Cardiomyocyte-Specific Modified mRNA for Myocardial Regeneration in a Porcine Myocardial Infarction Model

The inability of adult mammalian cardiomyocytes (CMs) to undergo cell renewal following ischemic injury stands as a major impediment to heart function recovery and contributes to heart failure (HF). Current therapeutic approaches do not offer an effective approach of reactivating CM cell division to prevent HF in humans. Recognizing this challenge, gene therapy has emerged as a promising approach to promote CM proliferation and enhance cardiac repair. A particularly notable approach is the utilization of modified mRNA (modRNA), which provides a safe, controlled, transient, non-viral, and clinically relevant means of gene delivery.

The study was conducted using a novel cardiomyocyte specific modRNA translational system (CM SMRTs) that could transiently drive the target protein translation in mammalian cardiomyocyte with high specificity. The CM SMRTs consists of two modRNA constructs: one codes for target gene and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the CM-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses target gene translation in non-cardiomyocytes, but is itself suppressed by endogenous miR-1 and -208 in CMs, thereby facilitating CM-specific target gene expression.

During this dissertation study, we first demonstrated that CM SMRTs drove efficient and exclusive protein translation in pig cardiomyocytes. Then, we have investigated two target genes for cardiomyocyte regeneration: cyclin D2 (CCND2), and pyruvate kinase muscle isozyme M2 (PKM2). We demonstrated that direct injection of human CCND2- or PKM2-CM SMRTs after ischemic/reperfusion (I/R) injury could elevate CM proliferation markers (Ki67 and PH3), and reactivate cardiomyocyte cell division at 3 days or 7 days after delivery. In a long-term I/R study we show that CCND2- or PKM2-CM SMRTs improved left ventricle (LV) ejection fraction (EF) and reduced cardiac LV scar formation at one or two months post-delivery, compared to vehicle control. In addition, CCND2 and PKM2 treatment increased mononuclear CM cell population while limiting cardiac hypertrophy without causing cardiac arrythmia.

In conclusion, this study demonstrated that intramyocardial injections of the CCND2- or PKM2-CM SMRTs could effectively promote CM proliferation, reduce infarct size, and enhance cardiac performance in mammalian hearts with MI, without increasing the risk of arrhythmia.