Abstract
Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy to date have focused on mouse models of disease. Here, we investigate two methods of allele-specific silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of disease. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy’s effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. We find a dissociation between allelic-specificity and functional improvements between the two tested therapeutic strategies, suggesting a more complex method of allelic control underlying HCM-associated transcripts.
Author summary Allele-specific silencing, whereby a therapeutic molecule is used to lower the expression of just one of the two copies or alleles of a gene, may be a potential therapeutic strategy in diseases caused by a single mutation. In this paper, we examine two such strategies in hypertrophic cardiomyopathy, a disease characterized by an overgrowth of the left-ventricular heart muscle as well as contractile dysfunction. We used a human cell model of disease, creating induced pluripotent stem cell derived cardiomyocytes from a patient with HCM caused by a single base pair change in just one allele of the gene MYH7. We used two strategies to silence the disease-associated copy of MYH7, both focused on reducing RNA expression from the mutated allele, as well as state-of-the-art biophysical techniques for measuring contractility. We found that one silencing strategy, which reduced expression of both the disease-associated and the healthy alleles of MYH7, showed great improvements in contractility between treated and untreated cells. Our second strategy, which silenced only the disease-associated copy of MYH7, showed more modest improvements in contractility. This suggests that the disease mechanism underlying this type of hypertrophic cardiomyopathy may be more complex than just presence or absence of the mutated RNA.
Footnotes
Financial Support A. Dainis received support from the NSF Graduate Research Fellowship Program. E. Ashley received funding from NIH Director’s New Innovator Award DP2 OD004613 and is supported by NIH U24 award 1U24EB023674-01 and NIH U01 award 1U01HG007708. B. Pruitt received support from AHA 1205987-120-UAKOD as well as NIH 1R21HL13099301. A. Ribeiro received support from AHA Fellowship 14POST18360018. J.C. Wu is supported by NIH R01 HL130020 and NIH R01 HL126527. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Disclosures E. Ashley is a Founder of Personalis and DeepCell, Inc, and an advisor for SequenceBio. M. Wheeler is an equity partner in Personalis, Inc and has been a consultant to MyoKardia.