Double-strand break repair pathways differentially affect processing and transduction by dual AAV vectors

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• Recombinant adeno-associated viral vectors (rAAV) are widely used for gene delivery.
• Current rAAV vectors have limited DNA carrying capacity, restricting therapeutic applications.
• Dual trans-splicing vectors expand payload size by concatenating rAAV genomes, but transduction efficiency is often suboptimal.

Purpose of the Study:

• To identify host factors regulating dual rAAV vector transduction efficiency.
• To investigate the role of DNA repair pathways in rAAV genome concatenation.
• To explore strategies for improving large transgene delivery using rAAV vectors.

Main Methods:

• Performed a genome-wide screen to identify host cell regulators of dual vector transduction.
• Assessed the impact of Homologous Recombination (HR) pathway factors on rAAV concatenation and transgene expression.
• Utilized gene depletion and pharmacological inhibition of HR factors BRCA1 and Rad51.

Main Results:

• Discovered that Homologous Recombination (HR) is inhibitory to dual rAAV vector transduction.
• Depletion or inhibition of HR factors BRCA1 and Rad51 significantly increased transgene reconstitution.
• Demonstrated enhanced concatenation and expression of large split transgenes mediated by rAAV upon HR inhibition.

Conclusions:

• Host DNA repair pathways, specifically HR, play a critical role in limiting dual rAAV vector efficacy.
• Inhibition of HR factors like BRCA1 and Rad51 can improve rAAV-mediated delivery of large genetic payloads.
• Pharmacological targeting of DNA repair mechanisms offers a promising strategy to enhance gene therapy vector capacity.