433269 AAV Ancestral Reconstruction Library Enables Selection of Broadly Infectious Viral Variants

Monday, November 9, 2015: 3:55 PM
151D/E (Salt Palace Convention Center)
Jorge Santiago-Ortiz1, David Ojala2, Oscar Westesson3, John Weinstein3, Sophie Wong3, Andrew Steinsapir3, Sanjay Kumar4, Ian Holmes3 and David V. Schaffer5, (1)Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, (2)Chemical and Biomolecular Engineering, University of California, Berkeley, (3)University of California, Berkeley, Berkeley, CA, (4)Bioengineering, University of California, Berkeley, Berkeley, CA, (5)Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. Enhanced vectors are required to extend these landmark successes to other indications, however, and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties, and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, ancestral AAVs were more thermostable than modern serotypes and did not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Finally, ancestral variants mediated 19-31 fold higher gene expression in muscle compared to AAV1, a clinically utilized serotype for muscle delivery, highlighting their promise for gene therapy. The putative mutational tolerance and evolvability of this library could be further harnessed in directed evolution studies to overcome gene therapy challenges such as targeted gene delivery and immune evasion.

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See more of this Session: Nucleic Acid Delivery
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division