Directed Cell Fusion for the Treatment of Myocardial Infarction

描述(申请人提供)：每年心肌梗死的发病率接近100万，但目前还没有治疗方法来恢复发作后失去的肌肉功能。干细胞募集和/或移植是提高心脏再生潜力的潜在策略。然而，最近的经验告诉我们，干细胞在移植和采用心肌细胞的功能特征方面犹豫不决。这一建议是基于我们的发现，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，通过诱导供体干细胞和受体心肌细胞之间的融合，可以促进心肌梗死后的心肌修复。在此背景下，细胞融合将促进干细胞向功能性心肌细胞的植入和分化。我们建议利用Moloney小鼠白血病病毒(MuLV)的独特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。在其他产物中，MuLV编码包膜蛋白SU(表面分子，gp70)和TM(跨膜分子，p15E)。这些蛋白质形成一个复合体，它介导两个脂质体膜的结合，并在这样做的过程中，促进包含在膜中的细胞质成分的混合。在目的I中，我们建议评估gp70/p15E诱导间充质干细胞(MSCs)与心肌细胞(CM)融合的体外效果。这将通过从野生型小鼠中分离骨髓来源的MSCs和心肌细胞来完成。我们将用编码小鼠内源性逆转录病毒融合蛋白(MuLV gp70和MuLV p15E)的载体转染MSCs，条件是最大限度地提高融合频率，最大限度地减少同基因白细胞的免疫反应。对于疗效研究，我们将确定融合产品的生存能力以及它们维持肌节组织和肌肉功能基线水平的能力。在AIM II中，我们将用产生gp70/p15E的MSCs治疗心肌梗死区域。我们将建立小鼠急性心肌梗死模型，并检测一次输注gp70/p15E转化的同基因MSCs对心肌再生的影响。我们将监测注射的MSCs与常驻心肌细胞融合的程度，并通过活细胞跟踪、心室横断面细胞的表型和功能评估来确定细胞融合的有利和不利影响，确定心室心肌细胞外基质的组成，并评估左心室几何和功能随时间的变化。我们预计，MSC-CM融合将促进MSCs的植入和分化，进而促进受损心肌的再生。公共卫生相关性：每年有近100万人发生心肌梗死(即心脏病发作)，但目前还没有治疗方法来恢复这种事件后丧失的心脏功能。干细胞可以为恢复丧失的心脏功能的有效治疗提供基础。我们建议利用从健康组织中的干细胞功能中获得的见解和经病毒优化的工具来提高干细胞在心脏病发作后用于修复心脏的效用。

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.