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CRISPR-Cas13d Nuclease in Hypertrophic Cardiomyopathy
Quick Takes
- A high precision CRISPR Cas13d variant with single nucleotide variation was developed that also showed low collateral cleavage in in vitro studies.
- In mouse HCM models with MYH6 variants (equivalent to human MYH7 HCM models), application of hp CRISPR Cas13d using an adenovirus vector mitigated HCM phenotype without adverse effects.
Study Questions:
What is the efficacy of RNA nuclease Cas13 in heterogenous MYH7 hypertrophic cardiomyopathy (HCM) animal models and in vitro analyses?
Methods:
The authors developed a high precision (hp) Cas13d variant that specifically cleaves MYH7 variant RNA containing one single nucleotide variant (SNV). The cleavage precision of this hpCas13d was evaluated first in in vitro assays. This was followed by assessing efficacy in two HCM mouse models with distinct MYH7 SNVs that received the hpCas13d delivered via an adenovirus vector to the cardiomyocytes.
Results:
Since wild-type Cas13d cannot distinguish and specifically cleave heterozygous MYH7 allele with a SNV, several different variants were generated on wild-type Cas13d. One variant with three amino acid substitutions (TNR variant) exhibited highest allelic discrimination with low collateral cleavage activity among all variants and was called hpCas13d variant. In vitro studies showed that the TNR substitutions increased SNV discrimination by dampening the RNase activity.
Next, two different MYH6 HCM mouse models (analogous to human MYH7) were treated with adeno-associated virus 9 (AAV9) that expresses hpCas13d in the cardiac-specific troponin T promoter. Neonatal mouse models were injected with cyclosporine A with hpCas13d. Hyperdynamic cardiac function seen in MYH6 HCM mouse models was normalized with hpCas13d with decrease in left ventricular wall thickness. Histological analyses showed smaller cardiomyocyte with hpCas13d treatment compared to without and electrocardiographic evidence of prolonged QTc also corrected.
Conclusions:
In vitro and mouse model analyses suggest that hpCas13d can efficiently cleave a point mutant allele while preserving the wild-type allele and mitigate HCM phenotype.
Perspective:
More recently there has been an increased focus on developing gene therapies for inherited cardiomyopathies. Currently, most gene therapies are editor-based therapies using adenovirus vectors that raise safety concerns due to the involved DNA manipulation and up to 30% of genetic variants cannot be edited with this technology. Instead of manipulating DNA, RNA nuclease CRISPR-Cas13d cleaves RNA, which is reversible and safer for long-term adenovirus vector delivery. In this study, the authors demonstrate that an hp Cas13d with a high discrimination ability can enable efficient SNV-specific knock down. While not all inherited cardiomyopathies are monogenic, CRISPR-Cas13 tools may open up new avenues to treat inherited cardiomyopathies.
Clinical Topics: Heart Failure and Cardiomyopathies
Keywords: Genetic Therapy, Hypertrophic Cardiomyopathy
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