Evolving membrane-associated accessory protein variants for improved adeno-associated virus production

Adam J Schieferecke; Hyuncheol Lee; Aleysha Chen; Vindhya Kilaru; Justin Krish Williams; David V Schaffer

doi:10.1016/j.ymthe.2023.12.015

Evolving membrane-associated accessory protein variants for improved adeno-associated virus production

Mol Ther. 2024 Feb 7;32(2):340-351. doi: 10.1016/j.ymthe.2023.12.015. Epub 2023 Dec 18.

Authors

Adam J Schieferecke¹, Hyuncheol Lee², Aleysha Chen³, Vindhya Kilaru³, Justin Krish Williams⁴, David V Schaffer⁵

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
² California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA.
³ Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.
⁴ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
⁵ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA; Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address: schaffer@berkeley.edu.

PMID: 38115579
PMCID: PMC10861973 (available on 2025-02-07)
DOI: 10.1016/j.ymthe.2023.12.015

Abstract

Manufacturing sufficient adeno-associated virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, although the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 open reading frame (ORF) (AAV2-ΔMAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1 × 10⁶ MAAP protein variants to five rounds of packaging selection into the AAV2-ΔMAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk-selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a frameshifted C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

Keywords: AAV2; AAV6; AAV9; MAAP; adeno-associated virus; directed evolution; gene therapy; manufacturing; membrane-associated accessory protein.

MeSH terms

Capsid / metabolism
Capsid Proteins* / genetics
Capsid Proteins* / metabolism
Dependovirus* / metabolism
Genetic Vectors / genetics
Serogroup
Transgenes

Substances

Capsid Proteins