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

Area of Science:

• Genetics
• Molecular Biology
• Oncology

Background:

• Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is an inherited cancer syndrome caused by loss-of-function mutations in the fumarate hydratase (FH) gene.
• A specific pathogenic variant in intron 9 of the FH gene disrupts normal splicing, leading to the inclusion of a cryptic exon and a premature termination codon, ultimately causing FH deficiency.
• Restoring functional FH protein expression is crucial for therapeutic intervention in HLRCC patients with this specific splicing defect.

Purpose of the Study:

• To identify and test strategies for modulating FH gene splicing to exclude the cryptic exon.
• To evaluate the efficacy of genome editing and antisense oligonucleotides (ASOs) in correcting the splicing defect.
• To assess the potential of splicing modulation as a therapeutic approach for HLRCC.

Main Methods:

• Development of a minigene GFP reporter system to mimic the FH splicing defect.
• Application of CRISPR-Cas9 genome editing to target and correct the splicing abnormality.
• Utilized antisense oligonucleotides (ASOs) to promote the skipping of the cryptic exon.
• Validation in patient-derived fibroblasts to demonstrate allele-specific modulation of FH mRNA isoforms.

Main Results:

• Both CRISPR-Cas9 and ASOs successfully induced the skipping of the cryptic exon in a reporter cell line.
• ASOs demonstrated the ability to alter the balance of FH mRNA isoforms derived from both reference and variant alleles in patient cells.
• The developed reporter system effectively recapitulated the splicing defect observed in HLRCC patients.

Conclusions:

• Splicing modulation strategies, including ASOs and genome editing, show promise for correcting the FH splicing defect in HLRCC.
• Antisense oligonucleotides offer a potential therapeutic avenue for HLRCC caused by non-coding FH mutations.
• These findings support the development of targeted therapies aimed at restoring FH function in HLRCC.