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Brief Article
Synthetic chemically modified mRNA-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction Chien-Ling Huang, Anne-Laure Leblond, Elizebeth C Turner, Arun HS Kumar, Kenneth Martin, Derek Whelan, Donnchadh M O’Sullivan, and Noel M Caplice Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5006239 • Publication Date (Web): 14 Jan 2015 Downloaded from http://pubs.acs.org on January 20, 2015
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Molecular Pharmaceutics
Synthetic chemically modified mRNA-based delivery of cytoprotective factor promotes early cardiomyocyte survival post-acute myocardial infarction Chien-Ling Huanga, Anne-Laure Leblonda, Elizebeth C Turnera, Arun HS Kumara, Kenneth Martina, Derek Whelana, Donnchadh M O’Sullivana, Noel M Caplicea,* a
Centre for Research in Vascular Biology (CRVB), Biosciences Institute, University College Cork, Cork, Ireland
KEYWORDS. Cytoprotective; insulin-like growth factor-1; modified mRNA; myocardial infarction
ABSTRACT: To extend the temporal window for cytoprotection in cardiomyocytes undergoing apoptosis after hypoxia and myocardial infarction (MI), a synthetic chemically modified mRNA (modRNA) was used to drive delivery of insulinlike growth factor-1 (IGF1) within the area at risk in a in vivo murine model of MI. Delivery of IGF1 modRNA, with polyethylenimine-based nanoparticle, augmented secreted and cell associated IGF1 promoting cardiomyocyte survival and abrogating cell apoptosis under hypoxia-induced apoptosis conditions. Translation of modRNA-IGF1 was sufficient to induce downstream increases in Akt and Erk phosphorylation. Downregulation of IGF1 specific miRNA-1 and -133 but not miR-145 expression, was also confirmed. As proof of concept, intramyocardial delivery of modRNA-IGF1 but not control modRNA-GFP significantly decreased TUNEL-positive cells, augmented Akt phosphorylation, and decreased caspase-9 activity within the infarct border zone (BZ) at 24 hours post-MI. These findings demonstrate the potential for an extended cytoprotective effect of transient IGF1 driven by synthetic modRNA delivery.
Introduction Cardiomyocyte death post myocardial infarction (MI) is associated with significant morbidity and mortality due to shock, heart failure and lethal arrhythmia1,2. Cytoprotection of these cells in the infarct and border zones early post MI offers a potentially propitious target but to date in vivo cardiomyocyte anti-apoptosis therapy has been limited by delivery strategies of recombinant protein derived agents with short half-life in the circulation3; or problematic as a result of systemic release and inactivation4. Since the timeframe for acute cardiomyocyte death post MI can extend out to 2-3 days post infarction5 it may be desirable to deliver more sustained therapeutic cytoprotection over this timeframe and to decrease the possibility of side-effects caused by long-term release. In this regard synthetic chemically modified mRNA (modRNA) exhibits auspicious delivery and expression characteristics with respect to targeting of the myocardium6 mediating efficient, and extended (for days) protein expression in cardiomyocytes without causing an immune response7-11. In this study our objective was to demonstrate proof of concept that transient modRNA expression of a candidate cytoprotective factor, insulin-like growth factor-1 (IGF1) could be induced in cardiomyocytes following hypoxia.
We also tested whether in the setting of experimental acute MI such a strategy mediates significant inhibition of in vivo hypoxia-induced cardiomyocyte apoptosis. Results and Discussion Acute and sub-acute cell death post myocardial infarction has been identified as a therapeutic target for both cell therapy and regenerative medicine1,2. We and others have identified molecular pathways that can be targeted in early hours to days after infarction1-4. However, most intravascular therapies are limited by temporo-spatial constraints. Synthetic chemically modRNA has been proven as an efficient delivery molecule, and is a promising alternative to viral vectors and plasmid DNA, mediating efficient, transient protein expression that can be targeted intracellularly7-11. Whether a modRNA based transient expression of a cytoprotective factor could be applied as an immediate strategy to provide early cytoprotection within the heart following acute ischemic injury is currently unknown. Transient transfection of synthetic GFP modRNA (nuclear localizing green fluorescent protein, nGFP) as a tracking indicator, with polyethylenimine (jetPEI)-based nanoparticle which has previously been used to facilitate
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Molecular Pharmaceutics
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in vivo non-viral gene delivery into the heart12,13, showed efficient delivery and expression of modRNA derived protein and minimal cytotoxicity in HL-1 cardiomyocytes (43.7±4.6%, n=3; Figure 1A and Supplementary Figure IAB). Delivery of modRNA-IGF1 increased intracellular protein expression and secretion of IGF1 at 24 hours and peaked at 48 hours post-transfection (Figure 1B). These data show that modRNA delivery into cardiomyocytes is efficient in providing transient expression of intracellular and secreted IGF1 at the picogram to nanogram levels in vitro.
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of IGF1 protein in transfected heart slices (Veh: vehicle control, mIGF1: modRNA-IGF1). E) Percentage of nGFP positive cells in normoxic (Nor) and hypoxic (Hyp) conditions (Veh: vehicle control). *: p
