delivery-of-modified-mrna-in-a-myocardial-infarction-mouse-model

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising ...

Icahn School of Medicine at Mount Sinai

-Epicardial adipose tissue (EAT) volume and coronary artery disease are strongly associated, even after accounting for overall body mass. Despite its pathophysiological significance, the origin and paracrine signaling pathways that regulate EAT's formation and expansion are unclear.

An IGF1R-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury.

Modified mRNA (modRNA) is a new technology in the field of somatic gene transfer that has been used for the delivery of genes into different tissues, including the heart. Our group and others have shown that modRNAs injected into the heart are robustly translated into the encoded protein and can potentially improve outcome in heart injury models. However, the optimal compositions of the modRNA and the reagents necessary to achieve optimal expression in the heart have not been characterized yet. In this study, our aim was to elucidate those parameters by testing different nucleotide modifications, modRNA doses, and transfection reagents both in vitro and in vivo in cardiac cells and tissue. Our results indicate that optimal cardiac delivery of modRNA is with N1-Methylpseudouridine-5'-Triphosphate nucleotide modification and achieved using 0.013 μg modRNA/mm/500 cardiomyocytes (CMs) transfected with positively charged transfection reagent in vitro and 100 μg/mouse heart (1.6 μg modRNA/μL in 60 μL total) sucrose-citrate buffer in vivo. We have optimized the conditions for cardiac delivery of modRNA in vitro and in vivo. Using the described methods and conditions may allow for successful gene delivery using modRNA in various models of cardiovascular disease.

Optimizing Cardiac Delivery of Modified mRNA.

Adult mammalian hearts have a very limited regeneration capacity, due largely to a lack of cardiomyocyte (CM) proliferation. It was recently reported that epicardial, but not myocardial, follistatin-like 1 (Fstl1) activates CM proliferation and cardiac regeneration after myocardial infarction (MI). Furthermore, bacterially synthesized human FSTL 1 (hFSTL1) was found to induce CM proliferation, whereas hFSTL1 synthesized in mammals did not, suggesting that post-translational modifications (e.g., glycosylation) of the hFSTL1 protein affect its regenerative activity. We used modified mRNA (modRNA) technology to investigate the possible role of specific hFSTL1 N-glycosylation sites in the induction, by hFSTL1, of CM proliferation and cardiac regeneration. We found that the mutation of a single site (N180Q) was sufficient and necessary to increase the proliferation of rat neonatal and mouse adult CMs in vitro and after MI in vivo, respectively. A single administration of the modRNA construct encoding the N180Q mutant significantly increased cardiac function, decreased scar size, and increased capillary density 28 days post-MI. Overall, our data suggest that the delivery of N180Q hFSTL1 modRNA to the myocardium can mimic the beneficial effect of epicardial hFSTL1, triggering marked CM proliferation and cardiac regeneration in a mouse MI model.

Ablation of a Single N-Glycosylation Site in Human FSTL 1 Induces Cardiomyocyte Proliferation and Cardiac Regeneration.

For more than a decade, Sca-1 cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive.

Cardiac Sca-1 Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair.

Rodent surgical animal models of heart failure (HF) are critically important for understanding the proof of principle of the cellular alterations underlying the development of the disease as well as evaluating therapeutics. Robust, reproducible rodent models are a prerequisite to the development of pharmacological and molecular strategies for the treatment of HF in patients. Due to the absence of standardized guidelines regarding surgical technique and clear criteria for HF progression in rats, objectivity is compromised. Scientific publications in rats rarely fully disclose the actual surgical details, and technical and physiological challenges. This lack of reporting is one of the main reasons that the outcomes specified in similar studies are highly variable and associated with unnecessary loss of animals, compromising scientific assessment. This review details rat circulatory and coronary arteries anatomy, the surgical details of rat models that recreate the HF phenotype of myocardial infarction, ischemia/reperfusion, left and right ventricular pressure, and volume overload states, and summarizes the technical and physiological challenges of creating HF. The purpose of this article is to help investigators understand the underlying issues of current HF models in order to reduce variable results and ensure successful, reproducible models of HF.

