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AAV ancestral reconstruction library enables selection of broadly infectious viral variants

Gene Therapy volume 22, pages 934–946 (2015)Cite this article

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Abstract

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications 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, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.

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Acknowledgements

We are grateful to Professor Brent Reynolds (University of Florida) for kindly providing the L0 human GBM tumor-initiating cells. This work was supported by the National Institutes of Health grant (R01EY022975). DSO is supported by a National Science Foundation Graduate Fellowship and JSO is supported by a National Science Foundation Graduate Fellowship and a UC Berkeley Graduate Division Fellowship. IH and OW were supported by the National Human Genome Research Institute grant (HG004483).

Author Contributions

DSO and JSO designed the project and carried out the experimental work, analyzed and interpreted the data, and wrote and edited the manuscript. OW and JRW designed the project and edited the manuscript. SYW carried out GBM cell culture and edited the manuscript. AS assisted with molecular cloning. SK edited the manuscript. IH designed the project, analyzed and interpreted the data, supervised the project and edited the manuscript. DVS designed the project, interpreted the data, supervised the project through all stages and edited the manuscript.

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  1. J Santiago-Ortiz and D S Ojala: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, USA

    J Santiago-Ortiz, D S Ojala, A Steinsapir & D V Schaffer

  2. Department of Bioengineering, University of California, Berkeley, CA, USA

    O Westesson, S Y Wong, S Kumar, I Holmes & D V Schaffer

  3. Department of Chemistry, University of California, Berkeley, CA, USA

    J R Weinstein

  4. The Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA

    D V Schaffer

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  1. J Santiago-Ortiz
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Correspondence to D V Schaffer.

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DVS is an inventor on patents involving AAV directed evolution.

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Santiago-Ortiz, J., Ojala, D., Westesson, O. et al. AAV ancestral reconstruction library enables selection of broadly infectious viral variants. Gene Ther 22, 934–946 (2015). https://doi.org/10.1038/gt.2015.74

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