Publications by authors named "Lior Zangi"

37 Publications

Direct Reprogramming Induces Vascular Regeneration Post Muscle Ischemic Injury.

Mol Ther 2021 Jul 28. Epub 2021 Jul 28.

Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029. Electronic address:

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail of four cardiac-reprogramming genes (Gata4 (G), Mef2c (M), Tbx5 (T) and Hand2 (H)) together with three reprogramming-helper genes (Dominant Negative (DN)-TGFβ, DN-Wnt8a and Acid ceramidase (AC)), termed 7G-modRNA, to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de-novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.
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http://dx.doi.org/10.1016/j.ymthe.2021.07.014DOI Listing
July 2021

Therapeutic Delivery of Pip4k2c-Modified mRNA Attenuates Cardiac Hypertrophy and Fibrosis in the Failing Heart.

Adv Sci (Weinh) 2021 05 12;8(10):2004661. Epub 2021 Mar 12.

Cardiovascular Research Center Icahn School of Medicine at Mount Sinai New York NY 10029 USA.

Heart failure (HF) remains a major cause of morbidity and mortality worldwide. One of the risk factors for HF is cardiac hypertrophy (CH), which is frequently accompanied by cardiac fibrosis (CF). CH and CF are controlled by master regulators mTORC1 and TGF-, respectively. Type-2-phosphatidylinositol-5-phosphate-4-kinase-gamma (Pip4k2c) is a known mTORC1 regulator. It is shown that Pip4k2c is significantly downregulated in the hearts of CH and HF patients as compared to non-injured hearts. The role of Pip4k2c in the heart during development and disease is unknown. It is shown that deleting Pip4k2c does not affect normal embryonic cardiac development; however, three weeks after TAC, adult Pip4k2c mice has higher rates of CH, CF, and sudden death than wild-type mice. In a gain-of-function study using a TAC mouse model, Pip4k2c is transiently upregulated using a modified mRNA (modRNA) gene delivery platform, which significantly improve heart function, reverse CH and CF, and lead to increased survival. Mechanistically, it is shown that Pip4k2c inhibits TGF1 via its N-terminal motif, Pip5k1, phospho-AKT 1/2/3, and phospho-Smad3. In sum, loss-and-gain-of-function studies in a TAC mouse model are used to identify Pip4k2c as a potential therapeutic target for CF, CH, and HF, for which modRNA is a highly translatable gene therapy approach.
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http://dx.doi.org/10.1002/advs.202004661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132051PMC
May 2021

Specific Modified mRNA Translation System.

Circulation 2020 Dec 21;142(25):2485-2488. Epub 2020 Dec 21.

Cardiovascular Research Center (A.M., A.A.K., E.C., Y.S., L.Z.), Icahn School of Medicine at Mount Sinai, New York.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.047211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768930PMC
December 2020

In Vitro Synthesis of Modified RNA for Cardiac Gene Therapy.

Methods Mol Biol 2021 ;2158:281-294

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Modified mRNA (modRNA) is a promising new gene therapy approach that has safely and effectively delivered genes into different tissues, including the heart. Current efforts to use DNA-based or viral gene therapy to induce cardiac regeneration postmyocardial infarction (MI) or in heart failure (HF) have encountered key challenges, e.g., genome integration and delayed and noncontrolled expression. By contrast, modRNA is a transient, safe, non-immunogenic, and controlled gene delivery method that is not integrated into the genome. For most therapeutic applications, especially in regenerative medicine, the ability to deliver genes to the heart transiently and with control is vital for achieving therapeutic effect. Additionally, modRNA synthesis is comparatively simple and inexpensive compared to other gene delivery methods (e.g., protein), though a simple, clear in vitro transcription (IVT) protocol for synthesizing modRNA is needed for it to be more widely used. Here, we describe a simple and improved step-by-step IVT protocol to synthesize modRNA for in vitro or in vivo applications.
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http://dx.doi.org/10.1007/978-1-0716-0668-1_21DOI Listing
March 2021

Lung-derived HMGB1 is detrimental for vascular remodeling of metabolically imbalanced arterial macrophages.

