Correction of aberrant splicing caused by intronic CAPN3 pathogenic variants using RNA-targeted therapeutic strategies in limb-girdle muscular dystrophy type R1.

BACKGROUND: Pre-mRNA splicing is a highly precise process, and it is estimated that approximately 9%-11% of pathogenic variants in patients with rare genetic diseases are caused by non-coding variants that disrupt this mechanism. Developing targeted strategies to correct such splicing defects represents a promising therapeutic avenue. In this proof-of-concept study, we demonstrate the feasibility of rescuing distinct aberrant splicing patterns using tailored RNA-targeted approaches.

RESULTS: We focused on two disease-causing intronic pathogenic variants in the CAPN3 gene (c.1193 + 30G > A and c.1354 + 5G > A), each leading to aberrant 5' splice site selection and premature termination codons. Using a faithful cellular minigene model, we designed and evaluated two variant-specific corrective strategies: a splice-switching oligonucleotide (SSO) to block a gained cryptic donor site (c.1193 + 30G > A), which restored canonical transcript levels to approximately 75% of wild-type; and an engineered U1 snRNA with compensatory base substitutions to restore a weakened canonical 5' splice site (c.1354 + 5G > A), which increased correct splicing from ~ 10% to nearly 60%.

CONCLUSIONS: This work establishes a versatile therapeutic framework, providing compelling in vitro validation that precisely targeted RNA-based strategies can be successfully adapted to correct different types of splicing defects, offering a promising blueprint for the treatment of splicing-deficient genetic disorders.