Surgical and physiological challenges in the development of left and right heart failure in rat models.

Synthetic modified RNA (modRNA) is a novel vector for gene transfer to the heart and other organs. modRNA can mediate strong, transient protein expression with minimal induction of the innate immune response and risk for genome integration. modRNA is already being used in several human clinical trials, and its use in basic and translational science is growing. Due to the complexity of preparing modRNA and the high cost of its reagents, there is a need for an improved, cost-efficient protocol to make modRNA. Here we show that changing the ratio between anti-reverse cap analog (ARCA) and N1-methyl-pseudouridine (N1mΨ), favoring ARCA over N1mΨ, significantly increases the yield per reaction, improves modRNA translation, and reduces its immunogenicity . This protocol will make modRNA preparation more accessible and financially affordable for basic and translational research.

Optimizing Modified mRNA  Synthesis Protocol for Heart Gene Therapy.

Inhibition of pulmonary fibrosis (PF) by restoring sarco/endoplasmic reticulum calcium ATPase 2a isoform (SERCA2a) expression using targeted gene therapy may be a potentially powerful new treatment approach for PF. Here, we found that SERCA2a expression was significantly decreased in lung samples from patients with PF and in the bleomycin (BLM) mouse model of PF. In the BLM-induced PF model, intratracheal aerosolized adeno-associated virus serotype 1 (AAV1) encoding for human SERCA2a (AAV1.hSERCA2a) reduces lung fibrosis and associated vascular remodeling. SERCA2a gene therapy also decreases right ventricular pressure and hypertrophy in both prevention and curative protocols. In vitro, we observed that SERCA2a overexpression inhibits fibroblast proliferation, migration, and fibroblast-to-myofibroblast transition induced by transforming growth factor β (TGF-β1). Thus, pro-fibrotic gene expression is prevented by blocking nuclear factor κB (NF-κB)/interleukin-6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3) activation. This effect is signaled toward an inhibitory mechanism of small mother against decapentaplegic (SMAD)/TGF-β signaling through the repression of OTU deubiquitinase, ubiquitin aldehyde binding 1 (OTUB1) and Forkhead box M1 (FOXM1). Interestingly, this cross-inhibition leads to an increase of SKI and SnoN expression, an auto-inhibitory feedback loop of TGF-β signaling. Collectively, our results demonstrate that SERCA2a gene transfer attenuates bleomycin (BLM)-induced PF by blocking the STAT3/FOXM1 pathway and promoting the SNON/SKI Axis. Thus, SERCA2a gene therapy may be a potential therapeutic target for PF.

AAV1.SERCA2a Gene Therapy Reverses Pulmonary Fibrosis by Blocking the STAT3/FOXM1 Pathway and Promoting the SNON/SKI Axis.

Sphingolipids have recently emerged as a biomarker of recurrence and mortality after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues during acute MI, as demonstrated by several groups, is associated with higher cell death rates in the left ventricle and deteriorated cardiac function. Ceramidase, the only enzyme known to hydrolyze proapoptotic ceramide, generates sphingosine, which is then phosphorylated by sphingosine kinase to produce the prosurvival molecule sphingosine-1-phosphate. We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after MI.

Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction.

The adult mammalian heart has limited regenerative capacity, mostly attributable to postnatal cardiomyocyte cell cycle arrest. In the last 2 decades, numerous studies have explored cardiomyocyte cell cycle regulatory mechanisms to enhance myocardial regeneration after myocardial infarction. Pkm2 (Pyruvate kinase muscle isoenzyme 2) is an isoenzyme of the glycolytic enzyme pyruvate kinase. The role of Pkm2 in cardiomyocyte proliferation, heart development, and cardiac regeneration is unknown.

Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration.

