Abstract
Introduction: The adult mammalian heart has limited regenerative capacity due to postnatal cardiomyocyte (CM) cell cyclearrest. With the success of modified mRNA (modRNA) vaccine against SARS-CoV-2, modRNA technology offers efficient, transient, safe, nonimmunogenic, and controlled mRNA delivery without any risk of genomic integration. Using our previously reported cardiomyocyte specific modRNA translation system (CM SMRTs), we investigated the myocardial regenerative potential and therapeutic outcome of transient and specific cardiomyocyte overexpression of cyclin D2 (CCND2) in a pig model of myocardial infarction (MI). Methods: Modified mRNA was synthesized by in vitro transcription. AMI was induced by occlusion of the left anterior descending coronary artery for 60 minutes with reperfusion. Pigs were randomly assigned to vehicle group (1ml sucrose-citrate buffer, n=6), nGFP-CM SMRTs group (7.5mg CM-specific nuclear GFP modRNA, n=7)and CCND2-CM SMRTs group (7.5mg CM-specific CCND2 modRNA, n=8). CM cell cycle activity was measured 3 days after injection by immunostaining for proliferation marker Ki67, M-phase marker phospho-histone H3 (PH3) and cytokinetic marker aurora B kinase (AuB). Cardiac function was evaluated by echo and cardiac MRI (cMRI) on day 28; infarct size was determined via late gadolinium enhancement cMRI; arrhythmia incidence was assessed via implanted loop recorders. Results: The short-term in vivo study demonstrated a highly specific overexpression of targeted genes in CMs. CCND2-CM SMRTs significantly promoted CM cell cycle activation that was evidenced by significant increase of cell cycle markers compared with nGFP-CM SMRTs group: Ki67 (4.21±0.34% vs. 0.81±0.05%), PH3 (0.51±0.06% vs. 0.13±0.03%), and Symmetric AuB (0.035±0.007/mm 2 vs. 0.000±0.000/mm 2 ), 3 days after AMI and modRNA treatment. The long-term follow-up of the animals also showed improved heart function (EF: 50.34±1.94% vs. 38.60±1.18%) and decreased scar size (8.80±1.07% vs. 22.77±2.77%). The frequency of arrhythmic events in animals between all groups were similar. Conclusion: The results in large animal model of AMI demonstrate remuscularization and therapeutic potential of cardiomyocyte-specific overexpression of CCND2 via modRNA.
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