mRNA-Based Protein Replacement Therapy for the Heart
Authors: Ajit Magadum, Keerat Kaur, Lior Zangi
Journal: Molecular Therapy (2018)

Myocardial infarction (MI) and heart failure (HF) are the leading causes of death in the United States and in most other industrialized nations. MI leads to a massive loss of cardiomyocytes (CMs), which are replaced with non-CM cells, leading to scarring and, in most cases, HF. The adult mammalian heart has a low intrinsic regenerative capacity, mainly because of cell-cycle arrest in CMs. No effective treatment promoting heart regeneration is currently available. Recent efforts to use DNA-based or viral gene therapy approaches to induce cardiac regeneration post-MI or in HF conditions have encountered major challenges, mostly because of the poor and uncontrolled delivery of the introduced genes. Modified mRNA (modRNA) is a safe, non-immunogenic, efficient, transient, local, and controlled nucleic acid delivery system that can overcome the obstacles to DNA-based or viral approaches for cardiac gene delivery. We here review the use of modRNA in cardiac therapy, to induce cardioprotection and vascular or cardiac regeneration after MI. We discuss the current challenges in modRNA-based cardiac treatment, which will need to be overcome for the application of such treatment to ischemic heart disease. Myocardial infarction (MI) and heart failure (HF) are the leading causes of death in the United States and in most other industrialized nations. MI leads to a massive loss of cardiomyocytes (CMs), which are replaced with non-CM cells, leading to scarring and, in most cases, HF. The adult mammalian heart has a low intrinsic regenerative capacity, mainly because of cell-cycle arrest in CMs. No effective treatment promoting heart regeneration is currently available. Recent efforts to use DNA-based or viral gene therapy approaches to induce cardiac regeneration post-MI or in HF conditions have encountered major challenges, mostly because of the poor and uncontrolled delivery of the introduced genes. Modified mRNA (modRNA) is a safe, non-immunogenic, efficient, transient, local, and controlled nucleic acid delivery system that can overcome the obstacles to DNA-based or viral approaches for cardiac gene delivery. We here review the use of modRNA in cardiac therapy, to induce cardioprotection and vascular or cardiac regeneration after MI. We discuss the current challenges in modRNA-based cardiac treatment, which will need to be overcome for the application of such treatment to ischemic heart disease. Despite advances in curative and preventive medicine, heart failure (HF) remains the leading cause of mortality and hospitalization worldwide.1Savarese G. Lund L.H. Global public health burden of heart failure.Card. Fail. Rev. 2017; 3: 7-11Google Scholar, 2Bui A.L. Horwich T.B. Fonarow G.C. Epidemiology and risk profile of heart failure.Nat. Rev. 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Franco S. et al.American Heart Association Statistics Committee and Stroke Statistics SubcommitteeHeart disease and stroke statistics—2014 update: a report from the American Heart Association.Circulation. 2014; 129: e28-e292Google Scholar Traditional approaches for dealing with end-stage HF are often not feasible, due to the limited number of hearts available for transplantation. Preclinical trials have reported improvements in patient outcomes,5Hajjar R.J. Zsebo K. Deckelbaum L. Thompson C. Rudy J. Yaroshinsky A. Ly H. Kawase Y. Wagner K. Borow K. et al.Design of a phase 1/2 trial of intracoronary administration of AAV1/SERCA2a in patients with heart failure.J. Card. Fail. 2008; 14: 355-367Google Scholar, 6Greenberg B. Butler J. Felker G.M. Ponikowski P. Voors A.A. Desai A.S. Barnard D. Bouchard A. Jaski B. Lyon A.R. et al.Calcium upregulation by percutaneous administration of gene therapy in patients with cardiac disease (CUPID 2): a randomised, multinational, double-blind, placebo-controlled, phase 2b trial.Lancet. 