent Protein (GFP) across the mouse CNS and selected peripheral tissues after intrathecal (i.t.) or intravenous (i.v.) delivery of equivalent doses of single-stranded AAV9 vector. After i.t. delivery, GFP immunoreactivity (-ir) was observed in spinal neurons, primary afferent fibers and corresponding primary sensory neurons at all spinal levels. Robust transduction was seen in small and large dorsal root ganglion (DRG) neurons as well as trigeminal and vagal primary afferent neurons. Transduction efficiency in sensory ganglia was substantially lower in i.v. treated mice. In brain, i.v. delivery yielded GFP-immunoreactivity (-ir) primarily in spinal trigeminal tract, pituitary, and scattered isolated neurons and astrocytes. In contrast, after i.t. delivery, GFP-ir was widespread throughout CNS, with greater intensity and more abundant neuropil-like staining at 6 weeks compared to 3 weeks. Brain regions with prominent GFP-ir included cranial nerve nuclei, ventral pons, cerebellar cortex, hippocampus, pituitary, choroid plexus, and selected nuclei of midbrain, thalamus and hypothalamus. In cortex, GFP-ir was associated with blood vessels, and was seen in both neurons and astrocytes. In the periphery, GFP-ir in colon and ileum was present in the enteric nervous system in both i.v. and i.t. treated mice. Liver and adrenal cortex, but not adrenal medulla, also showed abundant GFP-ir after both routes of delivery. In summary, i.t. delivery yielded higher transduction efficiency in sensory neurons and the CNS. The observation of comparable gene transfer to peripheral tissues using the two routes indicates that a component of i.t. delivered vector is redistributed from the subarachnoid space to the systemic circulation. PMID:24959122

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

    PubMed

    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

  19. Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

    PubMed Central

    Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

    2014-01-01

    ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will

  20. 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.

    PubMed

    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

  1. Prevalence of Anti–Adeno-Associated Virus Serotype 8 Neutralizing Antibodies and Arylsulfatase B Cross-Reactive Immunologic Material in Mucopolysaccharidosis VI Patient Candidates for a Gene Therapy Trial

    PubMed Central

    Ferla, Rita; Claudiani, Pamela; Savarese, Marco; Kozarsky, Karen; Parini, Rossella; Scarpa, Maurizio; Donati, Maria Alice; Sorge, Giovanni; Hopwood, John J.; Parenti, Giancarlo; Fecarotta, Simona; Nigro, Vincenzo; Sivri, Hatice Serap; Van Der Ploeg, Ans; Andria, Generoso; Brunetti-Pierri, Nicola

    2015-01-01

    Abstract Recombinant vectors based on adeno-associated virus serotype 8 (AAV8) have been successfully used in the clinic and hold great promise for liver-directed gene therapy. Preexisting immunity against AAV8 or the development of antibodies against the therapeutic transgene product might negatively affect the outcomes of gene therapy. In the prospect of an AAV8-mediated, liver-directed gene therapy clinical trial for mucopolysaccharidosis VI (MPS VI), a lysosomal storage disorder caused by arylsulfatase B (ARSB) deficiency, we investigated in a multiethnic cohort of MPS VI patients the prevalence of neutralizing antibodies (Nab) to AAV8 and the presence of ARSB cross-reactive immunologic material (CRIM), which will either affect the efficacy of gene transfer or the duration of phenotypic correction. Thirty-six MPS VI subjects included in the study harbored 45 (62.5%) missense, 13 (18%) nonsense, 9 (12.5%) frameshift (2 insertions and 7 deletions), and 5 (7%) splicing ARSB mutations. The detection of ARSB protein in 24 patients out of 34 (71%) was predicted by the type of mutations. Preexisting Nab to AAV8 were undetectable in 19/33 (58%) analyzed patients. Twelve out of 31 patients (39%) tested were both negative for Nab to AAV8 and CRIM-positive. In conclusion, this study allows estimating the number of MPS VI patients eligible for a gene therapy trial by intravenous injections of AAV8. PMID:25654180

  2. Assessment of brain metabolite correlates of adeno-associated virus-mediated over-expression of human alpha-synuclein in cortical neurons by in vivo (1) H-MR spectroscopy at 9.4 T.

    PubMed

    Cuellar-Baena, Sandra; Landeck, Natalie; Sonnay, Sarah; Buck, Kerstin; Mlynarik, Vladimir; In 't Zandt, René; Kirik, Deniz

    2016-06-01

    In this study, we used proton-localized spectroscopy ((1) H-MRS) for the acquisition of the neurochemical profile longitudinally in a novel rat model of human wild-type alpha-synuclein (α-syn) over-expression. Our goal was to find out if the increased α-syn load in this model could be linked to changes in metabolites in the frontal cortex. Animals injected with AAV vectors encoding for human α-syn formed the experimental group, whereas green fluorescent protein expressing animals were used as the vector-treated control group and a third group of uninjected animals were used as naïve controls. Data were acquired at 2, 4, and 8 month time points. Nineteen metabolites were quantified in the MR spectra using LCModel software. On the basis of 92 spectra, we evaluated any potential gender effect and found that lactate (Lac) levels were lower in males compared to females, while the opposite was observed for ascorbate (Asc). Next, we assessed the effect of age and found increased levels of GABA, Tau, and GPC+PCho. Finally, we analyzed the effect of treatment and found that Lac levels (p = 0.005) were specifically lower in the α-syn group compared to the green fluorescent protein and control groups. In addition, Asc levels (p = 0.05) were increased in the vector-injected groups, whereas glucose levels remained unchanged. This study indicates that the metabolic switch between glucose-lactate could be detectable in vivo and might be modulated by Asc. No concomitant changes were found in markers of neuronal integrity (e.g., N-acetylaspartate) consistent with the fact that α-syn over-expression in cortical neurons did not result in neurodegeneration in this model. We acquired the neurochemical profile longitudinally in a rat model of human wild-type alpha-synuclein (α-syn) over-expression in cortical neurons. We found that Lactate levels were reduced in the α-syn group compared to the control groups and Ascorbate levels were increased in the vector-injected groups. No changes were found in markers of neuronal integrity consistent with the fact that α-syn over-expression did not result in frank neurodegeneration. PMID:26811128

  3. A retrograde adeno-associated virus for collecting ribosome-bound mRNA from anatomically defined projection neurons

    PubMed Central

    Cook-Snyder, Denise R.; Jones, Alexander; Reijmers, Leon G.

    2015-01-01

    The brain contains a large variety of projection neurons with different functional properties. The functional properties of projection neurons arise from their connectivity with other neurons and their molecular composition. We describe a novel tool for obtaining the gene expression profiles of projection neurons that are anatomically defined by the location of their soma and axon terminals. Our tool utilizes adeno-associated virus serotype 9 (AAV9), which we found to retrogradely transduce projection neurons after injection at the site of the axon terminals. We used AAV9 to express Enhanced Green Fluorescent Protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a), which enables the immunoprecipitation of EGFP-tagged ribosomes and associated mRNA with a method known as Translating Ribosome Affinity Purification (TRAP). To achieve high expression of the EGFP-L10a protein in projection neurons, we placed its expression under control of a 1.3 kb alpha-calcium/calmodulin-dependent protein kinase II (Camk2a) promoter. We injected the AAV9-Camk2a-TRAP virus in either the hippocampus or the bed nucleus of the stria terminalis (BNST) of the mouse brain. In both brain regions the 1.3 kb Camk2a promoter did not confer complete cell-type specificity around the site of injection, as EGFP-L10a expression was observed in Camk2a-expressing neurons as well as in neuronal and non-neuronal cells that did not express Camk2a. In contrast, cell-type specific expression was observed in Camk2a-positive projection neurons that were retrogradely transduced by AAV9-Camk2a-TRAP. Injection of AAV9-Camk2a-TRAP into the BNST enabled the use of TRAP to collect ribosome-bound mRNA from basal amygdala projection neurons that innervate the BNST. AAV9-Camk2a-TRAP provides a single-virus system that can be used for the molecular profiling of anatomically defined projection neurons in mice and other mammalian model organisms. In addition, AAV9-Camk2a-TRAP may enable the discovery of protein synthesis

  4. Adeno-Associated Virus Type 2 Rep68 Can Bind to Consensus Rep-Binding Sites on the Herpes Simplex Virus 1 Genome

    PubMed Central

    Seyffert, Michael; Glauser, Daniel L.; Tobler, Kurt; Georgiev, Oleg; Vogel, Rebecca; Vogt, Bernd; Agúndez, Leticia; Linden, Michael; Büning, Hildegard; Ackermann, Mathias

    2015-01-01

    Adeno-associated virus type 2 is known to inhibit replication of herpes simplex virus 1 (HSV-1). This activity has been linked to the helicase- and DNA-binding domains of the Rep68/Rep78 proteins. Here, we show that Rep68 can bind to consensus Rep-binding sites on the HSV-1 genome and that the Rep helicase activity can inhibit replication of any DNA if binding is facilitated. Therefore, we hypothesize that inhibition of HSV-1 replication involves direct binding of Rep68/Rep78 to the HSV-1 genome. PMID:26292324

  5. Incorporation of Adeno-Associated Virus in a Calcium Phosphate Coprecipitate Improves Gene Transfer to Airway Epithelia In Vitro and In Vivo

    PubMed Central

    Walters, Robert W.; Duan, Dongsheng; Engelhardt, John F.; Welsh, Michael J.

    2000-01-01

    Adeno-associated virus (AAV) is inefficient at infecting differentiated airway epithelia because of a lack of receptors at the apical surface. We hypothesized that incorporation of AAV in a calcium phosphate coprecipitate would circumvent this barrier. Interestingly, coprecipitation of AAV type 2 improved gene transfer to differentiated human airway epithelia in vitro and to the mouse lung in vivo. These results suggest that delivery of AAV as a CaPi coprecipitate may significantly enhance its utility for gene transfer to the airway epithelia in vivo. PMID:10590145

  6. Adeno-Associated Virus-Like Particles as New Carriers for B-Cell Vaccines: Testing Immunogenicity and Safety in BALB/c Mice

    PubMed Central

    Manzano-Szalai, Krisztina; Thell, Kathrin; Willensdorfer, Anna; Weghofer, Margit; Pfanzagl, Beatrix; Singer, Josef; Ritter, Mirko; Stremnitzer, Caroline; Flaschberger, Ingo; Michaelis, Uwe

    2014-01-01

    Abstract Adeno-associated viruses (AAVs) are established vectors for gene therapy of different human diseases. AAVs are assembled of 60 capsomers, which can be genetically modified, allowing high-density display of short peptide sequences at their surface. The aim of our study was to evaluate the immunogenicity and safety of an adeno-associated virus-like particle (AAVLP)-displayed B-cell peptide epitope taking ovalbumin (OVA) as a model antigen or allergen from egg, respectively. An OVA-derived B-cell epitope was expressed as fusion protein with the AAV-2 capsid protein of VP3 (AAVLP-OVA) and for control, with the nonrelated peptide TP18 (AAVLP-TP18). Cellular internalization studies revealed an impaired uptake of AAVLP-OVA by mouse BMDC, macrophages, and human HeLa cells. Nevertheless, BALB/c mice immunized subcutaneously with AAVLP-OVA formed similarly high titers of OVA-specific IgG1 compared to mice immunized with the native OVA. The extent of the immune response was independent whether aluminum hydroxide or water in oil emulsion was used as adjuvant. Furthermore, in mice immunized with native OVA, high OVA-specific IgE levels were observed, which permitted OVA-specific mast-cell degranulation in a β-hexosaminidase release assay, whereas immunizations with AAVLP-OVA rendered background IgE levels only. Accordingly, OVA-immunized mice, but not AAVLP-OVA immunized mice, displayed an anaphylactic reaction with a significant drop of body temperature upon intravenous OVA challenge. From this mouse model, we conclude that AAVLPs that display B-cell epitope peptides on their surface are suitable vaccine candidates, especially in the field of allergy. PMID:25247267

  7. A role for XLF in DNA repair and recombination in human somatic cells.

    PubMed

    Fattah, Farjana Jahan; Kweon, Junghun; Wang, Yongbao; Lee, Eu Han; Kan, Yinan; Lichter, Natalie; Weisensel, Natalie; Hendrickson, Eric A

    2014-03-01

    Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor. PMID:24461734

  8. A role for XLF in DNA repair and recombination in human somatic cells

    PubMed Central

    Fattah, Farjana; Kweon, Junghun; Wang, Yongbao; Lee, Eu Han; Kan, Yinan; Lichter, Natalie; Weisensel, Natalie; Hendrickson, Eric A.

    2014-01-01

    Classic non-homologous end-joining (C-NHEJ) is required for the repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian cells and plays a critical role in lymphoid V(D)J recombination. A core C-NHEJ component is the DNA ligase IV co-factor, Cernunnos/XLF (hereafter XLF). In patients, mutations in XLF cause predicted increases in radiosensitivity and deficits in immune function, but also cause other less well-understood pathologies including neural disorders. To characterize XLF function(s) in a defined genetic system, we used a recombinant adeno-associated virus-mediated gene targeting strategy to inactivate both copies of the XLF locus in the human HCT116 cell line. Analyses of XLF-null cells (which were viable) showed that they were highly sensitive to ionizing radiation and a radiomimetic DNA damaging agent, etoposide. XLF-null cells had profound DNA DSB repair defects as measured by in vivo plasmid end-joining assays and were also dramatically impaired in their ability to form either V(D)J coding or signal joints on extrachromosomal substrates. Thus, our somatic XLF-null cell line recapitulates many of the phenotypes expected from XLF patient cell lines. Subsequent structure:function experiments utilizing the expression of wild-type and mutant XLF cDNAs demonstrated that all of the phenotypes of an XLF deficiency could be rescued by the overexpression of a wild-type XLF cDNA. Unexpectedly, mutant forms of XLF bearing point mutations at amino acid positions L115 and L179, also completely complemented the null phenotype suggesting, in contrast to predictions to the contrary, that these mutations do not abrogate XLF function. Finally, we demonstrate that the absence of XLF causes a small, but significant, increase in homologous recombination, implicating XLF in DSB pathway choice regulation. We conclude that human XLF is a non-essential, but critical, C-NHEJ-repair factor. PMID:24461734

  9. Recombinant adeno-associated virus type 2-mediated gene delivery into the Rpe65-/- knockout mouse eye results in limited rescue

    PubMed Central

    Lai, Chooi-May; Yu, Meaghan JT; Brankov, Meliha; Barnett, Nigel L; Zhou, Xiaohuai; Redmond, T Michael; Narfstrom, Kristina; Rakoczy, P Elizabeth

    2004-01-01

    Background Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65-/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function. Methods rAAV.RPE65 was injected into the subretinal space of Rpe65-/- knockout mice and control mice. Histological and immunohistological analyses were performed to evaluate any rescue of photoreceptors and to determine longevity and pattern of transgene expression. Electron microscopy was used to examine ultrastructural changes, and electroretinography was used to measure changes in visual function following rAAV.RPE65 injection. Results rAAV-mediated RPE65 expression was detected for up to 18 months post injection. The delivery of rAAV.RPE65 to Rpe65-/- mouse retinas resulted in a transient improvement in the maximum b-wave amplitude under both scotopic and photopic conditions (76% and 59% increase above uninjected controls, respectively) but no changes were observed in a-wave amplitude. However, this increase in b-wave amplitude was not accompanied by any slow down in photoreceptor degeneration or apoptotic cell death. Delivery of rAAV.RPE65 also resulted in a decrease in retinyl ester lipid droplets and an increase in short wavelength cone opsin-positive cells, suggesting that the recovery of RPE65 expression has long-term benefits for retinal health. Conclusion This work demonstrated the potential benefits of using the Rpe65-/- mice to study the effects and mechanism of rAAV.RPE65-mediated gene delivery into the retina. Although the functional recovery in this model was not as robust as in the dog model, these experiments provided important clues about the long-term physiological benefits of restoration of RPE65 expression in the retina. PMID:15109394

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

    PubMed

    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

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

    PubMed

    Stitelman, David H; Brazelton, Tim; Bora, Archana; Traas, Jeremy; Merianos, Demetri; Limberis, Maria; Davey, Marcus; Flake, Alan W

    2014-01-01

    Efficient gene transfer to muscle stem cells (satellite cells) has not been achieved despite broad transduction of skeletal muscle by systemically administered adeno-associated virus serotype 2/9 (AAV-9) in mice. We hypothesized that cellular migration during fetal development would make satellite cells accessible for gene transfer following in utero intravascular injection. We injected AAV-9 encoding green fluorescent protein (GFP) marker gene into the vascular space of mice ranging in ages from post-coital day 12 (E12) to postnatal day 1 (P1). Satellite cell transduction was examined using: immunohistochemistry and confocal microscopy, satellite cell migration assay, myofiber isolation and FACS analysis. GFP positive myofibers were detected in all mature skeletal muscle groups and up to 100% of the myofibers were transduced. We saw gestational variation in cardiac and skeletal muscle expression. E16 injection resulted in 27.7 ± 10.0% expression in satellite cells, which coincides with the timing of satellite cell migration, and poor satellite cell expression before and after satellite cell migration (E12 and P1). Our results demonstrate that efficient gene expression is achieved in differentiated myofibers and satellite cells after injection of AAV-9 in utero. These findings support the potential of prenatal gene transfer for muscle based treatment strategies. PMID:26015979

  12. Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome

    PubMed Central

    Pasta, Saloni; Akhile, Omoye; Tabron, Dorothy; Ting, Flora; Shackleton, Cedric; Watson, Gordon

    2015-01-01

    Smith Lemli Opitz syndrome (SLOS) is an inherited malformation and mental retardation metabolic disorder with no cure. Mutations in the last enzyme of the cholesterol biosynthetic pathway, 7-dehydrocholesterol reductase (DHCR7), lead to cholesterol insufficiency and accumulation of its dehyrdocholesterol precursors, and contribute to its pathogenesis. The central nervous system (CNS) constitutes a major pathophysiological component of this disorder and remains unamenable to dietary cholesterol therapy due to the impenetrability of the blood brain barrier (BBB). The goal of this study was to restore sterol homeostasis in the CNS. To bypass the BBB, gene therapy using an adeno-associated virus (AAV-8) vector carrying a functional copy of the DHCR7 gene was administered by intrathecal (IT) injection directly into the cerebrospinal fluid of newborn mice. Two months post-treatment, vector DNA and DHCR7 expression was observed in the brain and a corresponding improvement of sterol levels seen in the brain and spinal cord. Interestingly, sterol levels in the peripheral nervous system also showed a similar improvement. This study shows that IT gene therapy can have a positive biochemical effect on sterol homeostasis in the central and peripheral nervous systems in a SLOS animal model. A single dose delivered three days after birth had a sustained effect into adulthood, eight weeks post-treatment. These observations pave the way for further studies to understand the effect of biochemical improvement of sterol levels on neuronal function, to provide a greater understanding of neuronal cholesterol homeostasis, and to develop potential therapies. PMID:26347274

  13. Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

    PubMed

    Raissadati, Alireza; Jokinen, Janne J; Syrjälä, Simo O; Keränen, Mikko A I; Krebs, Rainer; Tuuminen, Raimo; Arnaudova, Ralica; Rouvinen, Eeva; Anisimov, Andrey; Soronen, Jarkko; Pajusola, Katri; Alitalo, Kari; Nykänen, Antti I; Lemström, Karl

    2013-11-01

    Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies. PMID:24102821

  14. Construction and gene expression analysis of a single-stranded DNA minivector based on an inverted terminal repeat of adeno-associated virus.

