Antisense oligonucleotides as a potential therapeutic strategy in hypertrophic cardiomyopathy caused by MYBPC3 variants

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Sociedad Española de Cardiología Background/Introduction Non-sense or frameshift mutations in the MYBPC3 gene encoding cardiac myosin-binding protein C (cMyBP-C) are a major cause for inherited forms of Hypertrophic Cardiomyopathy (HCM), the most frequent inherited cardiac disease, affecting 0.2% of the general population and represents the leading cause for sudden cardiac death in young athletes. During the last decade, RNA-based therapeutic approaches have been studied for neuromuscular disorders and recently became to be considered to envision causal therapies for cardiomyopathies, utilizing antisense oligonucleotide mediated exon skipping, which attempts to reframe mutated mybpc3 transcripts, resulting in shortened but functional protein. MYBPC3 gene has a total of 34 coding exons and, exons 2, 22, and 24-27 are ideal targets for skipping. Purpose We aim to design and evaluate antisense oligonucleotides (AONs) for efficient exon-skipping in targeted MYBPC3 exons. Methods We designed two specific AONs to mask the MYBPC3 exon 26 and confirmed successfully mediated exon skipping without disrupting the MYBPC3 reading frame. We chose a previously identified MYBPC3 c.2671insG/p.R891Afs*160 variant in exon 26, which causes a frameshift mutation leading to truncated MYBPC3 in HCM. Primary Human Cardiac Myocytes (PHCM) (Promocell) were transfected with the splice-site targeting AONs. Results In this study we designed two chemically modified AONs (AON1 and AON2) through the addition of a 2′-O-methyl RNA phosphorothioate backbone, targeting exonic splicing enhancers (ESEs) or splice sites (SSs) can induce skipping of MYBPC3 exon 26. We evaluated AON1 (i26-3SS) and AON2 (ESE)-treated Primary Human Cardiac Myocytes (PHCM) show 26-27 exons skipping in comparison to control samples. The identity of the exon 26-27 skipped bands detected by RT-PCR was confirmed by Sanger sequencing and preservation of both nucleotide sequences of MYBPC3 exons 25 and 28 were observed. These results indicate that AONs specifically for mutated MYBPC3 target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping MYBPC3-based HCM. In addition, skipping exons 26 and 27 we eliminate a total of 22 possible exonic mutations, which represent 7.2% of the total known MYBPC3 exonic mutations. Conclusion(s) These results demonstrate that disruption of the MYBPC3 reading frame due to a truncating HCM mutation can be restored by exon skipping in cardiomyocytes in vitro indicating RNA-based strategies as a potential treatment option for HCM.Exon skipping scheme