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Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design

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Science  29 Nov 2019:
Vol. 366, Issue 6469, pp. 1139-1143
DOI: 10.1126/science.aaw2900

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The fitness landscape of AAV

Adeno-associated virus (AAV) is an important gene therapy vector. Using tools from synthetic biology, Ogden et al. provide a comprehensive view of how sequence changes in capsid proteins affect AAV properties. After saturation mutagenesis of the AAV2 capsid gene, the resulting library was subjected to multiplexed phenotypic analyses, including virus production, immunity, thermostability, and biodistribution. The mutant distribution to major organs in mice revealed dominant trends affecting in vivo delivery. Moreover, the findings uncovered a viral accessory protein with a role in viral production. Finally, a model built from the capsid fitness landscape enabled machine-guided design of useful variants with much higher efficiency than random mutagenesis.

Science, this issue p. 1139

Abstract

Adeno-associated virus (AAV) capsids can deliver transformative gene therapies, but our understanding of AAV biology remains incomplete. We generated the complete first-order AAV2 capsid fitness landscape, characterizing all single-codon substitutions, insertions, and deletions across multiple functions relevant for in vivo delivery. We discovered a frameshifted gene in the VP1 region that expresses a membrane-associated accessory protein that limits AAV production through competitive exclusion. Mutant biodistribution revealed the importance of both surface-exposed and buried residues, with a few phenotypic profiles characterizing most variants. Finally, we algorithmically designed and experimentally verified a diverse in vivo targeted capsid library with viability far exceeding random mutagenesis approaches. These results demonstrate the power of systematic mutagenesis for deciphering complex genomes and the potential of empirical machine-guided protein engineering.

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