550. Developing an Optimal Insulin Augmentation Therapy to Improve AAV Gene Transfer to Skeletal Muscle and Liver in Mice

Abstract

We previously reported a novel finding that showed an insulin augmentation therapy can improve rAAV transduction of skeletal muscle and liver in cultured cells and mice. The insulin augmentation therapy in this earlier report was given just prior to AAV administration and was sustained for 28 days. The use of a long-term insulin augmentation was to determine if mice showed early signs of insulin resistance due to prolonged hyperinsulinemia. From the initial study we concluded that long-term insulin augmentation therapy improved AAV gene transfer to skeletal muscle and liver but that the mice showed a lack of elevated insulin activity and an increase in cortisol over time which are signs of insulin resistance. Additionally, our initial findings also suggested that an acute insulin augmentation therapy during the window of AAV administration would be sufficient without the concern for developing insulin resistance. The purpose of this new study was to investigate if an acute insulin augmentation therapy during the window of AAV delivery was sufficient to improve AAV transduction of skeletal muscle and liver. Additionally, we wanted to determine if an insulin augmentation therapy given after AAV infection would also enhance AAV gene transfer. Another aspect of this follow up study was to determine if an insulin augmentation therapy protocol would improve AAV transduction of lung tissue in mice. Finally, we wanted to investigate if fasting mice (which promotes reduced insulin activity) would result in sub-optimal AAV gene transfer. Our results showed an acute insulin augmentation therapy (4 hr treatment) given 30 minutes prior to intramuscular (IM) injection of AAV1-CMV-sc-hFIX or liver-directed delivery (via splenic capsule injection) of AAV8-CMV-sc-hFIX significantly improved (up to 5 Fold) sustained transduction for the length of the study (56 days).View Large Image | Download PowerPoint SlideThe acute therapy showed a minor (but significant) 1.7 Fold increase in AAV1-CMV-EGFP transduction in lung. In-vitro AAV2-CMV-LacZ transduction of HuH7 (human liver) and differentiated C2C12 (mice myofibers) also showed improvement with an acute insulin treatment. A 2hr treatment (5 ug/ml) resulted in a 2.7 fold increase while 8hr, 24hr & 72hr treatments had a 3.5 fold increase and giving insulin 24 hrs after AAV infection had no impact of AAV transduction.View Large Image | Download PowerPoint SlideFinally, prolonged fasting will reduce insulin levels which could impact AAV gene transfer. Fasting mice for 12 hrs prior to and 4 hrs after IM delivery of AAV1-CMV-sc-hFIX or at weekly intervals after administration timed around blood collection did not have an impact on AAV transduction of skeletal muscle.