2016; 387: 1178-1186Google Scholar but prognosis remains poor, and there is, therefore, an urgent need for new approaches to the prevention and treatment of HF. During HF, billions of cardiomyocytes (CMs) are progressively lost, and fibrotic non-functional scar tissue develops, significantly reducing the pumping capacity of the heart muscle. The remaining CMs have a limited intrinsic regenerative capacity and cannot, therefore, replace the lost CMs. Cardiac regeneration studies have shown that dividing CMs are abundant in the fetus, but rapidly lost after birth.7Porrello E.R. Mahmoud A.I. Simpson E. Hill J.A. Richardson J.A. Olson E.N. Sadek H.A. Transient regenerative potential of the neonatal mouse heart.Science. 2011; 331: 1078-1080Google Scholar Cell-based therapies with exogenous cells, such as bone marrow cells, cardiac progenitor cells, and other self-renewing stem cells, have been developed to improve heart function. However, little meaningful improvement has been reported for these treatments, owing to the limited interaction between the various progenitor cells and the myocardium environment during myocardial infarction (MI). In the last two decades, our understanding of the molecular pathways and genes involved in the disease has improved, and gene therapy has emerged as a possible treatment for HF. Given the limited site specificity of pharmacological inhibitors, gene therapy is an exciting prospect for more precise targeting of the signaling pathways involved in disease progression. The gene therapy approaches currently being developed for HF aim: (1) to increase the proliferation or contractility of endogenous CMs; (2) to reprogram cardiac fibroblasts to develop into beneficial cardiac cell types, such as endothelial cells (ECs) or CMs; and (3) to increase capillary density by activating endogenous ECs or progenitors. Recent studies have reported reactivation of the CM cell cycle following protein delivery to the myocardium, either by direct injection or via patch delivering the protein to the epicardium. CM proliferation has been reported following the delivery of NRG1 protein via intraperitoneal (i.p.) injections,8Engel F.B. Schebesta M. Duong M.T. Lu G. Ren S. Madwed J.B. Jiang H. Wang Y. Keating M.T. p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes.Genes Dev. 2005; 19: 1175-1187Google Scholar intramyocardial (IM) injections of agrin,9Bassat E. Mutlak Y.E. Genzelinakh A. Shadrin I.Y. Baruch Umansky K. Yifa O. Kain D. 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Periostin induces proliferation of differentiated cardiomyocytes and promotes cardiac repair.Nat. Med. 2007; 13: 962-969Google Scholar Also, Hajjar and coworkers13del Monte F. Hajjar R.J. Harding S.E. Overwhelming evidence of the beneficial effects of SERCA gene transfer in heart failure.Circ. Res. 2001; 88: E66-E67Google Scholar, 14Kho C. Lee A. Jeong D. Oh J.G. Chaanine A.H. Kizana E. Park W.J. Hajjar R.J. SUMO1-dependent modulation of SERCA2a in heart failure.Nature. 2011; 477: 601-605Google Scholar have shown that the adeno-associated virus (AAV)-mediated delivery of Serca2a13del Monte F. Hajjar R.J. Harding S.E. Overwhelming evidence of the beneficial effects of SERCA gene transfer in heart failure.Circ. Res. 2001; 88: E66-E67Google Scholar or SUMO14Kho C. Lee A. Jeong D. Oh J.G. Chaanine A.H. Kizana E. Park W.J. Hajjar R.J. SUMO1-dependent modulation of SERCA2a in heart failure.Nature. 2011; 477: 601-605Google Scholar improves cardiac function post-MI and in HF condition via elevation of endogenous CMs contractility. Another successful avenue of gene therapy for heart repair is the genetic in situ reprogramming of cardiac fibroblasts into CMs. Pioneering work by Srivastava and coworkers15Ieda M. Fu J.D. Delgado-Olguin P. Vedantham V. Hayashi Y. Bruneau B.G. Srivastava D. