In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector

• In silico ancestral sequence reconstruction leads to infectious viral particles
• Anc80, an ancestor of AAV1, 2, 8, and 9, is a potent in vivo gene therapy vector
• The putative evolutionary lineage of AAV is functionally restored
• Ancestral sequence reconstruction elucidates complex structure-function relations

SummaryAdeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina.

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