Enhancing Mitochondrial Biogenesis with a CRISPR/ndCas9 Adenoviral Vector System in Cardiomyocytes

Abstract

Mitochondrial dysfunction contributes to heart failure and the genes involved in mitochondrial biogenesis and quality control, such as the peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM), are downregulated in a guinea pig model of heart failure and sudden cardiac death. Here, we apply RNA-guided nuclease-dead Cas9 (ndCas9) technology as an activator to target sequences upstream of the transcriptional start site to promote transcription of PGC-1α or TFAM. To facilitate gene transfer to cardiomyocytes we developed an adenoviral gene delivery system for expression of the CRISPR/ndCas9 activating complex and a targeting gRNA to modulate expression of any gene of interest in differentiated cardiomyocytes, with our initial focus on activating the metabolic gene program.We use ndCas9 fused to the activation domain VP64 and guide RNAs for activation of target genes. Proof-of-principle evidence was obtained by activating a cyclic AMP response element (CRE)-driven luciferase reporter gene and activation of endogenous CRE-regulable genes (PCK1 and PGC-1α). We have successfully upregulated a reporter gene (luciferase) and endogenous CRE-regulated genes PCK1 and PGC-1α in the same reporter cell line with ndCas9-VP64, as evidenced by significant increases in both mRNA and protein expression levels. Next, we constructed and delivered ndCas9-gRNA adenoviral vectors to target TFAM in the rat genome that are efficiently expressed by the host cells (NRVMs and H9c2 cells) and showed increased expression of TFAM in H9c2 cells. Importantly, an increase in mtDNA nucleoids was evoked by TFAM activation. For the first time, we show that the CRISPR/ndCas9 system can be used as a powerful tool for modulating transcription of native genes involved in mitochondrial biogenesis in cardiac cells, opening the door to developing novel therapeutic approaches for heart failure.