Abstract 231: Identification of a Titin Enhancer using hiPSC-Derived Cardiomyocytes and CRISPR/Cas9 Genome Editing

Abstract

Dilated cardiomyopathy (DCM), a disorder that occurs in 1:250 individuals, is associated with rates of mortality of 20% within 5 years of diagnosis and is a leading cause for heart failure and cardiac transplantation. Mutations in the massive sarcomere protein titin (encoded by TTN ) are the most common genetic cause of DCM, occurring in 10-20% of cases. As dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency, we predict that strategies to increase the expression of the wild type (WT) TTN allele might attenuate the damaging effects of TTNtv. We used bioinformatic analyses to identify a putative TTN enhancer within intron 1. To confirm its function, we deleted 658 bp from intron 1 that encompasses the putative TTN enhancer in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing. We used qPCR and RNA sequencing of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs) and demonstrated that a homozygous deletion in this region leads to decreased TTN gene expression compared to the WT control (0.344 fold change, p < 0.001), and also to decreased expression of other sarcomeric genes such as TNNT2 (0.074 fold change, p < 0.001), MYH6 (0.18 fold change, p < 0.001), MYH7 (0.008 fold change, p < 0.001), and ACTN2 (0.118 fold change, p < 0.001). The expression of transcription factors (TF) that have binding sites in this region is also affected, such as MEIS2 (0.4 fold change, p < 0.001) and KLF6 (0.33 fold change, p < 0.001), both of which are known to be involved in cardiogenesis. These TF may act as a link between the deletion in TTN Intron 1 and a decreased transcription of other cardiac-relevant genes. We also utilized Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) on WT hiPSC-CMs to identify open regions of chromatin that are accessible for TF binding. This provided additional evidence that this 658 bp region in TTN intron 1 has enhancer activity. Ongoing studies are aiming to refine the TTN Intron 1 enhancer by further studies using CRISPR/Cas9, CRISPR droplet sequencing (CROP-Seq), and luciferase-based enhancer activity assays. If confirmed, we expect that increasing the activity of this 658 bp region with small molecules may provide a novel therapeutic target for DCM caused by TTNtv.