Nat Commun 2020 08 27;11(1):4311. Epub 2020 Aug 27.

Division of Vascular Surgery, Department of Surgery, New York University Langone Health, New York, NY, USA.

Pulmonary disease increases the risk of developing abdominal aortic aneurysms (AAA). However, the mechanism underlying the pathological dialogue between the lungs and aorta is undefined. Here, we find that inflicting acute lung injury (ALI) to mice doubles their incidence of AAA and accelerates macrophage-driven proteolytic damage of the aortic wall. ALI-induced HMGB1 leaks and is captured by arterial macrophages thereby altering their mitochondrial metabolism through RIPK3. RIPK3 promotes mitochondrial fission leading to elevated oxidative stress via DRP1. This triggers MMP12 to lyse arterial matrix, thereby stimulating AAA. Administration of recombinant HMGB1 to WT, but not Ripk3 mice, recapitulates ALI-induced proteolytic collapse of arterial architecture. Deletion of RIPK3 in myeloid cells, DRP1 or MMP12 suppression in ALI-inflicted mice repress arterial stress and brake MMP12 release by transmural macrophages thereby maintaining a strengthened arterial framework refractory to AAA. Our results establish an inter-organ circuitry that alerts arterial macrophages to regulate vascular remodeling.
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http://dx.doi.org/10.1038/s41467-020-18088-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453029PMC
August 2020

Modified mRNA as a Therapeutic Tool for the Heart.

Cardiovasc Drugs Ther 2020 12 21;34(6):871-880. Epub 2020 Aug 21.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Despite various clinical modalities available for patients, heart disease remains among the leading causes of mortality and morbidity worldwide. Genetic medicine, particularly mRNA, has broad potential as a therapeutic. More specifically, mRNA-based protein delivery has been used in the fields of cancer and vaccination, but recent changes to the structural composition of mRNA have led the scientific community to swiftly embrace it as a new drug to deliver missing genes to injured myocardium and many other organs. Modified mRNA (modRNA)-based gene delivery features transient but potent protein translation and low immunogenicity, with minimal risk of insertional mutagenesis. In this review, we compared and listed the advantages of modRNA over traditional vectors for cardiac therapy, with particular focus on using modRNA therapy in cardiac repair. We present a comprehensive overview of modRNA's role in cardiomyocyte (CM) proliferation, cardiac vascularization, and prevention of cardiac apoptosis. We also emphasize recent advances in modRNA delivery strategies and discuss the challenges for its clinical translation.
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http://dx.doi.org/10.1007/s10557-020-07051-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7441140PMC
December 2020

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model.

J Vis Exp 2020 06 11(160). Epub 2020 Jun 11.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai;

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 treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.
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http://dx.doi.org/10.3791/60832DOI Listing
June 2020

Probing myeloid cell dynamics in ischaemic heart disease by nanotracer hot-spot imaging.

Nat Nanotechnol 2020 05 20;15(5):398-405. Epub 2020 Apr 20.

BioMedical Engineering and Imaging Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

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.
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http://dx.doi.org/10.1038/s41565-020-0642-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416336PMC
May 2020

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

Mol Ther Methods Clin Dev 2020 Jun 31;17:622-633. Epub 2020 Mar 31.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

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.
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http://dx.doi.org/10.1016/j.omtm.2020.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150433PMC
June 2020

Pkm2 Regulates Cardiomyocyte Cell Cycle and Promotes Cardiac Regeneration.

Circulation 2020 04 11;141(15):1249-1265. Epub 2020 Feb 11.

Cardiovascular Research Center (A.M, N.S., A.A.K., I.M., T.M. K.B., M.T.K.S., E.C., Y.S., J.G.O., P.L, A.G.-S., C.K., M.M., L.Z.), Icahn School of Medicine at Mount Sinai, New York.

