Genome-wide activation screens to increase adeno-associated virus production.

Autor:	Barnes CR; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA., Lee H; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA., Ojala DS; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA., Lewis KK; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA., Limsirichai P; Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA., Schaffer DV; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA 94720, USA.; California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA 94720, USA.; Department of Bioengineering, University of California, Berkeley, Berkeley, CA 94720, USA.; Department of Cell and Molecular Biology, University of California, Berkeley, Berkeley, CA 94720, USA.; Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA 94720, USA.
Jazyk:	angličtina
Zdroj:	Molecular therapy. Nucleic acids [Mol Ther Nucleic Acids] 2021 Jul 16; Vol. 26, pp. 94-103. Date of Electronic Publication: 2021 Jul 16 (Print Publication: 2021).
DOI:	10.1016/j.omtn.2021.06.026
Abstrakt:	We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging. Competing Interests: C.R.B., D.S.O., and D.V.S. are inventors on patents related to cell lines for increased production of AAV. D.V.S. is a co-founder of 4D Molecular Therapeutics. (© 2021 The Authors.)
Databáze:	MEDLINE
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