Direct cardiac reprogramming via combined CRISPRa-mediated endogenous Gata4 activation and exogenous Mef2c and Tbx5 expression

Peisen Huang; Jun Xu; Benjamin Keepers; Yifang Xie; David Near; Yangxi Xu; James Rock Hua; Brian Spurlock; Shea Ricketts; Jiandong Liu; Li Wang; Li Qian

Molecular Therapy: Nucleic Acids (Dec 2024)

Direct cardiac reprogramming via combined CRISPRa-mediated endogenous Gata4 activation and exogenous Mef2c and Tbx5 expression

Peisen Huang,
Jun Xu,
Benjamin Keepers,
Yifang Xie,
David Near,
Yangxi Xu,
James Rock Hua,
Brian Spurlock,
Shea Ricketts,
Jiandong Liu,
Li Wang,
Li Qian

Affiliations

Peisen Huang: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Jun Xu: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Benjamin Keepers: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Yifang Xie: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
David Near: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Yangxi Xu: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
James Rock Hua: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Brian Spurlock: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Shea Ricketts: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Jiandong Liu: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
Li Wang: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Corresponding author: Li Wang, McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Li Qian: McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Corresponding author: Li Qian, McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Journal volume & issue: Vol. 35, no. 4
p. 102390

Abstract

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Direct cardiac reprogramming of fibroblasts into induced cardiomyocytes (iCMs) can be achieved by ectopic expression of cardiac transcription factors (TFs) via viral vectors. However, risks like genomic mutations, viral toxicity, and immune response limited its clinical application. Transactivation of endogenous TFs emerges as an alternative approach that may partially mitigate some of the risks. In this study, we utilized a modified CRISPRa/dCas9 strategy to transactivate endogenous reprogramming factors MEF2C, GATA4, and TBX5 (MGT) to induce iCMs from both mouse and human fibroblasts. We identified single-guide RNAs (sgRNAs) targeting promoters and enhancers of the TFs capable of activating various degrees of endogenous gene expression. CRISPRa-mediated Gata4 activation, combined with exogenous expression of Mef2c and Tbx5, successfully converted fibroblasts into iCMs. Despite extensive sgRNA screening, transactivation of Mef2c and Tbx5 via CRISPRa remained less effective, potentially due to de novo epigenetic barriers. While future work and refined technologies are needed to determine whether cardiac reprogramming could be achieved solely through CRISPRa activation of endogenous factors, our findings provide proof of concept that reliance on exogenous TFs for reprogramming can be reduced through CRISPRa-mediated activation of endogenous factors, particularly Gata4, offering a novel strategy to convert scar-forming fibroblasts into iCMs for regenerative purposes.

Published in Molecular Therapy: Nucleic Acids

ISSN: 2162-2531 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Medicine: Therapeutics. Pharmacology
Website: https://www.cell.com/molecular-therapy-family/nucleic-acids/latest-content

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