Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy

Ping Yang; Yingmei Lou; Zilong Geng; Zhizhao Guo; Shuo Wu; Yige Li; Kaiyuan Song; Ting Shi; Shasha Zhang; Junhao Xiong; Alex F Chen; Dali Li; William T Pu; Lintai Da; Yan Zhang; Kun Sun; Bing Zhang

doi:10.1161/CIRCULATIONAHA.123.067890

Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic Cardiomyopathy

Circulation. 2024 May 16. doi: 10.1161/CIRCULATIONAHA.123.067890. Online ahead of print.

Authors

Ping Yang^#¹, Yingmei Lou^#¹, Zilong Geng^#¹, Zhizhao Guo^#¹, Shuo Wu¹, Yige Li¹, Kaiyuan Song¹, Ting Shi², Shasha Zhang¹, Junhao Xiong¹, Alex F Chen¹, Dali Li¹, William T Pu^{3

4}, Lintai Da⁵, Yan Zhang^#⁶, Kun Sun^#¹, Bing Zhang^#¹

Affiliations

¹ Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Engineering Research Center of Techniques and Instruments for Diagnosis and Treatment of Congenital Heart Disease, Institute for Developmental and Regenerative Medicine, Xin Hua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China (P.Y., Y. Lou, Z. Geng, Z. Guo, S.W., Y. Li, K.S., S.Z., J.X., A.F.C., L.D., K.S., B.Z.).
² State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China (T.S.).
³ Department of Cardiology, Boston Children's Hospital, Harvard Medical School, MA (W.T.P.).
⁴ Harvard Stem Cell Institute, Harvard University, MA (W.T.P.).
⁵ Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China (D.L.).
⁶ School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China (Y.Z.).

^# Contributed equally.

PMID: 38752340
DOI: 10.1161/CIRCULATIONAHA.123.067890

Abstract

Background: Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored.

Methods: We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs.

Results: Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy.

Conclusions: Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.

Keywords: cardiomyopathy, hypertrophic; cardiomyopathy, hypertrophic, familial; therapeutics.