Background: 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.

Methods: We investigated the effect of Pkm2 in cardiomyocytes through models of loss (cardiomyocyte-specific Pkm2 deletion during cardiac development) or gain using cardiomyocyte-specific Pkm2 modified mRNA to evaluate Pkm2 function and regenerative affects after acute or chronic myocardial infarction in mice.

Results: Here, we identify Pkm2 as an important regulator of the cardiomyocyte cell cycle. We show that Pkm2 is expressed in cardiomyocytes during development and immediately after birth but not during adulthood. Loss of function studies show that cardiomyocyte-specific Pkm2 deletion during cardiac development resulted in significantly reduced cardiomyocyte cell cycle, cardiomyocyte numbers, and myocardial size. In addition, using cardiomyocyte-specific Pkm2 modified RNA, our novel cardiomyocyte-targeted strategy, after acute or chronic myocardial infarction, resulted in increased cardiomyocyte cell division, enhanced cardiac function, and improved long-term survival. We mechanistically show that Pkm2 regulates the cardiomyocyte cell cycle and reduces oxidative stress damage through anabolic pathways and β-catenin.

Conclusions: We demonstrate that Pkm2 is an important intrinsic regulator of the cardiomyocyte cell cycle and oxidative stress, and highlight its therapeutic potential using cardiomyocyte-specific Pkm2 modified RNA as a gene delivery platform.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241614PMC
April 2020

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

Circulation 2020 03 29;141(11):916-930. Epub 2020 Jan 29.

Cardiovascular Research Center (Y.H., E.Y., M.M.Ż., E.C., N.S., M.T.K.S., R.K., A.A.K., K.K., A.M., N.H., L.Z., A.F, M.G.K.), Icahn School of Medicine at Mount Sinai, New York.

Background: 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.

Methods: We performed transcriptomic, sphingolipid, and protein analyses to evaluate sphingolipid metabolism and signaling post-MI. We investigated the effect of altering ceramide metabolism through a loss (chemical inhibitors) or gain (modified mRNA [modRNA]) of AC function post hypoxia or MI.

Results: We found that several genes involved in de novo ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1, and C24) levels had significantly increased 24 hours after MI. AC inhibition after hypoxia or MI resulted in reduced AC activity and increased cell death. By contrast, enhancing AC activity via AC modRNA treatment increased cell survival after hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival, and smaller scar size than control mice 28 days post-MI. We attributed the improvement in heart function post-MI after AC modRNA delivery to decreased ceramide levels, lower cell death rates, and changes in the composition of the immune cell population in the left ventricle manifested by lowered abundance of proinflammatory detrimental neutrophils.

Conclusions: Our findings suggest that transiently altering sphingolipid metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post-MI, thereby highlighting the therapeutic potential of AC modRNA in ischemic heart disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.041882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135928PMC
March 2020

Optimizing Modified mRNA Synthesis Protocol for Heart Gene Therapy.

Mol Ther Methods Clin Dev 2019 Sep 30;14:300-305. Epub 2019 Jul 30.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

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.
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http://dx.doi.org/10.1016/j.omtm.2019.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722299PMC
September 2019

mRNA-Based Protein Replacement Therapy for the Heart.

Mol Ther 2019 04 6;27(4):785-793. Epub 2018 Dec 6.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ymthe.2018.11.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453506PMC
April 2019

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

Circulation 2018 12;138(25):2919-2930

Riley Heart Research Center and Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis (L. Zhang, F.Y., C.-L.C.).

Background: 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.

Methods: Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1 cells.

Results: With these novel genetic tools, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1 cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury.

Conclusions: Our study provides definitive insights into the nature of cardiac resident Sca-1 cells. The observations challenge the current dogma that cardiac resident Sca-1 cells are intrinsic stem cells for myocardial development, renewal, and repair, and suggest that the mechanisms of transplanted Sca-1 cells in heart repair need to be reassessed.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.035200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366943PMC
December 2018

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

Mol Ther Nucleic Acids 2018 Dec 1;13:133-143. Epub 2018 Sep 1.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.omtn.2018.08.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171324PMC
December 2018

[GENE THERAPY POTENTIAL AS A TREATMENT FOR HEART FAILURE].