    PubMed

    Ping, Han; Liu, Xiaomei; Zhu, Dongqin; Li, Taiming; Zhang, Chun

    2015-04-01

    The plasmid vectors currently used for nonviral gene transfer have the disadvantage of carrying a bacterial backbone and an antibiotic resistance gene, which may cause side effects. The adeno-associated virus (AAV) genome is a linear single-stranded DNA (ssDNA) molecule with palindromic inverted terminal repeat (ITR) sequences forming double-stranded DNA (dsDNA) hairpin (HP) structures at each end. Based on the AAV genome, we constructed an AAV-ITR ssDNA minivector that consists of a GFP expression cassette flanked by both ITR sequences of 125 nucleotides. The minivectors were produced by digestion of the parental plasmids followed by denaturation. The self-complementary inverted T-shaped HP structure of the minivector was automatically formed. The HEK 293T cells were transfected with the AAV-ITR ssDNA minivector, plasmid, and dsDNA expression cassette. The results showed that AAV-ITR ssDNA minivector had relatively low gene expression efficiency in vitro. However, we found that the GFP expression efficiency of the D sequence-deleted AAV-ITR ssDNA minivector was significantly increased and was similar to those obtained with the plasmid and dsDNA expression cassette. Our data suggest that the AAV-ITR ssDNA minivector may be a new type of gene expression vector for gene therapy besides the virus and plasmid. PMID:25555376

  15. Long-Term Robust Myocardial Transduction of the Dog Heart from a Peripheral Vein by Adeno-Associated Virus Serotype-8

    PubMed Central

    Pan, Xiufang; Yue, Yongping; Zhang, Keqing; Lostal, William; Shin, Jin-Hong

    2013-01-01

    Abstract Molecular intervention using noninvasive myocardial gene transfer holds great promise for treating heart diseases. Robust cardiac transduction from peripheral vein injection has been achieved in rodents using adeno-associated virus (AAV) serotype-9 (AAV-9). However, a similar approach has failed to transduce the heart in dogs, a commonly used large animal model for heart diseases. To develop an effective noninvasive method to deliver exogenous genes to the dog heart, we employed an AAV-8 vector that expresses human placental alkaline phosphatase reporter gene under the transcriptional regulation of the Rous sarcoma virus promoter. Vectors were delivered to three neonatal dogs at the doses of 1.35×1014, 7.14×1014, and 9.06×1014 viral genome particles/kg body weight via the jugular vein. Transduction efficiency and overall safety were evaluated at 1.5, 2.5, and 12 months postinjection. AAV delivery was well tolerated and dog growth was normal. Blood chemistry and internal organ histology were unremarkable. Widespread skeletal muscle transduction was observed in all dogs without T-cell infiltration. Encouragingly, whole heart myocardial transduction was achieved in two dogs that received higher doses and cardiac expression lasted for at least 1 year. In summary, peripheral vein AAV-8 injection may represent a simple heart gene transfer method in large mammals. Further optimization of this gene delivery strategy may open the door for a readily applicable gene therapy method to treat many heart diseases. PMID:23551085

  16. A 110-kDa nuclear shuttle protein, nucleolin, specifically binds to adeno-associated virus type 2 (AAV-2) capsid.

    PubMed

    Qiu, J; Brown, K E

    1999-05-10

    A 110-kDa protein was copurified with adeno-associated virus type 2 (AAV-2) virions after CsCl density gradient isopycnic centrifugation. Amino acid sequence of peptides derived from this protein after tryptic digestion, monoclonal antibody production, and Western blot analysis showed that the copurified protein was the major nucleolar phosphoprotein, human nucleolin. Virus overlay assays demonstrated that AAV-2 capsid specifically bound to the human nucleolin, and immunoprecipitation studies confirmed the in vitro binding of nucleolin and intact AAV-2 capsids but not denatured viral proteins. Double-immunofluorescence staining of infected cells showed that AAV capsid and nucleolin were colocalized in both cytoplasm and nucleus. In addition, when cytoplasmic and nuclear fractions were extracted from AAV-infected KB cells at different time points postinfection, immunoprecipitation data and Western blotting showed that AAV capsid formation and nucleolin interact specifically and share their subcellular localization in infected cells. With the known functions of nucleolin in the synthesis of rRNA and ribosome assembly, binding to single-stranded DNA, and acting as a shuttle between cytoplasm and nucleolus, our data showing that AAV-2 capsid binds specifically to nucleolin both in vitro and in vivo suggest a key role of nucleolin in AAV-2 replication, particularly in capsid assembly. PMID:10329548

  17. 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.

    PubMed

    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

  18. Systemic Trans-splicing adeno-associated viral delivery efficiently transduces the heart of adult mdx mouse, a model for duchenne muscular dystrophy.

    PubMed

    Ghosh, Arkasubhra; Yue, Yongping; Shin, Jin-Hong; Duan, Dongsheng

    2009-11-01

    Trans-splicing adeno-associated viral (tsAAV) vectors hold great promise for delivering large therapeutic genes. One potential application is in the treatment of Duchenne muscular dystrophy (DMD). In this case, it is necessary to transduce whole body muscle. We demonstrated body-wide AAV-9 tsAAV transduction in normal neonatal mice. However, it was not clear whether such an approach would work in diseased mice. In this study we delivered the AAV-9 alkaline phosphatase (AP) tsAAV vector (3 x 10(12) vector genome particles per vector per mouse, tail vein injection) to 2-month-old mdx mice, the most widely used DMD model. Four months later, we observed widespread AP expression in the heart. It reached the same level as we have seen in normal neonatal puppy. Interestingly, myocardial transduction correlated with beta-myosin heavy chain expression but not with LamR, the putative AAV-9 receptor. AP expression was also detected in various skeletal muscles but at levels much lower than in normal newborn mice. Despite the existing inflammatory milieu, we did not see any appreciable increase in CD4(+) and CD8(+) T cells and macrophages in striated muscles after systemic tsAAV infection. In summary, our results have paved the way for tsAAV-mediated gene therapy for Duchenne cardiomyopathy. PMID:19627234

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

    SciTech Connect

    Cho, Mi Ae; Yashar, Parham; Kim, Suk Kyoung; Noh, Taewoong; Gillam, Mary P.; Lee, Eun Jig Jameson, J. Larry

    2008-07-04

    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 {beta}-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.

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

    PubMed

    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

  1. Selective Cleavage of AAVS1 Substrates by the Adeno-Associated Virus Type 2 Rep68 Protein Is Dependent on Topological and Sequence Constraints

    PubMed Central

    Lamartina, Stefania; Ciliberto, Gennaro; Toniatti, Carlo

    2000-01-01

    The adeno-associated virus type 2 (AAV-2) Rep78 and Rep68 proteins are required for replication of the virus as well as its site-specific integration into a unique site, called AAVS1, of human chromosome 19. Rep78 and Rep68 initiate replication by binding to a Rep binding site (RBS) contained in the AAV-2 inverted terminal repeats (ITRs) and then specifically nicking at a nearby site called the terminal resolution site (trs). Similarly, Rep78 and Rep68 are postulated to trigger the integration process by binding and nicking RBS and trs homologues present in AAVS1. However, Rep78 and Rep68 cleave in vitro AAVS1 duplex-linear substrates much less efficiently than hairpinned ITRs. In this study, we show that the AAV-2 Rep68 endonuclease activity is affected by the topology of the substrates in that it efficiently cleaves in vitro in a site- and strand-specific manner the AAVS1 trs only if this sequence is in a supercoiled (SC) conformation. DNA sequence mutagenesis in the context of SC templates allowed us to elucidate for the first time the AAVS1 trs sequence and position requirements for Rep68-mediated cleavage. Interestingly, Rep68 did not cleave SC templates containing RBS from other sites of the human genome. These findings have intriguing implications for AAV-2 site-specific integration in vivo. PMID:10982325

  2. Differential Type I Interferon-dependent Transgene Silencing of Helper-dependent Adenoviral vs. Adeno-associated Viral Vectors In Vivo

    PubMed Central

    Suzuki, Masataka; Bertin, Terry K; Rogers, Geoffrey L; Cela, Racel G; Zolotukhin, Irene; Palmer, Donna J; Ng, Philip; Herzog, Roland W; Lee, Brendan

    2013-01-01

    We previously dissected the components of the innate immune response to Helper-dependent adenoviral vectors (HDAds) using genetic models, and demonstrated that multiple pattern recognition receptor signaling pathways contribute to this host response to HDAds in vivo. Based on analysis of cytokine expression profiles, type I interferon (IFN) mRNA is induced in host mouse livers at 1 hour post-injection. This type I IFN signaling amplifies cytokine expression in liver independent of the nature of vector DNA sequences after 3 hours post-injection. This type I IFN signaling in response to HDAds administration contributes to transcriptional silencing of both HDAd prokaryotic and eukaryotic DNA in liver. This silencing occurs early and is mediated by epigenetic modification as shown by in vivo chromatin immunoprecipitation (ChIP) with anti-histone deacetylase (HDAC) and promyelocytic leukemia protein (PML). In contrast, self-complementary adeno-associated viral vectors (scAAVs) showed significantly lower induction of type I IFN mRNA in liver compared to HDAds at both early and late time points. These results show that the type I IFN signaling dependent transgene silencing differs between AAV and HDAd vectors after liver-directed gene transfer. PMID:23319058

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

    PubMed

    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

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

    PubMed

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

    2016-01-01

    Recombinant AAV (rAAV) vectors are a suitable vector for gene therapy studies because of desired characteristics such as low immunogenicity, transfection of non-dividing and dividing cells, and long-term expression of the transgene. In this study, the large-scale production of single stranded (ss) and self-complementary (sc) AAV9 carrying the human survival motor neuron (SMN) gene (AAV9-SMN) suitable for in vivo gene therapy studies of SMA was described. SMN cDNA has been cloned into pAAV-CB6-PI and pAAVsc-CB6-PI with and without its specific UTRs, respectively. Both plasmids bear CMV enhancer/beta-actin (CB) promoter, CMV IE enhancer, and polyadenylation signal sequences. 2.5 μg of constructed pAAV-CB6-PI-SMN and pAAVsc-CB6-PI-SMN cause to, respectively, 4.853- and 2.321-fold increases in SMN protein levels in transfected cells compared to untransfected cells. Ss and scAAV9-SMN vectors were also produced from these plasmids by transient transfection of HEK293 cells using CaCl2 solution. The silver staining and electron microscopy analysis demonstrated good quality of both isolated vectors, ssAAV9-SMN and scAAV9-SMN, with the titers of 2.00E+13 and 1.00E+13 GC/ml. The results of this study show that, the plasmid containing UTR elements causes to twice more SMN gene expression in transfected cells. The quality control results show that both produced ss and scAAV9-SMN are suitable for in vivo studies. PMID:26607476

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

    PubMed Central

    RASHNONEJAD, Afrooz; CHERMAHINI, Gholamhossein AMINI; Li, Shaoyong; OZKINAY, Ferda; GAO, Guangping

    2016-01-01

    Recombinant AAV (rAAV) vectors are a suitable vector for gene therapy studies because of desired characteristics such as low immunogenicity, transfection of non-dividing and dividing cells, and long-term expression of the transgene. In this study, the large-scale production of single stranded (ss) and self-complementary (sc) AAV9 carrying the human survival motor neuron (SMN) gene (AAV9-SMN) suitable for in vivo gene therapy studies of SMA was described. SMN cDNA has been cloned into pAAV-CB6-PI and pAAVsc-CB6-PI with and without its specific UTRs, respectively. Both plasmids bear CMV enhancer/beta-actin (CB) promoter, CMV IE enhancer, and polyadenylation signal sequences. 2.5 μg of constructed pAAV-CB6-PI-SMN and pAAVsc-CB6-PI-SMN cause to, respectively, 4.853- and 2.321-fold increases in SMN protein levels in transfected cells compared to untransfected cells. Ss and scAAV9-SMN vectors were also produced from these plasmids by transient transfection of HEK293 cells using CaCl2 solution. The silver staining and electron microscopy analysis demonstrated good quality of both isolated vectors, ssAAV9-SMN and scAAV9- SMN, with the titers of 2.00E+13 and 1.00E+13 GC/ml. The results of this study show that, the plasmid containing UTR elements causes to twice more SMN gene expression in transfected cells. The quality control results show that both produced ss and scAAV9-SMN are suitable for in vivo studies. PMID:26607476

  6. Identification and Characterization of Nuclear and Nucleolar Localization Signals in the Adeno-Associated Virus Serotype 2 Assembly-Activating Protein

    PubMed Central

    Earley, Lauriel F.; Kawano, Yasuhiro; Adachi, Kei; Sun, Xiao-Xin; Dai, Mu-Shui

    2014-01-01

    ABSTRACT Assembly-activating protein (AAP) of adeno-associated virus serotype 2 (AAV2) is a nucleolar-localizing protein that plays a critical role in transporting the viral capsid VP3 protein to the nucleolus for assembly. Here, we identify and characterize AAV2 AAP (AAP2) nuclear (NLS) and nucleolar (NoLS) localization signals near the carboxy-terminal region of AAP2 (amino acid positions 144 to 184) (AAP2144–184). This region contains five basic-amino-acid-rich (BR) clusters, KSKRSRR (AAP2BR1), RRR (AAP2BR2), RFR (AAP2BR3), RSTSSR (AAP2BR4), and RRIK (AAP2BR5), from the amino terminus to the carboxy terminus. We created 30 AAP2BR mutants by arginine/lysine-to-alanine mutagenesis or deletion of AAP2BRs and 8 and 1 green fluorescent protein (GFP)-AAP2BR and β-galactosidase–AAP2BR fusion proteins, respectively, and analyzed their intracellular localization in HeLa cells by immunofluorescence microscopy. The results showed that AAP2144–184 has redundant multipartite NLSs and that any combinations of 4 AAP2BRs, but not 3 or less, can constitute a functional NLS-NoLS; AAP2BR1 and AAP2BR2 play the most influential role for nuclear localization, but either one of the two AAP2BRs is dispensable if all 4 of the other AAP2BRs are present, resulting in 3 different, overlapping NLS motifs; and the NoLS is shared redundantly among the five AAP2BRs and functions in a context-dependent manner. AAP2BR mutations not only resulted in aberrant intracellular localization, but also attenuated AAP2 protein expression to various degrees, and both of these abnormalities have a significant negative impact on capsid production. Thus, this study reveals the organization of the intermingling NLSs and NoLSs in AAP2 and provides insights into their functional roles in capsid assembly. IMPORTANCE Adeno-associated virus (AAV) has become a popular and successful vector for in vivo gene therapy; however, its biology has yet to be fully understood. In this regard, the recent discovery of

  7. Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity

    PubMed Central

    Tseng, Yu-Shan; Gurda, Brittney L.; Chipman, Paul; McKenna, Robert; Afione, Sandra; Chiorini, John A.; Muzyczka, Nicholas; Olson, Norman H.; Baker, Timothy S.; Kleinschmidt, Jürgen

    2014-01-01

    ABSTRACT The clinical utility of the adeno-associated virus (AAV) gene delivery system has been validated by the regulatory approval of an AAV serotype 1 (AAV1) vector for the treatment of lipoprotein lipase deficiency. However, neutralization from preexisting antibodies is detrimental to AAV transduction efficiency. Hence, mapping of AAV antigenic sites and engineering of neutralization-escaping vectors are important for improving clinical efficacy. We report the structures of four AAV-monoclonal antibody fragment complexes, AAV1-ADK1a, AAV1-ADK1b, AAV5-ADK5a, and AAV5-ADK5b, determined by cryo-electron microscopy and image reconstruction to a resolution of ∼11 to 12 Å. Pseudoatomic modeling mapped the ADK1a epitope to the protrusions surrounding the icosahedral 3-fold axis and the ADK1b and ADK5a epitopes, which overlap, to the wall between depressions at the 2- and 5-fold axes (2/5-fold wall), and the ADK5b epitope spans both the 5-fold axis-facing wall of the 3-fold protrusion and portions of the 2/5-fold wall of the capsid. Combined with the six antigenic sites previously elucidated for different AAV serotypes through structural approaches, including AAV1 and AAV5, this study identified two common AAV epitopes: one on the 3-fold protrusions and one on the 2/5-fold wall. These epitopes coincide with regions with the highest sequence and structure diversity between AAV serotypes and correspond to regions determining receptor recognition and transduction phenotypes. Significantly, these locations overlap the two dominant epitopes reported for autonomous parvoviruses. Thus, rather than the amino acid sequence alone, the antigenic sites of parvoviruses appear to be dictated by structural features evolved to enable specific infectious functions. IMPORTANCE The adeno-associated viruses (AAVs) are promising vectors for in vivo therapeutic gene delivery, with more than 20 years of intense research now realized in a number of successful human clinical trials that

  8. Enhanced efficacy of combination therapy with adeno-associated virus-delivered pigment epithelium-derived factor and cisplatin in a mouse model of Lewis lung carcinoma

    PubMed Central

    HE, SHA-SHA; WU, QIN-JIE; GONG, CHANG YANG; LUO, SHUN-TAO; ZHANG, SHUANG; LI, MENG; LU, LIAN; WEI, YU-QUAN; YANG, LI

    2014-01-01

    Pigment epithelium-derived factor (PEDF) is a potent inhibitor of angiogenesis, and the antitumor effect of adeno-associated virus (AAV)-mediated PEDF expression has been demonstrated in a range of animal models. The combined treatment of low-dose chemotherapy and gene therapy inhibits the growth of solid tumors more effectively than current traditional therapies or gene therapy alone. In the present study, the effect of treatment with an AAV2 vector harboring the human PEDF (hPEDF) gene in combination with low-dose cisplatin on the growth of Lewis lung carcinoma (LLC) in mice was assessed. LLC cells were infected with AAV-enhanced green fluorescent protein (EGFP) in the presence or absence of cisplatin, and then the effect of cisplatin on AAV-mediated gene expression was evaluated by image and flow cytometric analysis. Tumor growth, survival time, vascular endothelial growth factor (VEGF) expression, microvessel density (MVD) and apoptotic index were analyzed in C57BL/6 mice treated with AAV-hPEDF, cisplatin or cisplatin plus AAV-hPEDF. The results of the present study provide evidence that cisplatin treatment is able to enhance AAV-mediated gene expression in LLC cells. In addition, the combined treatment of cisplatin plus AAV-hPEDF markedly prolonged the survival time of the mice and inhibited tumor growth, resulting in significant suppression of tumor angiogenesis and induction of tumor apoptosis in vivo, and also protected against cisplatin-related toxicity. These findings suggest that combination of AAV-hPEDF and cisplatin has potential as a novel therapeutic strategy for lung cancer. PMID:24714917

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

    PubMed

    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

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

    PubMed Central

    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

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

    PubMed

    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

  12. A novel polyethyleneimine-coated adeno-associated virus-like particle formulation for efficient siRNA delivery in breast cancer therapy: preparation and in vitro analysis

