Allele-specific silencing ameliorates restrictive cardiomyopathy due to a human myosin regulatory light chain mutation
Authors: Zaleta-Rivera Kathia , Dainis Alexandra , Ribeiro Alexandre J. S. , Sanchez-Cordero Pablo , Rubio Gabriel , Shang Ching , Liu Jing , Finsterbach Thomas , Sinha Nikita , Jain Nikhil , Hajjar Roger , Kay Mark A. , Szczesna-Cordary Danuta , Pruitt Beth L. , Wheeler Matthew T. , Ashley Euan A. 
Published: February 2019

AbstractBackgroundRestrictive cardiomyopathy (RCM) is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model.MethodsAAV9-M7.8L shRNA was selected from a pool of RNAi oligonucleotides containing the SNV in different positions to specifically target the mutated allele causative of RCM by FACS screening. Two groups of RLC-N47K transgenic mice were injected with a single dose of AAV9-M7.8L shRNA at 3 days of age and at 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine VO2max and left ventricular mass. At the end of treatment, heart, lung, liver and kidney tissue was harvested to determine viral tropism and for transcriptome and proteomic analysis. Cardiomyocytes were isolated for single cell studies.ResultsOne time injection of AAV9-M7.8L RNAi in 3-day-old humanized RLC mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed 16 weeks post-injection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight and attenuated a pathological increase in left ventricular mass (LVM). Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of the maximal contraction velocity with marked reduction in hypercontractility as well as relaxation kinetics and improved time to maximal calcium reuptake velocity. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II (CAMKII) and the transcription activator Brg1 were reduced suggesting recovery towards a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts while endogenous microRNAs were preserved suggesting the RNAi pathway was not saturated.ConclusionsOur results show the feasibility, efficacy, and safety of RNAi therapeutics directed at human restrictive cardiomyopathy. This is a promising step towards targeted therapy for a prevalent human disease.Clinical PerspectiveWhat is new?Restrictive cardiomyopathy due to a mutation of human MYL2 modeled in cells and mice can be treated with RNA interferenceReduction of disease-causing allele improves function at the cellular and organ levelOff target effects evaluated and not found to be significantWhat are the clinical implications?Allele-specific RNA silencing of human alleles may be effective in treating inherited restrictive cardiomyopathyRNA therapies targeting individual mutations may need to be developed prior to consideration of clinical translation