Periodic Reporting for period 1 - REGeRNA (Cardiac regeneration by mRNA-triggered proliferation of cardiomyocytes)
Reporting period: 2022-09-01 to 2024-02-29
Heart failure is recognized as a major health problem worldwide both in terms of patient management and related costs. Its main underlying origin is the loss of millions of functional cardiomyocytes. Even though several treatments improve heart function and clinical outcomes, they all fail to address this root cause. Replacing the lost cardiomyocytes with new contractile cells is a real challenge because, unlike many non-mammalian species, the adult human heart does not regenerate spontaneously. An option that emerged over the last two decades to replenish the pool of functional cardiomyocytes is cell therapy, based on the premise that the exogenously transplanted cells would engraft in the recipient myocardium and couple with their host counterparts to make a functionally effective myocardial syncytium. Overall, the outcomes of most trials have been disappointing, and despite occasional hints of efficacy, few have conclusively demonstrated stable and durable cardiomyocyte engraftment and clear evidence of true myocardial ‘regeneration’.
The main objective of the REGeRNA project is thus to develop a synthetic messenger Ribonucleic Acid (mRNA) able to trigger the proliferation of endogenous (quiescent) cardiomyocytes and functionalized in such a way that the product is expressed in cardiomyocytes only, thereby minimizing cellular off-target effects. This construct should be conveyed to the heart via lipid nanoparticles (LNP), either by direct myocardial administration or systemically (in which case the LNPs should be externally decorated with cardiac-specific receptors to selectively target the heart).
The main objective of the REGeRNA project is thus to develop a synthetic messenger Ribonucleic Acid (mRNA) able to trigger the proliferation of endogenous (quiescent) cardiomyocytes and functionalized in such a way that the product is expressed in cardiomyocytes only, thereby minimizing cellular off-target effects. This construct should be conveyed to the heart via lipid nanoparticles (LNP), either by direct myocardial administration or systemically (in which case the LNPs should be externally decorated with cardiac-specific receptors to selectively target the heart).
The REGeRNA Project started in September 2022, putting together 6 partners from 5 European countries. The project is progressing well towards its objectives. The first tools required for an accurate tracking of cardiomyocyte cycle events have been developed. A key step has been the integration of a Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) reporter system into human induced pluripotent stem cells (hiPSC) with a cardiac-specific promoter, allowing the fluorescent signal to enable tracking of cardiomyocyte division. 3D cardiac microtissues containing hiPSC-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells have also been developed, assuming that penetration of these constructs by the LNP-enclosed mRNAs would more closely mimic the in vivo situation planned to be tested at a later stage.
After having optimized procedures and assay readouts, an extensive screening of several pro-proliferation mRNA candidates has been performed, and the candidates deemed the most efficient have been selected, using the above mentioned FUCCI system. This selection was thus based on the proliferation of cardiomyocytes in 2D cultures, and these results have been validated with the EdU cell proliferation assay.
In parallel, we have initiated the encapsulation of mRNAs in liposomes, taken as initial surrogates for LNPs, and validated their internalization in both 2D and 3D cardiac models. We are now evaluating the expression of the encoded protein after transfecting an mRNA-LNPs construction into the 3D model. We have also started to investigate strategies to allow a systemic administration of the future construct (LNP enclosing mRNA) bypassing an hepatic clearance by the mononuclear phagocyte system.
After having optimized procedures and assay readouts, an extensive screening of several pro-proliferation mRNA candidates has been performed, and the candidates deemed the most efficient have been selected, using the above mentioned FUCCI system. This selection was thus based on the proliferation of cardiomyocytes in 2D cultures, and these results have been validated with the EdU cell proliferation assay.
In parallel, we have initiated the encapsulation of mRNAs in liposomes, taken as initial surrogates for LNPs, and validated their internalization in both 2D and 3D cardiac models. We are now evaluating the expression of the encoded protein after transfecting an mRNA-LNPs construction into the 3D model. We have also started to investigate strategies to allow a systemic administration of the future construct (LNP enclosing mRNA) bypassing an hepatic clearance by the mononuclear phagocyte system.
The project could have a major impact on the management of severe heart failure by allowing a partial restoration of a contractile cell pool through the re-induction of tissue-resident cardiomyocyte proliferation. In parallel, the development of a systemic delivery of LNP overcoming the issue of hepatic macrophage uptake, thus allowing a substantial cardiac homing, would improve the efficiency of organ targeting of any intravenously delivered therapeutics. The mechanism for mitigating phagocytic events would be common to all applications while only the nature of the LNP-bound peptides would remain specific for the targeted organ and thus the disease under consideration.
A web site has been developed (https://www.regerna.eu//(opens in new window)) and regularly updated with all the information relevant to the project.
A web site has been developed (https://www.regerna.eu//(opens in new window)) and regularly updated with all the information relevant to the project.