Direct Reprogramming of Human Fibroblasts into Functional Cardiomyocytes by Non-Integrating Cardiogenic Nanoparticles

摘要 人多能干细胞在患者特异性体细胞重新编程后的产生 干细胞分化为更特化的细胞类型为人类打开了新的令人兴奋的机会 再生医学。将成纤维细胞直接重编程为各种类型的细胞，包括神经细胞和 使用不同生物活性因子的心肌细胞被成功地应用于小鼠和人类细胞。 然而，在人类细胞和成人成纤维细胞中重新编程仍然效率低下，还需要进一步的努力。 而对小鼠和人心肌细胞直接重编程的确切分子机制在很大程度上是 未知，这些研究通常使用携带不同心脏重编程因子的病毒载体 进入宿主细胞基因组，导致这些心脏特异转录因子的结构性表达。一位少校 这种方法的缺点是病毒DNA随机整合到细胞基因组中，并可能改变正常基因 表达模式或触发癌基因异常表达导致癌症和/或其他有害后果。 我们在Stemgenics的合作者开发了一种创新的专利保护技术，使用非集成 功能化纳米颗粒以~15%的比例将成熟细胞重新编程为自体多能干细胞 重新编程的效率和完整的基因组。Stemgenics对纳米技术重新编程的重要方面 在重新编程系统中没有任何基因组整合元件，因此产生的细胞保留了它们的 天然完整的基因组。这项技术与人成纤维细胞的直接重编程相结合，使其具有 心肌细胞为患者提供了生成个性化功能心肌细胞的独特机会 患有心肌病或心力衰竭疾病。在此SBIR第一阶段提案中，我们将1)生成非集成 生物相容纳米颗粒与共价连接的心脏特异性重编程因子功能化，2)优化 直接成纤维细胞到心肌细胞的重编程效率，以及3)所得到的产物的功能特性 人类心肌细胞。我们建议的结果将使我们能够进一步进行更全面的第二阶段研究 直接重编程修复纤维化瘢痕对体内直接重编程ICM的评价 并将为直接对患者进行不同治疗药物的疗效评估提供宝贵的新工具- 具有完整基因组的特定成纤维细胞来源的心肌细胞。

Abstract Generation of human pluripotent stem cells upon reprogramming of patient-specific somatic cells and subsequent differentiation of the stem cells into more specialized cell types opened new exciting opportunities for human regenerative medicine. Direct reprogramming of fibroblasts to various cell types including neural cells and cardiomyocytes using different sets of bioactive factors was successfully demonstrated for mouse and human cells. However, reprogramming in human cells and adult fibroblasts remains inefficient, and further efforts are needed. While the precise molecular mechanisms of direct reprogramming to mouse and human cardiomyocytes are largely unknown, these studies commonly use viral vectors harboring different cardiac reprogramming factors that integrate into the host cell genome leading to constitutive expression of these cardiac-specific transcription factors. A major drawback of this approach is that viral DNA randomly integrates into the cell genome and may alter the normal gene expression pattern or trigger abnormal oncogene expression leading to cancer and/or other detrimental consequences. Our collaborators at Stemgenics have developed an innovative patent-protected technology using non-integrating functionalized nanoparticles to reprogram mature cells into autologous pluripotent stem cells with ~15% reprogramming efficiency and intact genome. The important aspect of the Stemgenics reprogramming nanotechnology is the absence of any genome integrating elements in the reprogramming system, thus the resulting cells preserve their native intact genome. This technology combined with direct reprogramming of human fibroblasts to functional cardiomyocytes presents unique opportunities for generation of personalized functional cardiomyocytes for patients with cardiomyopathy or heart failure diseases. In this SBIR Phase I proposal, we will 1) generate non-integrating biocompatible nanoparticles functionalized with covalently linked cardiac-specific reprogramming factors, 2) optimize direct fibroblast-to-cardiomyocyte reprogramming efficiency, and 3) characterize functional properties of the resultant human cardiomyocytes. The outcome of our proposal will permit further, more comprehensive Phase II studies for evaluation of directly reprogrammed iCM in vivo by direct reprogramming of fibrotic scars in animal models of heart injury, and will provide invaluable novel tools for efficacy evaluation of different therapeutic drugs directly on patient- specific fibroblast-derived cardiomyocytes with intact genome.