TY - JOUR T1 - Extracellular vesicle microRNA cargo engineering reveals critical mechanisms underlying therapeutic efficacy JF - bioRxiv DO - 10.1101/2022.01.31.478505 SP - 2022.01.31.478505 AU - Lindsey M. Euscher AU - Kyle I. Mentkowski AU - Touba Tarvirdizadeh AU - Isabella Julian AU - Karan Bhatt AU - Lisa Eagler AU - Jennifer K. Lang Y1 - 2022/01/01 UR - http://biorxiv.org/content/early/2022/01/31/2022.01.31.478505.abstract N2 - Background Extracellular vesicles (EVs) are key mediators of intercellular communication and function to transfer biological cargo, including microRNA (miR), from donor to recipient cells. EVs isolated from cardiosphere-derived cells (CDCs) have demonstrated therapeutic efficacy in pre-clinical models of ischemic heart disease, highlighting them as promising vectors for the treatment of CVD. Importantly, it has not yet been established whether miR cargo is necessary for the observed therapeutic benefit of CDC-EVs following acute MI (AMI).Methods CDCs were transfected with siRNA against Drosha, the initial endonuclease in the miRNA biogenesis pathway, to generate miR depleted DROSHA-EVs. EVs were characterized by size, morphology, and protein/miR expression. The role of EV miRNA on cardiac target cell apoptosis, proliferation and angiogenesis was examined using a series of in vitro assays. Mice with acute MI underwent delivery of human CDC EVs, DROSHA-EVs and placebo in a double-blind study. LVEF was assessed by echo at 1- and 28-days post-MI and tissue samples processed for assessment of histological endpoints. In vitro sufficiency assays were performed using a combinatorial approach with individual candidate miRs to identify clusters exhibiting synergistic efficacy.Results DROSHA-EVs exhibited global downregulation of miRNA cargo but were otherwise indistinguishable from wild-type CDC-EVs. miR cargo was responsible for mediating the beneficial effects of human CDC-EV treatment on cardiomyocyte apoptosis, fibroblast proliferation and angiogenesis in vitro. DROSHA-EVs were unable to promote recovery following AMI on a functional or histological level, highlighting the critical role of EV miRNAs in cardioprotection following ischemic injury. A potentially therapeutic miR cluster, miR-146a-370-126a, was identified which acted synergistically to reduce cardiomyocyte apoptosis and was sufficient to render inert EVs into therapeutic vectors.Conclusions These results demonstrate for the first time that miRNAs are required for the regenerative potential of CDC-EVs following AMI and identify a novel miR cluster with therapeutic implications.Competing Interest StatementThe authors have declared no competing interest.AMIacute myocardial infarctionCDCcardiosphere-derived cellCMcardiomyocyteCMPcardiomyocyte specific peptideEFejection fractionEVsextracellular vesiclesHUVEChuman umbilical vein endothelial celllncRNAlong non-coding RNALVleft ventricleMImyocardial infarctionmiRmicroRNANHDFnormal human dermal fibroblastNTANanoparticle Tracking AnalysissnRNAsmall nuclear RNAsnoRNAsmall nucleolar RNA ER -