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.Cell. 2010; 142: 375-386Google Scholar showed that fibroblasts could undergo cardiac reprogramming to become beating CMs following direct virus-mediated IM delivery and overexpression of the cardiac myocyte transcription factors Gata4, Mef2c, and Tbx5 (GMT). This approach is promising as an alternative to cell-based regeneration therapies, but the efficiency of in vivo reprogramming remained low, and there is also a potential risk of viral genome insertions associated with the use of viral vectors. Olson and coworkers16Song K. Nam Y.J. Luo X. Qi X. Tan W. Huang G.N. Acharya A. Smith C.L. Tallquist M.D. Neilson E.G. et al.Heart repair by reprogramming non-myocytes with cardiac transcription factors.Nature. 2012; 485: 599-604Google Scholar improved in vivo reprograming by using Hand2 as an additional reprogramming factor, together with GMT. In addition to its use to stimulate CM proliferation and reprogramming, gene therapy has also been used to induce myocardial repair by enhancing angiogenesis and inducing cardiovascular regeneration. Over the last decade, preclinical studies have reported revascularization in ischemic heart following the direct delivery of vascular endothelial growth factors (VEGFs) by various methods.17Henry T.D. Annex B.H. McKendall G.R. Azrin M.A. Lopez J.J. Giordano F.J. Shah P.K. Willerson J.T. Benza R.L. Berman D.S. et al.VIVA InvestigatorsThe VIVA trial: vascular endothelial growth factor in ischemia for vascular angiogenesis.Circulation. 2003; 107: 1359-1365Google Scholar, 18Hendel R.C. Henry T.D. Rocha-Singh K. Isner J.M. Kereiakes D.J. Giordano F.J. Simons M. Bonow R.O. Effect of intracoronary recombinant human vascular endothelial growth factor on myocardial perfusion: evidence for a dose-dependent effect.Circulation. 2000; 101: 118-121Google Scholar, 19Sato K. Wu T. Laham R.J. Johnson R.B. Douglas P. Li J. Sellke F.W. Bunting S. Simons M. Post M.J. Efficacy of intracoronary or intravenous VEGF165 in a pig model of chronic myocardial ischemia.J. Am. Coll. Cardiol. 2001; 37: 616-623Google Scholar, 20Hedman M. Hartikainen J. Syvänne M. Stjernvall J. Hedman A. Kivelä A. Vanninen E. Mussalo H. Kauppila E. Simula S. et al.Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation. 2003; 107: 2677-2683Google Scholar, 21Stewart D.J. Kutryk M.J. Fitchett D. Freeman M. Camack N. Su Y. Della Siega A. Bilodeau L. Burton J.R. Proulx G. Radhakrishnan S. NORTHERN Trial InvestigatorsVEGF gene therapy fails to improve perfusion of ischemic myocardium in patients with advanced coronary disease: results of the NORTHERN trial.Mol. Ther. 2009; 17: 1109-1115Google Scholar, 22Stewart D.J. Hilton J.D. Arnold J.M. Gregoire J. Rivard A. Archer S.L. Charbonneau F. Cohen E. Curtis M. Buller C.E. et al.Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment.Gene Ther. 2006; 13: 1503-1511Google Scholar, 23Losordo D.W. Vale P.R. Symes J.F. Dunnington C.H. Esakof D.D. Maysky M. Ashare A.B. Lathi K. Isner J.M. Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia.Circulation. 1998; 98: 2800-2804Google Scholar Widely used vectors provided robust and consistent gene expression leading to neovascularization, but this expression was often accompanied by undesirable effects, such as the induction of edema or angioma due to the prolonged expression of VEGF, the elicitation of an immune response against the vector, or a potential risk of genomic integration.20Hedman M. Hartikainen J. Syvänne M. Stjernvall J. Hedman A. Kivelä A. Vanninen E. Mussalo H. Kauppila E. Simula S. et al.Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation. 