Development of the Clinical Application of miRNA with Proregenerative Effect on the Heart

Abstract

Rationale: Cardiovascular disorders are the first cause of mortality and morbidity worldwide. This is due, at least in part, to the poor regenerative capacity of the heart and the lack of drugs able to foster cardiac regeneration. Our recent work has identified a few human microRNAs (miRNAs), in particular hsa-miR-199a-3p, able to stimulate proliferation of cardiomyocytes and, once expressed in the mouse heart using viral vectors, to induce cardiac regeneration after myocardial infarction. Objective: As a first step towards clinical translation, in this study we assess the efficacy of the pro-regenerative miR-199a after myocardial infarction, delivered using an AAV vector in a pig model of ischemia-reperfusion and as a synthetic RNA molecule after a single intracardiac injection in mice. Methods and Results: We evaluated the efficacy of miR-199a in a pig model of ischemiareperfusion after direct myocardial injection of an AAV6 vectors carrying the miR-199a precursor. Cardiac function was evaluated at days 2 and 28 by gadolinium-enhanced cardiac magnetic resonance imaging (cMRI). The results showed significant reduction of infarct size and increased cardiac function in the animals treated with AAV6 miR-199a at one month after treatment. Histological analysis uncovered a significant increase in cardiomyocyte proliferation in the infarct border zone, paralleled by the expression of markers of cardiomyocyte de-differentiation. Despite these remarkably positive signs of cardiac regeneration, pigs treated with AAV6-miR-199a died of sudden death at weeks 7-8 after treatment. In three of these pigs, clusters of small, proliferating cells with a phenotype of undifferentiated myogenic progenitors were apparent. In parallel, we comparatively analyzed the efficacy different lipid formulations in delivering hsa-miR-199a-3p as a naked RNA mimic in both primary neonatal rat cardiomyocytes and in vivo. We established a transfection protocol allowing persistence of miR-199a-3p mimics, carrying different chemical modifications, for at least 12 days after a single intracardiac injection, with minimal dispersion to other organs and long-term preservation of miRNA functional activity, as assessed by monitoring the expression of two direct mRNA targets. We administered this synthetic formulation immediately after myocardial infarction in mice and found that a single intracardiac injection was sufficient to reduce scar size and improve global cardiac function up to two months. Histology confirmed reduced scar extension and immunofluorescence showed increased cardiomyocyte proliferation in the miR-199a-3p mimic injected animals. Conclusions: AAV6-miR-199a injection after ischemia-reperfusion in pigs significantly improves cardiac function and reduce scar size. However, AAV6-driven miR-199a persistent expression in the pig myocardium is fraught with safety issues. A single administration of mir-199a-3p mimic is sufficient to stimulate adult mouse cardiac repair and restoration of cardiac function. Together, these results are concordant in indicating that a miRNA-based therapy aimed at inducing cardiomyocyte proliferation might be pursued to stimulate cardiac regeneration, however that the duration of the miRNA effect needs to be tightly controlled.