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Specific Modified mRNA Translation System

2020; Lippincott Williams & Wilkins; Volume: 142; Issue: 25 Linguagem: Inglês

10.1161/circulationaha.120.047211

1524-4539

Ajit Magadum, Ann Kurian, Elena Chepurko, Yassine Sassi, Roger J. Hajjar, Lior Zangi,

CRISPR and Genetic Engineering

HomeCirculationVol. 142, No. 25Specific Modified mRNA Translation System Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBSpecific Modified mRNA Translation System Ajit Magadum, PhD, Ann Anu Kurian, BSc, Elena Chepurko, DVM, Yassine Sassi, PhD, Roger J. Hajjar, MD and Lior Zangi, PhD Ajit MagadumAjit Magadum Cardiovascular Research Center (A.M., A.A.K., E.C., Y.S., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Department of Genetics and Genomic Sciences (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Black Family Stem Cell Institute (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Search for more papers by this author , Ann Anu KurianAnn Anu Kurian Cardiovascular Research Center (A.M., A.A.K., E.C., Y.S., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Department of Genetics and Genomic Sciences (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Black Family Stem Cell Institute (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Search for more papers by this author , Elena ChepurkoElena Chepurko Cardiovascular Research Center (A.M., A.A.K., E.C., Y.S., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Department of Genetics and Genomic Sciences (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Black Family Stem Cell Institute (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Search for more papers by this author , Yassine SassiYassine Sassi Cardiovascular Research Center (A.M., A.A.K., E.C., Y.S., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Search for more papers by this author , Roger J. HajjarRoger J. Hajjar Phospholamban Foundation, Amsterdam, The Netherlands (R.J.H.). Search for more papers by this author and Lior ZangiLior Zangi Lior Zangi, PhD, Icahn School of Medicine at Mount Sinai, Hess Building 7th Floor, Room 107, One Gustave L. Levy Place, Box 1030, New York, NY 10029. Email E-mail Address: [email protected] Cardiovascular Research Center (A.M., A.A.K., E.C., Y.S., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Department of Genetics and Genomic Sciences (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Black Family Stem Cell Institute (A.M., A.A.K., E.C., L.Z.), Icahn School of Medicine at Mount Sinai, New York. Search for more papers by this author Originally published21 Dec 2020https://doi.org/10.1161/CIRCULATIONAHA.120.047211Circulation. 2020;142:2485–2488Adult mammalian hearts cannot regenerate after myocardial infarction (MI): cardiomyocytes (CMs) that die after MI are replaced with a fibrotic scar that compromises heart function and induces heart failure. Gene delivery through modified mRNA (modRNA) is a safe, transient, nonimmunogenic, local, controlled platform that rapidly translates genes in heart cells after MI.1 We recently demonstrated that our specific modRNA translation system (SMRTs) delivers potent intracellular genes (eg, cell cycle–promoting Pkm2 [pyruvate kinase muscle isoenzyme 2]), which are beneficial when expressed in 1 cell type (CM), but not others (non-CM), exclusively to CMs.2 Here we show how SMRTs allows modRNA translation only in CMs or non-CMs both in culture and after MI.SMRTs may reduce several CM-specific microRNAs (cmsmiRs) that are detrimental after MI. miR208a encodes using the same intron as the CM-specific marker Myh6 and induces hypertrophy by upregulating β-myosin heavy chain.3 Elevated muscle-specific miR-1 increases cell death.4 CM-specific miR-199a impairs autophagy and promotes cardiac hypertrophy through mTOR [mammalian target of rapamycin] activation.5 We created 4 inactive human CD25 (reporter gene ihCD25) modRNAs with or without recognition elements for these cardiac-detrimental cmsmiRs (Figure A). We transfected these modRNAs into neonatal rat heart cells and immunostained them for hCD25 1 day later in vitro (Figure B). In parallel, we delivered these modRNAs to adult mouse hearts 4 days after MI in vivo and collected the hearts 2 days later (Figure C). All animal procedures accorded with institutional guidelines. Our results (Figure D through F) show that CD25 modRNA carrying miR1 or miR208 recognition elements were translated only in non-CMs, whereas CD25 modRNA with or without miR199 recognition elements were translated in both CMs and non-CMs, in vitro (Figure D, upper) and in vivo (Figure D, lower). These data suggest that miR1 and miR208 recognition elements can significantly inhibit modRNA translation in CMs, in vitro (Figure E), and in vivo (Figure F); however, our results do not necessarily mean that CMs express or function more in miR1/miR208 than other cmsmiRs.Download figureDownload PowerPointFigure. Development of CM- and non–CM-specific modRNA expression platform.A, Construction design for identifying CM-specific miRs (cmsmiRs) using inactive human CD25 (ihCD25) modRNA with or without recognition elements for known cmsmiRs posttransfection. B and C, Experimental timeline for evaluating cmsmiRs expression in neonatal rat heart cells in vitro (B) and in an adult mouse MI model in vivo (C). D, Representative images of CD25 immunostaining (red) after delivery of ihCD25 modRNA with or without recognition elements for different miRs posttransfection in neonatal rat heart cells in vitro (Upper) and mouse MI model in vivo (Lower). E and F, Quantification of the in vitro (E) and in vivo (F) experiments (n=3). G, Construct