    PubMed Central

    Shao, Wei; Paul, Arghya; Abbasi, Sana; Chahal, Parminder S; Mena, Jimmy A; Montes, Johnny; Kamen, Amine; Prakash, Satya

    2012-01-01

    Background Systemic delivery of small interfering RNA (siRNA) is limited by its poor stability and limited cell-penetrating properties. To overcome these limitations, we designed an efficient siRNA delivery system using polyethyleneimine-coated virus-like particles derived from adeno-associated virus type 2 (PEI-AAV2-VLPs). Methods AAV2-VLPs were produced in insect cells by infection with a baculovirus vector containing three AAV2 capsid genes. Using this method, we generated well dispersed AAV2-VLPs with an average diameter of 20 nm, similar to that of the wild-type AAV2 capsid. The nanoparticles were subsequently purified by chromatography and three viral capsid proteins were confirmed by Western blot. The negatively charged AAV2-VLPs were surface-coated with PEI to develop cationic nanoparticles, and the formulation was used for efficient siRNA delivery under optimized transfection conditions. Results PEI-AAV2-VLPs were able to condense siRNA and to protect it from degradation by nucleases, as confirmed by gel electrophoresis. siRNA delivery mediated by PEI-AAV2-VLPs resulted in a high transfection rate in MCF-7 breast cancer cells with no significant cytotoxicity. A cell death assay also confirmed the efficacy and functionality of this novel siRNA formulation towards MCF-7 cancer cells, in which more than 60% of cell death was induced within 72 hours of transfection. Conclusion The present study explores the potential of virus-like particles as a new approach for gene delivery and confirms its potential for breast cancer therapy. PMID:22619514

  13. Hydrodynamic Limb Vein Injection of Adeno-Associated Virus Serotype 8 Vector Carrying Canine Myostatin Propeptide Gene into Normal Dogs Enhances Muscle Growth

    PubMed Central

    Qiao, Chunping; Li, Juan; Zheng, Hui; Bogan, Janet; Li, Jianbin; Yuan, Zhenhua; Zhang, Cheng; Bogan, Dan; Kornegay, Joe

    2009-01-01

    Abstract Inhibition or blockade of myostatin, a negative growth factor of skeletal muscle, enhances muscle growth and therefore is considered a promising strategy for the treatment of muscle-wasting diseases such as the muscular dystrophies. Previously, we showed that myostatin blockade in both normal and dystrophin-deficient mdx mice by systemic delivery of the myostatin propeptide (MPRO) gene by an adeno-associated virus serotype 8 (AAV8) vector could enhance muscle growth and ameliorate dystrophic lesions. Here, we further investigate whether the muscle growth effect of myostatin blockade can be achieved in dogs by gene transfer. First, we cloned the canine MPRO gene, packaged it in the AAV8 vector, and showed robust muscle-enhancing effects after systemic delivery into neonatal mice. This vector was then further tested in two 3-month-old normal dogs (weighing 9.7 and 6.3 kg). The vector was delivered to one limb by hydrodynamic vein injection, and the contralateral limb served as a control. The delivery procedure was safe, without discernible adverse effects. AAV vector DNA and MPRO gene expression were detected by quantitative polymerase chain reaction, Western blotting, and immunofluorescence staining of muscle biopsies. Overexpression of MPRO resulted in enhanced muscle growth without a cytotoxic T lymphocytic immune response, as evidenced by larger myofibers in multiple muscles, increased muscle volume determined by magnetic resonance imaging, and the lack of CD4+ and CD8+ T cell infiltration in the vector-injected limbs. Our preliminary study thus supports further investigation of this therapeutic strategy in the dystrophin-deficient golden retriever muscular dystrophy dog model. PMID:18828709

  14. Deficiency in MyD88 Signaling Results in Decreased Antibody Responses to an Adeno-Associated Virus Vector in Murine Pompe Disease

    PubMed Central

    Zhang, Ping; Luo, Xiaoyan; Bird, Andrew; Li, Songtao

    2012-01-01

    Abstract We have previously shown that antibody and T cell responses limit the efficacy of an adeno-associated virus (AAV) pseudotype 8 (2/8) vector containing the universally active cytomegalovirus enhancer/chicken β-actin regulatory cassette (AAV2/8-CBhGAA) in treating murine Pompe disease. However, the innate immune responses to AAV2/8-CBhGAA are largely unknown. In this study, we investigated acute immune responses to AAV2/8-CBhGAA and the role of MyD88/TRIF signaling pathway in shaping adaptive immune responses to this vector. We showed here that a small and transient increase in CXCL-1 and IL-1β expression in livers of acid-α-glucosidase knockout (GAAKO) mice 6 h following injection with AAV2/8-CBhGAA. There was a robust antibody response to GAA in wild-type mice injected with this vector. In contrast, the anti-GAA IgG1 response was diminished in MyD88KO mice, and showed a trend toward a decrease in TRIFKO mice. In addition, the vector genome and GAA activity were significantly higher in MyD88KO livers compared with wild-type livers, suggesting reduced cytotoxic T cell responses. Importantly, elevated CD4+ T cells were detected by immunohistochemistry in MyD88KO livers. When adoptively transferred to wild-type mice, these CD4+ T cells have an ability to suppress antibody responses against AAV2/8-CBhGAA and to prevent further immunization against rhGAA. Our study suggests that the MyD88 deficiency leads to the suppression of deleterious immune responses to AAV2/8-CBhGAA, which has implications for gene therapy in Pompe disease. PMID:23514839

  15. Deficiency in MyD88 Signaling Results in Decreased Antibody Responses to an Adeno-Associated Virus Vector in Murine Pompe Disease.

    PubMed

    Zhang, Ping; Luo, Xiaoyan; Bird, Andrew; Li, Songtao; Koeberl, Dwight D

    2012-06-01

    We have previously shown that antibody and T cell responses limit the efficacy of an adeno-associated virus (AAV) pseudotype 8 (2/8) vector containing the universally active cytomegalovirus enhancer/chicken β-actin regulatory cassette (AAV2/8-CBhGAA) in treating murine Pompe disease. However, the innate immune responses to AAV2/8-CBhGAA are largely unknown. In this study, we investigated acute immune responses to AAV2/8-CBhGAA and the role of MyD88/TRIF signaling pathway in shaping adaptive immune responses to this vector. We showed here that a small and transient increase in CXCL-1 and IL-1β expression in livers of acid-α-glucosidase knockout (GAAKO) mice 6 h following injection with AAV2/8-CBhGAA. There was a robust antibody response to GAA in wild-type mice injected with this vector. In contrast, the anti-GAA IgG1 response was diminished in MyD88KO mice, and showed a trend toward a decrease in TRIFKO mice. In addition, the vector genome and GAA activity were significantly higher in MyD88KO livers compared with wild-type livers, suggesting reduced cytotoxic T cell responses. Importantly, elevated CD4(+) T cells were detected by immunohistochemistry in MyD88KO livers. When adoptively transferred to wild-type mice, these CD4(+) T cells have an ability to suppress antibody responses against AAV2/8-CBhGAA and to prevent further immunization against rhGAA. Our study suggests that the MyD88 deficiency leads to the suppression of deleterious immune responses to AAV2/8-CBhGAA, which has implications for gene therapy in Pompe disease. PMID:23514839

  16. Adeno-associated virus type 2 rep gene-mediated inhibition of basal gene expression of human immunodeficiency virus type 1 involves its negative regulatory functions.

    PubMed Central

    Oelze, I; Rittner, K; Sczakiel, G

    1994-01-01

    Adeno-associated virus type 2 (AAV-2), a human parvovirus which is apathogenic in adults, inhibits replication and gene expression of human immunodeficiency virus type 1 (HIV-1) in human cells. The rep gene of AAV-2, which was shown earlier to be sufficient for this negative interference, also down-regulated the expression of heterologous sequences driven by the long terminal repeat (LTR) of HIV-1. This effect was observed in the absence of the HIV-1 transactivator Tat, i.e., at basal levels of LTR-driven transcription. In this work, we studied the involvement of functional subsequences of the HIV-1 LTR in rep-mediated inhibition in the absence of Tat. Mutated LTRs driving an indicator gene (cat) were cointroduced into human SW480 cells together with rep alone or with double-stranded DNA fragments or RNA containing sequences of the HIV-1 LTR. The results indicate that rep strongly enhances the function of negative regulatory elements of the LTR. In addition, the experiments revealed a transcribed sequence element located within the TAR-coding sequence termed AHHH (AAV-HIV homology element derived from HIV-1) which is involved in rep-mediated inhibition. The AHHH element is also involved in down-regulation of basal expression levels in the absence of rep, suggesting that AHHH also contributes to negative regulatory functions of the LTR of HIV-1. In contrast, positive regulatory elements of the HIV-1 LTR such as the NF kappa B and SP1 binding sites have no significant influence on the rep-mediated inhibition. Images PMID:8289357

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

    PubMed Central

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

    1997-01-01

    The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences. PMID:9151837

  18. Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

    PubMed Central

    Arbetman, Alejandra E.; Lochrie, Michael; Zhou, Shangzhen; Wellman, Jennifer; Scallan, Ciaran; Doroudchi, Mohammad M.; Randlev, Britta; Patarroyo-White, Susannah; Liu, Tongyao; Smith, Peter; Lehmkuhl, Howard; Hobbs, Lea Ann; Pierce, Glenn F.; Colosi, Peter

    2005-01-01

    Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies. PMID:16306595

  19. X-Linked Inhibitor of Apoptosis Protein-Mediated Attenuation of Apoptosis, Using a Novel Cardiac-Enhanced Adeno-Associated Viral Vector

    PubMed Central

    Piacentino III, Valentino; Milano, Carmelo A.; Bolanos, Michael; Schroder, Jacob; Messina, Emily; Cockrell, Adam S.; Jones, Edward; Krol, Ava; Bursac, Nenad; Mao, Lan; Devi, Gayathri R.; Samulski, R. Jude

    2012-01-01

    Abstract Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or β-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector. PMID:22339372

  20. Efficient retrograde transport of adeno-associated virus type 8 to spinal cord and dorsal root ganglion after vector delivery in muscle.

    PubMed

    Zheng, Hui; Qiao, Chunping; Wang, Chi-Hsien; Li, Juan; Li, Jianbin; Yuan, Zhenhua; Zhang, Cheng; Xiao, Xiao

    2010-01-01

    The peripheral nervous system (PNS), including peripheral nerves and dorsal root ganglion (DRG), is involved in numerous neurological disorders, such as peripheral neuropathies (diabetic neuropathy, chronic pain, etc.) and demyelination diseases (multiple sclerosis, congenital muscular dystrophy, Charcot-Marie-Tooth disease, etc.). Effective clinical interventions for those diseases are very limited. Gene therapy represents a novel therapeutic strategy for the PNS diseases, especially with simply and minimally invasive delivery methods. Previously, we have shown that adeno-associated virus type 8 (AAV8) can efficiently transduce muscles body wide by a simple intraperitoneal injection in neonatal mice. In this study, we investigated the capacity of AAV8 in transducing PNS in neonatal mice by intraperitoneal injection and also in adult mice by intramuscular injection. Efficient and long-term gene transfer was found in the white matter of the spinal cord, DRG neurons, and peripheral nerves in both groups, treated either as neonates or as adults, particularly neonates. In the adult mice injected with AAV8 in tibialis anterior and gastrocnemius muscles in one of the hind legs, more neurons were transduced in the lower part of the spinal cord than in the upper part; the DRG neurons were transduced more on the vector-injected side than in the contralateral uninjected side. Few cells in the gray matter of the spinal cord were transduced regardless of the delivery methods and age of the mice. These results support the mechanism of vector retrograde transport and suggest that AAV8 crosses blood-nerve barrier poorly. Our finding should have important implications in gene therapy for peripheral neurological disorders. PMID:19719401

  1. Cell-Type-Specific Characteristics Modulate the Transduction Efficiency of Adeno-Associated Virus Type 2 and Restrain Infection of Endothelial Cells

    PubMed Central

    Pajusola, Katri; Gruchala, Marcin; Joch, Hana; Lüscher, Thomas F.; Ylä-Herttuala, Seppo; Büeler, Hansruedi

    2002-01-01

    Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated. PMID:12388714

  2. Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells.

    PubMed

    Pajusola, Katri; Gruchala, Marcin; Joch, Hana; Lüscher, Thomas F; Ylä-Herttuala, Seppo; Büeler, Hansruedi

    2002-11-01

    Adeno-associated viruses (AAVs) are promising vectors for various gene therapy applications due to their long-lasting transgene expression and wide spectrum of target cells. Recently, however, it has become apparent that there are considerable differences in the efficiencies of transduction of different cell types by AAVs. Here, we analyzed the efficiencies of transduction and the transport mechanisms of AAV type 2 (AAV-2) in different cell types, emphasizing endothelial cells. Expression analyses in both cultured cells and the rabbit carotid artery assay showed a remarkably low level of endothelial cell transduction in comparison to the highly permissive cell types. The study of the endosomal pathways of AAV-2 with fluorescently labeled virus showed clear targeting of the Golgi area in permissive cell lines, but this phenomenon was absent in the endothelial cell line EAhy-926. On the other hand, the response to the block of endosomal acidification by bafilomycin A1 also showed differences among the permissive cell types. We also analyzed the effect of proteasome inhibitors on endothelial cells, but their impact on the primary cells and in vivo was not significant. On the contrary, analysis of the expression pattern of heparan sulfate proteoglycans (HSPGs), the primary receptors of AAV-2, revealed massive deposits of HSPG in the extracellular matrix of endothelial cells. The matrix-associated receptors may therefore compete for virus binding and reduce transduction in endothelial cells. Accordingly, in endothelial cells detached from their matrix, AAV-2 transduction was significantly increased. Altogether, these results point to a more complex cell-type-specific mode of transduction of AAV-2 than previously appreciated. PMID:12388714

  3. The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

    PubMed Central

    Rahman, Shamim H.; Bobis-Wozowicz, Sylwia; Chatterjee, Debanjana; Gellhaus, Katharina; Pars, Kaweh; Heilbronn, Regine; Jacobs, Roland

    2013-01-01

    Abstract Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord–derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3′-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3′-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications. PMID:23072634

  4. The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing

    PubMed Central

    Raupp, Christina; Naumer, Matthias; Müller, Oliver J.; Gurda, Brittney L.; Agbandje-McKenna, Mavis

    2012-01-01

    Adeno-associated virus (AAV) has attracted considerable interest as a vector for gene therapy owing its lack of pathogenicity and the wealth of available serotypes with distinct tissue tropisms. One of the most promising isolates for vector development, based on its superior gene transfer efficiency to the liver in small animals compared to AAV type 2 (AAV2), is AAV8. Comparison of the in vivo gene transduction of rAAV2 and rAAV8 in mice showed that single amino acid exchanges in the 3-fold protrusions of AAV8 in the surface loops comprised of residues 581 to 584 and 589 to 592 to the corresponding amino acids of AAV2 and vice versa had a strong influence on transduction efficiency and tissue tropism. Surprisingly, not only did conversion of AAV8 to AAV2 cap sequences increase the transduction efficiency and change tissue tropism but so did the reciprocal conversion of AAV2 to AAV8. Insertion of new peptide motifs at position 590 in AAV8 also enabled retargeting of AAV8 capsids to specific tissues, suggesting that these sequences can interact with receptors on the cell surface. However, a neutralizing monoclonal antibody that binds to amino acids 588QQNTA592 of AAV8 does not prevent cell binding and virus uptake, indicating that this region is not necessary for receptor binding but rather that the antibody interferes with an essential step of postattachment processing in which the 3-fold protrusion is also involved. This study supports a multifunctional role of the 3-fold region of AAV capsids in the infection process. PMID:22718833

  5. Long-Term Rescue of Retinal Structure and Function by Rhodopsin RNA Replacement with a Single Adeno-Associated Viral Vector in P23H RHO Transgenic Mice

    PubMed Central

    Mao, Haoyu; Gorbatyuk, Marina S.; Rossmiller, Brian; Hauswirth, William W.

    2012-01-01

    Abstract Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adeno-associated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease. PMID:22289036

  6. Mapping the Structural Determinants Responsible for Enhanced T Cell Activation to the Immunogenic Adeno-Associated Virus Capsid from Isolate Rhesus 32.33

    PubMed Central

    Mays, Lauren E.; Wang, Lili; Tenney, Rebeca; Bell, Peter; Nam, Hyun-Joo; Lin, Jianping; Gurda, Brittney; Van Vliet, Kim; Mikals, Kyle; Agbandje-McKenna, Mavis

    2013-01-01

    Avoiding activation of immunity to vector-encoded proteins is critical to the safe and effective use of adeno-associated viral (AAV) vectors for gene therapy. While commonly used serotypes, such as AAV serotypes 1, 2, 7, 8, and 9, are often associated with minimal and/or dysfunctional CD8+ T cell responses in mice, the threshold for immune activation appears to be lower in higher-order species. We have modeled this discrepancy within the mouse by identifying two capsid variants with differential immune activation profiles: AAV serotype 8 (AAV8) and a hybrid between natural rhesus isolates AAVrh32 and AAVrh33 (AAVrh32.33). Here, we aimed to characterize the structural determinants of the AAVrh32.33 capsid that augment cellular immunity to vector-encoded proteins or those of AAV8 that may induce tolerance. We hypothesized that the structural domain responsible for differential immune activation could be mapped to surface-exposed regions of the capsid, such as hypervariable regions (HVRs) I to IX of VP3. To test this, a series of hybrid AAV capsids was constructed by swapping domains between AAV8 and AAVrh32.33. By comparing their ability to generate transgene-specific T cells in vivo versus the stability of transgene expression in the muscle, we confirmed that the functional domain lies within the VP3 portion of the capsid. Our studies were able to exclude the regions of VP3 which are not sufficient for augmenting the cellular immune response, notably, HVRs I, II, and V. We have also identified HVR IV as a region of interest in conferring the efficiency and stability of muscle transduction to AAVrh32.33. PMID:23720715

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

    PubMed Central

    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

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

    PubMed Central

    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

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

    PubMed

    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

  10. Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

    PubMed

    Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

    2014-04-01

    Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

  11. Long-Term Efficacy Following Readministration of an Adeno-Associated Virus Vector in Dogs with Glycogen Storage Disease Type Ia

    PubMed Central

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

    2012-01-01

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

  12. Long-term efficacy following readministration of an adeno-associated virus vector in dogs with glycogen storage disease type Ia.