Harefuah 2018 Feb;157(2):112-116

Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY, USA.

Introduction: Advances in understanding the molecular biology of heart failure, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed heart failure within the reach of gene-based therapy. During the last decade the concept of delivering cDNA encoding a therapeutic gene to failing cardiomyocytes has moved from hypothesis to the bench of preclinical applications and clinical trials. However, despite significant promise, several obstacles exist, which are described in this review. We anticipate that advances in the field will improve gene therapy in heart failure in future clinical approaches.
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February 2018

Synthetic MicroRNAs Stimulate Cardiac Repair.

Circ Res 2017 04;120(8):1222-1223

From the Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York.

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http://dx.doi.org/10.1161/CIRCRESAHA.117.310863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834342PMC
April 2017

Optimizing Cardiac Delivery of Modified mRNA.

Mol Ther 2017 06 4;25(6):1306-1315. Epub 2017 Apr 4.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ymthe.2017.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474881PMC
June 2017

Modified mRNA as a therapeutic tool to induce cardiac regeneration in ischemic heart disease.

Wiley Interdiscip Rev Syst Biol Med 2017 01 2;9(1). Epub 2016 Dec 2.

Cardiovascular Research Center, Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Ischemic heart disease (IHD) is a leading cause of morbidity and mortality in developed countries. Current pharmacological and interventional therapies provide significant improvement in the life quality of patient; however, they are mostly symptom-oriented and not curative. A high disease and economic burden of IHD requires the search for new therapeutic strategies to significantly improve patients' prognosis and quality of life. One of the main challenges during IHD is the massive loss of cardiomyocytes that possess minimal regenerative capacity. Recent understanding of the pathophysiological mechanisms underlying IHD, as well as new therapeutic approaches provide new hope for patients suffering from IHD. Synthetic modified mRNA (modRNA) is a new gene delivery vector that is increasingly used in in vivo applications. modRNA is a relatively stable, non-immunogenic, highly-expressed molecule that has been shown to mediate high and transient expression of proteins in different type of cells and tissues including cardiomyocytes. modRNA properties, together with its expression kinetics in the heart make it an attractive option for the treatment of IHD, especially after myocardial infarction. In this review we discuss the role of gene therapy in cardiac regeneration as an approach to treat IHD; traditional and innovative gene delivery methods; and focus specifically on modRNA structure, mode of delivery, and its use for the induction of endogenous regenerative capacity, mainly in the context of IHD. WIREs Syst Biol Med 2017, 9:e1367. doi: 10.1002/wsbm.1367 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wsbm.1367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880206PMC
January 2017

Synthesis of Modified mRNA for Myocardial Delivery.

Methods Mol Biol 2017 ;1521:127-138

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Cardiac gene therapy shows tremendous promise in combating the growing problem of heart disease. Modified mRNA (modRNA) is a novel gene delivery system used in vitro or in vivo to achieve transient expression of therapeutic proteins in a heterogeneous population of cells. Incorporation of specific modified nucleosides enables modRNA to be translated efficiently without triggering antiviral and innate immune responses. ModRNA has been shown to be effective at delivering short-term robust gene expression to the heart and its use in the field of cardiac gene therapy is expanding. Here, we describe a stepwise protocol for the synthesis of modRNA for in vivo myocardial delivery.
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http://dx.doi.org/10.1007/978-1-4939-6588-5_8DOI Listing
January 2018

Insulin-Like Growth Factor 1 Receptor-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury.

Circulation 2017 Jan 1;135(1):59-72. Epub 2016 Nov 1.

