Targeting a pathogenic cryptic exon that drives HLRCC to induce exon skipping

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) arises from the loss of fumarate hydratase (FH) activity, leading to the development of cutaneous and uterine leiomyomas as well as early-onset type 2 papillary renal cell carcinoma. Recently, we identified a pathogenic intronic variant in the FH gene that disrupts splicing by creating a novel splice acceptor site, resulting in the aberrant inclusion of a cryptic exon. This exon introduces a premature termination codon, leading to loss of enzymatic function. To restore FH expression, we aimed to identify strategies to exclude the cryptic exon from the mature mRNA. To this end, we generated a mini-gene GFP reporter system that recapitulates the splicing defect observed in patients. We employed CRISPR-Cas9-mediated genome editing and antisense oligonucleotides (ASOs) to modulate splicing and were able to show that both strategies can successfully promoted exon skipping in a reporter cell line. Furthermore, we were able to shift the balance between the different FH mRNA isoforms in patient derived fibroblasts using ASOs. These findings support the potential of splicing modulation as a therapeutic approach for HLRCC-associated FH non-coding loss-of-function mutations. Overall design: Sequencing of sgRNA cassettes from genomic DNA extracted from cells before and after sorting for GFP-positive cells—expression of GFP reporters on splicing.