Restoration of endogenous transcription factor activity by nuclease-deficient Cas9VPR prevents cardiac failure upon stress II
Cardiac remodeling is linked to maladaptive transcriptional networks, but identifying and targeting key factors with potential to reverse these processes is still a challenge. We employed network-based analysis of single cell heart transcriptomes to compute the transcription factor activities triggering heart failure. We found that the regulation of KLF15 activity is a major driver of cardiomyocyte failure. Through the use of CRISPR/nuclease-deficient (d)Cas9-based transcriptional regulation, we demonstrated that restoration of endogenous Klf15 expression and activity in cardiomyocytes was sufficient to mitigate pathological transcriptional reprogramming and preserved cardiac function in mice and human myocardial models. We also showed that KLF15 is a downstream targetable factor in the broad TGFß-mediated stress response in human cardiomyocytes, controlling transcriptional embryonalization. Lastly, we engineered a therapeutically applicable mini-dCas9VPR variant suitable for adeno-associated viral delivery, eliciting endogenous KLF15 transcriptional activation in human cardiomyocytes. This case study presents a therapeutic strategy for restoring the stress-induced downregulation of essential genes to prevent heart failure with broader implications for other disease contexts. Overall design: Engineered heart muscle (EHM) were generated by combining CRISPRa hiPSC-cardiomyocytes (3.85x10^5) transduced with either non-targeted control gRNAs (NT) or KLF15 gRNAs and with human foreskin fibroblasts (1.65x10^5) in a bovine collagen matrix. These EHM were cast into a 48-well plate (myriamed GmbH) and cultured initially in IMDM (Gibco) supplemented with 4% B27 minus insulin, 100 ng/mL IGF-1, 5 ng/mL VEGF165, 10 ng/mL FGF-2, 100 U/mL penicillin, 100 µg/mL streptomycin, and 5 ng/mL TGF-ß1 for 3 days. From day 4 onwards, TGF-ß1 was removed. Starting from day 14, the medium was switched to alpha-MEM (Gibco) with 4% B27 minus insulin, 1% MEM non-essential amino acids, 100 ng/mL IGF-1, 5 ng/mL VEGF165, 10 ng/mL FGF-2, 100 U/mL penicillin, and 100 µg/mL streptomycin. On day 28, EHM were either cultured on flexible, silicone poles (control, healthy = Flex) or subjected to mechanical stress by culturing them on rigid metal poles (stressed = Fix) for an additional 7 days. Total RNA was extracted for RNA sequencing to analyze gene expression changes under the different experimental conditions (NT vs. KLF15 gRNA and Flex vs. Fix).