From Cardiovascular Research Center, Department of Genetics and Genomic Sciences, and Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York (L.Z., N.S., Y.H.); Department of Cardiology, Boston Children's Hospital, MA (L.Z., M.S.O., L.Y.Y., Q.M., W.T.P.); Cardiovascular and Metabolic Diseases Innovative Medicine Biotech Unit, AstraZeneca, Möllndal, Sweden (D.S., Q.-D.W.); The State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (B.Z.); Department of Genetics (W.L.C.), Harvard Stem Cell Institute (W.E., W.T.P.), Harvard Medical School, and Cardiovascular Research Center, Massachusetts General Hospital (M.A.), Harvard Medical School, Boston, MA; and Department of Cell and Molecular Biology and Medicine, Karolinska Institutet, Stockholm, Sweden (K.R.C.).

Background: Epicardial adipose tissue 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 epicardial adipose tissue's formation and expansion are unclear.

Methods: We used a novel modified mRNA-based screening approach to probe the effect of individual paracrine factors on epicardial progenitors in the adult heart.

Results: Using 2 independent lineage-tracing strategies in murine models, we show that cells originating from the Wt1 mesothelial lineage, which includes epicardial cells, differentiate into epicardial adipose tissue after myocardial infarction. This differentiation process required Wt1 expression in this lineage and was stimulated by insulin-like growth factor 1 receptor (IGF1R) activation. IGF1R inhibition within this lineage significantly reduced its adipogenic differentiation in the context of exogenous, IGF1-modified mRNA stimulation. Moreover, IGF1R inhibition significantly reduced Wt1 lineage cell differentiation into adipocytes after myocardial infarction.

Conclusions: Our results establish IGF1R signaling as a key pathway that governs epicardial adipose tissue formation in the context of myocardial injury by redirecting the fate of Wt1 lineage cells. Our study also demonstrates the power of modified mRNA -based paracrine factor library screening to dissect signaling pathways that govern progenitor cell activity in homeostasis and disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.022064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5195872PMC
January 2017

How to make a cardiomyocyte.

Development 2014 Dec 18;141(23):4418-31. Epub 2014 Nov 18.

Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, 7 Divinity Avenue, Cambridge, MA 02138, USA Department of Cell and Molecular Biology and Medicine, Karolinska Institutet, 35 Berzelius Vag, Stockholm 171 77, Sweden

During development, cardiogenesis is orchestrated by a family of heart progenitors that build distinct regions of the heart. Each region contains diverse cell types that assemble to form the complex structures of the individual cardiac compartments. Cardiomyocytes are the main cell type found in the heart and ensure contraction of the chambers and efficient blood flow throughout the body. Injury to the cardiac muscle often leads to heart failure due to the loss of a large number of cardiomyocytes and its limited intrinsic capacity to regenerate the damaged tissue, making it one of the leading causes of morbidity and mortality worldwide. In this Primer we discuss how insights into the molecular and cellular framework underlying cardiac development can be used to guide the in vitro specification of cardiomyocytes, whether by directed differentiation of pluripotent stem cells or via direct lineage conversion. Additional strategies to generate cardiomyocytes in situ, such as reactivation of endogenous cardiac progenitors and induction of cardiomyocyte proliferation, will also be discussed.
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http://dx.doi.org/10.1242/dev.091538DOI Listing
December 2014

Synthetic chemically modified mRNA (modRNA): toward a new technology platform for cardiovascular biology and medicine.

Cold Spring Harb Perspect Med 2014 Oct 9;5(1):a014035. Epub 2014 Oct 9.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138.