Modified mRNA (modRNA) is a gene-delivery platform for transiently introducing a single gene or several genes of interest to different cell types and tissues. modRNA is considered to be a safe vector for gene transfer, as it negligibly activates the innate immune system and does not compromise the genome integrity. The use of modRNA in basic and translational science is rising, due to the clinical potential of modRNA. We are currently using modRNA to induce cardiac regeneration post-ischemic injury. Major obstacles in using modRNA for cardiac ischemic disease include the need for the direct and single administration of modRNA to the heart and the inefficient translation of modRNA due to its short half-life. Modulation of the 5' untranslated region (5' UTR) to enhance translation efficiency in ischemic cardiac disease has great value, as it can reduce the amount of modRNA needed per delivery and will achieve higher and longer protein production post-single delivery. Here, we identified that 5' UTR, from the fatty acid metabolism gene carboxylesterase 1D (Ces1d), enhanced the translation of firefly luciferase (Luc) modRNA by 2-fold in the heart post-myocardial infarction (MI). Moreover, we identified, in the Ces1d, a specific RNA element (element D) that is responsible for the improvement of modRNA translation and leads to a 2.5-fold translation increment over Luc modRNA carrying artificial 5' UTR, post-MI. Importantly, we were able to show that 5' UTR Ces1d also enhances modRNA translation in the liver, but not in the kidney, post-ischemic injury, indicating that Ces1d 5' UTR and element D may play a wider role in translation of protein under an ischemic condition.

Optimization of 5' Untranslated Region of Modified mRNA for Use in Cardiac or Hepatic Ischemic Injury.

Ischaemic heart disease evokes a complex immune response. However, tools to track the systemic behaviour and dynamics of leukocytes non-invasively in vivo are lacking. Here, we present a multimodal hot-spot imaging approach using an innovative high-density lipoprotein-derived nanotracer with a perfluoro-crown ether payload (F-HDL) to allow myeloid cell tracking by F magnetic resonance imaging. The F-HDL nanotracer can additionally be labelled with zirconium-89 and fluorophores to detect myeloid cells by in vivo positron emission tomography imaging and optical modalities, respectively. Using our nanotracer in atherosclerotic mice with myocardial infarction, we observed rapid myeloid cell egress from the spleen and bone marrow by in vivo F-HDL magnetic resonance imaging. Concurrently, using ex vivo techniques, we showed that circulating pro-inflammatory myeloid cells accumulated in atherosclerotic plaques and at the myocardial infarct site. Our multimodality imaging approach is a valuable addition to the immunology toolbox, enabling the study of complex myeloid cell behaviour dynamically.

Article	Year
An IGF1R-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury. Circulation\| PubMed ID: 27803039	2016
Optimizing Cardiac Delivery of Modified mRNA. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 28389322	2017
Ablation of a Single N-Glycosylation Site in Human FSTL 1 Induces Cardiomyocyte Proliferation and Cardiac Regeneration. Molecular therapy. Nucleic acids\| PubMed ID: 30290305	2018
Cardiac Sca-1 Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair. Circulation\| PubMed ID: 30566018	2018
Surgical and physiological challenges in the development of left and right heart failure in rat models. Heart failure reviews\| PubMed ID: 30903356	2019
Optimizing Modified mRNA Synthesis Protocol for Heart Gene Therapy. Molecular therapy. Methods & clinical development\| PubMed ID: 31508439	2019
AAV1.SERCA2a Gene Therapy Reverses Pulmonary Fibrosis by Blocking the STAT3/FOXM1 Pathway and Promoting the SNON/SKI Axis. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 31879190	2020
Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction. Circulation\| PubMed ID: 31992066	2020
Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration. Circulation\| PubMed ID: 32078387	2020
Optimization of 5' Untranslated Region of Modified mRNA for Use in Cardiac or Hepatic Ischemic Injury. Molecular therapy. Methods & clinical development\| PubMed ID: 32300609	2020
Probing myeloid cell dynamics in ischaemic heart disease by nanotracer hot-spot imaging. Nature nanotechnology\| PubMed ID: 32313216	2020

Elena Chepurko

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