An essential requisite to pursue the goal of achieving cardiac regeneration is the identification of genes capable of stimulating the proliferation of cardiac muscle cells. We pursue this goal through a series of high throughput screenings aimed to identify small RNAs that stimulate proliferation of cardiomyocytes. We started with a collection (library) of human microRNA mimics, namely small regulatory RNAs that mimic the function of endogenously expressed microRNAs (over 2,000 molecules), to then move to the screening of targeted collections of short interfering RNAs (siRNAs, namely small RNAs each one repressing the expression of a specific cellular gene). In total, we screened over 6,000 different regulatory RNAs for their effect on the proliferation of cardiomyocytes. The overall outcome of these studies was the identification of 4 microRNA mimics and 5 siRNAs that stimulate the proliferation of mouse, rat and human cardiomyocytes.
Based on this information, we tested the efficacy of the same molecules at stimulating cardiac regeneration after myocardial infarction first in mice and later in pigs. We delivered the genes coding for the microRNAs using viral vectors based on the adeno-associated virus (AAV), which target cardiomyocytes efficiently, and found that two of the microRNAs were very effective at stimulating cardiac regeneration in mice and one also in pigs. To progress towards clinical translation of these findings, next we explored the possibility of administering these small RNAs as synthetic molecules, the effect of which can be modulated by chemical modification to last 2-3 weeks and then disappear (expression is instead permanent using AAV vectors). We explored the efficacy of over 40 different lipid nanoparticle (LNP) formulations, obtained using the same technology as the COVID-19 vaccines from BioNTech/Pfizer and Moderna, for cardiomyocyte-specific RNA delivery, and eventually identified an LNP that is particularly effective. Using this LNP, we confirmed that all the four identified pro-proliferative miRNAs induce cardiac regeneration in mice and that two of them (miR-199a-3p and miR-1825) are also effective in pigs.
This translational work was helped by an ERC Proof-of-Concept grant. The patent relative to the use of these microRNAs was licensed from King’s to Heqet Therapeutics, a start-up that now aims to progress these findings towards a clinical trial in patients with acute myocardial infarction.
Parallel to this work, we also explored the mechanisms by the microRNAs and siRNAs we identified stimulate cardiomyocyte proliferation. We found that these molecules, through different mechanisms, all act by activating YAP, a factor that controls gene expression in the cells during cardiac development, and by changing the cardiac muscle cell architecture, thus promoting division of these cells.
