Current approaches to transfer genes in vivo employ neither recombinant viral vectors or non-viral delivery systems. Adeno-associated viral (AAV) vectors are non-pathogenic integrating vectors that infect both dividing and non-dividing cells. Since all viral genes are removed (96 percent of the viral genome), packaging of foreign DNA up to 5 kb can be incorporated into these vectors. Recently Dr. Samulski and his colleagues demonstrated transduction of rAAV vector expressing B- galactosidase after direct injection in rodent muscle. Vector DNA and transduced gene expression was detected for over one year without significant immune response. There was no evidence of vector toxicity in any animal treated suggesting the ability to safely and stably transfer sequences into muscle as an attractive platform for gene therapy. These studies have been extended into a large animal model (hemophilic FIX dog) using FIX sequences in place of B-galactosidase. Evidence for stable gene expression now out to 16 weeks suggests successful long term vector delivery. Analysis of vector spread was localized to site of injection and draining lymph node with infiltrating immune cells at the site of injection. Ironically this infiltration correlated with residual inactivated A helper virus and not AAV transduced cells, suggesting further improvement in vector production is still required, a focus of this grant. Analysis of gene expression follows an atypical expression curve with onset of expression 10 to 21 days after vector delivery. In addition, gene expression appears to climb with time suggesting that molecular events that may be related to viral replication are taking place. These observations of stable long term gene expression, assumed to be obtained by vector integration, is in contrast to low level replication over four months. While these results demonstrate the first example of AVV productive gene therapy in a large animal, better elucidation of the molecular fate of the vector genome is required in order for safe translation into a clinical setting. Finally, these results strongly suggest that muscle is an attractive site for AAV transduction, and that further analysis of vector preps carrying various modified FIX genes devoid of helper Ad proteins will provide meaningful preclinical information that should facilitate AAV gene therapy for this genetic disorder.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Medical Biochemistry Study Section (MEDB)
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Mckeon, Catherine T
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Lehmann, Thorsten G; Luedde, Tom; Schwabe, Robert F et al. (2006) Minimizing oxidative stress by gene delivery of superoxide dismutase accelerates regeneration after transplantation of reduced-size livers in the rat. Liver Transpl 12:550-9
Stilwell, Jackie L; Samulski, Richard Jude (2004) Role of viral vectors and virion shells in cellular gene expression. Mol Ther 9:337-46
Lehmann, Thorsten G; Wheeler, Michael D; Froh, Matthias et al. (2003) Effects of three superoxide dismutase genes delivered with an adenovirus on graft function after transplantation of fatty livers in the rat. Transplantation 76:28-37
Rabinowitz, Joseph E; Rolling, Fabienne; Li, Chengwen et al. (2002) Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol 76:791-801
Chao, H; Walsh, C E (2001) Induction of tolerance to human factor VIII in mice. Blood 97:3311-2
Lehmann, T G; Wheeler, M D; Schwabe, R F et al. (2000) Gene delivery of Cu/Zn-superoxide dismutase improves graft function after transplantation of fatty livers in the rat. Hepatology 32:1255-64
Chao, H; Liu, Y; Rabinowitz, J et al. (2000) Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors. Mol Ther 2:619-23
Chao, H; Mao, L; Bruce, A T et al. (2000) Sustained expression of human factor VIII in mice using a parvovirus-based vector. Blood 95:1594-9
Malik, A K; Monahan, P E; Allen, D L et al. (2000) Kinetics of recombinant adeno-associated virus-mediated gene transfer. J Virol 74:3555-65