794. Evaluation of Gene Transfer Efficacy Mediated by AAV1 and AAV6 Vectors in Skeletal Muscle of Adult Mice Following Different Routes of Administration

Abstract

Top of pageAbstract Skeletal muscle represents an important target tissue for gene therapy application due to the large number of genetic muscular disorders including Duchene Muscular Dystrophy. In addition the muscle may be also used as a delivery platform to express therapeutic proteins into the bloodstream for non-muscular metabolic disorders. Adeno-Associated Virus 1-based vector (rAAV1) is often considered as the most efficient AAV serotype to deliver genes into the muscles. In this study, we compared the performance of AAV pseudotyped (based on AAV2 genome) vectors expressing a secreted form of the murine alkaline phosphatase, (mSEAP) with capsid from serotype 1 and 6, in mouse skeletal muscle using different delivery methods: direct injection into individual muscle (intramuscular, IM), local limb distribution of vectors via the circulation (intra-arterial delivery, IA) or systemic delivery (intravenous administration, IV). We injected the maximal volume that each of these route allowed. We analyzed two parameters: the levels of a reporter circulating protein (), a relevant parameter for a depot organ strategy, and the extent of fiber transduction in the muscles, a relevant parameter in view of a muscular disorder therapeutic approach. We compared rAAV1 and rAAV6 encoding mSEAP in a dose response study following IM administration in adult C57Bl/6 mice. Both vectors showed strong and equivalent levels of transduction at the highest doses (6x108 vg and 3x109 vg), while at the lowest dose injected (1.5x1010 vg), rAAV6 was 3-fold more efficient than rAAV1 in transducing muscle, suggesting a differential threshold of efficiency at sub-optimal doses. In addition, the potential for systemic gene transfer after IV of rAAV1 and rAAV6 at the whole body level was investigated. For the two doses tested (1|[times]|1011 vg and 3|[times]|1011 vg). AAV6 vector led to 3-fold higher levels of circulating mSEAP levels than rAAV1. In addition, a widespread transduction of both skeletal and cardiac tissues was observed with rAAV6 after histochemical detection of mSEAP. However, in term of circulating protein level, IV administration was less efficient than IM injection since the same level of circulating mSEAP was achieved with 3|[times]|1011 vg using IV delivery compared to 3|[times]|109 vg for IM delivery. We have also evaluated intra-arterial delivery via the femoral artery of rAAV1 and rAAV6 at two doses (1|[times]|1011 vg and 3|[times]|1011 vg). This route yielded a 10 fold higher mSEAP levels in the serum than IV and led to robust transgene expression pattern in the hind limb muscles. Finally, in an attempt to increase gene transfer efficacy in muscle after IV delivery, we co-injected the VEGF, with either rAAV1 or rAAV6. For both vectors, the presence of VEGF did not show a positive impact on transgene expression levels. In conclusion, our results show that despite the high similarity between AAV1 and AAV6 capsid sequences, rAAV6 performance for muscle transduction are superior to rAAV1 with all IM, IA or IV delivery routes. This finding prompts us to consider AAV6 vectors as one of the most efficient viral agent to deliver gene in skeletal muscle and further clinical applications.