317. HH67.2, a Promising Novel Adeno-Associated Virus (AAV) Vector, Shows Improved Muscle Specificity and Infectivity Compared To AAV9

Abstract

Currently, AAV9 serotype is considered a robust heart and muscle gene delivery vehicle widely used in preclinical studies and in a clinical trial for spinal muscle atrophy (SMA). However, the systemic delivery of AAV9 with large vector doses leads to undesirable effects such as ectopic gene expression in other tissues. These shortcomings may be overcome by using certain strategies such as the development of novel AAV vectors with muscle-specific tropism. Previously, our lab identified a novel muscle specific AAV vector, HH67, by DNA shuffling and in vivo selection. Similar to other evolved tissue-specific vectors, the HH67 vector showed reduced liver tropism, but its transduction efficiency in heart and muscle tissues was still lower than that of AAV9. We set out to introduce site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of AAV capsids which has been shown to significantly increase gene transfer efficiency for certain AAV serotypes. In this study, we investigated whether two capsid mutations in HH67, Y445F and Y731F (named HH67.2), would enhance gene delivery to the muscle while maintaining low non-specific transgene expression. We found that the HH67.2 mutant had a significant increase in transgene delivery to the muscle compared to AAV9 after systemic delivery in C57BL/6 mice via tail-vein injection. In the muscle, expression of the LacZ reporter gene (2.5 × 10^5±65413 units/μg HH67.2 vs 7.2 × 10^4±8211 units/μg AAV9; p=0.0049) and the DNA genome copy number (4.50±2.07 VG/cell HH67.2 vs 1.15±0.197 VG/cell AAV9; p=0.0118) both increased 3.5-fold compared to AAV9. Furthermore, liver and other non-specific organs showed up to an 11-fold decrease in HH67.2 vector copy number (11.8±2.5 VG/cell HH67.2 liver vs 126.8±39.6 VG/cell AAV9 liver; p<0.0001). However, HH67.2 and AAV9 had no significant difference in gene expression in the heart. LacZ expression results were confirmed by vector genome copy number via quantitative PCR and X-Gal staining. Finally, we also introduced the same Y-F mutations in three similarly shuffled and in vivo selected AAV capsids such as M41, H43 and H48, but the mutations decreased infectivities of all these capsids. Our results suggest that HH67.2 is a novel AAV vector with enhanced muscle specificity and infectivity compared to AAV9 and could be a promising vehicle to deliver therapeutic genes to skeletal muscle or heart.**This work was part of Carrie Martin and Christina Mayer's research project for the Honors Program at the UNC Eshelman School of Pharmacy.