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| Safe, Efficient, and Durable Transduction of Human and Canine Saphenous Vein Grafts (SVG) Using a Modified Adeno-associated Viral (AAV) Vector |
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Mani A. Daneshmand1, Nestor R. Villamizar1, Roberto J. Manson1, Thomas Mulhearn2, Christopher D. Kontos2, Carmelo A. Milano1, Dawn Bowles1 1 Surgery, Duke University Medical Center, Durham, NC; 2 Medicine, Division of Cardiology, Duke University Medical Center, Durham, NC Objective: The effectiveness of SVGs in coronary artery bypass surgery is compromised by a high incidence of failure. Gene therapy with anti-proliferative agents limits intimal hyperplasia and SVG failure in experimental models. As a gene delivery agent, AAV vectors are notable for their beneficial safety profile and long-term gene expression. The purpose of this study was to determine the optimal conditions for AAV-mediated SVG transduction in anticipation of clinical trials. Methods: Cultured human aortic smooth muscle cells (HASMC) (n=4/group) and segments of human SV (n=3-7/group) were utilized to evaluate the transduction efficiency of a panel of luciferase-encoding AAV vectors (AAV serotypes 1-9 or SASTG, a modified capsid vector). Human SVs were infected with AAV ex vivo, and residual infectious virus DNA present in serial flushes was quantified by real-time PCR. In vivo, AAV transduction efficiency was evaluated using a canine carotid artery to jugular vein fistula model. SV segments transduced with either AAV2- or SASTG-luciferase (1 x 1012 total viral particles/vein) were analyzed for luciferase expression by luminometry and bioluminescence imaging 30 or 90 days after gene delivery (n=5/vector type for both time points). Results: In HASMC and cultured SV segments the SASTG vector induced significantly greater luciferase transgene activity relative to other AAV serotypes (See Figure for comparison to AAV2; P<0.05). Greater than 98% of residual vector was removed from the human vein following a single ex vivo PBS flush. In vivo, robust luciferase expression was detected in SVG 30 days after delivery and was sustained at 90 days. Notably, within the same animal, SASTG treated grafts achieve on average 3.7-fold higher transgene expression compared to grafts treated with AAV2 (p=0.0486) (See Figure). Conclusion: This is the first study to evaluate the transduction properties of different AAV vectors in large caliber veins. In all model systems, a novel genetically modified AAV capsid (SASTG) led to significantly greater transgene expression relative to other AAV serotypes. 90-day expression was observed in vivo, reflecting the longest documented transgene expression in SVG. Little residual infectious virus was detectable prior to anastomosis, supporting the safety of this approach. These findings pave the way for the use of AAV vectors as a safe and effective means of gene delivery for the prevention of vein graft disease.  Transduction (as measured by relative light units) of AAV2 or SASTG luciferase vectors in either cultured human aortic smooth muscle cells (HASMC) or in vivo in canine saphenous vein grafts (SVG). Back to 2010 Annual Meeting Back to Program Outline Back to Main Program |
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