CRISPR screen for rAAV production implicates genes associated with infection

Area of Science:

• Molecular Biology
• Gene Therapy Manufacturing
• Virology

Background:

• Recombinant adeno-associated virus (rAAV) vectors are crucial for gene therapy, with several approved treatments and many more in development.
• Current rAAV manufacturing processes are expensive, time-consuming, and labor-intensive, hindering wider clinical application.
• Identifying host factors that can enhance rAAV production is essential for improving manufacturing efficiency.

Purpose of the Study:

• To identify host genes that can be targeted to increase recombinant adeno-associated virus (rAAV) particle yield.
• To explore the role of host cell machinery in viral vector production and assembly.
• To develop strategies for more efficient and cost-effective rAAV manufacturing.

Main Methods:

• Conducted a genome-wide CRISPR knockout screen in HEK 293 cells to identify genes affecting rAAV production.
• Utilized an antibody against intact AAV2 capsids and flow cytometry for high-throughput screening.
• Analyzed the impact of specific gene knockouts on rAAV yield.

Main Results:

• Knockout of heparan sulfate biosynthesis genes, previously linked to rAAV infectivity, decreased rAAV production.
• Disruption of several vesicular trafficking proteins significantly increased rAAV yields in producer cells.
• Demonstrated that host proteins involved in viral infection can be repurposed for viral assembly.

Conclusions:

• Host cell genetic manipulation can enhance recombinant adeno-associated virus (rAAV) production.
• Vesicular trafficking pathways represent potential targets for optimizing viral vector manufacturing.
• Findings contribute to developing more efficient and scalable gene therapy production methods.