    PubMed

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

    2012-04-01

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

  13. Three Year Follow-Up after Unilateral Subretinal Delivery of Adeno-Associated Virus in Patients with Leber Congenital Amaurosis Type 2

    PubMed Central

    Testa, Francesco; Maguire, Albert M; Rossi, Settimio; Pierce, Eric A; Melillo, Paolo; Marshall, Kathleen; Banfi, Sandro; Surace, Enrico M.; Sun, Junwei; Acerra, Carmela; Wright, J. Fraser; Wellman, Jennifer; High, Katherine A; Auricchio, Alberto; Bennett, Jean; Simonelli, Francesca

    2012-01-01

    Objective The aim of the current study is to show the clinical data of long-term (3 year) follow-up of five patients affected by Leber Congenital Amaurosis type 2 (LCA2) treated with a single unilateral injection of AAV2-hRPE65v2. Design clinical trial Participants five LCA2 patients with RPE65 gene mutations Methods After informed consent and confirmation of trial eligibility criteria, the eye with worse visual function was selected for subretinal delivery of Adeno-Associated Virus (AAV2-hRPE65v2). Subjects were evaluated before and after surgery at designated follow-up visits (1, 2, 3, 14, 30, 60, 90, 180, 270, 365 days, 1.5 years and 3 years) by complete ophthalmic examination. Efficacy for each subject was monitored with best corrected visual acuity, kinetic visual field, nystagmus testing and pupillary light reflex. Main Outcome Measures best corrected visual acuity, kinetic visual field, nystagmus testing and pupillary light reflex. Results The data showed a statistically significant improvement of best corrected visual acuity between baseline and 3 years after treatment in the treated eye (p<0.001). In all patients we observed an enlargement of the areas of visual field, which remained stable till 3 years post injection (average values: baseline 1058 deg2 vs 3 years post treatment: 4630 deg2) and a reduction of the nystagmus frequency compared to baseline at the 3 year time-point. Furthermore, a statistically significant difference was observed in the pupillary constriction of the treated eye (p<0.05) compared to the untreated eye in three patients at 1 and 3-year time-points. No patients suffered serious adverse events related to the vector in the 3 year post-injection period. Conclusions The long-term follow-up data (3 years) on the 5-patient Italian cohort involved in the LCA2 gene therapy clinical trial clearly showed a stability of improvement in visual and retinal function that had been achieved a few months after treatment. Longitudinal data analysis

  14. Adeno-associated virus-2-mediated TGF-β1 microRNA transfection inhibits adhesion formation after digital flexor tendon injury.

    PubMed

    Wu, Y F; Mao, W F; Zhou, Y L; Wang, X T; Liu, P Y; Tang, J B

    2016-02-01

    Adhesion formation after digital flexor tendon injury greatly affects gliding function of the tendon, which is a major clinical complication after hand surgery. Transforming growth factor beta 1 (TGF-β1) has a critical role in adhesion formation during tendon healing. Persistent regulation of TGF-β1 through application of microRNA (miRNA) specifically inhibiting the function of TGF-β1 (TGF-β1-miRNA) holds promise for treatment of such a complication. Adeno-associated virus (AAV) was used to transfer TGF-β1-miRNA to the chicken digital flexor tendons, which had been injured and surgically repaired. Four doses of AAV2-TGF-β1-miRNA (2 × 10(11), 2 × 10(10), 2 × 10(9) and 2 × 10(8) vector genomes (vg)) were used to determine the transfection efficiency. At postoperative 3 weeks, we found a positive correlation between the administered AAV2-TGF-β1-miRNA doses and transfection efficiency. The transfection rate ranged from 10% to 77% as the doses increased. Production of TGF-β1 protein in the tendons decreased on increasing vector dosage. When 2 × 10(11) and 2 × 10(10) vg were injected into the tendon, gliding excursion of the repaired tendon and work of flexion of chicken toes were significantly increased and adhesion score decreased 6 and 8 weeks later, indicating the improvement of tendon gliding and decreases in adhesion formations. However, the ultimate strength of the tendons transfected at the dose of 2 × 10(10) vg was 12-24% lower than that of the control tendons. The results of this study demonstrate that application of TGF-β1-miRNA had a mixed impact on tendon healing: adhesion around the tendon is reduced but strength of the tendon healing is adversely affected. Future studies should aim at maintaining the beneficial effects of reducing tendon adhesions, while eliminating the adverse effects of decreasing the healing strength. PMID:26381218

  15. High-Throughput Functional MicroRNA Profiling Using Recombinant AAV-Based MicroRNA Sensor Arrays

    PubMed Central

    Tian, Wenhong; Dong, Xiaoyan; Wu, Xiaobing; Wu, Zhijian

    2014-01-01

    There is a lack of methods for high-throughput functional microRNA (miRNA) profiling. In this chapter, we describe a recombinant adeno-associated virus-based miRNA sensor array (miRNA Asensor array), which is able to profile functional miRNAs in cultured cells. The preparation of an miRNA Asensor array and its usage are discussed. PMID:24026702

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

    PubMed Central

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

    2016-01-01

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

  17. Virus-Mediated Knockdown of Nav1.3 in Dorsal Root Ganglia of STZ-Induced Diabetic Rats Alleviates Tactile Allodynia

    PubMed Central

    Tan, Andrew M; Samad, Omar A; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2015-01-01

    Diabetic neuropathic pain affects a substantial number of people and represents a major public health problem. Available clinical treatments for diabetic neuropathic pain remain only partially effective and many of these treatments carry the burden of side effects or the risk of dependence. The misexpression of sodium channels within nociceptive neurons contributes to abnormal electrical activity associated with neuropathic pain. Voltage-gated sodium channel Nav1.3 produces tetrodotoxin-sensitive sodium currents with rapid repriming kinetics and has been shown to contribute to neuronal hyperexcitability and ectopic firing in injured neurons. Suppression of Nav1.3 activity can attenuate neuropathic pain induced by peripheral nerve injury. Previous studies have shown that expression of Nav1.3 is upregulated in dorsal root ganglion (DRG) neurons of diabetic rats that exhibit neuropathic pain. Here, we hypothesized that viral-mediated knockdown of Nav1.3 in painful diabetic neuropathy would reduce neuropathic pain. We used a validated recombinant adeno-associated virus (AAV)-shRNA-Nav1.3 vector to knockdown expression of Nav1.3, via a clinically applicable intrathecal injection method. Three weeks following vector administration, we observed a significant rate of transduction in DRGs of diabetic rats that concomitantly reduced neuronal excitability of dorsal horn neurons and reduced behavioral evidence of tactile allodynia. Taken together, these findings offer a novel gene therapy approach for addressing chronic diabetic neuropathic pain. PMID:26101954

  18. Virus-Mediated Knockdown of Nav1.3 in Dorsal Root Ganglia of STZ-Induced Diabetic Rats Alleviates Tactile Allodynia.

    PubMed

    Tan, Andrew M; Samad, Omar A; Dib-Hajj, Sulayman D; Waxman, Stephen G

    2015-01-01

    Diabetic neuropathic pain affects a substantial number of people and represents a major public health problem. Available clinical treatments for diabetic neuropathic pain remain only partially effective and many of these treatments carry the burden of side effects or the risk of dependence. The misexpression of sodium channels within nociceptive neurons contributes to abnormal electrical activity associated with neuropathic pain. Voltage-gated sodium channel Nav1.3 produces tetrodotoxin-sensitive sodium currents with rapid repriming kinetics and has been shown to contribute to neuronal hyperexcitability and ectopic firing in injured neurons. Suppression of Nav1.3 activity can attenuate neuropathic pain induced by peripheral nerve injury. Previous studies have shown that expression of Nav1.3 is upregulated in dorsal root ganglion (DRG) neurons of diabetic rats that exhibit neuropathic pain. Here, we hypothesized that viral-mediated knockdown of Nav1.3 in painful diabetic neuropathy would reduce neuropathic pain. We used a validated recombinant adeno-associated virus (AAV)-shRNA-Nav1.3 vector to knockdown expression of Nav1.3, via a clinically applicable intrathecal injection method. Three weeks following vector administration, we observed a significant rate of transduction in DRGs of diabetic rats that concomitantly reduced neuronal excitability of dorsal horn neurons and reduced behavioral evidence of tactile allodynia. Taken together, these findings offer a novel gene therapy approach for addressing chronic diabetic neuropathic pain. PMID:26101954

  19. Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABAA receptors in cortical pyramidal cells

    PubMed Central

    Sumegi, Mate; Fukazawa, Yugo; Matsui, Ko; Lorincz, Andrea; Eyre, Mark D; Nusser, Zoltan; Shigemoto, Ryuichi

    2012-01-01

    Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABAARγ277Ilox) in which the γ2 subunit of the GABAA receptor (GABAAR) was mutated to become zolpidem insensitive (γ277I) and used viral vectors to swap γ277I with wild-type, zolpidem-sensitive γ2 subunits (γ277F). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABAARγ2−/− mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 (AU1γ277F) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the AU1γ277F subunit (Cre-2A-AU1γ277F) into the neocortex of GABAARγ277Ilox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the AU1γ277F subunits in perisomatic GABAergic synapses. In line with this, miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ277I and AU1γ277F GABAARs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that

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

    PubMed Central

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

    2016-01-01

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

  1. 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.

    PubMed

    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

  2. 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

    PubMed Central

    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

  3. Comprehensive Small RNA-Seq of Adeno-Associated Virus (AAV)-Infected Human Cells Detects Patterns of Novel, Non-Coding AAV RNAs in the Absence of Cellular miRNA Regulation.

    PubMed

    Stutika, Catrin; Mietzsch, Mario; Gogol-Döring, Andreas; Weger, Stefan; Sohn, Madlen; Chen, Wei; Heilbronn, Regine

    2016-01-01

    Most DNA viruses express small regulatory RNAs, which interfere with viral or cellular gene expression. For adeno-associated virus (AAV), a small ssDNA virus with a complex biphasic life cycle miRNAs or other small regulatory RNAs have not yet been described. This is the first comprehensive Illumina-based RNA-Seq analysis of small RNAs expressed by AAV alone or upon co-infection with helper adenovirus or HSV. Several hotspots of AAV-specific small RNAs were detected mostly close to or within the AAV-ITR and apparently transcribed from the newly identified anti-p5 promoter. An additional small RNA hotspot was located downstream of the p40 promoter, from where transcription of non-coding RNAs associated with the inhibition of adenovirus replication were recently described. Parallel detection of known Ad and HSV miRNAs indirectly validated the newly identified small AAV RNA species. The predominant small RNAs were analyzed on Northern blots and by human argonaute protein-mediated co-immunoprecipitation. None of the small AAV RNAs showed characteristics of bona fide miRNAs, but characteristics of alternative RNA processing indicative of differentially regulated AAV promoter-associated small RNAs. Furthermore, the AAV-induced regulation of cellular miRNA levels was analyzed at different time points post infection. In contrast to other virus groups AAV infection had virtually no effect on the expression of cellular miRNA, which underscores the long-established concept that wild-type AAV infection is apathogenic. PMID:27611072

  4. Employing a Gain-of-Function Factor IX Variant R338L to Advance the Efficacy and Safety of Hemophilia B Human Gene Therapy: Preclinical Evaluation Supporting an Ongoing Adeno-Associated Virus Clinical Trial

    PubMed Central

    Sun, Junjiang; Gui, Tong; Hu, Genlin; Hannah, William B.; Wichlan, David G.; Wu, Zhijian; Grieger, Joshua C.; Li, Chengwen; Suwanmanee, Thipparat; Stafford, Darrel W.; Booth, Carmen J.; Samulski, Jade J.; Kafri, Tal; McPhee, Scott W.J.

    2015-01-01

    Abstract Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose–response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2–500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8+ T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX–/– mice 8–10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma

  5. Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels.

    PubMed Central

    Kyöstiö, S R; Owens, R A; Weitzman, M D; Antoni, B A; Chejanovsky, N; Carter, B J

    1994-01-01

    The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p5 and p19 promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p19 transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and p19 mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p5 and p19 mRNA accumulation was inhibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 showed that the ability of Rep78 to decrease p5 and p19 mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p5 and p19 transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein with a mutation in the putative nucleoside triphosphate binding site inhibited expression from p5 but not from p19, suggesting that the regulation of p5 transcript levels by Rep78 and Rep68 differs from that of p19. A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not required for negative regulation of p5 and p19 transcript levels and that the regulation of p19 mRNA levels by Rep78 did not require the presence

  6. Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

    PubMed

    Monahan, Paul E; Sun, Junjiang; Gui, Tong; Hu, Genlin; Hannah, William B; Wichlan, David G; Wu, Zhijian; Grieger, Joshua C; Li, Chengwen; Suwanmanee, Thipparat; Stafford, Darrel W; Booth, Carmen J; Samulski, Jade J; Kafri, Tal; McPhee, Scott W J; Samulski, R Jude

    2015-02-01

    Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity

  7. Recombinant AAV as a Platform for Translating the Therapeutic Potential of RNA Interference

    PubMed Central

    Borel, Florie; Kay, Mark A; Mueller, Christian

    2014-01-01

    RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed. PMID:24352214

  8. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

  9. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    SciTech Connect

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  10. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

    SciTech Connect

    Brodzik, R.; Bandurska, K.; Deka, D.; Golovkin, M.; Koprowski, H. . E-mail: h_koprowski@jefferson.edu

    2005-12-16

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.

  11. Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta.

    PubMed

    Brodzik, R; Bandurska, K; Deka, D; Golovkin, M; Koprowski, H

    2005-12-16

    Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP. PMID:16236249

  12. Intranasal Delivery of Recombinant NT4-NAP/AAV Exerts Potential Antidepressant Effect.

    PubMed

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

    2016-06-01

    The present study was designed to construct a recombinant adeno-associated virus (rAAV) which can express NAP in the brain and examine whether this virus can produce antidepressant effects on C57 BL/6 mice that had been subjected to open field test and forced swimming test, via nose-to-brain pathway. When the recombinant plasmid pGEM-T Easy/NT4-NAP was digested by EcoRI, 297 bp fragments can be obtained and NT4-NAP sequence was consistent with the designed sequence confirmed by DNA sequencing. When the recombinant plasmid pSSCMV/NT4-NAP was digested by EcoRI, 297 bp fragments is visible. Immunohistochemical staining of fibroblasts revealed that expression of NAP was detected in NT4-NAP/AAV group. Intranasal delivery of NT4-NAP/AAV significantly reduced immobility time when the FST was performed after 1 day from the last administration. The effects observed in the FST could not be attributed to non-specific increases in activity since intranasal delivery of NT4-NAP/AAV did not alter the behavior of the mice during the open field test. The results indicated that a recombinant AAV vector which could express NAP in cells was successfully constructed and NAP may be a potential target for therapeutic action of antidepressant treatment. PMID:26846142

  13. Homologous recombination is required for AAV-mediated gene targeting

    PubMed Central

    Vasileva, Ana; Linden, R. Michael; Jessberger, Rolf

    2006-01-01

    High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ∼5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR. PMID:16822856

  14. Ebola virus mediated infectivity is restricted in canine and feline cells.

    PubMed

    Han, Ziying; Bart, Stephen M; Ruthel, Gordon; Vande Burgt, Nathan H; Haines, Kathleen M; Volk, Susan W; Vite, Charles H; Freedman, Bruce D; Bates, Paul; Harty, Ronald N

    2016-01-15

    Ebolaviruses and marburgviruses belong to the Filoviridae family and often cause severe, fatal hemorrhagic fever in humans and non-human primates. The magnitude of the 2014 outbreak in West Africa and the unprecedented emergence of Ebola virus disease (EVD) in the United States underscore the urgency to better understand the dynamics of Ebola virus infection, transmission and spread. To date, the susceptibility and possible role of domestic animals and pets in the transmission cycle and spread of EVD remains unclear. We utilized infectious VSV recombinants and lentivirus pseudotypes expressing the EBOV surface glycoprotein (GP) to assess the permissiveness of canine and feline cells to EBOV GP-mediated entry. We observed a general restriction in EBOV-mediated infection of primary canine and feline cells. To address the entry mechanism, we used cells deficient in NPC1, a host protein implicated in EBOV entry, and a pharmacological blockade of cholesterol transport, to show that an NPC1-dependent mechanism of EBOV entry is conserved in canine and feline cells. These data demonstrate that cells of canine and feline origin are susceptible to EBOV GP mediated infection; however, infectivity of these cells is reduced significantly compared to controls. Moreover, these data provide new insights into the mechanism of EBOV GP mediated entry into cells of canine and feline origin. PMID:26711035

  15. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  16. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  17. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  18. Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors

    PubMed Central

    Zeltner, Nadja; Kohlbrenner, Erik; Clément, Nathalie; Weber, Thomas; Linden, R. Michael

    2010-01-01

    Viral vectors derived from adeno-associated viruses (AAV) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently demonstrated immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a “ceiling” for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and more strikingly only approximately one of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy. PMID:20336156

  19. Recombinant AAV-directed gene therapy for type I glycogen storage diseases

    PubMed Central

    Chou, JY; Mansfield, BC

    2011-01-01

    Introduction Glycogen storage disease (GSD) type Ia and Ib are disorders of impaired glucose homeostasis affecting the liver and kidney. GSD-Ib also affects neutrophils. Current dietary therapies cannot prevent long-term complications. In animal studies, recombinant adeno-associated virus (rAAV) vector-mediated gene therapy can correct or minimize multiple aspects of the disorders, offering hope for human gene therapy. Areas covered A summary of recent progress in rAAV-mediated gene therapy for GSD-I; strategies to improve rAAV-mediated gene delivery, transduction efficiency and immune avoidance; and vector refinements that improve expression. Expert opinion rAAV-mediated gene delivery to the liver can restore glucose homeostasis in preclinical models of GSD-I, but some long-term complications of the liver and kidney remain. Gene therapy for GSD-Ib is less advanced than for GSD-Ia and only transient correction of myeloid dysfunction has been achieved. A question remains whether a single rAAV vector can meet the expression efficiency and tropism required to treat all aspects of GSD-I, or if a multi-prong approach is needed. An understanding of the strengths and weaknesses of rAAV vectors in the context of strategies to achieve efficient transduction of the liver, kidney, and hematopoietic stem cells is required for treating GSD-I. PMID:21504389

  20. Kupffer Cells Support Hepatitis B Virus-Mediated CD8+ T Cell Exhaustion via Hepatitis B Core Antigen-TLR2 Interactions in Mice.

    PubMed

    Li, Min; Sun, Rui; Xu, Long; Yin, Wenwei; Chen, Yongyan; Zheng, Xiaodong; Lian, Zhexiong; Wei, Haiming; Tian, Zhigang

    2015-10-01

    Hepatitis B virus (HBV) persistence is a fundamental process in chronic HBV infection and a key factor in all related liver diseases; however, the mechanisms have yet to be elucidated. We studied the role of TLR2 in HBV persistence using a well-established HBV-carrier mouse model generated by hydrodynamically injecting a phospho-adeno-associated virus/HBV1.2 plasmid into mice. We found that a genetic deficiency in TLR2 improves HBV elimination, whereas activating TLR2 led to more stable HBV persistence, suggesting that TLR2 activation is critical in HBV persistence. Furthermore, we noted that TLR2 activation could inhibit CD8(+) T cell function, causing the exhaustion phenotype in HBV-carrier mice, because TLR2 deficiency might rescue CD8(+) T cell function in a cellular adoptive experiment. TLR2 expression on Kupffer cells (KCs) was upregulated in HBV-carrier mice, which accounts for HBV persistence, because the difference in anti-HBV immunity between HBV-carrier wild-type and Tlr2(-/-) mice did not exist after KC depletion. In addition, similar to TLR2 deficiency, after KC depletion, CD8(+) T cells were more efficiently activated in HBV-carrier mice, leading to rapid HBV elimination. KCs produced more IL-10 upon TLR2 activation in response to direct hepatitis B core Ag stimulation, and the elevated IL-10 inhibited CD8(+) T cell function in HBV-carrier mice, because IL-10 deficiency or anti-IL-10R treatment resulted in CD8(+) T cells with stronger antiviral function. In conclusion, KCs support liver tolerance by inducing anti-HBV CD8(+) T cell exhaustion via IL-10 production after TLR2 activation by hepatitis B core Ag stimulation. PMID:26304988

  1. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria

    PubMed Central

    Harding, CO; Gillingham, MB; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, DD

    2009-01-01

    Novel recombinant adeno-associated virus vectors pseudo-typed with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pahenu2 mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5±2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism. PMID:16319949

  2. Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria.

