681. Increasing Insulin Activity During AAV (Adeno-Associated Virus) Administration to Muscle Improves Gene Transfer in Mice

25+ years of AAV (adeno-associated virus) gene therapy research has resulted in optimized: 1- delivery routes, 2- vectors, 3- genomes and 4- immunosuppression. These studies have yielded the first westernized AAV gene therapy drug to treat LPLD (lipoprotein Lipase Deficiency) and the promise of future AAV gene therapy drugs to treat hemophilia B and other genetic disorders. Interestingly, we have little understanding of how hormones regulate these AAV drug/future drug candidates. Our studies show that hormone management has a place in improving AAV gene therapy. Since liver and skeletal muscle are strong responders to insulin and these tissues are common targets for these AAV treatments, our studies focused on how insulin regulates AAV gene therapy. We tested insulin supplementation in: HepG2 (liver), differentiated myocytes, HEK293 (kidney) and A549 (lung epithelial). HepG2 and myocytes showed elevated INSR (insulin receptor) mRNA levels (6 to 9-fold respectively) compared to HEK293 and A549 cells. The elevated INSR levels paralleled a significant 2.5 and 3 fold increase in AAV2-CMV-LacZ (M.O.I of 5e3)gene transfer to HepG2 and myocytes respectively when media was supplemented with insulin. Insulin enhanced gene transfer was not seen in HEK293 and A549 cells. Additionally, using HI glucose (4.5 g/L) and LOW glucose (1.0 g/L) medias, we showed that these results were not influenced by glucose availability. When comparing INSR levels in C57BL/6 mouse tissue, liver and skeletal muscle showed a 10-Fold and 22-Fold increase respectively compared to lung tissue. In these mice, intra-muscular (I. M.) gene delivery using AAV1-CMV-schFIX (dose: 3.0e10 v.g.) was significantly improved 4 to 5 fold (figure 1) using insulin augmentation protocols: either a subcutaneous insulin pellet which released 4 U/kg per day or a high carbohydrate (70%) diet. View Large Image | Download PowerPoint SlideThe insulin augmentation protocols began 1.0 hr prior to AAV administration and continued for 4 weeks after. AAV delivery occurred during the window of a significant drop (3 fold) in blood glucose indicating an increase in insulin activity. The glucose levels had returned to normal 7 days later even though the insulin augmentation protocols continued for 4 weeks likely due to antagonistic feedback responses to long-term hyperinsulinemic conditions (i.e. cortisol release). The insulin augmentation protocols only demonstrated an increase in insulin activity at the onset of vector delivery but not after, yet elevated hFIX levels were sustained throughout the study, even after the insulin augmentation protocols ended. This indicates that insulin activity does not impact sustained transgene expression and may only regulate AAV uptake, intracellular trafficking or both. Data from liver directed studies using AAV8-CMV-schFIX are pending.