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 (<10 bp) in cells, including CMs, but efficiency of larger DNA insertion is low.
Recent work has developed a template-jumping (TJ) PE method, which mimics the genomic insertion
mechanism of retrotransposons to achieve efficient large DNA insertion in cells. This aim will measure the
efficiency of targeted DNA insertion by TJ-PE (vs. canonical PE) to rewrite the 18-bp RBM20 cluster in RBM20-
HEK293T cells and isogenic induced pluripotent stem cell (iPSCs) expressing nine distinct RBM20 cluster
variants. Edited and unedited iPSCs will be differentiated into CMs to characterize the molecular consequences
of each pathogenic variant and the extent to which TJ-PE-mediated rewriting reverses these defects. Aim 2 will
investigate the toxicity of genomic editing in CMs. The RT domain of PE bears inherent risks of genotoxicity and
a low affinity for dNTP substrates, a critical consideration in CMs due to their limited regenerative capacity and
low dNTP environment. Recent work has developed a template-jumping DNA polymerase prime editor (DPE)
with a higher affinity for dNTPs and may or may not be less genotoxic than RT. This aim will test the RBM20
cluster rewriting efficiency of TJ DPE (vs TJ PE) in RBM20-HEK293T reporter lines, isogenic iPSCs, and
differentiated CMs. Following delivery to CMs, genotoxicity and phenotypic changes will also be determined.
This work will inform the development of an efficient, safe PE platform to treat genetically diverse cases of
RBM20-DCM and provide the fellow with training in therapeutic gene editing and genetic cardiac disease biology.
