Abstract 498: Krueppel-like Factor 15 Modulation: A Crispr-based Re-establishment of Homeostatic Transcription in Diseased Mouse and Human Cardiomyocytes

Abstract

An altered transcriptomic profile of cardiomyocytes is a hallmark of cardiac remodelling and heart failure. Re-activation of gene programs necessary for myocardial homeostasis and inhibition of deleterious signalling pathways, such as Wnt/beta-catenin signalling, is therefore therapeutically desirable, however challenging to achieve tissue-specifically. We identified Krueppel-like factor 15 (KLF15) as a cardiac specific inhibitor of Wnt/beta-catenin signalling, lost during heart failure progression in the mouse and human heart. To re-establish endogenous Klf15 expression, we designed a cardiomyocyte specific CRISPR activation mouse model expressing enzymatically inactive Cas9 (dCas9) fused to a transcriptional activator (VPR) under Myh6 promoter control. The dCas9VPR protein is directed by guide RNAs (gRNA), delivered by AAV9, to the promoter region of Klf15 and induces gene expression. We tested activation of Klf15 in the adult myocardium by injecting 15 weeks old Myh6-dCas9VPR mice systemically with AAV9 carrying Klf15 gRNAs and increasing Klf15 transcript levels 8 weeks later up to 1.6 fold compared to controls. Since we found a 0.5 fold change of Klf15 expression upon transverse aortic constriction compared to sham operated mice, we expect the CRISPR activation induced Klf15 activation to be sufficient to re-establish Klf15 expression. As a translational approach and since we demonstrated KLF15-WNT regulation in human cardiomyocytes, we integrated dCas9VPR into human induced pluripotent stem cells (hIPSC) by targeted genome editing at the AAVS1 safe harbour locus and confirmed dCas9VPR expression in hIPSC and in hIPSC-derived cardiomyocytes (hIPSC-CM). We tested KLF15 gRNAs in HEK293 cells and selected the most efficient candidate gRNAs to test Klf15 activation in dCas9VPR-IPSC resulting in an up to 1.7 fold activation compared to controls. For gene activation in dCas9VPR hIPSC-CM, we are currently testing lentivirus constructs to deliver up to 3 gRNAs simultaneously to tailor KLF15 expression. With these prerequisites, we aim to re-activate master regulators of normal cardiomyocyte function in failing cardiomyocytes at physiologically relevant levels, such as KLF15, to test potential therapeutic targets for the prevention of heart failure progression.