Over the past two decades, a host of new molecular pathways have been uncovered that guide mammalian heart development and disease. The ability to genetically manipulate these pathways in vivo have largely been dependent on the generation of genetically engineered mouse model systems or the transfer of exogenous genes in a variety of DNA vectors (plasmid, adenoviral, adeno-associated viruses, antisense oligonucleotides, etc.). Recently, a new approach to manipulate the gene program of the adult mammalian heart has been reported that will quickly allow the high-efficiency expression of virtually any protein in the intact heart of mouse, rat, porcine, nonhuman primate, and human heart cells via the generation of chemically modified mRNA (modRNA). The technology platform has important implications for delineating the specific paracrine cues that drive human cardiogenesis, and the pathways that might trigger heart regeneration via the rapid generation of modRNA libraries of paracrine factors for direct in vivo administration. In addition, the strategy can be extended to a variety of other cardiovascular tissues and solid organs across multiple species, and recent improvements in the core technology have supported moving toward the first human studies of modRNA in the next 2 years. These recent advances are reviewed along with projections of the potential impact of the technology for a host of other biomedical problems in the cardiovascular system.
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http://dx.doi.org/10.1101/cshperspect.a014035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4292072PMC
October 2014

Cardiovascular regenerative therapeutics via synthetic paracrine factor modified mRNA.

Stem Cell Res 2014 Nov 8;13(3 Pt B):693-704. Epub 2014 Jul 8.

Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, USA; Department of Cell and Molecular Biology and Medicine, Karolinska Institute, Stockholm, Sweden. Electronic address:

The heart has a limited capacity for regeneration following injury. Recent strategies to promote heart regeneration have largely focused on autologous and allogeneic cell-based therapy, where the transplanted cells have been suggested to secrete unknown paracrine factors that are envisioned to promote endogenous repair and/or mobilization of endogenous heart progenitors. Here, we discuss the importance of paracrine mechanisms in facilitating replication of endogenous epicardial progenitor cells in the adult heart and signaling their subsequent reactivation and de novo differentiation into functional cell types such as endothelial cells and cardiomyocytes. Moreover, we discuss the use of a novel modified RNA technology in delivering such therapeutic paracrine factors into myocardium following injury. These studies suggest that modified mRNA may be a valuable experimental tool for the precise in vivo identification of paracrine factors and their downstream signaling that may promote heart repair, cardiac muscle replication, and/or heart progenitor mobilization. In addition, these studies lay the foundation for a new clinically tractable technology for a cell-free approach to promote heart regeneration.
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http://dx.doi.org/10.1016/j.scr.2014.06.007DOI Listing
November 2014

Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies.

Nat Med 2014 Jun 11;20(6):616-23. Epub 2014 May 11.

1] Department of Cardiology, Boston Children's Hospital, Boston, Massachusetts, USA. [2] Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.

Study of monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combined patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene encoding tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS 'heart-on-chip' tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole-cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies and advances iPSC-based in vitro modeling of cardiomyopathy.
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http://dx.doi.org/10.1038/nm.3545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172922PMC
June 2014

Driving vascular endothelial cell fate of human multipotent Isl1+ heart progenitors with VEGF modified mRNA.

Cell Res 2013 Oct 10;23(10):1172-86. Epub 2013 Sep 10.

1] Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA 02114, USA [2] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 01238, USA [3] Stem Cell and Regenerative Medicine Consortium, LKS Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong SAR, China.

Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.
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http://dx.doi.org/10.1038/cr.2013.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790234PMC
October 2013

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.

Nat Biotechnol 2013 Oct 8;31(10):898-907. Epub 2013 Sep 8.

1] Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Cardiovascular Research Center, Massachusetts General Hospital, Boston, Massachusetts, USA. [3] Department of Cardiology, Children's Hospital Boston, Boston, Massachusetts, USA. [4] Immune Disease Institute and Program in Cellular and Molecular Medicine, Children's Hospital Boston, Boston, Massachusetts, USA. [5] Boston and Harvard Stem Cell Institute, Cambridge, Massachusetts, USA. [6].

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) results in the expansion and directed differentiation of endogenous heart progenitors in a mouse myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors and temporal control with VEGF inhibitors revealed the greatly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long-term organ repair.
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http://dx.doi.org/10.1038/nbt.2682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4058317PMC
October 2013

A HCN4+ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells.

Nat Cell Biol 2013 Sep 25;15(9):1098-106. Epub 2013 Aug 25.

1] Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA [2] Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA.

