Genetic modulation of RNA splicing rescues BRCA2 function in mutant cells

This study provides proof of principle for the therapeutic potential of splicing modulation as a strategy to rescue BRCA2 function in mutant cells. Variants in the hereditary cancer-associated BRCA1 and BRCA2 genes can alter RNA splicing, producing transcripts that encode internally truncated yet potentially functional proteins. However, few studies have quantitatively analyzed variant-specific splicing isoforms. Here, we investigated cells heterozygous and homozygous for the BRCA2 :c.681+5G>C variant. Using droplet digital RT–PCR, we identified two variant-specific mRNA isoforms. The predominant transcript is out-of-frame, contains a premature termination codon, and is degraded via the nonsense-mediated mRNA decay pathway. In addition, we detected a novel minor isoform encoding an internally truncated protein lacking non-essential domains. Homozygous mutant cells expressed low levels of BRCA2 protein and were defective in DNA repair. Using CRISPR-Cas9 gene editing, we induced the production of in-frame transcripts in mutant cells, which resulted in increased protein expression, enhanced RAD51 focus formation, and reduced chromosomal breaks after exposure to genotoxic agents. Our findings highlight the therapeutic potential of splicing modulation to restore BRCA2 function in mutant cells, offering a promising strategy to prevent cancer development.