251. Defining Conserved Structural Components of the AAV Capsid That Enable Tissue-Specific Transduction Following Systemic Administration

We have previously correlated the destabilization of hydrogen bond networks within recombinant adeno-associated viral (rAAV) capsid surface loop variable region 1 (VR1) to transduction efficiency following intramuscular injection. To investigate the utility of this finding for systemic applications, the capsid structures of rAAV serotypes 1 through 9 were computationally analyzed and VR1 residues participating in hydrogen bonding were individually deleted. Mutant capsids were assessed for tissue tropism and transduction efficiency following intravenous administration into mice. Ex vivo luciferase assays revealed increased muscle transduction and targeting in five of the nine serotypes (rAAV1, rAAV6, rAAV7, rAAV8, rAAV9); cardiac and skeletal muscle transduction were enhanced by up to 170-fold and 9027-fold over parental serotypes, while hepatic transduction was decreased up to 427-fold. VR1 stability also appeared critical to the remaining serotypes, as VR1 deletion mutations rendered rAAV2 and rAAV3b defective in transduction, and rAAV4 and rAAV5 defective in virion production. Intriguingly, amino acid insertions into VR1 produced an opposing phenotype: rAAV1, rAAV2, rAAV3b, and rAAV6 capsids could instead be targeted to transduce the liver with high efficiency, increasing hepatic transduction up to 132-fold. Finally, VR1 mutation synergized with established tyrosine-to-phenylalanine mutations to further increase transduction efficiency while maintaining preferential cardiac/skeletal muscle targeting.