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

Abstract

Dilated cardiomyopathy is the second most common cause for heart failure with no cure except a high-risk heart transplantation. Approximately 30% of 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 employ a combination of the viral 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 dilated cardiomyopathy. Using optimized conditions, we repair >70% of cardiomyocytes in two Rbm20 knock-in mouse models that we have generated to serve as an in vivo platform of our editing strategy. Treatment of juvenile mice restores 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 reach wild-type levels. Single-nuclei RNA sequencing uncovers restoration of the transcriptional profile across all major cardiac cell types and whole-genome sequencing reveals 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

“…9,10 Recently, Rbm20 R636Q mice, as well as Rbm20 P635L mice, were also generated and characterized by the Steinmetz group with similar findings of a DCM-like phenotype with cardiac dysfunction and premature mortality. 27 Interestingly, a comparison of heterozygous Rbm20 P635L and Rbm20 R636Q mice revealed greater cardiac dysfunction in the latter, which correlated with RBM20 nuclear exclusion and sarcoplasmic granule formation. 27 This is noteworthy as it may explain our previous observation that Rbm20 S639G mice develop more severe disease than Rbm20 S637A mice.…”

“…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][13][14][15][16][17][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).…”

“…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][13][14][15][16][17][18]25,27 have provided further support for the pathogenesis of variants in this portion of the protein.…”

“…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 In our own studies, analysis of Rbm20 S637A mice revealed marked systolic dysfunction and dilation of the left ventricle in both heterozygous and homozygous variant carriers by 8 weeks of age, consistent with the development of a DCM-like phenotype in these mice.…”

“…13,14,27 More recently, Rbm20 P635L mice (analogous to P635L in humans) have been generated. 27,28 In our own studies, analysis of Rbm20 S637A mice revealed marked systolic dysfunction and dilation of the left ventricle in both heterozygous and homozygous variant carriers by 8 weeks of age, consistent with the development of a DCM-like phenotype in these mice. 10 Homozygous Rbm20 S637A mice also exhibited premature mortality with ≈34% of mice dying within 100 days of birth, which mimics the early onset and high mortality associated with pathogenic variants in RBM20 in humans.…”