A single prime editing strategy for correcting diverse mutations responsible for RBM20-associated dilated cardiomyopathy

PROJECT SUMMARY Familial dilated cardiomyopathy (DCM) is a heritable disorder characterized by progressive enlargement of the heart’s ventricles and impaired contraction, leading to early-onset heart failure and heart transplantation at a young age. A significant subset of inherited DCM cases is attributed to mutations in an 18-bp segment of the

arginine-/serine-rich (RS) domain of the RNA binding motif protein 20 (RBM20) gene. These mutations lead to aberrant splicing of cardiac genes critical to contractility and calcium signaling, causing deterioration of cardiac function. Approved medical treatments offer symptomatic relief, but there remains an unmet need for CRISPR

gene editing therapies that correct the genetic causes of RBM20-induced DCM to halt disease progression. This project seeks to develop a gene editing strategy that replaces the entire 18-bp RBM20 pathogenic cluster with a synonymous DNA sequence (i.e., rewriting) to offer a universal treatment for all RBM20-DCM patients

with RS-domain mutations. This approach is conceivable with prime editing (PE) – which uses an RNA-guided Cas9 nickase fused to reverse transcriptase (RT) to mediate base substitutions, genomic insertions, or deletions. PE-mediated base substitution corrected an RBM20 mutation to reverse phenotypes in DCM cells, but targeted

DNA insertion by PE to rewrite the RBM20 pathogenic cluster has not been tested. The ideal PE platform for RBM20-DCM will maximize DNA insertion efficiency in cardiomyocytes (CMs) while minimizing genotoxicity. Aim 1 will establish the efficiency of a PE method for rewriting the RBM20 pathogenic cluster. Canonical PE

effectively inserts small sequences (