mRNA Therapeutics Encoding Multiple Reparative Factors using Polyplex Nanomicelle Attenuating Myocardial Infarction

Background: Gene therapy for heart failure has been explored using single factors such as angiogenic agents; however, clinical success remains limited. In this study, we investigated an alternative approach by directly administering mRNA encoding multiple genes that are transiently upregulated during cardiac recovery. Using a model in which heart function is restored through the administration of extracellular vesicles secreted by human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM-derived EVs), we aimed to evaluate the potential of multi-gene mRNA therapy for heart failure, being delivered using polyplex nanomicelles, which serve as an effective delivery system with superior mRNA stabilization and target tissue penetration properties. This study aimed to evaluate the therapeutic efficacy of mRNA encoding multiple cardiac-reparative factors on cardiac dysfunction in heart failure mouse model using nanomicelles. Methods: Multiple cardiac-reparative factors involved in cardiac functional recovery were identified through comprehensive RNA sequencing data of myocardial infarction (MI) model animals treated with hiPSC-CM-derived EVs. MI model mice were created by left coronary artery ligation, followed by injection of mRNA encoding those factors (1 µg/each factor), an equal amount of Luciferase (Luc) -mRNA as a control, or PBS into the border zone. Cardiac function was evaluated at two and four weeks later by echocardiography and subsequent histological analysis. Results: Comprehensive RNA sequencing of myocardial tissue treated with hiPSC-CM-derived EVs showed that five genes (Hgf, Igf1, Pdgfb, Cxcl12, Tgfb1) were notably upregulated on day 3 post-treatment may be involved in the therapeutic effect. To evaluate their potential, an mRNA cocktail encoding these factors was administered to the border zone of mouse heart with impaired cardiac function due to MI. The five-factor mRNA group exhibited significantly mitigated cardiac dilation and improved contractility at two weeks post-treatment compared with the other groups, with this trend persisting at four weeks. Subsequent histological analysis was performed four weeks after treatment. Sirius red staining revealed a significant reduction in fibrosis in the five-factor mRNA group compared with the other groups. Additionally, CD31 staining of capillary endothelial cells was conducted to assess angiogenesis in the border zone, revealing a significant increase in capillary density in the five-factor mRNA group. Conclusion: Five cardiac-reparative factors (Hgf, Igf1, Pdgfb, Cxcl12, Tgfb1) were identified from RNA sequencing data of MI myocardial tissue treated with hPSC-CM-derived EVs. Administration of mRNA cocktails encoding these factors to the MI heart achieved an increase in angiogenesis and an inhibition of fibrosis, resulting in improvement of cardiac function.