Abstract P1128: CRISPR-mediated Activation Of DLK1 Induces A Regenerative State In Engineered Human Myocardium

Abstract

Background: The human heart displays limited potential to recover from injury. Delta-like non-canonical Notch ligand-1 ( DLK1 ) is an imprinted gene strongly expressed during embryo-fetal development. Expression in postnatal organs is low but re-expression commonly occurs in regenerative organs after injury. We hypothesized that enhancing DLK1 expression in engineered human myocardium (EHM) may induce a regenerative response. Methods and Results: RNA sequencing of muscle biopsies from non-failing (n=14) and failing (n=12) human hearts demonstrated a reduced expression of DLK1 in heart failure. In addition, serum levels of soluble DLK1 were reduced in patients with heart failure and correlated to left ventricular ejection fraction. Similar to patient samples, DLK1 expression was reduced in EHM with simulated heart failure. To test if DLK1 restoration prevents contractile dysfunction, we made use of a CRISPRa transgenic iPSC cell line, which stably expresses an enzymatic inactive dCas9 complex to specifically increase and modulate DLK1 expression. Cardiomyocyte-specific DLK1 activation ( DLK1 a) in EHM made with primary cardiac fibroblasts in a collagen hydrogel enhanced global DLK1 expression levels to 12±5 fold (n=5) of non-target (NT) control in simulated heart failure. While DLK1 a EHM showed comparable baseline function, contractile dysfunction by simulated heart failure was attenuated in DLK1 a EHM (Force of contraction: -13±5 % vs -45±5 % in DLK1 a vs NT EHM, n=8, p<0.05). RNA sequencing identified a regenerative transcriptome pattern in DLK1 a EHM with differential expression of cell motility, extracellular matrix, cell cycle and metabolism-related processes. Conclusion: Reactivation of the developmental gene DLK1 induces a regenerative state and may protect from heart failure.