Non-viral delivery of cardiac reprogramming factors using oriP/EBNA-1 vectors (2014)

Undergraduate: Chuner Guo

Faculty Advisor: Li Qian
Department: Biology

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We and others reported the generation of mouse induced cardiomyocytes (iCMs) from cardiac fibroblasts (CFs) or tail tip fibroblasts (TTFs) using cardiac reprogramming factors Gata4, Mef2c and Tbx5 (GMT), which marked an exciting path to regenerating the damaged heart. However, current reprograming approaches involve the introduction of GMT via retroviral transduction, which delivers the factors by genomic integration. Under this approach, adverse effects such as disruption of biologically important endogenous genes cannot be ruled out. Our study aims to develop an non-viral and integration-free delivery method for iCM generation. We intend to take advantage of episomal oriP/EBNA-1 vectors, which are large plasmids that reside and replicate in the host nuclei extrachromosomally, thus resolving the issue of integration. To this end, we generated episomal vector constructs containing the reprogramming factors Gata4, Mef2c, and Tbx5. Using optimized electroporation conditions for primary mouse fibroblasts, we show that the episomal system is able to confer robust expression in mouse cells. In addition, reprogrammed cells express reporter gene as well as cardiac markers in a manner comparable to iCM generation by retroviral transduction. Taken together, our data suggest that the episomal system can be used as an alternative approach for safer generation of iCMs, taking iCM technology closer to future clinical applications.