2003; 107: 2677-2683Google Scholar, 21Stewart D.J. Kutryk M.J. Fitchett D. Freeman M. Camack N. Su Y. Della Siega A. Bilodeau L. Burton J.R. Proulx G. Radhakrishnan S. NORTHERN Trial InvestigatorsVEGF gene therapy fails to improve perfusion of ischemic myocardium in patients with advanced coronary disease: results of the NORTHERN trial.Mol. Ther. 2009; 17: 1109-1115Google Scholar, 22Stewart D.J. Hilton J.D. Arnold J.M. Gregoire J. Rivard A. Archer S.L. Charbonneau F. Cohen E. Curtis M. Buller C.E. et al.Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121) (AdVEGF121) versus maximum medical treatment.Gene Ther. 2006; 13: 1503-1511Google Scholar Nevertheless, these studies have provided useful insight and support for the use of gene therapy to repair the injured myocardium, although a number of hurdles remain to be overcome for this therapeutic approach to be considered successful. Gene therapy can be defined as the transplantation of normal genes into cells to replace missing or defective genes, with the aim of correcting genetic disorders or promoting inactive beneficial mechanisms or pathways. With improvements in our understanding of cardiac disease, interest is growing in the use of gene therapies to treat coronary heart disease. The ultimate goal of gene therapies is the expression of protein of interest, and the most feasible way of achieving this goal is to introduce the corresponding protein directly into the myocardium. Direct protein delivery overcomes the difficulties of translation within the cell, thereby offering a potential advantage in terms of higher levels of expression, dose regulation, and control over a viral-based gene therapy approach. However, the short half-life and instability of injected proteins, the lack of use of this approach for intracellular proteins (e.g., transcription factors), and possible immunogenicity due to minor histocompatibility antigens are problems that must be overcome for this approach to be therapeutically successful. However, approaches based on the insertion of nucleic acids, which can be translated into proteins within the cardiac cells, can circumvent the challenges of protein therapy. In recent decades, considerable advances have been made toward the delivery of nucleic acids into the heart by viral and non-viral vectors. Lentiviral vectors are favored for cardiac gene therapy by many researchers because they can transduce non-dividing CMs. However, their chief advantage—their integration into the host genome, ensuring sustained gene expression—also entails a risk of compromising the genome and tumorigenesis. The exceptional transduction efficiencies of adenoviruses and AAVs have resulted in these vectors being the most widely used for cardiovascular applications. Adenoviruses transfer genes efficiently into the myocardium in large animals,24French B.A. Mazur W. Geske R.S. Bolli R. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors.Circulation. 1994; 90: 2414-2424Google Scholar but expression is transient, and these viruses trigger a strong immune response.25Muruve D.A. The innate immune response to adenovirus vectors.Hum. Gene Ther. 2004; 15: 1157-1166Google Scholar AAVs have low immunogenicity and are a widely used alternative for gene delivery to the heart. These nonpathogenic vectors ensure cardiac tropism without integration into the host genome and have been used in a recent study26Lin Z. von Gise A. Zhou P. Gu F. Ma Q. Jiang J. Yau A.L. Buck J.N. Gouin K.A. van Gorp P.R. et al.Cardiac-specific YAP activation improves cardiac function and survival in an experimental murine MI model.Circ. Res. 2014; 115: 354-363Google Scholar in which persistent Yap-associated protein expression resulted in CM proliferation and regeneration post-MI. AAV gene delivery peak levels of expression are reached about 4 weeks after delivery27Chu D. Sullivan C.C. Weitzman M.D. Du L. Wolf P.L. Jamieson S.W. Thistlethwaite P.A. Direct comparison of efficiency and stability of gene transfer into the mammalian heart using adeno-associated virus versus adenovirus vectors.J. Thorac. Cardiovasc. Surg. 2003; 126: 671-679Google Scholar and continue for up to 11 months.28Vassalli G. Büeler H. Dudler J. von Segesser L.K. Kappenberger L. Adeno-associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors.Int. J. Cardiol. 