    PubMed

    Harding, C O; Gillingham, M B; Hamman, K; Clark, H; Goebel-Daghighi, E; Bird, A; Koeberl, D D

    2006-03-01

    Novel recombinant adeno-associated virus vectors pseudotyped with serotype 8 capsid (rAAV2/8) have recently shown exciting promise as effective liver-directed gene transfer reagents. We have produced a novel liver-specific rAAV2/8 vector expressing the mouse phenylalanine hydroxylase (Pah) cDNA and have administered this vector to hyperphenylalaninemic PAH-deficient Pah(enu2) mice, a model of human phenylketonuria (PKU). Our hypothesis was that this vector would produce sufficient hepatocyte transduction frequency and PAH activity to correct blood phenylalanine levels in murine PKU. Portal vein injection of recombinant AAV2/8 vector into five adult Pah(enu2) mice yielded complete and stable (up to 17 weeks) correction of serum phenylalanine levels. Liver PAH activity was corrected to 11.5+/-2.4% of wild type liver activity and was associated with a significant increase in phenylalanine clearance following parenteral phenylalanine challenge. Although questions of long-term safety and stability of expression remain, recombinant AAV2/8-mediated, liver-directed gene therapy is a promising novel treatment approach for PKU and allied inborn errors of metabolism. PMID:16319949

  3. Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity.

    PubMed

    Ge, Jingping; Liu, Ying; Jin, Liying; Gao, Dongni; Bai, Chengle; Ping, Wenxiang

    2016-08-10

    Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle disease virus (NDV) which poses a substantial threat to China's poultry industry. Conventional live vaccines against NDV are available, but they can revert to virulent strains and do not protect against mutant strains of the virus. Therefore, there is a critical unmet need for a novel vaccine that is safe, efficacious, and cost effective. Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or HN genes. To optimize antigen expression, we tested the incorporation of multiple regulatory elements including: (1) truncated vesicular stomatitis virus G protein (VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV Serotype II), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo efficacy of the viruses, we vaccinated chickens with each construct and characterized the cellular and humoral immune response to challenge with virulent NDV (F48E9). All of the vaccine constructs provided some level of protection (62.5-100% protection). The F-series of vaccines provided a greater degree of protection (87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a robust cellular and humoral response subtle differences in efficacy were observed. The combination of the WPRE and VSV-GED regulatory elements enhanced the immune response and increased antigen expression. The ITRs effectively increased the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus system is a promising platform for NDV vaccine development that combines the immunostimulatory benefits of a recombinant virus vector with the non-replicating benefits of a DNA vaccine. PMID:27015979

  4. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

    PubMed Central

    Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

    2016-01-01

    With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

  5. Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector.

    PubMed

    Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

    2016-01-01

    With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

  6. Comparison of Transduction Efficiency of Recombinant AAV Serotypes 1, 2, 5, and 8 in the Rat Nigrostriatal System

    PubMed Central

    McFarland, Nikolaus R.; Lee, Jeng-Shin; Hyman, Bradley T.; McLean, Pamela J.

    2009-01-01

    Enhanced delivery and expression of genes in specific neuronal systems is critical for the development of genetic models of neurodegenerative disease and potential gene therapy. Recent discovery of new recombinant adeno-associated viral (rAAV) capsid serotypes has resulted in improved transduction efficiency, but expression levels, spread of transgene, and potential toxicity can differ depending on brain region and among species. We compared the transduction efficiency of titer-matched rAAV 2/1, 2/5 and 2/8 to the commonly used rAAV2/2 in the rat nigrostriatal system via expression of the reporter transgene, enhanced green fluorescent protein (EGFP). Newer rAAV serotypes 2/1, 2/5 and 2/8 demonstrated marked increase in transduction and spread of EGFP expression in dopaminergic nigrostriatal neurons and projections to the striatum and globus pallidus compared to rAAV2/2 at 2 weeks post injection. The number of nigral cells transduced was greatest for rAAV2/1, but for serotypes 2/5 and 2/8 was still 2 to 3-fold higher than that for 2/2. Enhanced transduction did not cause an increase in glial cell response or toxicity. New rAAV serotypes thus promise improved gene delivery to nigrostriatal system with the potential for better models and therapeutics for Parkinson disease and other neurodegenerative disorders. PMID:19250335

  7. Systemic Administration of a Recombinant AAV1 Vector Encoding IGF-1 Improves Disease Manifestations in SMA Mice

    PubMed Central

    Tsai, Li-Kai; Chen, Chien-Lin; Ting, Chen-Hung; Lin-Chao, Sue; Hwu, Wuh-Liang; Dodge, James C; Passini, Marco A; Cheng, Seng H

    2014-01-01

    Spinal muscular atrophy is a progressive motor neuron disease caused by a deficiency of survival motor neuron. In this study, we evaluated the efficacy of intravenous administration of a recombinant adeno-associated virus (AAV1) vector encoding human insulin-like growth factor-1 (IGF-1) in a severe mouse model of spinal muscular atrophy. Measurable quantities of human IGF-1 transcripts and protein were detected in the liver (up to 3 months postinjection) and in the serum indicating that IGF-1 was secreted from the liver into systemic circulation. Spinal muscular atrophy mice administered AAV1-IGF-1 on postnatal day 1 exhibited a lower extent of motor neuron degeneration, cardiac and muscle atrophy as well as a greater extent of innervation at the neuromuscular junctions compared to untreated controls at day 8 posttreatment. Importantly, treatment with AAV1-IGF-1 prolonged the animals' lifespan, increased their body weights and improved their motor coordination. Quantitative polymerase chain reaction and western blot analyses showed that AAV1-mediated expression of IGF-1 led to an increase in survival motor neuron transcript and protein levels in the spinal cord, brain, muscles, and heart. These data indicate that systemically delivered AAV1-IGF-1 can correct several of the biochemical and behavioral deficits in spinal muscular atrophy mice through increasing tissue levels of survival motor neuron. PMID:24814151

  8. Assessment of Tropism and Effectiveness of New Primate-Derived Hybrid Recombinant AAV Serotypes in the Mouse and Primate Retina

    PubMed Central

    Lipinski, Daniel M.; Singh, Mandeep S.; Mouravlev, Alexandre; You, Qisheng; Barnard, Alun R.; Hankins, Mark W.; During, Matthew J.; MacLaren, Robert E.

    2013-01-01

    Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4−/− mouse which is a model for Stargardt disease and in the Pde6brd1/rd1 mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4−/− mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments. PMID:23593201

  9. Enhancing Transgene Expression from Recombinant AAV8 Vectors in Different Tissues Using Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element

    PubMed Central

    Wang, Lizheng; Wang, Zixuan; Zhang, Fangfang; Zhu, Rui; Bi, Jinpeng; Wu, Jiaxin; Zhang, Haihong; Wu, Hui; Kong, Wei; Yu, Bin; Yu, Xianghui

    2016-01-01

    Adeno-associated virus (AAV) vectors have been utilized extensively in gene therapy and gene function studies, as strong transgene expression is a prerequisite for positive outcomes. AAV8 was reported as the most efficient AAV serotype for transduction of the liver, brain and muscle compared with other serotypes. However, AAV8-mediated transduction of human hepatocytes is rather poor with approximately 20-fold lower efficiency compared with that of mouse hepatocytes. Therefore, we applied the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) to enhance AAV8-mediated transgene expression driven by a combination promoter (CAG promoter) with a CMV-IE enhancer and chicken beta-actin promoter for a more efficient viral vector. Transgene expression from recombinant AAV8 (rAAV8) vectors harboring a red fluorescent protein (RFP) reporter gene with or without WPRE were evaluated in vitro and in vivo. The results demonstrated that WPRE improved AAV8-mediated RFP expression in different cell lines with clear increases of transgene expression in the liver, brain or muscle of animals. The findings of this study will help to substantially reduce the quantity of viral particles that must be injected in order to reach a therapeutic level of transgene expression in gene therapy. Consequently, such dose reductions may lessen the potential risks associated with high doses of viral vectors. PMID:27076785

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

    NASA Astrophysics Data System (ADS)

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

    2014-07-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes

    PubMed Central

    von Jonquieres, Georg; Fröhlich, Dominik; Klugmann, Claudia B.; Wen, Xin; Harasta, Anne E.; Ramkumar, Roshini; Spencer, Ziggy H. T.; Housley, Gary D.; Klugmann, Matthias

    2016-01-01

    Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene

  13. Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes.

    PubMed

    von Jonquieres, Georg; Fröhlich, Dominik; Klugmann, Claudia B; Wen, Xin; Harasta, Anne E; Ramkumar, Roshini; Spencer, Ziggy H T; Housley, Gary D; Klugmann, Matthias

    2016-01-01

    Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene

  14. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  15. Assessment of toxicity and biodistribution of recombinant AAV8 vector–mediated immunomodulatory gene therapy in mice with Pompe disease

    PubMed Central

    Wang, Gensheng; Young, Sarah P; Bali, Deeksha; Hutt, Julie; Li, Songtao; Benson, Janet; Koeberl, Dwight D

    2014-01-01

    A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 1013 vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4+ (but not CD8+) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease. PMID:26015962

  16. Assessment of toxicity and biodistribution of recombinant AAV8 vector-mediated immunomodulatory gene therapy in mice with Pompe disease.

    PubMed

    Wang, Gensheng; Young, Sarah P; Bali, Deeksha; Hutt, Julie; Li, Songtao; Benson, Janet; Koeberl, Dwight D

    2014-01-01

    A preclinical safety study was conducted to evaluate the short- and long-term toxicity of a recombinant adeno-associated virus serotype 8 (AAV2/8) vector that has been developed as an immune-modulatory adjunctive therapy to recombinant human acid α-glucosidase (rhGAA, Myozyme) enzyme replacement treatment (ERT) for patients with Pompe disease (AAV2/8-LSPhGAApA). The AAV2/8-LSPhGAApA vector at 1.6 × 10(13) vector particles/kg, after intravenous injection, did not cause significant short- or long-term toxicity. Recruitment of CD4(+) (but not CD8(+)) lymphocytes to the liver was elevated in the vector-dosed male animals at study day (SD) 15, and in group 8 animals at SD 113, in comparison to their respective control animals. Administration of the vector, either prior to or after the one ERT injection, uniformly prevented the hypersensitivity induced by subsequent ERT in males, but not always in female animals. The vector genome was sustained in all tissues through 16-week postdosing, except for in blood with a similar tissue tropism between males and females. Administration of the vector alone, or combined with the ERT, was effective in producing significantly increased GAA activity and consequently decreased glycogen accumulation in multiple tissues, and the urine biomarker, Glc4, was significantly reduced. The efficacy of the vector (or with ERT) was better in males than in females, as demonstrated both by the number of tissues showing significantly effective responses and the extent of response in a given tissue. Given the lack of toxicity for AAV2/8LSPhGAApA, further consideration of clinical translation is warranted in Pompe disease. PMID:26015962

  17. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  18. Similar Therapeutic Efficacy Between a Single Administration of Gene Therapy and Multiple Administrations of Recombinant Enzyme in a Mouse Model of Lysosomal Storage Disease

    PubMed Central

    Ferla, Rita; Claudiani, Pamela; Cotugno, Gabriella; Saccone, Paola; De Leonibus, Elvira

    2014-01-01

    Abstract Enzyme replacement therapy (ERT) has become the standard of care for several lysosomal storage disorders (LSDs). Despite ERT's undisputed efficacy, the requirement for multiple and costly administrations as well as ERT's limited improvement of some LSD manifestations prompts the search for better therapies. Using a mouse model of mucopolysaccharidosis VI, we compared the efficacy of a single intravascular administration of an adeno-associated viral vector targeting liver to weekly infusions of human recombinant enzyme at the same doses used in mucopolysaccharidosis VI patients. While gene therapy results in increased and stable levels of circulating enzyme up to 1 year after vector administration, ERT has typical peak-and-drop serum kinetics. Both therapies similarly reduced glycosaminoglycan levels in urine and tissues including heart valves and myocardium, with gene therapy improving skeletal skull abnormalities slightly better, although not significantly, than ERT. Both therapies seem to similarly improve animal motor performance, with gene therapy possibly associated with less animal distress. Thus, a single vector administration that converts liver into a factory organ for systemic secretion of therapeutic proteins is at least as effective as ERT in a mouse model of LSD, potentially eliminating problems with compliance and costs. Only testing in humans will prove whether this holds true in a clinical setting. PMID:24725025

  19. Temperature-sensitive miR-483 is a conserved regulator of recombinant protein and viral vector production in mammalian cells.

    PubMed

    Emmerling, Verena V; Fischer, Simon; Stiefel, Fabian; Holzmann, Karlheinz; Handrick, René; Hesse, Friedemann; Hörer, Markus; Kochanek, Stefan; Otte, Kerstin

    2016-04-01

    Cell engineering and bioprocess optimizations such as low temperature cultivation represent powerful tools to improve cellular performance and product yields of mammalian production cells. Besides monoclonal antibodies (mABs), novel biotherapeutic formats such as viral vectors will gain increasing importance. Here, we demonstrate that similar to Chinese hamster ovary (CHO) cells, product yields of recombinant adeno-associated virus (rAAV) producing HeLa cells can be markedly increased by low temperature cultivation. MicroRNAs (miRNAs) are small non-coding RNAs that critically regulate cell phenotypes. We thus investigated differential miRNA expression in response to mild hypothermia in CHO and HeLa production cells. We discovered miR-483 to be substantially up-regulated upon temperature down-shift in both cell types. Functional validation experiments revealed that introduction of miR-483 mimics led to a significant increase in both rAAV and mAB production in HeLa and CHO cells, respectively. Furthermore, inhibition of miR-483 up-regulation during mild hypothermia significantly decreased product yields, suggesting that miR-483 is a key regulator of cellular productivity in mammalian cells. In addition, miRNA target gene identification indicated that miR-483 might regulate genes directly involved in cellular survival and protein expression. Our results highlight that miR-483 is a valuable tool for product-independent engineering of mammalian production cells. PMID:26461143

  20. Similar therapeutic efficacy between a single administration of gene therapy and multiple administrations of recombinant enzyme in a mouse model of lysosomal storage disease.

    PubMed

    Ferla, Rita; Claudiani, Pamela; Cotugno, Gabriella; Saccone, Paola; De Leonibus, Elvira; Auricchio, Alberto

    2014-07-01

    Enzyme replacement therapy (ERT) has become the standard of care for several lysosomal storage disorders (LSDs). Despite ERT's undisputed efficacy, the requirement for multiple and costly administrations as well as ERT's limited improvement of some LSD manifestations prompts the search for better therapies. Using a mouse model of mucopolysaccharidosis VI, we compared the efficacy of a single intravascular administration of an adeno-associated viral vector targeting liver to weekly infusions of human recombinant enzyme at the same doses used in mucopolysaccharidosis VI patients. While gene therapy results in increased and stable levels of circulating enzyme up to 1 year after vector administration, ERT has typical peak-and-drop serum kinetics. Both therapies similarly reduced glycosaminoglycan levels in urine and tissues including heart valves and myocardium, with gene therapy improving skeletal skull abnormalities slightly better, although not significantly, than ERT. Both therapies seem to similarly improve animal motor performance, with gene therapy possibly associated with less animal distress. Thus, a single vector administration that converts liver into a factory organ for systemic secretion of therapeutic proteins is at least as effective as ERT in a mouse model of LSD, potentially eliminating problems with compliance and costs. Only testing in humans will prove whether this holds true in a clinical setting. PMID:24725025

  1. Forelimb Treatment in a Large Cohort of Dystrophic Dogs Supports Delivery of a Recombinant AAV for Exon Skipping in Duchenne Patients

    PubMed Central

    Le Guiner, Caroline; Montus, Marie; Servais, Laurent; Cherel, Yan; Francois, Virginie; Thibaud, Jean-Laurent; Wary, Claire; Matot, Béatrice; Larcher, Thibaut; Guigand, Lydie; Dutilleul, Maeva; Domenger, Claire; Allais, Marine; Beuvin, Maud; Moraux, Amélie; Le Duff, Johanne; Devaux, Marie; Jaulin, Nicolas; Guilbaud, Mickaël; Latournerie, Virginie; Veron, Philippe; Boutin, Sylvie; Leborgne, Christian; Desgue, Diana; Deschamps, Jack-Yves; Moullec, Sophie; Fromes, Yves; Vulin, Adeline; Smith, Richard H; Laroudie, Nicolas; Barnay-Toutain, Frédéric; Rivière, Christel; Bucher, Stéphanie; Le, Thanh-Hoa; Delaunay, Nicolas; Gasmi, Mehdi; Kotin, Robert M; Bonne, Gisèle; Adjali, Oumeya; Masurier, Carole; Hogrel, Jean-Yves; Carlier, Pierre; Moullier, Philippe; Voit, Thomas

    2014-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients. PMID:25200009

  2. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  3. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  4. Dre - Cre Sequential Recombination Provides New Tools for Retinal Ganglion Cell Labeling and Manipulation in Mice

    PubMed Central

    Shi, Melody; Liu, Pinghu; Dong, Lijin; Parmhans, Nadia; Popescu, Octavian; Badea, Tudor Constantin

    2014-01-01

    Background Genetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations. Methods and Findings We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3aCKOCre and Brn3bCKOCre. When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3aCKOCre or Brn3bCKOCre is removed and replaced with a Cre recombinase, generating Brn3aCre and Brn3bCre knock-in alleles. Surprisingly both Brn3aCre and Brn3bCre knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the Brn3a and Brn3b genes. We use the Brn3aCre and Brn3bCre alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs. Conclusions Dre recombinase effectively recombines the Brn3aCKOCre and Brn3bCKOCre alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3aCre and Brn3bCre alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early

  5. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  6. Recombination in electron coolers

    NASA Astrophysics Data System (ADS)

    Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.

    2000-02-01

    An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.

  7. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

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

    PubMed Central

    D’Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field. PMID:27069952

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

    PubMed Central

    Ling, Chen; Yin, Zifei; Li, Jun; Zhang, Daniel; Aslanidi, George; Srivastava, Arun

    2016-01-01

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

  10. [Recombinant antibodies against bioweapons].

