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Updated: Sep 9, 2025

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

Siddhardha S Maligireddy1, Mariana D Mandler1, Judith C Lunger1

  • 1Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Molecular Therapy. Nucleic Acids
|September 2, 2025
PubMed
Summary

Strategies to restore fumarate hydratase (FH) expression were explored for Hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Splicing modulation using CRISPR-Cas9 and antisense oligonucleotides (ASOs) successfully excluded a cryptic exon, offering therapeutic potential.

Keywords:
ASOCRISPR-Cas9FHHLRCCMT: Oligonucleotides: Therapies and Applicationssplicing

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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.