2003; 90: 229-238Google Scholar Despite the predominance of AAVs over other gene delivery vectors, the production of neutralizing antibodies against the AAV capsid, delayed pharmacokinetics, and limited gene packaging capacity of these vectors restrict their use in cardiac gene therapy.29Mingozzi F. High K.A. Immune responses to AAV vectors: overcoming barriers to successful gene therapy.Blood. 2013; 122: 23-36Google Scholar The delivery of naked plasmid DNA overcomes the risk of immune responses and oncogenesis, because of the absence of the viral vector. Plasmid DNA displays impressive organ specificity, but transfection efficiency is low. The recent elucidation of the role of microRNAs (miRNAs) and long noncoding RNAs in cardiac repair and regeneration has provided new hope for innovative therapy.30Eulalio A. Mano M. Dal Ferro M. Zentilin L. Sinagra G. Zacchigna S. Giacca M. Functional screening identifies miRNAs inducing cardiac regeneration.Nature. 2012; 492: 376-381Google Scholar A recent review by Hermans-Beijnsberger et al.31Hermans-Beijnsberger S. van Bilsen M. Schroen B. Long non-coding RNAs in the failing heart and vasculature.Noncoding RNA Res. 2018; 3: 118-130Google Scholar summarized newly found long non-coding RNAs involved in the cellular process during development of cardiovascular disease (CVD). These non-coding RNAs can efficiently suppress the target mRNA post-transcriptionally by promoting mRNA degradation or inhibiting translation. Despite the successful results obtained in vitro, systems for delivering them to the heart in vivo have yet to be optimized. Furthermore, therapeutic miRNAs may have off-target effects, resulting in potential risk of oncogenesis.32Rane S. He M. Sayed D. Vashistha H. Malhotra A. Sadoshima J. Vatner D.E. Vatner S.F. Abdellatif M. Downregulation of miR-199a derepresses hypoxia-inducible factor-1alpha and Sirtuin 1 and recapitulates hypoxia preconditioning in cardiac myocytes.Circ. Res. 2009; 104: 879-886Google Scholar, 33Lin Z. Pu W.T. Strategies for cardiac regeneration and repair.Sci. Transl. Med. 2014; 6: 239rv1Google Scholar There is, therefore, an urgent need to explore clinically relevant approaches for enhancing cardiac regeneration and maintaining correct heart function both during and immediately after ischemic injury. Different gene therapies have proved inefficient due to a short half-life, production of neutralizing antibodies, or a poor transduction capacity. By contrast, mRNA-based therapies are highly promising for the treatment of various human disorders. The delivery of mRNA to the cell has significant advantages over the use of protein or DNA-based delivery systems: (1) the use of mRNA transfection overcomes the need for nuclear localization or for transcription of the gene of interest in the patient's cells; (2) the introduction of mRNA into cells is safe under physiological conditions, because mRNA does not integrate into the host genome; and (3) the effect of the mRNA is transient, minimizing the risk of mutagenesis after mRNA therapy (Figure 1). Successful direct mRNA transfer was first reported about three decades ago, when Wolff et al.34Wolff J.A. Malone R.W. Williams P. Chong W. Acsadi G. Jani A. Felgner P.L. Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Google Scholar demonstrated the delivery of mRNA and its translation into protein in mouse skeletal muscle. After a few initial successes with mRNA therapy, research into mRNA structure and delivery methods continued, but the use of mRNA therapy was limited to vaccine development, because of problems of instability and immunogenicity. Within cells, mRNA is prone to cleavage by RNase and can trigger the innate immune system via Toll-like receptors (TLRs) 7 and 8 (which recognize single-stranded RNA) or TLR3 (which recognizes double-stranded RNA), leading to an increase in cytokine levels and associated toxicity. In 2008, pioneering work by Karikó et al.35Karikó K. Muramatsu H. Welsh F.A. Ludwig J. Kato H. Akira S. Weissman D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.Mol. Ther. 2008; 16: 1833-1840Google Scholar addressed these issues and provided a platform for mRNA therapy in genetic, regenerative medicine, immunotherapeutics, and cancer. The study showed that replacing the uridine residues in mRNA with the naturally occurring modified nucleoside pseudouridine (hence the name modified mRNA [modRNA]) enhanced translation, due to changes in the secondary structure of the mRNA limiting its recognition by the TLRs and nucleases.35Karikó K. Muramatsu H. Welsh F.A. Ludwig J. Kato H. Akira S. Weissman D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.Mol. Ther. 2008; 16: 1833-1840Google Scholar, 36Kormann M.S. Hasenpusch G. Aneja M.K. Nica G. Flemmer A.W. Herber-Jonat S. Huppmann M. Mays L.E. Illenyi M. Schams A. et al.Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.Nat. Biotechnol. 2011; 29: 154-157Google Scholar The use of modRNA has since been on the increase in genetic medicine, for protein-replacement therapies and the treatment of genetic diseases. The efficiency of modRNA delivery in vivo has been increased by enhancing the stability of the mRNA and increasing translational efficiency by capping the molecule with the 3′-O-Me-m7G(5′)ppp(5′)G Anti Reverse Cap Analog (ARCA) at its 5′ end.37Kondrat J. Sultana N. Zangi L. Synthesis of modified mRNA for myocardial delivery.Methods Mol. Biol. 2017; 1521: 127-138Google Scholar, 38Mockey M. Gonçalves C. Dupuy F.P. Lemoine F.M. Pichon C. Midoux P. mRNA transfection of dendritic cells: synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level.Biochem. Biophys. Res. Commun. 2006; 340: 1062-1068Google Scholar The uses of modRNA technology as a model for cardiac repair are listed in Table 1.Table 1Published Studies for the Use of modRNA Technology as a Model of Cardiac Repair: modRNA as a Therapeutic Strategy for Cardiac Vascularization and RegenerationNo.PublicationGene(s)RoleCellular Process or DiseaseDelivery MaterialAnimal1Zangi et al.45Zangi L. Lui K.O. von Gise A. Ma Q. Ebina W. Ptaszek L.M. Später D. Xu H. Tabebordbar M. Gorbatov R. et al.Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.Nat. Biotechnol. 2013; 31: 898-907Google ScholarVEGFadirects the fate of heart progenitor cells and induces vascular regeneration after MIcellular fate switch post-MIRNAiMAXmice2Lui et al.46Lui K.O. Zangi L. Silva E.A. Bu L. Sahara M. Li R.A. Mooney D.J. Chien K.R. Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA.Cell Res. 2013; 23: 1172-1186Google ScholarVEGFaVEGF-A promotes not only the endothelial specification but also engraftment, proliferation, and survival (reduced apoptosis) of the human Isl1+ progenitors in vivoVEGFa promotes Isl1+ to endothelial cell fate, proliferation and survival of Isl1+ progenitorsRNAiMAXmice3Huang et al.52Huang C.L. Leblond A.L. Turner E.C. Kumar A.H. Martin K. Whelan D. O'Sullivan D.M. Caplice N.M. Synthetic chemically modified mrna-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction.Mol. Pharm. 