260. AAV Ancestral Reconstruction Library Enables Selection of Broadly Infectious Viral Variants

Adeno-associated virus (AAV) vectors have demonstrated clinical efficacy in treating several diseases. Improved vectors may extend these landmark successes to other indications, and protein engineering approaches are enhancing the gene delivery properties of AAV vectors to address such unmet medical needs. To access new viral sequences with potentially enhanced infectious properties, and to gain insights into AAV's evolutionary history, we computationally designed and experimentally characterized an ancestral reconstruction of the AAV capsid. To this end, we first generated a phylogenetic tree of AAV sequences through Bayesian Markov chain Monte Carlo simulations, then selected a node for reconstruction based on its relative confidence value and proximity to AAV serotypes of clinical interest. We next used HandAlign, a Markov chain Monte Carlo alignment sampler, to predict the ancestral sequence of the most likely alignment at the selected node. This generated a reconstruction where most amino acids were predicted with high confidence, while 32 of the positions were predicted with low confidence. In order to address uncertainty in these positions, we synthesized the inferred ancestral capsid as a large combinatorial library incorporating degenerate residues at the 32 variable amino acid sites. We then characterized the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to six rounds of selection on a panel of representative cell lines. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general the selected ancestral libraries were broadly infectious across the cell line panel, indicating that they favor promiscuity over specificity. Unlike many current serotypes, these ancestral AAVs do not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Additionally, ancestral libraries were found to be as susceptible to neutralizing antibodies as extant serotypes, suggesting that such ancestral variant candidates exhibit immunogenic properties similar to modern descendants. Interestingly, ancestral AAVs retained infectivity at temperatures that completely ablated infectivity of modern serotypes; this higher thermostability may have supported increased evolvability through mutational tolerance. Finally, selected variants mediated 19-31 fold higher gene expression in muscle compared to AAV1, a serotype clinically utilized for muscle delivery, highlighting their potential promise for gene therapy.