    PubMed

    Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique

    2009-12-01

    The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695

  11. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  12. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  13. Construction of PR39 recombinant AAV under control of the HRE promoter and the effect of recombinant AAV on gene therapy of ischemic heart disease

    PubMed Central

    SUN, LIJUN; HAO, YUEWEN; NIE, XIAOWEI; ZHANG, XUEXIN; YANG, GUANGXIAO; WANG, QUANYING

    2012-01-01

    The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the

  14. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  15. Successful Interference with Cellular Immune Responses to Immunogenic Proteins Encoded by Recombinant Viral Vectors

    PubMed Central

    Sarukhan, Adelaida; Camugli, Sabine; Gjata, Bernard; von Boehmer, Harald; Danos, Olivier; Jooss, Karin

    2001-01-01

    Vectors derived from the adeno-associated virus (AAV) have been successfully used for the long-term expression of therapeutic genes in animal models and patients. One of the major advantages of these vectors is the absence of deleterious immune responses following gene transfer. However, AAV vectors, when used in vaccination studies, can result in efficient humoral and cellular responses against the transgene product. It is therefore important to understand the factors which influence the establishment of these immune responses in order to design safe and efficient procedures for AAV-based gene therapies. We have compared T-cell activation against a strongly immunogenic protein, the influenza virus hemagglutinin (HA), which is synthesized in skeletal muscle following gene transfer with an adenovirus (Ad) or an AAV vector. In both cases, cellular immune responses resulted in the elimination of transduced muscle fibers within 4 weeks. However, the kinetics of CD4+ T-cell activation were markedly delayed when AAV vectors were used. Upon recombinant Ad (rAd) gene transfer, T cells were activated both by direct transduction of dendritic cells and by cross-presentation of the transgene product, while upon rAAV gene transfer T cells were only activated by the latter mechanism. These results suggested that activation of the immune system by the transgene product following rAAV-mediated gene transfer might be easier to control than that following rAd-mediated gene transfer. Therefore, we tested protocols aimed at interfering with either antigen presentation by blocking the CD40/CD40L pathway or with the T-cell response by inducing transgene-specific tolerance. Long-term expression of the AAV-HA was achieved in both cases, whereas immune responses against Ad-HA could not be prevented. These data clearly underline the importance of understanding the mechanisms by which vector-encoded proteins are recognized by the immune system in order to specifically interfere with them and

  16. Recombinant AAV9-TLK1B Administration Ameliorates Fractionated Radiation-Induced Xerostomia

    PubMed Central

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

    2013-01-01

    Abstract Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviral-mediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (1011 vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline- and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; p<0.01). Salivary gland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection

  17. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  18. Recombinant renewable polyclonal antibodies

    PubMed Central

    Ferrara, Fortunato; D’Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew RM

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products. PMID:25530082

  19. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  20. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  1. Oligonucleotide recombination in bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Today, there are more than 1,500 completed or draft bacterial genome sequences available for public access. To functionally analyze these genomes and to test the hypotheses that are generated from the sequence information we require new and generically useful tools. Recombineering (genetic engineer...

  2. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  3. Recombinant vaccines against leptospirosis.

    PubMed

    Dellagostin, Odir A; Grassmann, André A; Hartwig, Daiane D; Félix, Samuel R; da Silva, Éverton F; McBride, Alan J A

    2011-11-01

    Leptospirosis is an important neglected infectious disease that occurs in urban environments, as well as in rural regions worldwide. Rodents, the principal reservoir hosts of pathogenic Leptospira spp., and other infected animals shed the bacteria in their urine. During occupational or even recreational activities, humans that come into direct contact with infected animals or with a contaminated environment, particularly water, are at risk of infection. Prevention of urban leptospirosis is largely dependent on sanitation measures that are often difficult to implement, especially in developing countries. Vaccination with inactivated whole-cell preparations (bacterins) has limited efficacy due to the wide antigenic variation of the pathogen. Intensive efforts towards developing improved recombinant vaccines are ongoing. During the last decade, many reports on the evaluation of recombinant vaccines have been published. Partial success has been obtained with some surface-exposed protein antigens. The combination of protective antigens and new adjuvants or delivery systems may result in the much-needed effective vaccine. PMID:22048111

  4. Recombinant influenza vaccines.

    PubMed

    Sedova, E S; Shcherbinin, D N; Migunov, A I; Smirnov, Iu A; Logunov, D Iu; Shmarov, M M; Tsybalova, L M; Naroditskiĭ, B S; Kiselev, O I; Gintsburg, A L

    2012-10-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery platform for a variety of genetic vaccines. Adenoviruses can efficiently penetrate the human organism through mucosal epithelium, thus providing long-term antigen persistence and induction of the innate immune response. This review provides an overview of the practicability of the production of new recombinant influenza cross-protective vaccines on the basis of adenoviral vectors expressing hemagglutinin genes of different influenza strains. PMID:23346377

  5. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  6. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  7. Virus-Mediated Chemical Changes in Rice Plants Impact the Relationship between Non-Vector Planthopper Nilaparvata lugens Stål and Its Egg Parasitoid Anagrus nilaparvatae Pang et Wang

    PubMed Central

    Gao, Guanchun; Zhou, Xiaojun; Zheng, Xusong; Sun, Yujian; Yang, Yajun; Tian, Junce; Lu, Zhongxian

    2014-01-01

    In order to clarify the impacts of southern rice black-streaked dwarf virus (SRBSDV) infection on rice plants, rice planthoppers and natural enemies, differences in nutrients and volatile secondary metabolites between infected and healthy rice plants were examined. Furthermore, the impacts of virus-mediated changes in plants on the population growth of non-vector brown planthopper (BPH), Nilaparvata lugens, and the selectivity and parasitic capability of planthopper egg parasitoid Anagrus nilaparvatae were studied. The results showed that rice plants had no significant changes in amino acid and soluble sugar contents after SRBSDV infection, and SRBSDV-infected plants had no significant effect on population growth of non-vector BPH. A. nilaparvatae preferred BPH eggs both in infected and healthy rice plants, and tended to parasitize eggs on infected plants, but it had no significant preference for infected plants or healthy plants. GC-MS analysis showed that tridecylic aldehyde occurred only in rice plants infected with SRBSDV, whereas octanal, undecane, methyl salicylate and hexadecane occurred only in healthy rice plants. However, in tests of behavioral responses to these five volatile substances using a Y-tube olfactometer, A. nilaparvatae did not show obvious selectivity between single volatile substances at different concentrations and liquid paraffin in the control group. The parasitic capability of A. nilaparvatae did not differ between SRBSDV-infected plants and healthy plant seedlings. The results suggested that SRBSDV-infected plants have no significant impacts on the non-vector planthopper and its egg parasitoid, A. nilaparvatae. PMID:25141278

  8. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  9. Dielectronic recombination theory

    SciTech Connect

    LaGattuta, K.J.

    1991-12-31

    A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  10. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  11. Did the universe recombine?

    NASA Technical Reports Server (NTRS)

    Bartlett, James G.; Stebbins, Albert

    1991-01-01

    The Zel'dovich-Sunyaev model-independent arguments for the existence of a neutral hydrogen phase is reviewed in light of new limits on the Compton y parameter from COBE. It is concluded that with baryon densities compatible with standard cosmological nucleosynthesis, the universe could have remained fully ionized throughout its history without producing a detectable spectral distortion. It is argued that it is unlikely that spectral observations of the cosmic microwave background will ever require the universe to have recombined for flat cosmologies.

  12. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  13. Recombinant factor VIIa.

    PubMed

    Aitken, Michael G

    2004-01-01

    Human coagulation factor (F) VII is a single chain protease that circulates in the blood as a weakly active zymogen at concentrations of approximately 10 nmol/L. When converted to the active 2 chain form (FVIIa), it is a powerful initiator of haemostasis. Recombinant factor VIIa (rFVIIa, eptacog alfa, NovoSeven) is a genetically engineered product that was first introduced in 1988 for the treatment of patients with haemophilia A and B with high inhibitory antibody titres to factors VIII and IX. Recent reports in the form of case studies and series, and early trial data, have suggested a role for rFVIIa across a diverse range of indications including bleeding associated with trauma, surgery, thrombocytopaenia, liver disease and oral anticoagulant toxicity. This review describes the physiology of the coagulation pathway and in particular the role of recombinant factor VIIa. It will also focus on the emerging role of rFVIIa in both trauma and non-trauma bleeding and its potential use in the ED. PMID:15537408

  14. Unraveling recombination rate evolution using ancestral recombination maps

    PubMed Central

    Munch, Kasper; Schierup, Mikkel H; Mailund, Thomas

    2014-01-01

    Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it. PMID:25043668

  15. Algebraic theory of recombination spaces.

    PubMed

    Stadler, P F; Wagner, G P

    1997-01-01

    A new mathematical representation is proposed for the configuration space structure induced by recombination, which we call "P-structure." It consists of a mapping of pairs of objects to the power set of all objects in the search space. The mapping assigns to each pair of parental "genotypes" the set of all recombinant genotypes obtainable from the parental ones. It is shown that this construction allows a Fourier decomposition of fitness landscapes into a superposition of "elementary landscapes." This decomposition is analogous to the Fourier decomposition of fitness landscapes on mutation spaces. The elementary landscapes are obtained as eigenfunctions of a Laplacian operator defined for P-structures. For binary string recombination, the elementary landscapes are exactly the p-spin functions (Walsh functions), that is, the same as the elementary landscapes of the string point mutation spaces (i.e., the hypercube). This supports the notion of a strong homomorphism between string mutation and recombination spaces. However, the effective nearest neighbor correlations on these elementary landscapes differ between mutation and recombination and among different recombination operators. On average, the nearest neighbor correlation is higher for one-point recombination than for uniform recombination. For one-point recombination, the correlations are higher for elementary landscapes with fewer interacting sites as well as for sites that have closer linkage, confirming the qualitative predictions of the Schema Theorem. We conclude that the algebraic approach to fitness landscape analysis can be extended to recombination spaces and provides an effective way to analyze the relative hardness of a landscape for a given recombination operator. PMID:10021760

  16. Recombinant Human Erythropoietin

    PubMed Central

    Bartels, Claudia; Späte, Kira; Krampe, Henning

    2008-01-01

    Treatment of multiple sclerosis (MS) is still unsatisfactory and essentially non-existing for the progressive course of the disease. Recombinant human erythropoietin (EPO) may be a promising neuroprotective/neuroregenerative treatment of MS. In the nervous system, EPO acts anti-apoptotic, antioxidative, anti-inflammatory, neurotrophic and plasticity-modulating. Beneficial effects have been shown in animal models of various neurological and psychiatric diseases, including different models of experimental autoimmune encephalomyelitis. EPO is also effective in human brain disease, as shown in double-blind placebo-controlled clinical studies on ischemic stroke and chronic schizophrenia. An exploratory study on chronic progressive MS yielded lasting improvement in motor and cognitive performance upon high-dose long-term EPO treatment. PMID:21180577

  17. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  18. The recombination of genetic material

    SciTech Connect

    Low, K.B.

    1988-01-01

    Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

  19. Coalescent Simulation of Intracodon Recombination

    PubMed Central

    Arenas, Miguel; Posada, David

    2010-01-01

    The coalescent with recombination is a very useful tool in molecular population genetics. Under this framework, genealogies often represent the evolution of the substitution unit, and because of this, the few coalescent algorithms implemented for the simulation of coding sequences force recombination to occur only between codons. However, it is clear that recombination is expected to occur most often within codons. Here we have developed an algorithm that can evolve coding sequences under an ancestral recombination graph that represents the genealogies at each nucleotide site, thereby allowing for intracodon recombination. The algorithm is a modification of Hudson's coalescent in which, in addition to keeping track of events occurring in the ancestral material that reaches the sample, we need to keep track of events occurring in ancestral material that does not reach the sample but that is produced by intracodon recombination. We are able to show that at typical substitution rates the number of nonsynonymous changes induced by intracodon recombination is small and that intracodon recombination does not generally result in inflated estimates of the overall nonsynonymous/synonymous substitution ratio (ω). On the other hand, recombination can bias the estimation of ω at particular codons, resulting in apparent rate variation among sites and in the spurious identification of positively selected sites. Importantly, in this case, allowing for variable synonymous rates across sites greatly reduces the false-positive rate and recovers statistical power. Finally, coalescent simulations with intracodon recombination could be used to better represent the evolution of nuclear coding genes or fast-evolving pathogens such as HIV-1.We have implemented this algorithm in a computer program called NetRecodon, freely available at http://darwin.uvigo.es. PMID:19933876

  20. Delayed recombination and standard rulers

    SciTech Connect

    De Bernardis, Francesco; Melchiorri, Alessandro; Bean, Rachel; Galli, Silvia; Silk, Joseph I.; Verde, Licia

    2009-02-15

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  1. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  2. Controlled Release from Recombinant Polymers

    PubMed Central

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  3. Recombinant DNA means and method

    SciTech Connect

    Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.

    1987-05-19

    This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.

  4. Stable recombination hotspots in birds.

    PubMed

    Singhal, Sonal; Leffler, Ellen M; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M; Strand, Alva I; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N; Griffith, Simon C; McVean, Gil; Przeworski, Molly

    2015-11-20

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but it appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking the gene that encodes PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species: the zebra finch, Taeniopygia guttata, and the long-tailed finch, Poephila acuticauda. We found that both species have recombination hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, most hotspots are shared between the two species, and their conservation seems to extend over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  5. Recombination device for storage batteries

    DOEpatents

    Kraft, H.; Ledjeff, K.

    1984-01-01

    A recombination device including a gas-tight enclosure connected to receive the discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  6. Recombination device for storage batteries

    DOEpatents

    Kraft, Helmut; Ledjeff, Konstantin

    1985-01-01

    A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  7. [Antithrombotic recombinant antibodies].

    PubMed

    Muzard, Julien; Loyau, Stéphane; Ajzenberg, Nadine; Billiald, Philippe; Jandrot-Perrus, Martine

    2006-01-01

    Coronary syndromes, stroke and other ischaemic arterial diseases are the leading cause of death in the world and will probably remain it at least until 2020. Cardiovascular diseases kill 17 million people each year with an expected increase to 20 million in 2020 and 24 million in 2030. The global impact of recurrence and death during the 6 months following an acute coronary syndrome remains at 8-15% in the present state of medical practice. Acute ischaemic syndromes have a common aetiology that is the formation of a platelet-rich clot at the site of severe coronary stenosis and of eroded atherosclerotic plaques. Therapy consists of medical treatments associating thrombolysis, antiplatelet drugs, and the re-opening of the coronary artery by angioplasty. But these treatments do not prevent morbidity and mortality reaching 15% at 6 months. Finally the treatment of stroke is very limited. There is thus a real clinical need to improve existing treatments and to discover new molecules. Platelet activation is a critical step in ischaemic cardiovascular diseases. This is the reason why antiplatelet drugs are most often prescribed in these cases. Currently, only one recombinant antithrombotic antibody is used in therapy. This is a chimeric Fab, c7E3 or abciximab, which inhibits the final phase of platelet aggregation. Abciximab is prescribed in acute coronary syndromes treated by angioplasty. However, treatment by abciximab can induce severe complications, principally, hemorrages and thrombopenia. Other platelet receptors involved in the earlier steps of platelet activation, such as the phases of contact with and of activation by the subendothelium matrix, have been identified as potential targets for the development of antithrombotic antibodies and are described in this revue. PMID:17652972

  8. Genetic recombination in Streptomyces griseus.

    PubMed Central

    Parag, Y

    1978-01-01

    Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described. PMID:415037

  9. [Vaccine application of recombinant herpesviruses].

    PubMed

    Yokoyama, N; Xuan, X; Mikami, T

    2000-04-01

    Recently, genetic engineering using recombinant DNA techniques has been applied to design new viral vaccines in order to reduce some problems which the present viral vaccines have. Up to now, many viruses have been investigated for development of recombinant attenuated vaccines or live viral vectors for delivery of foreign genes coding immunogenic antigens. In this article, we introduced the new vaccine strategy using genetically engineered herpesviruses. PMID:10774221

  10. Combinatorics in Recombinational Population Genomics

    NASA Astrophysics Data System (ADS)

    Parida, Laxmi

    The work that I will discuss is motivated by the need for understanding, and processing, the manifestations of recombination events in chromosome sequences. In this talk, we focus on two related problems. First, we explore the very general problem of reconstructability of pedigree history. How plausible is it to unravel the history of a complete unit (chromosome) of inheritance? The second problem deals with reconstructing the recombinational history of a collection of chromosomes.

  11. Delayed recombination and cosmic parameters

    NASA Astrophysics Data System (ADS)

    Galli, Silvia; Bean, Rachel; Melchiorri, Alessandro; Silk, Joseph

    2008-09-01

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: γα<0.39 and γi<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  12. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  13. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  14. Delayed recombination and cosmic parameters

    SciTech Connect

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-09-15

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n{sub s}, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z{sub *}=1078{+-}11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1{sigma} to R=1.734{+-}0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: {epsilon}{sub {alpha}}<0.39 and {epsilon}{sub i}<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  15. Recombination Drives Vertebrate Genome Contraction

    PubMed Central

    Nam, Kiwoong; Ellegren, Hans

    2012-01-01

    Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process. PMID:22570634

  16. Recombination processes in ionised plasmas

    NASA Astrophysics Data System (ADS)

    Bastin, Robert

    The observational analysis of astrophysical plasmas relies on accurate calculations of the atomic processes involved. The recombination spectra of singly ionised oxygen (O il) and carbon (C il) present excellent tools for investigating regions such as planetary nebulae and H II regions. In this thesis, detailed treatments of the recombination processes of both O II and C II are presented. Using the R-matrix solution to the close coupling equations, I present the results of accurate photoionisation calculations. Bound state energy levels are determined and oscillator strengths calculated for both species. Recombination coefficients were evalu ated for low n and 1, for C II in LS-coupling, and 0 II in intermediate coupling, taking particular care to treat resonances effectively. Sample photoionisation cross-sections are presented for both species, and compared to previous work. A complete radiative-cascade model is treated for both species, in order to determine line emissivities under nebular conditions at a wide range of temperatures and densities. Collisional effects are treated for C II, along with, for the first time, the effects of high temperature dielectronic recombination, allowing the modelling of regions of much higher electron temperature than previous work. The O II calculations were performed under intermediate coupling for the first time, allowing the effects of non-statistical popula tions of the parent ion fine-structure levels and dielectronic recombination onto bound states within this fine-structure to be taken into account in line emissivities. Detailed comparison with previous theoretical work was made for both species. The application of the C II and 0 n recombination spectra to determining tempera ture and densities from the observed spectra of a number of ionised nebulae is considered. The potential for using the new recombination spectra as diagnostic tools to solve some of the key problems in the study of ionised nebulae is demonstrated.

  17. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  18. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    SciTech Connect

    Moriya, Takashi J.

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  19. Recombinant allergens for specific immunotherapy.

