Combinatorial miRNA1a/15b interference drives adult cardiac regeneration

BACKGROUND: Despite its promise, cardiac regenerative therapy remains clinically elusive due to the difficulty of spatio-temporal control of proliferative induction, and the need to coordinately reprogram multiple regulatory pathways to overcome the strict post-mitotic state of human adult cardiomyocytes. The present study was designed to identify a novel combinatorial miRNA therapy to address this unmet therapeutic need. METHODS: We performed a combinatorial miRNA interference screen specifically targeting cardiac-predominant miRNAs regulating key aspects of cardiomyocyte mitotic induction to cell-cycle completion, including sarcomerogenesis, metabolic and cell-cycle control pathways. Cardiomyocyte proliferation and cardiac function were assessed in human cardiac biopsies, human cardiac tissue mimetics and in mouse disease models. RESULTS: We identified combinatorial interference of miR-1a and miR-15b (LNA-1a/15b) as drivers of adult cardiomyocyte proliferation. Due to miR-1a/15b function on multiple processes modulating adult cardiomyocyte mitosis, its inhibition augmented adult cardiomyocyte cell-cycle completion and daughter cell formation, and improved contractility in in vitro 2D and 3D ischemic models, and in a mouse model of ST-segment elevation myocardial infarction (STEMI). Due to the cardiac-restricted pattern of miR-1a/15b expression, this strategy provides a feasible strategy for specific cardiomyocyte proliferative induction with minimal risk of neoplasm formation and off-target toxicity. CONCLUSIONS: Combinatorial miR-1a/15b inhibition drives mitotic re-entry in adult cardiomyocytes and improves cardiac function in response to myocardial infarction. Our data provides a novel and clinically feasible LNA-based anti-miR-1a/15b strategy to attenuate heart failure and highlights an underutilized therapeutic strategy for simultaneous co-regulation of multiple disease pathways through combinatorial miRNA interference. ### Competing Interest Statement T.Y., S.D. and J.K. are inventors on a patent application pertaining to the inhibition of miR-1a and miR-15b for the treatment of heart disease. ### Funding Statement This work was supported by the Messer Foundation, LOEWE Center for Cell and Gene Therapy and the European Innovation Council (GA: 822455) to J.K., and the SFB-TRR 267 (Non-coding RNA in the cardiovascular system) and the German Research Foundation (DFG) (Exc2026) to S.D. and J.K., and instrument grant support (INST 515/28-1 FUGG) to M.P., the European Research Council (Angiolnc) to S.D., and the Foundation for Pathobiochemistry and Molecular Diagnostics to P.M. Y.W. was supported by the China Scholarship Council (CSC) Grant #202108080020. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee of Goethe University Frankfurt Research Services gave ethical approval for this work. All necessary patient/participant consent has been obtained and patient/participant/sample identifiers cannot be used to identify individuals. Ethics committee of Regierungsprasidium Darmstadt gave ethical approval for the animal studies this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.