이현철 (UC Berkeley, Vivere Oncotherapies Inc) | 한빛사논문 > 한빛사

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조회695

이현철Hyuncheol Lee

UC Berkeley, Vivere Oncotherapies Inc

CV File 130 kb

Mol. Ther., Dec 18 2023, S1525-0016(23)00679-2 | https://doi.org/10.1016/j.ymthe.2023.12.015 Evolving Membrane-Associated Accessory Protein Variants for Improved Adeno-Associated Virus Production

Adam J. Schieferecke ^1,2∗, Hyuncheol Lee ^2∗, Aleysha Chen ³, Vindhya Kilaru³, Justin Krish Williams ¹, David.V. Schaffer Ph.D. ^1,2,3,4,5

¹Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA

²California Institute for Quantitative Biosciences, University of California, Berkeley, CA, 94720, USA

³Department of Bioengineering, University of California, Berkeley, CA, 94720, USA

⁴Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA

⁵Helen Wills Neuroscience Institute, University of California, Berkeley, CA, 94720, USA

∗Denotes co-first author

Corresponding author : David.V. Schaffer Ph.D.

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, though 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 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 1E6 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 novel C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

논문정보

형식Research article
게재일2023년 12월 (BRIC 등록일 2023-12-26)
연구진국외 연구진
분야 생명과학 > 생명과학 기타

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