Systemically Injected Exosomes Targeted to EGFR Deliver Antitumor MicroRNA to Breast Cancer Cells

The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported previously that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here we use genetic lineage tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became binucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. Heart failure affects over 14 million people worldwide and is a leading cause of death in adults and in children. Because postnatal cardiomyocytes (CMs) have little or no regenerative capacity, therapies are limited at present. The introduction of exogenous stem-cell-derived CMs holds promise, but also challenges, including delivery, integration, rejection and cellular maturation 1–3 . Reprogramming adult fibroblasts into induced pluripotent stem cells (iPSCs) that are similar to embryonic stem cells addresses some issues 4–6 , but others, including efficient directed differentiation into CMs and effective delivery, remain. A new generation of reprogramming technology involves transdifferentiating one adult somatic cell type directly into another 7–11 . We reported direct reprogramming of fibroblasts into CM-like cells in vitro by expressing three transcription factors: Gata4, Mef2c and Tbx5 (GMT) 7 . As observed in reprogramming to iPSCs, the percentage of fibroblast cells fully reprogrammed to beating CMs in vitro was small, but far more were partially reprogrammed, much like preiPSCs that can become fully pluripotent with additional stimuli 12 . We posited that cardiac fibroblasts may reprogram more fully in vivo in their native environment, which might promote survival, maturation, and coupling with neighbouring cells. If so, the vast pool of cardiac fibroblasts in the heart could serve as an endogenous source of new CMs for regenerative therapy.

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In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

Abstract 10466: In Vivo Reprogramming of Murine Cardiac Fibroblasts into Induced Cardiomyocytes

Paracrine mechanisms of stem cell reparative and regenerative actions in the heart.

BACKGROUND: MicroRNAs (miRs) are small noncoding RNAs that posttranscriptionally control gene expression. Small-animal studies suggest that miRs might offer novel therapeutic targets in cardiovascular diseases such as cardioprotection of murine hearts after myocardial infarction via miR-92a inhibitors. Because the functional benefits of miR-92a inhibitors in larger preclinical models are not known, we assessed the therapeutic efficacy of miR-92a inhibition in a porcine model of ischemia and reperfusion. METHODS AND RESULTS: Pigs (n=5 per group) underwent percutaneous ischemia/reperfusion (60 min/72 h or 7 days, respectively). Locked nucleic acid-modified antisense miR-92a (LNA-92a) was applied either regionally (antegrade or retrograde) with a catheter or systemically (intravenously). LNA-92a significantly (P<0.01) reduced miR-92a expression in the infarct zone regardless of the application venue. However, catheter-based delivery, but not intravenous infusion, of LNA-92a significantly (P<0.05) reduced the infarct size compared with control LNA-treated pigs, which correlated with an improved ejection fraction and left ventricular end-diastolic pressure (P<0.05). Histochemistry revealed that LNA-92a increased capillary density but decreased leukocyte influx and cardiac cell death. Complete loss of miR-92a in mice attenuated the infarct-related myocardial dysfunction to a larger extent than cardiomyocyte-specific miR-92a deletion. In vitro, LNA-92a protected against hypoxia/reoxygenation-induced cardiomyocyte cell death. CONCLUSIONS: Regional LNA-92a delivery reduces miR-92a levels and infarct size and postischemic loss of function. LNA-92a exerts cell-protective, proangiogenic, and anti-inflammatory effects. miR-92a inhibition might be a novel therapeutic tool to preserve cardiac function after ischemia.

Inhibition of MicroRNA-92a Protects Against Ischemia/Reperfusion Injury in a Large-Animal Model

Background— Prolonged myocardial ischemia results in cardiomyocyte loss despite successful revascularization. We have reported that retrograde application of embryonic endothelial progenitor cells (eEPCs) provides rapid paracrine protection against ischemia-reperfusion injury. Here, we investigated the role of thymosin β4 (Tβ4) as a mediator of eEPC-mediated cardioprotection. Methods and Results— In vitro, neonatal rat cardiomyocytes were subjected to hypoxia-reoxygenation in the absence or presence of eEPCs with or without Tβ4 short hairpin RNA (shRNA) transfection. In vivo, pigs (n=9 per group) underwent percutaneous left anterior descending artery occlusion for 60 minutes on day 1. After 55 minutes of ischemia, control eEPCs (5×106 cells) or cells transfected with Tβ4 shRNA when indicated or 15 mg Tβ4 alone were retroinfused into the anterior interventricular vein. Segmental endocardial shortening in the infarct zone at 150-bpm atrial pacing, infarct size (triphenyl tetrazolium chloride viability and m...

Thymosin β4 Is an Essential Paracrine Factor of Embryonic Endothelial Progenitor Cell–Mediated Cardioprotection

The epidermal growth factor receptor (EGFR) is upregulated within a high percentage of solid tumors and hence is an attractive target for tumor-targeted therapies including gene therapy. The natural EGFR ligand epidermal growth factor (EGF) has been used for this purpose, despite the risk of mitogenic effects due to EGFR activation. We have developed a fully synthetic, EGFR-targeted gene delivery system based on PEGylated linear polyethylenimine (LPEI), allowing evaluation of different EGFR-binding peptides in terms of transfection efficiency and EGFR activation. Peptide sequences directly derived from the human EGF molecule enhanced transfection efficiency with concomitant EGFR activation. Only the EGFR-binding peptide GE11, which has been identified by phage display technique, showed specific enhancement of transfection on EGFR-overexpressing tumor cells including glioblastoma and hepatoma, but without EGFR activation. EGFR targeting led to high levels of cell association of fluorescently label...

Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

The invention relates to genetically modified mesenchymal stem cells (MSCs) comprising one or more exogenous nucleic acid molecules, wherein said exogenous nucleic acid molecules comprise a therapeutic protein-encoding region operably linked to a hypoxia-induced promoter. The invention further relates to their use in the treatment of medical conditions associated with hypoxia or hypoxic tissue, such as tumour diseases, lung diseases, gastrointestinal diseases, ischemic diseases and infectious diseases.

Christine Spitzweg

Papers

Thymosin β4 Is an Essential Paracrine Factor of Embryonic Endothelial Progenitor Cell–Mediated Cardioprotection

Disconnecting the Yin and Yang Relation of Epidermal Growth Factor Receptor (EGFR)-Mediated Delivery: A Fully Synthetic, EGFR-Targeted Gene Transfer System Avoiding Receptor Activation

Hypoxia-induced expression of therapeutic proteins in mesenchymal stem cells

Rhenium-188 as an alternative to Iodine-131 for treatment of prostate cancer following tissue-specific sodium iodide symporter gene transfer

Systemic sodium iodide symporter (NIS) gene transfer in hepatocellular carcinoma using EGF-receptor targeted non-viral gene delivery vectors