    PubMed

    Cromwell, Oliver; Häfner, Dietrich; Nandy, Andreas

    2011-04-01

    Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient's sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years. PMID:21377719

  20. Stable recombination hotspots in birds

    PubMed Central

    Singhal, Sonal; Leffler, Ellen M.; Sannareddy, Keerthi; Turner, Isaac; Venn, Oliver; Hooper, Daniel M.; Strand, Alva I.; Li, Qiye; Raney, Brian; Balakrishnan, Christopher N.; Griffith, Simon C.; McVean, Gil; Przeworski, Molly

    2016-01-01

    The DNA-binding protein PRDM9 has a critical role in specifying meiotic recombination hotspots in mice and apes, but appears to be absent from other vertebrate species, including birds. To study the evolution and determinants of recombination in species lacking PRDM9, we inferred fine-scale genetic maps from population resequencing data for two bird species, the zebra finch Taeniopygia guttata and the long-tailed finch Poephila acuticauda. We find that both species have hotspots, which are enriched near functional genomic elements. Unlike in mice and apes, the two species share most hotspots, with conservation seemingly extending over tens of millions of years. These observations suggest that in the absence of PRDM9, recombination targets functional features that both enable access to the genome and constrain its evolution. PMID:26586757

  1. Recombinant snake venom prothrombin activators.

    PubMed

    Lövgren, Ann

    2013-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

  2. The Dissociative Recombination of OH(+)

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.

    1995-01-01

    Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

  3. Current Drive in Recombining Plasma

    SciTech Connect

    P.F. Schmit and N.J. Fisch

    2012-05-15

    The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.

  4. Selenium incorporation using recombinant techniques

    SciTech Connect

    Walden, Helen

    2010-04-01

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  5. Classifications and comparisons of multilocus recombination distributions

    PubMed Central

    Karlin, Samuel; Liberman, Uri

    1978-01-01

    Various classifications and representations of multilocus recombination structures are delineated based on generalized notions of linkage values and recombination rates. An important class of recombination distributions (called the count-location chiasma process) is parameterized by a distribution of the number of crossover events and, for each such crossover count, by a conditional distribution of crossover locations. A number of properties of this recombination structure are developed. A multilocus definition of a “natural” recombination range is set forth. Orderings among recombination distributions in the multilocus setting are also discussed. Comparisons are made in terms of complete linkage, free assortment and noninterference schemes serving as standards. PMID:16592601

  6. Improving recombinant protein purification yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  7. CRMAGE: CRISPR Optimized MAGE Recombineering

    PubMed Central

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten O. A.; Nielsen, Alex Toftgaard

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering. PMID:26797514

  8. Inhomogeneous recombinations during cosmic reionization

    NASA Astrophysics Data System (ADS)

    Sobacchi, Emanuele; Mesinger, Andrei

    2014-05-01

    By depleting the ionizing photon budget available to expand cosmic H II regions, recombining systems (or Lyman limit systems) can have a large impact during (and following) cosmic reionization. Unfortunately, directly resolving such structures in large-scale reionization simulations is computationally impractical. Instead, here we implement a subgrid prescription for tracking inhomogeneous recombinations in the intergalactic medium. Building on previous work parametrizing photoheating feedback on star formation, we present large-scale, seminumeric reionization simulations which self-consistently track the local (subgrid) evolution of both sources and sinks of ionizing photons. Our simple, single-parameter model naturally results in both an extended reionization and a modest, slowly evolving emissivity, consistent with observations. Recombinations are instrumental in slowing the growth of large H II regions, and damping the rapid rise of the ionizing background in the late stages of (and following) reionization. As a result, typical H II regions are smaller by factors of ˜2 to 3 throughout reionization. The large-scale (k ≲ 0.2 Mpc-1) ionization power spectrum is suppressed by factors of ≳2-3 in the second half of reionization. Therefore properly modelling recombinations is important in interpreting virtually all reionization observables, including upcoming interferometry with the redshifted 21cm line. Consistent with previous works, we find the clumping factor of ionized gas to be C H II ˜ 4 at the end of reionization.

  9. Dopamine-dependent neurodegeneration in rats induced by viral vector-mediated overexpression of the parkin target protein, CDCrel-1

    PubMed Central

    Dong, Zhizhong; Ferger, Boris; Paterna, Jean-Charles; Vogel, Denise; Furler, Sven; Osinde, Maribel; Feldon, Joram; Büeler, Hansruedi

    2003-01-01

    Mutations in the parkin gene are linked to autosomal-recessive juvenile parkinsonism (AR-JP). Parkin functions as a ubiquitin protein ligase in the degradation of several proteins, including the neuron-specific septin CDCrel-1. AR-JP-associated parkin mutations inhibit ubiquitination and degradation of CDCrel-1 and other parkin target proteins. Here we show that recombinant adeno-associated virus-mediated CDCrel-1 gene transfer to the substantia nigra of rats results in a rapid onset (6-10 days) of nigral and striatal CDCrel-1 expression that is followed by a progressive loss of nigral dopaminergic neurons and a decline of the striatal dopamine levels. In contrast, neurons of the globus pallidus are spared from CDCrel-1 toxicity. Furthermore, CDCrel-1 inhibits the release of dopamine from stably-transfected PC12 cells, and pharmacological inhibition of tyrosine hydroxylase and dopamine synthesis in rats prevents CDCrel-1-induced nigral neurodegeneration. These results show that CDCrel-1 overexpression exerts dopamine-dependent neurotoxicity and suggest that inhibition of dopamine secretion by CDCrel-1 may contribute to the development of AR-JP. PMID:14530399

  10. Role of Hypothalamic VGF in Energy Balance and Metabolic Adaption to Environmental Enrichment in Mice.

    PubMed

    Foglesong, Grant D; Huang, Wei; Liu, Xianglan; Slater, Andrew M; Siu, Jason; Yildiz, Vedat; Salton, Stephen R J; Cao, Lei

    2016-03-01

    Environmental enrichment (EE), a housing condition providing complex physical, social, and cognitive stimulation, leads to improved metabolic health and resistance to diet-induced obesity and cancer. One underlying mechanism is the activation of the hypothalamic-sympathoneural-adipocyte axis with hypothalamic brain-derived neurotrophic factor (BDNF) as the key mediator. VGF, a peptide precursor particularly abundant in the hypothalamus, was up-regulated by EE. Overexpressing BDNF or acute injection of BDNF protein to the hypothalamus up-regulated VGF, whereas suppressing BDNF signaling down-regulated VGF expression. Moreover, hypothalamic VGF expression was regulated by leptin, melanocortin receptor agonist, and food deprivation mostly paralleled to BDNF expression. Recombinant adeno-associated virus-mediated gene transfer of Cre recombinase to floxed VGF mice specifically decreased VGF expression in the hypothalamus. In contrast to the lean and hypermetabolic phenotype of homozygous germline VGF knockout mice, specific knockdown of hypothalamic VGF in male adult mice led to increased adiposity, decreased core body temperature, reduced energy expenditure, and impaired glucose tolerance, as well as disturbance of molecular features of brown and white adipose tissues without effects on food intake. However, VGF knockdown failed to block the EE-induced BDNF up-regulation or decrease of adiposity indicating a minor role of VGF in the hypothalamic-sympathoneural-adipocyte axis. Taken together, our results suggest hypothalamic VGF responds to environmental demands and plays an important role in energy balance and glycemic control likely acting in the melanocortin pathway downstream of BDNF. PMID:26730934

  11. Targeted Disruption of LDLR Causes Hypercholesterolemia and Atherosclerosis in Yucatan Miniature Pigs

    PubMed Central

    Rohret, Judy A.; Struzynski, Jason T.; Merricks, Elizabeth P.; Bellinger, Dwight A.; Rohret, Frank A.; Nichols, Timothy C.; Rogers, Christopher S.

    2014-01-01

    Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor (LDLR) gene in an effort to provide an improved large animal model of familial hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and size. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, male and female LDLR+/− pigs were generated. Subsequent breeding of heterozygotes produced LDLR−/− pigs. When fed a standard swine diet (low fat, no cholesterol), LDLR+/− pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while LDLR−/− pigs had considerably elevated levels. This severe hypercholesterolemia in homozygote animals resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. These phenotypes were more severe and developed over a shorter time when fed a diet containing natural sources of fat and cholesterol. LDLR-targeted Yucatan miniature pigs offer several advantages over existing large animal models including size, consistency, availability, and versatility. This new model of cardiovascular disease could be an important resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications. PMID:24691380

  12. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  13. Antibacterial activity of recombinant murine beta interferon.

    PubMed Central

    Fujiki, T; Tanaka, A

    1988-01-01

    Recombinant murine beta interferon was protective and therapeutic for mice against Listeria monocytogenes infection in vivo. The recombinant murine beta interferon caused enhanced H2O2 release by macrophages in vivo, but not in vitro. PMID:3343048

  14. Wnt11 Gene Therapy with Adeno-associated Virus 9 Improves Recovery from Myocardial Infarction by Modulating the Inflammatory Response

    PubMed Central

    Morishita, Yoshihiro; Kobayashi, Koichi; Klyachko, Ekaterina; Jujo, Kentaro; Maeda, Kengo; Losordo, Douglas W.; Murohara, Toyoaki

    2016-01-01

    Acute myocardial infarction induces activation of the acute phase response and infiltration of leukocytes to the infarcted area. Moreover, myocardium that is remote from ischemic area also becomes inflamed. Inflammatory reaction clears dead cells and matrix debris, while prolongation or expansion of the inflammatory response results in dysfunction following myocardial infarction. Wnt glycolipoproteins are best characterized as regulators of embryonic development. Recently several reports suggest that they also contribute to the inflammatory response in adult animals. However, the effects of Wnt proteins on myocardial infarction have not been explored. Here we show that Wnt11 expression leads to significant improvements of survival and cardiac function by suppressing infiltration of multiple kinds of inflammatory cells in infarcted heart. Wnt11 protein suppresses gene expression of inflammatory cytokines through the modulation of NF-κB in vitro. These results reveal a novel function of Wnt11 in the regulation of inflammatory response and provide a rationale for the use of Wnt11 to manipulate human diseases that are mediated by inflammation. PMID:26882996

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

    PubMed

    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

  16. Correction of glycogen storage disease type II by an adeno-associated virus vector containing a muscle-specific promoter.

    PubMed

    Sun, Baodong; Zhang, Haoyue; Franco, Luis M; Brown, Talmage; Bird, Andrew; Schneider, Ayn; Koeberl, Dwight D

    2005-06-01

    Glycogen storage disease type II (Pompe disease) causes death in infancy from cardiorespiratory failure due to acid alpha-glucosidase (GAA; acid maltase) deficiency. An AAV2 vector pseudotyped as AAV6 (AAV2/6 vector) transiently expressed high-level human GAA in GAA-knockout (GAA-KO) mice without reducing glycogen storage; however, in immunodeficient GAA-KO/SCID mice the AAV2/6 vector expressed high-level GAA and reduced the glycogen content of the injected muscle for 24 weeks. A CD4+/CD8+ lymphocytic infiltrate was observed in response to the AAV2/6 vector in immunocompetent GAA-KO mice. When a muscle-specific creatine kinase promoter was substituted for the CB promoter (AAV-MCKhGAApA), that AAV2/6 vector expressed high-level GAA and reduced glycogen content in immunocompetent GAA-KO mice. Muscle-restricted expression of hGAA provoked only a humoral (not cellular) immune response. Intravenous administration of a high number of particles of AAV-MCKhGAApA as AAV2/7 reduced the glycogen content of the heart and skeletal muscle and corrected individual myofibers in immunocompetent GAA-KO mice 24 weeks postinjection. In summary, persistent correction of muscle glycogen content was achieved with an AAV vector containing a muscle-specific promoter in GAA-KO mice, and this approach should be considered for muscle-targeted gene therapy in Pompe disease. PMID:15922959

  17. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA.

    PubMed Central

    Weitzman, M D; Kyöstiö, S R; Kotin, R M; Owens, R A

    1994-01-01

    AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration. Images PMID:8016070

  18. [Recombination in Drosophila in space flight].

    PubMed

    Filatova, L P; Vaulina, E N; Lapteva, N Sh; Grozdova, T Ia

    1988-04-01

    An experiment with Drosophila melanogaster males was performed aboard the Artificial Satellite "Kosmos-1667". Mutagenic effects of a 7-day space flight on intergene recombination in chromosome 2 were studied. The space flight factors decreased the frequency of recombination. A model experiment on a laboratory centrifuge demonstrated insignificant increase in recombination frequency caused by acceleration. PMID:3135244

  19. Selection of Recombinant Human Antibodies.

    PubMed

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  20. Chemical kinetics of geminal recombination

    SciTech Connect

    Levin, P.P.; Khudyakov, I.V.; Brin, E.F.; Kuz'min, V.A.

    1988-09-01

    The kinetics of geminal recombination of triplet radical pairs formed in photoreduction of benzophenone by p-cresol in glycerin solution was studied by pulsed laser photolysis. The experiments were conducted at several temperatures and in a constant magnetic field of H = 0.34 T. The parameters in six kinetic equations describing geminal recombination were determined with a computer. The values of the sums of the squares of the residual deviations of the approximation were obtained. It was found that the kinetics are best described by the functions proposed by Noyes and Shushin. It was shown that it is necessary to use the mutual diffusion coefficient of the radicals, which is significantly smaller than the sum of the estimations of the experimental values of the radical diffusion coefficients, for describing the kinetics due to the correlations of the molecular motions of the radicals in the cage.

  1. Recombination Catalysts for Hypersonic Fuels

    NASA Technical Reports Server (NTRS)

    Chinitz, W.

    1998-01-01

    The goal of commercially-viable access to space will require technologies that reduce propulsion system weight and complexity, while extracting maximum energy from the products of combustion. This work is directed toward developing effective nozzle recombination catalysts for the supersonic and hypersonic aeropropulsion engines used to provide such access to space. Effective nozzle recombination will significantly reduce rk=le length (hence, propulsion system weight) and reduce fuel requirements, further decreasing the vehicle's gross lift-off weight. Two such catalysts have been identified in this work, barium and antimony compounds, by developing chemical kinetic reaction mechanisms for these materials and determining the engine performance enhancement for a typical flight trajectory. Significant performance improvements are indicated, using only 2% (mole or mass) of these compounds in the combustor product gas.

  2. Surface recombination statistics at traps

    NASA Astrophysics Data System (ADS)

    Landsberg, P. T.; Abrahams, M. S.

    1983-09-01

    The Shockley-Read-Hall recombination statistics was recently generalised by Dhariwal, Kothari and Jain to include the effect of a finite time of relaxation before the captured carrier settles into its ground state, and by Landsberg to allow for Auger effects and so-called "extra" carriers supplied to the semiconductor from the outside. The combined result of these effects is studied here theoretically, together with the consideration of a simple distribution of trap states. It is found that the surface recombination velocity s has the usual minimum in the near intrinsic state and that s passes through a maximum as a function of excess electron concentration. Both extrema are enhanced if the trap states are distributed over an energy range. Experimental plots of s as a function of excess electron and hole concentrations should yield insight concerning the numerical importance of (a) Auger effects with the participation of traps and (b) relaxation times.

  3. Recombinant Toxins for Cancer Treatment

    NASA Astrophysics Data System (ADS)

    Pastan, Ira; Fitzgerald, David

    1991-11-01

    Recombinant toxins target cell surface receptors and antigens on tumor cells. They kill by mechanisms different from conventional chemotherapy, so that cross resistance to conventional chemotherapeutic agents should not be a problem. Furthermore, they are not mutagens and should not induce secondary malignancies or accelerate progression of benign malignancies. They can be mass-produced cheaply in bacteria as homogeneous proteins. Either growth factor-toxin fusions or antibody-toxin fusions can be chosen, depending on the cellular target.

  4. Introductory experiments in recombinant DNA.

    PubMed

    Tait, R C

    2000-07-01

    Nine practical exercises demonstrate the basic principles in recombinant DNA. The exercises explain the principles that DNA equals genes and that changes in DNA cause changes in genetic properties. The aim is to provide a teaching resource that can be used to illustrate the theory and applications of molecular biology to highschool students, undergraduate students, medics, dentists, doctors, nurses, life scientists, and anyone learning the basics of DNA technology. PMID:11471559

  5. Recombinant vector and eukaryotic host transformed thereby

    SciTech Connect

    Sugden, W.M.

    1987-08-11

    A recombinant plasmid is described comprising: a segment from a first plasmid which is not a lymphotrophic herpes virus segment and which facilitates the replication of the recombinant plasmid in a prokaryotic host; a segment from a lymphotrophic herpes virus which is linked to the first plasmid segment such that is a capable of assisting in maintaining the recombinant plasmid as a plasmid if the recombinant plasmid is inserted into a eukaryotic host that has been transformed by the lymphotrophic herpes virus; and a foreign eukaryotic gene component linked as part of the recombinant plasmid.

  6. A coalescent model of recombination hotspots.

    PubMed Central

    Wiuf, Carsten; Posada, David

    2003-01-01

    Recent experimental findings suggest that the assumption of a homogeneous recombination rate along the human genome is too naive. These findings point to block-structured recombination rates; certain regions (called hotspots) are more prone than other regions to recombination. In this report a coalescent model incorporating hotspot or block-structured recombination is developed and investigated analytically as well as by simulation. Our main results can be summarized as follows: (1) The expected number of recombination events is much lower in a model with pure hotspot recombination than in a model with pure homogeneous recombination, (2) hotspots give rise to large variation in recombination rates along the genome as well as in the number of historical recombination events, and (3) the size of a (nonrecombining) block in the hotspot model is likely to be overestimated grossly when estimated from SNP data. The results are discussed with reference to the current debate about block-structured recombination and, in addition, the results are compared to genome-wide variation in recombination rates. A number of new analytical results about the model are derived. PMID:12750351

  7. Nondisjunction of chromosome 15: Origin and recombination

    SciTech Connect

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. ); Langlois, S. ); Morris, M.A.; Malcolm, S.

    1993-09-01

    Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.

  8. Effect of gamma radiation on retroviral recombination.

    PubMed

    Hu, W S; Temin, H M

    1992-07-01

    To elucidate the mechanism(s) of retroviral recombination, we exposed virions to gamma radiation prior to infecting target cells. By using previously described spleen necrosis virus-based vectors containing multiple markers, recombinant proviruses were studied after a single round of retrovirus replication. The current models of retroviral recombination predict that breaking virion RNA should promote minus-strand recombination (forced copy-choice model), decrease or not affect plus-strand recombination (strand displacement/assimilation model), and shift plus-strand recombination towards the 3' end of the genome. However, we found that while gamma irradiation of virions reduced the amount of recoverable viral RNA, it did not primarily cause breaks. Thus, the frequency of selected recombinants was not significantly altered with greater doses of radiation. In spite of this, the irradiation did decrease the number of recombinants with only one internal template switch. As a result, the average number of additional internal template switches in the recombinant proviruses increased from 0.7 to 1.4 as infectivity decreased to 6%. The unselected internal template switches tended to be 5' of the selected crossover even in the recombinants from irradiated viruses, inconsistent with a plus-strand recombination mechanism. PMID:1602553

  9. Bacteriophage recombination systems and biotechnical applications.

    PubMed

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  10. Recombinant DNA technology in apple.

