(582c) Calibrating Schema-Guided Computational Design of Adeno-Associated Virus Chimeras

Adeno-associated virus (AAV) is a non-pathogenic member of the Parvoviridae virus family that is increasingly being exploited as a gene delivery vehicle for human gene therapy. While there is great promise in virus nanoparticle (VNP) gene therapy, it has yet to be established as the leading treatment platform due to cell targeting inefficiencies. Researchers have been slow to create VNPs with new functions, such as specific cell targeting, because 1) the effects of even single amino acid substitutions on virus capsid structures cannot yet be predicted and 2) the current throughput of combinatorial screening is a limiting factor in mining chimeric libraries for new structures and functions. To overcome these challenges, we are examining whether the SCHEMA algorithm can predict structural disruption in AAV capsids when highly divergent serotypes are recombined. Homologous recombination has contributed to AAV evolution, but the extent to which AAV capsid proteins (VP1, VP2, and VP3) tolerate amino acid substitutions created through this process remains poorly understood. To evaluate SCHEMA predictions, we created 17 chimeras by recombining divergent (~58% sequence identity) AAV serotypes 2 and 4 and analyzed the structures and functions of these chimeras. Interestingly, chimeric capsids that differed by more than one hundred residues from natural capsids still retained their ability to package viral genomes, a characteristic of properly assembled 60-subunit virus capsids. Even within our small chimera population, we observed structural and functional diversity. For example, chimeric capsids varied in their ability to assemble and protect genomes from nucleases. We also found that chimera structural disruption, calculated using the Schema algorithm, tended to correlate with capsid disruption: only the chimera with the lowest structural disruption completely protected its genome from nuclease digestion and retained the ability to transduce Cos-7 cells like wild-type AAV2 and AAV4. These results suggest that the sequence space accessed by recombination of highly divergent AAV serotypes is rich in genetically-encoded VNPs with diverse sequences and binding properties. Our findings indicate that Schema-guided recombination should be useful for creating libraries enriched in capsid-forming proteins with a range of functions for future gene therapy applications.