Regulatory Perspective for Biologics License Application of Recombinant Adeno-Associated Virus Products in China.

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates

Integration Frequency and Intermolecular Recombination of rAAV Vectors in Non-human Primate Skeletal Muscle and Liver

Adeno-Associated Virus Vectors in Clinical Trials

Self-complementary AAV Vectors; Advances and Applications

Sustained Enzymatic Correction by rAAV-Mediated Liver Gene Therapy Protects Against Induced Motor Neuropathy in Acute Porphyria Mice

Sexually Dimorphic Patterns of Episomal rAAV Genome Persistence in the Adult Mouse Liver and Correlation With Hepatocellular Proliferation

Analysis of Adeno-associated Virus Progenitor Cell Transduction in Mouse Lung

Efficacy of a Combined Intracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder

Optimization of the quality by design approach for gene therapy products: A case study for adeno-associated viral vectors

The delivery of recombinant genes to cardiomyocytes holds promise for the treatment of a variety of cardiovascular diseases. Previous gene transfer approaches that used direct injection of plasmid DNA or replication-defective adenovirus vectors have been limited by low transduction frequencies and transient transgene expression due to immune responses, respectively. In this report, we have tested the feasibility of using intramyocardial injection or intracoronary infusions of recombinant adeno-associated virus (rAAV) vectors to program transgene expression in murine cardiomyocytes in vivo. We constructed an rAAV containing the LacZ gene under the transcriptional control of the cytomegalovirus (CMV) promoter (AAVCMV-LacZ). We then injected 1x10(8) infectious units (IU) of this virus into the left ventricular myocardium of adult CD-1 mice. Control hearts were injected with the AdCMV-LacZ adenovirus vector. Hearts harvested 2, 4, and 8 weeks after AAVCMV-LacZ injection demonstrated stable beta-galactosidase (beta-gal) expression in large numbers of cardiomyocytes without evidence of myocardial inflammation or myocyte necrosis. In contrast, the AdCMV-LacZ-injected hearts displayed transient beta-gal expression, which was undetectable by 4 weeks after injection. Explanted C57BL/6 mouse hearts were also perfused via the coronary arteries with 1.5x10(9) IU of AAVCMV-LacZ and assayed 2, 4, and 8 weeks later for beta-gal expression. beta-Gal expression was detected in <1% of cardiomyocytes at 2 weeks after perfusion but was detected in up to 50% of cardiomyocytes 4 to 8 weeks after perfusion. Direct intramyocardial injection or coronary artery perfusion with rAAV vectors can be used to program stable transgene expression in cardiomyocytes in vivo. rAAV appears to represent a useful vector for the delivery of therapeutic genes to the myocardium.

Enhancement of gene transfer with recombinant adeno-associated virus (rAAV) vectors into primary B-cell chronic lymphocytic leukemia cells by CpG-oligodeoxynucleotides

Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors

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 ). Currently, rAAV vectors are being utilized in phase I/II clinical trials for cystic fibrosis, α-1 antitrypsin deficiency, Canavan's disease, Parkinson's disease, hemophilia, limb-girdle muscular dystrophy, arthritis, Batten's disease, and Leber's congenital amaurosis (Flotte et al., 1996 , 2004 ; Kay et al., 2000 ; Aitken et al., 2001 ; Wagner et al., 2002 ; Manno et al., 2003 ; Snyder and Francis, 2005 ; Maguire et al., 2008 ; Cideciyan et al., 2009 ). In this study, we present preclinical data to support the viability of an rAAV-based gene transfer strategy for cure of ADA-SCID. We report efficient transduction of a variety of postmitotic target tissues in vivo, subsequent human ADA (hADA) expression, and enhanced hADA secretion in tissues and blood, with increasing peripheral lymphocyte populations over time.

Production of research and clinical‐grade recombinant adeno‐associated virus vectors

Recombinant adeno-associated virus (rAAV) vectors were used in human trials as carriers of vaccines for HIV-1 after encouraging preclinical results. However, the clinical trials yielded disappointing results. Here we demonstrated that in mice, rAAV vectors expressing the gene encoding HIV-1 gag stimulated gag-specific CD8(+) T cells, but these T cells failed to expand after a booster immunization with a replication-defective adenoviral (Ad) vector also expressing gag. We tested rAAV vectors of different serotypes expressing HIV-1 gag for induction of transgene product-specific CD8(+) T cells and found that the immunoinhibitory effect of rAAV priming observed with different AAV serotypes was transgene product specific, was independent of the interval between prime and boost, and extended to boosts with vaccine modalities other than Ad vectors. rAAV vector-induced CD8(+) T cells proliferated poorly, produced low levels of IFN-gamma in response to gag stimulation, and upregulated immunoinhibitory molecules. These T cells did not protect efficiently against challenge with a surrogate pathogen. Finally, we showed that the impaired proliferative capacity of the T cells was caused by persistence of the antigen-encoding rAAV vectors and could be reversed by placing the CD8(+) T cells in an antigen-free environment. Our data suggest that rAAV vectors induce functionally impaired T cells and could dampen the immune response to a natural infection.