    PubMed

    Gessler, Cesare; Patocchi, Andrea

    2007-01-01

    This review summarizes the achievements of almost 20 years of recombinant DNA technology applied to apple, grouping the research results into the sections: developing the technology, insect resistance, fungal disease resistance, self-incompatibility, herbicide resistance, fire blight resistance, fruit ripening, allergens, rooting ability, and acceptance and risk assessment. The diseases fire blight, caused by Erwinia amylovora, and scab, caused by Venturia inaequalis, were and still are the prime targets. Shelf life improvement and rooting ability of rootstocks are also relevant research areas. The tools to create genetically modified apples of added value to producers, consumers, and the environment are now available. PMID:17522823

  11. Recombinant erythropoietin in clinical practice

    PubMed Central

    Ng, T; Marx, G; Littlewood, T; Macdougall, I

    2003-01-01

    The introduction of recombinant human erythropoietin (RHuEPO) has revolutionised the treatment of patients with anaemia of chronic renal disease. Clinical studies have demonstrated that RHuEPO is also useful in various non-uraemic conditions including haematological and oncological disorders, prematurity, HIV infection, and perioperative therapies. Besides highlighting both the historical and functional aspects of RHuEPO, this review discusses the applications of RHuEPO in clinical practice and the potential problems of RHuEPO treatment. PMID:12897214

  12. Annealing Vs. Invasion in Phage λ Recombination

    PubMed Central

    Stahl, M. M.; Thomason, L.; Poteete, A. R.; Tarkowski, T.; Kuzminov, A.; Stahl, F. W.

    1997-01-01

    Genetic recombination catalyzed by λ's Red pathway was studied in rec(+) and recA mutant bacteria by examining both intracellular λ DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec(+) cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of λ catalyzes recombination primarily by annealing. PMID:9383045

  13. Recombinant DNA production of spider silk proteins

    PubMed Central

    Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

    2013-01-01

    Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

  14. Recombinant bacteria for mosquito control.

    PubMed

    Federici, B A; Park, H-W; Bideshi, D K; Wirth, M C; Johnson, J J

    2003-11-01

    Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria. PMID:14506223

  15. Dissociative recombination in planetary ionospheres

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1993-01-01

    Ionization in planetary atmospheres can be produced by solar photoionization, photoelectron impact ionization, and, in auroral regions, by impact of precipitating particles. This ionization is lost mainly in dissociative recombination (DR) of molecular ions. Although atomic ions cannot undergo DR, they can be transformed locally through ion-molecule reactions into molecular ions, or they may be transported vertically or horizontally to regions of the atmosphere where such transformations are possible. Because DR reactions tend to be very exothermic, they can be an important source of kinetically or internally excited fragments. In interplanetary thermospheres, the neutral densities decrease exponentially with altitude. Below the homopause (or turbopause), the atmosphere is assumed to be throughly mixed by convection and/or turbulence. Above the homopause, diffusion is the major transport mechanism, and each species is distributed according to its mass, with the logarithmic derivative of the density with repect to altitude given approximately by -1/H, where H = kT/mg is the scale height. In this expression, T is the neutral temperature, g is the local acceleratiion of gravity, and m is the mass of the species. Thus lighter species become relatively more abundant, and heavier species less abundant, as the altitude increases. This variation of the neutral composition can lead to changes in the ion composition; furthermore, as the neutral densities decrease, dissociative recombination becomes more important relative to ion-neutral reactions as a loss mechanism for molecular ions.

  16. Human Insulin from Recombinant DNA Technology

    NASA Astrophysics Data System (ADS)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  17. Safety and Biodistribution Evaluation in CNGB3-Deficient Mice of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia.

    PubMed

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

    2016-03-01

    Applied Genetic Technologies Corporation (AGTC) is developing rAAV2tYF-PR1.7-hCNGB3, a recombinant adeno-associated virus (rAAV) vector expressing the human CNGB3 gene, for treatment of achromatopsia, an inherited retinal disorder characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. We report here results of a study evaluating safety and biodistribution of rAAV2tYF-PR1.7-hCNGB3 in CNGB3-deficient mice. Three groups of animals (n = 35 males and 35 females per group) received a subretinal injection in one eye of 1 μl containing either vehicle or rAAV2tYF-PR1.7-hCNGB3 at one of two dose concentrations (1 × 10(12) or 4.2 × 10(12) vg/ml) and were euthanized 4 or 13 weeks later. There were no test-article-related changes in clinical observations, body weights, food consumption, ocular examinations, clinical pathology parameters, organ weights, or macroscopic observations at necropsy. Cone-mediated electroretinography (ERG) responses were detected after vector administration in the treated eyes in 90% of animals in the higher dose group and 31% of animals in the lower dose group. Rod-mediated ERG responses were reduced in the treated eye for all groups, with the greatest reduction in males given the higher dose of vector, but returned to normal by the end of the study. Microscopic pathology results demonstrated minimal mononuclear cell infiltrates in the retina and vitreous of some animals at the interim euthanasia and in the vitreous of some animals at the terminal euthanasia. Serum anti-AAV antibodies developed in most vector-injected animals. No animals developed antibodies to hCNGB3. Biodistribution studies demonstrated high levels of vector DNA in vector-injected eyes but little or no vector DNA in nonocular tissue. These results support the use of rAAV2tYF-PR1.7-hCNGB3 in clinical studies in patients with achromatopsia caused by CNGB3 mutations. PMID:27003752

  18. Dielectronic recombination of tungsten ions

    NASA Astrophysics Data System (ADS)

    Li, Bowen; O’Sullivan, Gerry; Dong, Chenzhong; Chen, Ximeng

    2016-08-01

    Ab initio calculations of dielectronic recombination rate coefficients of Ne-, Pd- and Ag-like tungsten have been performed. Energy levels, radiative transition probabilities and autoionization rates were calculated using the Flexible Atomic Code. The contributions from different channels to the total rate coefficients are discussed. The present calculated rate coefficients are compared with other calculations where available. Excellent agreement has been found for Ne-like W while a large discrepancy was found for Pd-like W, which implies that more ab initio calculations and experimental measurements are badly needed. Further calculations demonstrated that the influence of configuration interaction is small while nonresonant radiative stabilizing (NRS) contribution to doubly excited non-autoionizing states are vital. The data obtained are expected to be useful for modeling plasmas for fusion applications, especially for the ITER community, which makes experimental verification even more essential.

  19. Steric effects on geminate recombinations

    SciTech Connect

    Traylor, T.G.; Taube, D.; Jongeward, K.A.; Magde, D. )

    1990-09-12

    Steric effects on the binding of isonitrile ligands to iron(II) porphyrins were investigated by picosecond flash photolysis. Two different types of steric effects were distinguished and characterized: (1) steric restrictions to porphyrin planarity and (2) blocking of the pathway for ligand approach. Heme planarity was restricted by coordinating 1,2-dimethylimidazole trans to the ligand binding site being investigated. Blocking of the binding site was explored by using adamantane heme 6,6-cyclophane, in which the adamantane moiety forms a cap over the binding site. Results of picosecond kinetic measurements demonstrate that the first effect, heme nonplanarity or trans strain, influences the bond-making step, whereas the second effect, ligand blocking, involves a conformational preequilibrium prior to bond making. Relevance of these findings for contact pair recombination, in general, and for heme protein ligation, in particular, are discussed.

  20. Modeling Interference in Genetic Recombination

    PubMed Central

    McPeek, M. S.; Speed, T. P.

    1995-01-01

    In analyzing genetic linkage data it is common to assume that the locations of crossovers along a chromosome follow a Poisson process, whereas it has long been known that this assumption does not fit the data. In many organisms it appears that the presence of a crossover inhibits the formation of another nearby, a phenomenon known as ``interference.'' We discuss several point process models for recombination that incorporate position interference but assume no chromatid interference. Using stochastic simulation, we are able to fit the models to a multilocus Drosophila dataset by the method of maximum likelihood. We find that some biologically inspired point process models incorporating one or two additional parameters provide a dramatically better fit to the data than the usual ``no-interference'' Poisson model. PMID:7713406

  1. Deleterious background selection with recombination

    SciTech Connect

    Hudson, R.R.; Kaplan, N.L.

    1995-12-01

    An analytic expression for the expected nucleotide diversity is obtained for a neutral locus in a region with deleterious mutation and recombination. Our analytic results are used to predict levels of variation for the entire third chromosome of Drosophila melanogaster. The predictions are consistent with the low levels of variation that have been observed at loci near the centromeres of the third chromosome of D. melanogaster. However, the low levels of variation observed near the tips of this chromosome are not predicted using currently available estimates of the deleterious mutation rate and of selection coefficients. If considerably smaller selection coefficients are assumed, the low observed levels of variation at the tips of the third chromosome are consistent with the background selection model. 33 refs., 4 figs., 1 tab.

  2. Recombinant prolylcarboxypeptidase activates plasma prekallikrein.

    PubMed

    Shariat-Madar, Zia; Mahdi, Fakhri; Schmaier, Alvin H

    2004-06-15

    The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment. PMID:14996700

  3. Fundamental Studies of Recombinant Hydrogenases

    SciTech Connect

    Adams, Michael W

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  4. RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

    PubMed Central

    Liao, C L; Lai, M M

    1992-01-01

    Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur

  5. Recombining without Hotspots: A Comprehensive Evolutionary Portrait of Recombination in Two Closely Related Species of Drosophila

    PubMed Central

    Smukowski Heil, Caiti S.; Ellison, Chris; Dubin, Matthew; Noor, Mohamed A.F.

    2015-01-01

    Meiotic recombination rate varies across the genome within and between individuals, populations, and species in virtually all taxa studied. In almost every species, this variation takes the form of discrete recombination hotspots, determined in some mammals by a protein called PRDM9. Hotspots and their determinants have a profound effect on the genomic landscape, and share certain features that extend across the tree of life. Drosophila, in contrast, are anomalous in their absence of hotspots, PRDM9, and other species-specific differences in the determination of recombination. To better understand the evolution of meiosis and general patterns of recombination across diverse taxa, we present a truly comprehensive portrait of recombination across time, combining recently published cross-based contemporary recombination estimates from each of two sister species with newly obtained linkage-disequilibrium-based historic estimates of recombination from both of these species. Using Drosophila pseudoobscura and Drosophila miranda as a model system, we compare recombination rate between species at multiple scales, and we suggest that Drosophila replicate the pattern seen in human–chimpanzee in which recombination rate is conserved at broad scales. We also find evidence of a species-wide recombination modifier(s), resulting in both a present and historic genome-wide elevation of recombination rates in D. miranda, and identify broad scale effects on recombination from the presence of an inversion. Finally, we reveal an unprecedented view of the distribution of recombination in D. pseudoobscura, illustrating patterns of linked selection and where recombination is taking place. Overall, by combining these estimation approaches, we highlight key similarities and differences in recombination between Drosophila and other organisms. PMID:26430062

  6. Oligo Recombination in Gram Negative Bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Homologous recombination is important for bacterial survival because it simultaneously provides genomic stability as well as genomic plasticity. Of the mechanistic pathways for homologous recombination, those mediated by RecA are the most thoroughly characterized and are understood to be structural...

  7. Dissociative recombination of highly symmetric polyatomic ions.

    PubMed

    Douguet, Nicolas; Orel, Ann E; Greene, Chris H; Kokoouline, Viatcheslav

    2012-01-13

    A general first-principles theory of dissociative recombination is developed for highly symmetric molecular ions and applied to H(3)O(+) and CH(3)(+), which play an important role in astrophysical, combustion, and laboratory plasma environments. The theoretical cross sections obtained for the dissociative recombination of the two ions are in good agreement with existing experimental data from storage ring experiments. PMID:22324682

  8. High efficiency recombineering in lactic acid bacteria

    PubMed Central

    van Pijkeren, Jan-Peter; Britton, Robert A.

    2012-01-01

    The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to Gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lactic acid bacteria. Mutations were incorporated in the chromosome of Lactobacillus reuteri and Lactococcus lactis without selection at frequencies ranging between 0.4% and 19%. Whole genome sequence analysis showed that ssDNA recombineering is specific and not hypermutagenic. To highlight the utility of ssDNA recombineering we reduced the intrinsic vancomymycin resistance of L. reuteri >100-fold. By creating a single amino acid change in the d-Ala-d-Ala ligase enzyme we reduced the minimum inhibitory concentration for vancomycin from >256 to 1.5 µg/ml, well below the clinically relevant minimum inhibitory concentration. Recombineering thus allows high efficiency mutagenesis in lactobacilli and lactococci, and may be used to further enhance beneficial properties and safety of strains used in medicine and industry. We expect that this work will serve as a blueprint for the adaptation of ssDNA recombineering to other Gram-positive bacteria. PMID:22328729

  9. Oligonucleotide recombination in gram negative bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  10. Dielectronic recombination lines of C{sup +}

    SciTech Connect

    Sochi, Taha Storey, Peter J.

    2013-11-15

    The present paper presents atomic data generated to investigate the recombination lines of C II in the spectra of planetary nebulae. These data include energies of bound and autoionizing states, oscillator strengths and radiative transition probabilities, autoionization probabilities, and recombination coefficients. The R-matrix method of electron scattering theory was used to describe the C{sup 2+} plus electron system.

  11. Recombinant Vaccinia Virus: Immunization against Multiple Pathogens

    NASA Astrophysics Data System (ADS)

    Perkus, Marion E.; Piccini, Antonia; Lipinskas, Bernard R.; Paoletti, Enzo

    1985-09-01

    The coding sequences for the hepatitis B virus surface antigen, the herpes simplex virus glycoprotein D, and the influenza virus hemagglutinin were inserted into a single vaccinia virus genome. Rabbits inoculated intravenously or intradermally with this polyvalent vaccinia virus recombinant produced antibodies reactive to all three authentic foreign antigens. In addition, the feasibility of multiple rounds of vaccination with recombinant vaccinia virus was demonstrated.

  12. Recombinant Swinepox Virus for Veterinary Vaccine Development.

    PubMed

    Fan, Hong-Jie; Lin, Hui-Xing

    2016-01-01

    Poxvirus-vectors have been widely used in vaccine development for several important human and animal diseases; some of these vaccines have been licensed and used extensively. Swinepox virus (SPV) is well suited to develop recombinant vaccines because of its large packaging capacity for recombinant DNA, its host range specificity, and its ability to induce appropriate immune responses. PMID:26458836

  13. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  14. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  15. V(D)J recombination deficiencies.

    PubMed

    de Villartay, Jean-Pierre

    2009-01-01

    V(D)J recombination not only comprises the molecular mechanism that insures diversity of the immune system but also constitutes a critical checkpoint in the developmental program of B- and T-lymphocytes. The analysis of human patients with Severe Combined Immune Deficiency (SCID) has contributed to the understanding of the biochemistry of the V(D)J recombination reaction. The molecular study V(D)J recombination settings in humans, mice and in cellular mutants has allowed to unravel the process of Non Homologous End Joining (NHEJ), one of the key pathway that insure proper repair of DNA double strand breaks (dsb), whether they occur during V(D)J recombination or secondary to other DNA injuries. Two NHEJ factors, Artemis and Cernunnos, were indeed discovered through the study of human V(D)J recombination defective human SCID patients. PMID:19731800

  16. Biochemistry of Meiotic Recombination: Formation, Processing, and Resolution of Recombination Intermediates

    PubMed Central

    Ehmsen, Kirk T.

    2009-01-01

    Meiotic recombination ensures accurate chromosome segregation during the first meiotic division and provides a mechanism to increase genetic heterogeneity among the meiotic products. Unlike homologous recombination in somatic (vegetative) cells, where sister chromatid interactions prevail and crossover formation is avoided, meiotic recombination is targeted to involve homologs, resulting in crossovers to connect the homologs before anaphase of the first meiotic division. The mechanisms responsible for homolog choice and crossover control are poorly understood, but likely involve meiosis-specific recombination proteins, as well as meiosis-specific chromosome organization and architecture. Much progress has been made to identify and biochemically characterize many of the proteins acting during meiotic recombination. This review will focus on the proteins that generate and process heteroduplex DNA, as well as those that process DNA junctions during meiotic recombination, with particular attention to how recombination activities promote crossover resolution between homologs. PMID:20098639

  17. GENERATION OF RECOMBINANT BACULOVIRUS VIA LIPOSOME MEDIATED TRANSFECTION

    EPA Science Inventory

    Baculovirus expression vectors have become a popular method of producing recombinant proteins. Production of recombinant virus requires the transfection of both the native viral DNA and a transfer plasmid into insect cells where recombination takes place. While several methods of...

  18. Charge recombination and thermoluminescence in photosystem II.

    PubMed

    Rappaport, Fabrice; Cuni, Aude; Xiong, Ling; Sayre, Richard; Lavergne, Jérôme

    2005-03-01

    In the recombination process of Photosystem II (S(2)Q(A)(-)-->S(1)Q(A)) the limiting step is the electron transfer from the reduced primary acceptor pheophytin Ph(-) to the oxidized primary donor P(+) and the rate depends on the equilibrium constant between states S(2)PPhQ(A)(-) and S(1)P(+)Ph(-)Q(A). Accordingly, mutations that affect the midpoint potential of Ph or of P result in a modified recombination rate. A strong correlation is observed between the effects on the recombination rate and on thermoluminescence (TL, the light emission from S(2)Q(A)(-) during a warming ramp): a slower recombination corresponds to a large enhancement and higher temperature of the TL peak. The current theory of TL does not account for these effects, because it is based on the assumption that the rate-limiting step coincides with the radiative process. When implementing the known fact that the radiative pathway represents a minor leak, the modified TL theory readily accounts qualitatively for the observed behavior. However, the peak temperature is still lower than predicted from the temperature-dependence of recombination. We argue that this reflects the heterogeneity of the recombination process combined with the enhanced sensitivity of TL to slower components. The recombination kinetics are accurately fitted as a sum of two exponentials and we show that this is not due to a progressive stabilization of the charge-separated state, but to a pre-existing conformational heterogeneity. PMID:15653722

  19. Electron Recombination in a Dense Hydrogen Plasma

    SciTech Connect

    Jana, M.R.; Johnstone, C.; Kobilarcik, T.; Koizumi, G.M.; Moretti, A.; Popovic, M.; Tollestrup, A.V.; Yonehara, K.; Leonova, M.A.; Schwarz, T.A.; Chung, M.; /Unlisted /IIT, Chicago /Fermilab /MUONS Inc., Batavia /Turin Polytechnic

    2012-05-01

    A high pressure hydrogen gas filled RF cavity was subjected to an intense proton beam to study the evolution of the beam induced plasma inside the cavity. Varying beam intensities, gas pressures and electric fields were tested. Beam induced ionized electrons load the cavity, thereby decreasing the accelerating gradient. The extent and duration of this degradation has been measured. A model of the recombination between ionized electrons and ions is presented, with the intent of producing a baseline for the physics inside such a cavity used in a muon accelerator. Analysis of the data taken during the summer of 2011 shows that self recombination takes place in pure hydrogen gas. The decay of the number of electrons in the cavity once the beam is turned off indicates self recombination rather than attachment to electronegative dopants or impurities. The cross section of electron recombination grows for larger clusters of hydrogen and so at the equilibrium of electron production and recombination in the cavity, processes involving H{sub 5}{sup +} or larger clusters must be taking place. The measured recombination rates during this time match or exceed the analytic predicted values. The accelerating gradient in the cavity recovers fully in time for the next beam pulse of a muon collider. Exactly what the recombination rate is and how much the gradient degrades during the 60 ns muon collider beam pulse will be extrapolated from data taken during the spring of 2012.