Restoration of endogenous transcription factor activity by nuclease-deficient Cas9VPR prevents cardiac failure upon stress
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 preserve 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: Double transgenic Myh6-dCas9VPR/Klf15 knockout (KO) mice were generated by crossing Myh6-dCas9VPR mice with Klf15 KO mice. Wild-type C57BL/6N mice were used as controls. Mice (both male and female, 4-6 months old) were injected via the tail vein with either rAAV2/9-TRISPR-Klf15 or rAAV2/9-TRISPR-NT. Following injection, mice underwent transverse aortic constriction (TAC) or sham surgery. Pressure overload was confirmed by measuring the blood flow velocity ratio between the right and left carotid arteries. Echocardiography was conducted at 4 and 8 weeks post-surgery, and hearts were harvested at 12-16 weeks. Ventricular measurements were taken using a VisualSonics Vevo 2100 Imaging System. All procedures were performed in a blinded manner and in accordance with ethical guidelines from the Lower Saxony State Office for Consumer Protection and Food Safety.