Engineering RNA biodevices for precise modulation of fibroblasts to boost cardiac reprogramming

SUMMARY: Myocardial infarction (MI)-induced cardiovascular diseases remain a major global cause of death. Post-MI excessive fibrosis can lead to adverse remodeling and eventual heart failure. Unfortunately, there are limited therapies to prevent fibrosis and hinder heart failure progression. In vivo reprogramming techniques hold promise in converting resident fibroblasts into cardiac cells for heart regeneration through enforced cardiogenic gene expression. Advantages of in vivo reprogramming include direct gene delivery to resident fibroblasts, eliminating the need for surgical biopsy procedures, cell expansion, pluripotency induction, or auto-transplantation. However, challenges, including low efficiency and a lack of specific targeting, still need to be addressed. Besides optimizing the reprogramming factor cocktail, several molecular and epigenetic obstacles in reprogramming fibroblasts have been identified and demonstrated to enhance the conversion efficiency by using chemical inhibitors and RNAi approaches. Despite the promising results of inhibition approaches in vitro, a significant challenge emerges in safely and effectively translating these strategies for in vivo reprogramming, given that most targets are not exclusively expressed in fibroblasts. For instance, the pro-fibrotic TGFβ-Smad signaling, which is a well-studied reprogramming roadblock, is also present in cardiomyocytes and vascular cells. Therefore, achieving fine-tuned regulation of fibroblasts’ signaling pathways for reprogramming requires a nuanced strategy that leverages more advanced molecular or genetic targeting techniques. Notably, recent progress in synthetic biology has paved the way for novel research and development in medicine. A particularly noteworthy breakthrough comes from gene- editing technology, enabling precise binding or modifications to DNA (via CRISPR) or RNA (via ADAR) sequences. Our previous study demonstrated that the fibroblast lineage can be altered by the CRISPR-based activation of endogenous cardiac reprogramming factors for regenerative therapy. This accomplishment strongly motivates us to further explore the application of gene-editing techniques for in vivo reprogramming. To advance the translation of in vivo reprogramming strategies, this proposal aims to address challenges related to the efficiency and specificity of converting fibroblasts into cardiac cells. This will be accomplished by innovatively engineering RNA biodevices that integrate the CRISPR and ADAR technologies. Consequently, two specific aims are proposed in this exploratory study. In Aim-1, we will engineer a novel RNA scaffold to not only boost the activation of cardiogenic genes but also overcome a reprogramming obstacle via CRISPR-based epigenetic editing. In Aim-2, we will develop an ADAR-based system that can recognize fibroblast-specific RNAs (input) and then switch on effector proteins (output) to manipulate the targeted cell with unprecedented precision. Finally, this research will validate our novel concept of reprogramming fibroblast cell fate using advanced gene-editing techniques, offering more effective and specific approaches against excessive fibrosis and heart failure.