Directed Cell Fusion for the Treatment of Myocardial Infarction

描述（由申请人提供）：心肌梗塞的年发病率接近一百万，但目前尚无治疗方法可以恢复发作后失去的肌肉功能。干细胞招募和/或移植是增强心脏再生潜力的潜在策略。然而，最近的经验表明，干细胞对于移植和采用心肌细胞的功能特征犹豫不决。该提议基于我们的发现，即移植的干细胞与健康心脏组织中的心肌细胞融合，并有助于异种嵌合体心脏的功能。我们假设梗塞后的心肌修复可以通过诱导供体干细胞和受体心肌细胞之间的融合来增强。在这种情况下，细胞融合将促进植入并驱动干细胞分化为功能性心肌细胞。我们建议利用莫洛尼鼠白血病病毒（MuLV）独特的融合能力来开发一种提高干细胞和心肌细胞之间融合频率的方法。 MuLV 编码的产物包括包膜 (env) 蛋白 SU（表面分子，gp70）和 TM（跨膜分子，p15E）。这些蛋白质形成复合物，介导两个脂质体膜的结合，从而促进膜中所含细胞质成分的混合。在目标 I 中，我们建议评估 gp70/p15E 诱导间充质干细胞 (MSC) 和心肌细胞 (CM) 之间融合的体外功效。这将通过从野生型小鼠中分离骨髓来源的间充质干细胞和心肌细胞来实现。我们将根据发现的条件，用编码鼠内源性逆转录病毒融合蛋白（MuLV gp70 和 MuLV p15E）的载体转染 MSC，以最大限度地提高融合频率并最大限度地减少同基因白细胞的免疫反应。对于功效研究，我们将确定融合产品的可行性及其维持肌节组织和肌肉功能基线水平的能力。在目标 II 中，我们将用产生 gp70/p15E 的 MSC 治疗心肌梗塞区域。我们将在小鼠中诱导急性心肌梗死，并测试单次输注 gp70/p15E 转化的同基因 MSC 对心肌再生的影响。我们将监测注射的间充质干细胞与驻留心肌细胞融合的程度，并通过活细胞追踪、心室横截面细胞的表型和功能评估来确定细胞融合的有益和不利影响，确定心室心肌细胞外基质的组成，并评估左心室几何形状和功能随时间的变化。我们期望MSC-CM融合将促进MSC的植入和MSC的分化，从而促进受损心肌的再生。公共卫生相关性：每年有近百万人发生心肌梗塞（即心脏病发作），但在此类事件发生后尚无治疗方法可以恢复丧失的心脏功能。干细胞可以为恢复失去的心脏功能的有效疗法提供基础。我们建议利用从健康组织中干细胞功能获得的见解和病毒优化的工具来提高干细胞在攻击后修复心脏的效用。

DESCRIPTION (provided by applicant): The annual incidence of myocardial infarction is nearly one million and yet no therapy currently exists to restore lost muscle function following an attack. Stem cell recruitment and/or transplantation are potential strategies for boosting the regenerative potential of the heart. However recent experience has taught that stem cells are hesitant to engraft and adopt the functional traits of cardiomyocytes. This proposal is based on our finding that transplanted stem cells fuse with cardiomyocytes in healthy cardiac tissue and contribute to the function of the heart of xenogeneic chimeras. We hypothesize that myocardial repair following infarction could be enhanced by induction of fusion between donor stem cells and recipient cardiomyocytes. Cell fusion in this context would promote engraftment and drive differentiation of stem cells to functional cardiomyocytes. We propose to co-opt the unique fusion capabilities of the Moloney murine leukemia virus (MuLV) to develop a method for increasing the frequency of fusion between stem cells and cardiomyocytes. MuLV encodes, among other products, the envelope (env) proteins SU (surface molecule, gp70) and TM (transmembrane molecule, p15E). These proteins form a complex which mediates the binding of two liposomal membranes and in so doing, promotes the intermingling of the cytoplasmic components contained in the membranes. In Aim I, we propose to evaluate the in vitro efficacy of gp70/p15E-induced fusion between mesenchymal stem cells (MSCs) and cardiomyocytes (CM). This will be accomplished by isolating bone marrow-derived MSCs and cardiomyocytes from wild type mice. We will transfect MSCs with a vector encoding murine endogenous retroviral fusion proteins (MuLV gp70 and MuLV p15E) based on conditions found to maximize the frequency of fusion and minimize immune responses of syngeneic leukocytes. For efficacy studies, we will determine the viability of fusion products and their ability to maintain sarcomeric organization and baseline levels of muscle function. In Aim II, we will treat myocardial infarct zones with gp70/p15E-producing MSCs. We will induce acute myocardial infarction in mice and test the effects of a single infusion of gp70/p15E-transformed syngeneic MSCs on myocardial regeneration. We will monitor the extent to which injected MSCs fused with resident cardiomyocytes and identify both the beneficial and adverse effects of cell fusion via live cell tracking, phenotypic and functional assessment of the cells of ventricular cross-sections, determine the composition of the extracellular matrix of the myocardium of the ventricle and assess time-dependent changes in left ventricular geometry and function. We expect that MSC-CM fusion will promote engraftment of MSCs and differentiation of MSCs which will in turn promote regeneration of the injured myocardium. PUBLIC HEALTH RELEVANCE: Myocardial infarction (i.e., heart attack) occurs in nearly one million individuals every year and yet there exists no therapy to recover lost heart function after such an event. Stem cells could provide a basis for effective therapies to recover lost heart function. We propose to utilize insights gained from stem cell function in healthy tissue and tools optimized by viruses to advance the utility of stem cells for repair of the heart following an attack.