2015; 12: 991-996Google ScholarIGF-1anti-apoptosis, cardiomyocyte survival, augmented Akt phosphorylation, and decreased caspase-9 activityanti-apoptosis, cardiomyocyte survival post-MIpolyethylenimine-based nanoparticlemice4Turnbull et al.58Turnbull I.C. Eltoukhy A.A. Fish K.M. Nonnenmacher M. Ishikawa K. Chen J. Hajjar R.J. Anderson D.G. Costa K.D. Myocardial delivery of lipidoid nanoparticle carrying modRNA induces rapid and transient expression.Mol. Ther. 2016; 24: 66-75Google ScholarEGFPmodRNA delivery (direct myocardial or intracoronary administration) into rat and pig heartmodRNA expression in heartformulated lipidoid nanoparticles (FLNP)rat and pig5Turnbull et al.59Turnbull I.C. Eltoukhy A.A. Anderson D.G. Costa K.D. Lipidoid mRNA nanoparticles for myocardial delivery in rodents.Methods Mol. Biol. 2017; 1521: 153-166Google ScholarEGFPprotocollipidoid mRNA nanoparticles protocolformulated lipidoid nanoparticles (FLNP)rodents6Kondrat et al.37Kondrat J. Sultana N. Zangi L. Synthesis of modified mRNA for myocardial delivery.Methods Mol. Biol. 2017; 1521: 127-138Google Scholarprotocolmodified mRNA synthesisRNAiMAXmice7Sultana et al.51Sultana N. Magadum A. Hadas Y. Kondrat J. Singh N. Youssef E. Calderon D. Chepurko E. Dubois N. Hajjar R.J. Zangi L. Optimizing cardiac delivery of modified mRNA.Mol. Ther. 2017; 25: 1306-1315Google ScholarmodRNA delivery optimizationmodRNA delivery optimization, modRNA amount and time optimizationoptimal modRNA expressionsucrose-citrate buffermice8Zangi et al.53Zangi L. Oliveira M.S. Ye L.Y. Ma Q. Sultana N. Hadas Y. Chepurko E. Später D. Zhou B. Chew W.L. et al.Insulin-like growth factor 1 receptor-dependent pathway drives epicardial adipose tissue formation after myocardial injury.Circulation. 2017; 135: 59-72Google ScholarDN-IGF-1R, IGFRinhibition of adipogenic differentiation post-MIinhibition of adipogenic differentiation post-MIRNAiMAXmice9Carlsson et al.47Carlsson L. Clarke J.C. Yen C. Gregoire F. Albery T. Billger M. Egnell A.C. Gan L.M. Jennbacken K. Johansson E. et al.Biocompatible, purified VEGF-A mRNA improves cardiac function after intracardiac injection 1 week post-myocardial infarction in Swine.Mol. Ther. Methods Clin. Dev. 2018; 9: 330-346Google ScholarVEGFaincreased angiogenesis, improved heart function post-MI, reduced fibrosisincreased angiogenesis, improved heart function post-MI, reduced fibrosissucrose-citrate bufferpig, monkey10Singh et al.57Singh R.D. Hillestad M.L. Livia C. Li M. Alekseev A.E. Witt T.A. Stalboerger P.G. Yamada S. Terzic A. Behfar A. M3RNA drives targeted gene delivery in acute myocardial infarction.Tissue Eng. Part A. 2018; (Published online September 21, 2018)https://doi.org/10.1089/ten.tea.2017.0445Google ScholarEGFP, mCherry, FlucmodRNA delivery optimizationoptimal modRNA expression in heartalginate, nanomaterial encapsulatedmice and pig11Magadum et al.48Magadum A. Singh N. Kurian A.A. Sharkar M.T.K. Chepurko E. Zangi L. Ablation of a single N-glycosylation site in human FSTL 1 induces cardiomyocyte proliferation and cardiac regeneration.Mol. Ther. Nucleic Acids. 2018; 13: 133-143Google Scholarmutated FSTL1ablation of N180Q, N-glycosylation site of hFSTL1 by modRNA delivery increased CM proliferation, improved cardiac output, and reduced scar size post-MICM proliferation, decreased scar size, improved heart functionsucrose-citrate buffermice Open table in a new tab Immediately after MI, CMs and other cardiac cells such as ECs are lost due to occlusion of the coronary artery. A chain of events downstream leads to oxidative stress and inflammation, resulting in impaired pump function and, ultimately, HF. The remaining CMs in the heart have a very limited proliferative potential and are therefore unable to replace the lost cells. The damaged coronary vasculature also creates a hostile environment in which it is difficult for the remaining CMs to survive. Various strategies have been developed to try to reverse the situation by inducing regeneration of cardiac neovasculature and encouraging CMs to proliferat