Modulating TTN gene expression in human iPSC-CMs to investigate mechanisms and therapies in TTN-associated dilated cardiomyopathy

Abstract Background Familial dilated cardiomyopathy (DCM) is a common and harmful condition, and no available therapies target the underlying genetic mechanisms. Truncating variants in TTN are the most commonly identified genetic cause of DCM. However, it remains unclear how TTN variants drive disease. Contemporary thinking identifies haploinsufficiency, dominant negative effects, or a combination of both as likely driving mechanisms. There is competing evidence as to whether I-band and A-band mutations differ in severity or in the relative roles of haploinsufficiency and dominant-negative effects. Clarifying disease mechanisms will help pave the way to targeted genetic therapies. Purpose To modulate TTN gene expression in human-induced stem cell-derived cardiomyocyte (hiPSC-CM) models of TTN-associated DCM carrying I-band or A-band variants to provide insight into the comparative roles of haploinsufficiency and dominant negative mechanisms in DCM, and to identify novel therapeutic strategies. Methods We used CRISPR-Cas9 gene-editing to generate hiPSC lines with known disease-causing heterozygous truncating mutations in the I-band or A-band of TTN. These were screened for off-target mutations and pluripotency. hiPSCs were differentiated into hiPSC-CMs for phenotyping. We assayed TTN mRNA and titin protein levels. We trialled approaches for modulating TTN expression including CRISPR activation. Results Two hiPSC lines with heterozygous mutations in the I-band and A-band of TTN were generated using CRISPR/Cas-9 gene-editing. These hiPSC lines were successfully differentiated into hiPSC-CMs. Both cell lines demonstrated unchanged TTN mRNA expression compared to wildtype controls, but had reduced expression of full-length titin protein and identifiable truncated protein products. TTN mRNA expression was subsequently increased in these models using modulators of gene expression. Conclusions hiPSC-CM models of DCM with truncating mutations in the I-band or A-band of TTN were generated and phenotyped. These models were then used to trial gene expression modulation methods. This provides a human cellular system that can be used to better understand disease mechanisms of TTN-associated DCM, with the ultimate aim of establishing gene therapies for TTN-associated DCM.