A2 - AAV Vectors - Virology and Vectorology
453: CRISPR Screen for rAAV Production Implicates Genes Associated with Infection
Type: Poster Session
Poster Board Number: 453
Presentation Details
Session Title: Wednesday Posters: AAV Vectors - Virology and Vectorology
Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the growing gene therapy field, with five approved AAV-mediated gene therapies already on the market and numerous more in clinical trials. However, manufacturing rAAV vectors is an expensive, timely, and labor-intensive process that limits the commercial use of AAV-mediated gene therapies. To address this limitation, we screened producer cells for genes that could be targeted to increase rAAV yield. Specifically, we performed a CRISPR-based genome-wide knockout screen in HEK 293 cells using an antibody specific to intracellular, intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan-sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. These findings were validated in a secondary screen performed in producer cells that lack AAV receptor (AAVR). Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields that were further validated using RNP based approach. This approach yielded higher knockout efficiency and avoided the selection effect on cell growth and viability. In summary, our work presents new pathways that can be co-opted to improve rAAV production, with practical applications to the adoption of AAV-gene therapies more broadly.
(S.A. and E.O. contributed equally to the work)
Plain Language Summary
Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the gene therapy field. However, manufacturing rAAV vectors is an expensive, and labor-intensive process that limits its commercial use. To address this limitation, we performed a CRISPR-based genome-wide knockout screen in HEK293 cells using an antibody specific to intracellular, intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan-sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. These findings were validated in a secondary screen performed in producer cells that lack AAV receptor (AAVR). Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields that were further validated using RNP-based approach. In summary, our work presents new pathways that can be co-opted to improve rAAV production.
Sakshi Arora1, Emily O’Driscoll2, John Lang1, Beverly L. Davidson1, Ophir Shalem1
1Children's Hospital of Philadelphia, Philadelphia, PA,2Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA"
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