design and experimental timeline to analyze the inhibition of miR1 and miR208 target genes in mouse hearts 2 days after MI and delivery of modRNA with or without recognition elements for miR1 or miR208. H and I, qPCR evaluation of miR1 (H) and miR208 (I) target genes, 2 days after MI and delivery of modRNA with or without recognition elements for miR1 or miR208 (n=3). J, Construct design for SMRTs GFP non–CM-specific modRNA to be evaluated in neonatal rat heart cells in vitro. K, GFP immunostaining to evaluate nGFP modRNA expression (green) in neonatal rat CMs (red) 1 day after delivery of nGFP modRNA with or without CM-specific recognition elements (miR1-208) in vitro (similar timeline as in B). L, Quantification of the experiment described in K (n=3). M, Construct design for SMRTs Cre non–CM-specific modRNA to be evaluated in vivo (similar timeline as in C) using Rosa26mTmG reporter mice in MI model (on Cre expression, cells express GFP). N, GFP immunostaining to evaluate Cre modRNA expression (green) in non-CMs and CMs (red) 28 days after delivery of Cre modRNA with or without CM-specific recognition elements (miR1-208) in vivo. O, Quantification of the experiment described in N (n=3). P, SMRTs CM-specific GFP/Cre based on 2 modRNAs: 1 modRNA is a suppressor gene (L7AE) with miR1-208 recognition element upstream of its 3′-UTR, and in the second modRNA the gene of interest is regulated by a K-motif downstream of its 5′-UTR. On L7AE translation, the circuit modRNA binds to the K-motif and prevents translation of the gene of interest (Cre or nGFP). On transfection into CMs, L7AE is suppressed by endogenous miR1 or miR208. Lack of L7AE results in Cre or nGFP translation; however, in non-CM cells, L7AE is expressed and suppresses Cre or nGFP expression. Q, Rosa26mTmG reporter mice cotransfected with Cre-K alone or circuit modRNA (Cre-K+miR1-208 [CM-SMRTs Cre]) and analyzed 28 days after MI. R, Evaluation of different ratios of miR1-208 and nGFP k modRNA for delivery in an MI setting in vivo (similar timeline as in C). S, Sequence alignment of miR1 or miR208 in rat, mouse, and human. Unpaired 2-tailed t test for E, F, L, O, and S. One-way ANOVA, Tukey Multiple Comparison Test for H and I. ****P<0.0001, ***P<0.001, **P 20% of the transfected area 28 days after MI.2 This system has limitations, specifically that the expression efficiency remains low and warrants improvement. In parallel, using our adult mouse MI model, we show that transfecting nGFP k-motif generated nGFP translation in both CMs and non-CMs, yet nGFP k-motif cotransfection with L7Ae modRNA carrying miR1-208 (1:0.5 ratio) produced sufficient nGFP for exclusive translation in CMs (Figure R).miR1/miR208 sequence alignment in rats, mice, and humans show high similarity (Figure S), suggesting that SMRTs can also be used in humans. Accordingly, we designed 2 efficient SMRTs that translate mRNA exclusively in CMs or non-CMs in vitro and in vivo. Our SMRTs also reduced disadvantageous cmsmiRs in the heart after MI. This new system will help researchers evaluate function or tailor gene-of-interest treatment in different cell types after MI.AcknowledgmentsThe authors acknowledge N. Singh, M. M. Żak, K. Kaur, Y. Hadas, and N. Sultana for their help with this article. The authors thank Dr Patrick Soon-Shiong for advice regarding the title of the manuscript.Sources of FundingThis work was funded by a cardiology startup grant awarded to the Zangi laboratory and also by a National Institutes of Health grant (R01 HL142768-01).DisclosuresDrs Zangi and Magadum are inventors of a Provisional Patent (Cell-specific expression of modRNA, WO2018053414A1), which covers the results in this article.FootnotesThe data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure presented in this article.https://www.ahajournals.org/journal/circLior Zangi, PhD, Icahn School of Medicine at Mount Sinai, Hess Building 7th Floor, Room 107, One Gustave L. Levy Place, Box 1030, New York, NY 10029. Email lior.[email protected]eduReferences1. Sultana N, Magadum A, Hadas Y, Kondrat J, Singh N, Youssef E, Calderon D, Chepurko E, Dubois N, Hajjar RJ, et al.. Optimizing cardiac delivery of modified mRNA.Mol Ther. 2017; 25:1306–1315. doi: 10.1016/j.ymthe.2017.03.016CrossrefMedlineGoogle Scholar2. Magadum A, Singh N, Kurian AA, Munir I, Mehmood T, Brown K, Sharkar MTK, Chepurko E, Sassi Y, Oh JG, et al.. Pkm2 regulates cardiomyocyte cell cycle and promotes cardiac regeneration.Circulation. 2020; 141:1249–1265. doi: 10.1161/CIRCULATIONAHA.119.043067LinkGoogle Scholar3. Callis TE, Pandya K, Seok HY, Tang RH, Tatsuguchi M, Huang ZP, Chen JF, Deng Z, Gunn B, Shumate J, et al.. MicroRNA-208a is a regulator of cardiac hypertrophy and conduction in mice.J Clin Invest. 2009; 119:2772–2786. doi: 10.1172/JCI36154CrossrefMedlineGoogle Scholar4. Xu C, Lu Y, Pan Z, Chu W, Luo X, Lin H, Xiao J, Shan H, Wang Z, Yang B. The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes.J Cell Sci. 2007; 120pt 173045–3052. doi: 10.1242/jcs.010728CrossrefMedlineGoogle Scholar5. Li Z, Song Y, Liu L, Hou N, An X, Zhan D, Li Y, Zhou L, Li P, Yu L, et al.. miR-199a impairs autophagy and induces cardiac hypertrophy through mTOR activation.Cell Death Differ. 2017; 24:1205–1213. doi: 10.1038/cdd.2015.95CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails December 22, 2020Vol 142, Issue 25Article InformationMetrics Download: 875 © 2020 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.120.047211PMID: 33347328 Originally publishedDecember 21, 2020 KeywordsRNA, messengergenesmyocardial infarctionPDF download Advertisement SubjectsGene TherapyHeart FailureMyocardial InfarctionRemodeling