Evaluation of MYBPC3 trans -Splicing and Gene Replacement as Therapeutic Options in Human iPSC-Derived Cardiomyocytes

Abstract

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5′ or 3′ pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation.

Disease modeling of a mutation in α‐actinin 2 guides clinical therapy in hypertrophic cardiomyopathy

“…However, we provide several lines of evidence for this novel ACTN2 mutation to be HCM-causing: (i) It was segregated in three HCM-affected members of the family, (ii) it was not found in the gnomAD browser, assembling > 130,000 exome or whole-genome sequences from unrelated control individuals, and (iii) the patientderived hiPSC-CMs developed a phenotype, which was corrected in the isogenic control. Our disease modeling approach revealed HCM phenotypes in 2D-and 3D-cultured hiPSC-CMs that were also reported previously in hiPSC-CMs and animal models, such as myofibrillar disarray and CM hypertrophy (Lan et al, 2013;Dambrot et al, 2014;Tanaka et al, 2014;Ojala et al, 2016;Prondzynski et al, 2017), higher contraction force, longer relaxation time (Wijnker et al, 2016), and higher myofilament Ca 2+ sensitivity (Fraysse et al, 2012;Eschenhagen & Carrier, 2019). The fact that all parameters were significantly different between the HCM and the isogenic control line carrying the same genetic background provides evidence for the mutation to be causative.…”

Modelling of Genetic Cardiac Diseases

“…When HCM tissues carrying a mutation in MYBPC3 gene were compared with donor heart sample, no specific truncated MyBP-C peptides were detected, but the overall level of MyBP-C in myofibrils was significantly reduced [79]. Similar haploinsufficiency results were also shown in HCM hiPSC-CMs with mutation in MYBPC3 gene [25,80], and gene replacement in HCM hiPSC-CMs partially improves the haploinsufficiency and reduces cellular hypertrophy [80]. Similar to higher myofilament Ca 2+ sensitivity observed in isolated cardiac biopsies from HCM with E99K mutation in cardiac actin [81], in vitro model of HCM hiPSC-CMs carrying E99K mutation in cardiac actin demonstrated significantly stronger contraction and increased arrhythmogenic events [82] Furthermore, a study in HCM mice harboring I79N mutation in cTnT resulted in increased cardiac contractility, altered Ca 2+ transients, and remodeling of action potential [83].…”

An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility

“…Expression analysis on the nCounter SPRINT Profiler (NanoString) was performed as described before with 50 ng of total RNA and a customized expression CodeSet. 36 RNA sequencing was performed as described by Stenzig et al 20 from 500 ng RNA per sample on the Illumina HiSeq 4000 sequencing system. Pathway mapping was performed with the DAVID Functional Annotation tool.…”