Striated muscle-specific base editing enables correction of mutations causing dilated cardiomyopathy

Abstract

Dilated cardiomyopathy (DCM) is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of DCM patients harbor heritable mutations which are amenable to CRISPR-based gene therapy. However, challenges related to delivery of the editing complex and off-target concerns hamper the broad applicability of CRISPR agents in the heart. We employed a combination of the viral gene transfer vector AAVMYO with superior targeting specificity of heart muscle tissue and CRISPR base editors to repair patient mutations in the cardiac splice factor Rbm20, which cause aggressive and arrhythmogenic DCM. Using optimized conditions, we could improve splice defects in human iPSC-derived cardiomyocytes (iPSC-CMs) and repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restored the localization defect of RBM20 in 75% of cells and splicing of RBM20 targets including TTN. Three months after injection, cardiac dilation and ejection fraction reached wild-type levels. Single-nuclei RNA sequencing (snRNA-seq) uncovered restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing (WGS) revealed no evidence for aberrant off-target editing. Our study highlights the potential of base editors combined with AAVMYO to achieve gene repair for treatment of hereditary cardiac diseases.

Mechanisms of RBM20 Cardiomyopathy: Insights From Model Systems

“…9 We reported similar findings for mice carrying the S637A variant (analogous to S635A in humans). 10 Analysis of other variants in the NLS, including P633L, R634Q, and R636S (number based on the human sequence), in animal models and hiPSC-CMs, 12–18,25,27 have provided further support for the pathogenesis of variants in this portion of the protein.…”

“…Recent studies from our laboratory and others have confirmed that NLS-disrupting variants in exon 9 are causative in RBM20 cardiomyopathy using in vivo and in vitro model systems (Table 1). 9,10,12–18,25,27 For example, our laboratory generated mice harboring the S639G variant (analogous to S637G in humans) and showed that these mice develop a severe DCM with early onset heart failure that phenocopies disease in human patients with the same variant. 9 We reported similar findings for mice carrying the S637A variant (analogous to S635A in humans).…”

“…11,12 The discovery that these variants promote nuclear exclusion and sarcoplasmic accumulation in biomolecular condensates not only confirmed prior in vitro data 25 but also provided a novel putative disease paradigm in RBM20-associated DCM based on the formation of pathological biomolecular condensates (Figure 3). The formation of sarcoplasmic RBM20 condensates secondary to NLS variants has now been validated in human patient tissue, 26 as well as a growing list of additional models, including gene-edited animals and hiPSC-CMs 9,10,13–18,27,28 ; yet, many questions remain to be answered chief among which is to what extent these condensates contribute to the pathogenesis of RBM20 cardiomyopathy.…”

“…12 Three groups have independently generated Rbm20 R636Q mice (analogous to R634Q in humans). 13,14,27 More recently, Rbm20 P635L mice (analogous to P635L in humans) have been generated. 27,28…”

“…11,12,26 In the years since this discovery, these findings have been confirmed by our laboratory and others in additional rodent models and human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). 9,10,13–18,27,28 The finding that RS domain pathogenic variant knock-in animals recapitulate aspects of RBM20 cardiomyopathy not observed in knockout rodents was a significant breakthrough in the field as it not only unambiguously confirmed that these variants are causative in aggressive DCM but also implicated sarcoplasmic RBM20 condensates in the development of this devastating disease. Nevertheless, many questions remain to be answered.…”