In vivo CRISPR-mediated activation of cardiogenic genes to reprogram cardiac fibroblasts.

BACKGROUND: We previously demonstrated that fibroblasts can be reprogrammed through a proliferative progenitor stage to drive both cardiomyogenesis and neovascularization. However, it remains to be determined whether cardiac fibroblasts (CFs), the primary mediators of post-injury remodeling, retain the plasticity to be concurrently redirected into cardiovascular lineages within the in vivo environment.

METHODS: This study aimed to activate endogenous cardiogenic genes (Gata4, Nkx2.5, Tbx5, Isl1, and Smarcd3, referred to as GNTIS) in CFs using a CRISPR activation (CRISPRa) system. CFs isolated from fibroblast-specific transgenic dCas9 mice were used to validate cardiogenic gene activation mediated by single-guide RNAs and VP64 activators delivered via adeno-associated viral vectors (AAVs). The reprogramming potential of CFs into cardiovascular lineages was further evaluated in dCas9 mice subjected to myocardial infarction followed by administration of pooled AAVs.

RESULTS: Individual GNTIS sgRNAs effectively upregulated their respective targets in the transfected CFs. The pooled sgRNA GNTIS induced cardiac-like phenotypes in the CFs, as demonstrated by the Nkx2.5 cardiac progenitor reporter and increased cardiac differentiation markers. Subsequently, AAV-sgRNA GNTIS was assessed in vivo in dCas9 mice following myocardial infarction. While global cardiac function did not reach statistical significance, GNTIS activation improved key hemodynamic parameters, effectively preserving cardiac performance compared to controls. Immunostaining further revealed that GNTIS-activated CFs exhibited the potential for transdifferentiation into cardiomyocyte-like or vascular smooth muscle cell-like lineages within the infarcted heart, which was not observed in the control group.

CONCLUSION: This CRISPRa transgenic model provides a foundational proof of concept for in vivo reprogramming of CFs into cardiovascular cells.