# Pathogenic hotspots illuminate mechanism and therapeutic potential in arrhythmogenic cardiomyopathy

> **NIH NIH R01** · STANFORD UNIVERSITY · 2023 · $771,818

## Abstract

PROJECT SUMMARY
Recent exponential advancement of genome engineering technology has revived enthusiasm for its
implementation in genetic cardiomyopathies. This is especially promising for arrhythmogenic cardiomyopathy
(ACM), a cause of sudden cardiac death and end stage heart failure. Most early genome engineering therapies
have focused on gene replacement; however, a significant minority of ACM variants likely act via dominant
negative disease mechanisms that will not respond to gene replacement therapy. RNA Binding Motif 20
(RBM20) and plakophilin 2 (PKP2) are genes associated with deadly forms of ACM in which there are both
dominant negative and haploinsufficient pathogenic variants. Variants in these genes cause cardiomyopathy
and arrhythmia by disrupting global cardiomyocyte transcriptional splicing and desmosomal structure,
respectively. hat these variants are clustered in pathogenic hotspots that align to known and novel functional
protein domains, indicating that focused study of these hotspots can illuminate differential disease mechanisms
and potentially reduce the burden of therapeutic design. Our central hypothesis is that variants in pathogenic
hotspots of RBM20 and PKP2 have differential downstream mechanisms that converge on ACM disease
phenotypes, and that these pathogenic hotspots allow the design of a genome engineering strategy to edit
many pathogenic variants with a single reagent. In Aim 1, we will identify haploinsufficient vs. dominant
negative variants in RBM20. We then use high throughput genome engineering techniques to create a library
of these variants in induced pluripotent stem cell cardiomyocytes. We will apply a combination of single cell
library preparation and long read RNAseq to define the downstream consequences of each disease
mechanism on splicing of known and novel RBM20 targets. In Aim 2, we focus on a novel dominant negative
mechanism for C-terminal PKP2 truncating variants in which they lose their plasma membrane localization,
sequestering critical desmosome components in the cytoplasm. We will use variant effect mapping to define
downstream mechanisms of a library of pathogenic PKP2 truncating variants, and will define the role of a novel
PKP2 interactor on PKP2 membrane localization. In Aim 3, we will extend our work showing the feasibility of
single prime editing (PE) reagents for correction of multiple variants in a pathogenic hotspot in vitro: We will
design engineered prime editing (epe)gRNAs with the newest high efficiency PEmax construct for the PKP2 C-
terminus hotspot and dominant negative RBM20 RS domain hotspot in vitro. We will then use innovative
methods to package PEmax in AAVMYO to correct two pathogenic murine Rbm20 RS domain variants in vivo
using the same epegRNA. We will go on to measure the effect of this editing on deep ACM phenotypes. In
summary, this project will capitalize on our identification of pathogenic hotspots in RBM20 and PKP2 to provide
a comprehensive evaluation of varia...

## Key facts

- **NIH application ID:** 10633507
- **Project number:** 1R01HL168059-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Victoria Parikh
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $771,818
- **Award type:** 1
- **Project period:** 2023-04-05 → 2028-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10633507

## Citation

> US National Institutes of Health, RePORTER application 10633507, Pathogenic hotspots illuminate mechanism and therapeutic potential in arrhythmogenic cardiomyopathy (1R01HL168059-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10633507. Licensed CC0.

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