Genetic therapy for hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder, affecting ~1:500 individuals. Pathogenic variants in sarcomeric genes, particularly MYBPC3, drive myocardial hypertrophy, diastolic dysfunction, and elevated risk of sudden cardiac death. Current treatments mitigate symptoms but do not address the underlying genetic defect. Gene-editing strategies hold curative potential, yet conventional approaches show limited efficiency in non-dividing cardiomyocytes.

We sought to develop a targeted gene-editing therapy for Mybpc3-associated HCM using CRISPR/Cas9-mediated Homology-Independent Targeted Integration (HITI), which leverages non-homologous end joining to enable precise genomic insertion in post-mitotic cells. Our objective was to correct Mybpc3 mutations, restore MYBPC3 protein expression, and improve cardiac structure and function in heterozygous and homozygous Mybpc3 mutant mouse models.

Mybpc3 c.2373InsG mutant mice carrying a human pathogenic variant were crossed with Cas9-expressing mice. Two HITI strategies were designed: one targeting the mutation site and another replacing the entire affected exon. Guide RNAs and donor templates were packaged into AAV vectors and delivered in vitro and in vivo. Editing efficiency and therapeutic benefit were assessed by immunostaining, Western blotting, echocardiography, and histological analysis.

HITI-based editing achieved efficient genomic correction with partial restoration of MYBPC3 expression. Approximately 56% of cardiomyocytes exhibited successful editing in a dose‑dependent manner, accompanied by recovery of up to 53% of normal MYBPC3 protein levels and measurable improvements in cardiac function in vivo.

This study represents the first application of HITI to correct a genetic cardiomyopathy. These findings demonstrate that HITI enables precise gene repair in cardiomyocytes and holds strong therapeutic promise for HCM.