AAV-mediated genome editing is influenced by the formation of R-loops

Area of Science:

• Molecular Biology
• Genetics
• Gene Therapy

Background:

• Recombinant adeno-associated viral vectors (rAAV) are widely used for in vivo gene therapy due to their ability to stimulate homologous recombination (AAV-HR).
• However, rAAV integration throughout the genome poses safety concerns for gene editing.
• Genomic R-loops, DNA:RNA hybrids, are implicated in various genomic processes.

Purpose of the Study:

• To investigate the role of R-loops in rAAV integration and homologous recombination.
• To compare R-loop formation in a transformed cell line versus intact tissue.
• To explore R-loop modulation as a strategy to improve rAAV gene therapy safety and efficacy.

Main Methods:

• DNA-RNA immunoprecipitation sequencing (DRIP-seq) was performed in murine HEPA1-6 hepatoma cells and whole murine liver.
• Experiments involved genetic and pharmacological upregulation of R-loops.
• The Albumin gene was used as a model locus for genome editing in vitro and in vivo.

Main Results:

• R-loop upregulation enhanced AAV-HR in mice.
• The R-loop prone 3' end of the Albumin gene was efficiently edited by AAV-HR.
• A positive correlation was observed between previously reported off-target rAAV integration sites and R-loop enriched genomic regions.

Conclusions:

• High levels of R-loops in highly transcribed genes may promote rAAV vector genome integration.
• Modulating R-loops could improve the safety and efficacy of genome editing.
• These findings may help predict genomic regions susceptible to rAAV insertional mutagenesis.