Most of the mammalian heart is formed from mesodermal progenitors in the first and second heart fields (FHF and SHF), whereby the FHF gives rise to the left ventricle and parts of the atria and the SHF to the right ventricle, outflow tract and parts of the atria. Whereas SHF progenitors have been characterized in detail, using specific molecular markers, comprehensive studies on the FHF have been hampered by the lack of exclusive markers. Here, we present Hcn4 (hyperpolarization-activated cyclic nucleotide-gated channel 4) as an FHF marker. Lineage-traced Hcn4+/FHF cells delineate FHF-derived structures in the heart and primarily contribute to cardiomyogenic cell lineages, thereby identifying an early cardiomyogenic progenitor pool. As a surface marker, HCN4 also allowed the isolation of cardiomyogenic Hcn4+/FHF progenitors from human embryonic stem cells. We conclude that a primary purpose of the FHF is to generate cardiac muscle and support the contractile activity of the primitive heart tube, whereas SHF-derived progenitors contribute to heart cell lineage diversification.
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http://dx.doi.org/10.1038/ncb2824DOI Listing
September 2013

Deletion of cognate CD8 T cells by immature dendritic cells: a novel role for perforin, granzyme A, TREM-1, and TLR7.

Blood 2012 Aug 9;120(8):1647-57. Epub 2012 Jul 9.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Immature dendritic cells (imDCs) can have a tolerizing effect under normal conditions or after transplantation. However, because of the significant heterogeneity of this cell population, it is extremely difficult to study the mechanisms that mediate the tolerance induced or to harness the application of imDCs for clinical use. In the present study, we describe the generation of a highly defined population of imDCs from hematopoietic progenitors and the direct visualization of the fate of TCR-transgenic alloreactive CD4(+) and CD8(+) T cells after encountering cognate or noncognate imDCs. Whereas CD4(+) T cells were deleted via an MHC-independent mechanism through the NO system, CD8(+) T-cell deletion was found to occur through a unique MHC-dependent, perforin-based killing mechanism involving activation of TLR7 and signaling through Triggering Receptor-1 Expressed on Myeloid cells (TREM-1). This novel subpopulation of perforin-expressing imDCs was also detected in various lymphoid tissues in normal animals and its frequency was markedly enhanced after GM-CSF administration.
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http://dx.doi.org/10.1182/blood-2012-02-410803DOI Listing
August 2012

Embryonic pig pancreatic tissue for the treatment of diabetes: potential role of immune suppression with "off-the-shelf" third-party regulatory T cells.

Transplantation 2011 Feb;91(4):398-405

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Background: Xenogeneic embryonic pancreatic tissue can provide an attractive alternative for organ replacement therapy. However, immunological rejection represents a major obstacle. This study examines the potential of regulatory T cells (Tregs) in the prevention of E42 pancreas rejection.

Methods: To develop new approaches to combat rejection, we evaluated engraftment, growth, and development of E42 pig pancreatic tissue in mice treated with ex vivo expanded Tregs in combination with T-cell debulking and the conventional immunosuppressive drugs, rapamycin and FTY720.

Results: Transplantation of E42 pig pancreas into C57BL/6 mice immunosuppressed by this protocol resulted in complete rejection within less than 6 weeks. In contrast, additional treatment with a single infusion of ex vivo expanded third-party Tregs markedly delayed the onset of graft rejection to 10 weeks. The infusion of Tregs was associated with a significant reduction in CD4 and CD8 expansion in the lymph nodes and other peripheral organs at the priming stages after implantation. Freezing and thawing of the Tregs did not affect their efficacy, indicating the potential of Tregs banking.

Conclusion: Considering the technical difficulties encountered in the generation of Tregs from patients or from specific donors, our results demonstrate the feasibility of using "off-the-shelf" fresh or frozen third-party Tregs to control rejection in organ transplantation.
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http://dx.doi.org/10.1097/TP.0b013e318204be15DOI Listing
February 2011
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