Functional analysis of TTN uORFs reveals context-dependent translational regulation

Familial dilated cardiomyopathy (DCM) is a common condition with a high clinical burden, and no therapies that target the underlying genetic mechanisms. The leading genetic cause of DCM is heterozygous truncating variants in TTN, which are thought to drive disease through haploinsufficiency, where titin is reduced. This raises the possibility of treating disease through therapeutic upregulation of endogenous TTN expression. Upstream open reading frames (uORFs) are regulatory elements that repress translation and have been targeted to upregulate gene expression in a range of genes. Two uORFs in the TTN 5' untranslated region have been shown to repress expression in luciferase reporter assays, suggesting potential for therapeutic targeting. Here, we investigated the role of TTN uORFs in reporter systems and in the endogenous locus in a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. While disruption of uORFs increased expression in luciferase reporter gene assays, including in hiPSC-CMs, CRISPR-Cas9-mediated disruption of uORFs at the endogenous TTN locus in hiPSC-CMs did not increase titin protein expression. These findings indicate that uORF-mediated repression of TTN is context-dependent and not recapitulated in a disease-relevant model. This highlights the complex regulation of TTN expression and the importance of validating regulatory elements in their endogenous context.