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

摘要 在患者特异性体细胞重编程后产生人多能干细胞和随后的细胞分化。 干细胞分化为更特化的细胞类型为人类的发展提供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细胞类型，包括神经细胞， 使用不同组的生物活性因子成功地证明了小鼠和人细胞的心肌细胞。 然而，在人类细胞和成人成纤维细胞中的重编程仍然效率低下，需要进一步的努力。 虽然直接重编程到小鼠和人类心肌细胞的精确分子机制在很大程度上是未知的， 未知的是，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，导致这些心脏特异性转录因子的组成型表达。一个主要 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 表达模式或触发导致癌症和/或其他有害后果的异常癌基因表达。 我们在Stemgenics的合作者开发了一种创新的专利保护技术， 功能化纳米颗粒将成熟细胞重编程为自体多能干细胞， 重编程效率和完整的基因组。Stemgenics重新编程纳米技术的重要方面 在重编程系统中缺乏任何基因组整合元件，因此所得细胞保留了它们的 天然完整基因组。该技术与直接重编程人成纤维细胞以使其功能 心肌细胞为患者产生个性化功能心肌细胞提供了独特的机会 患有心肌病或心力衰竭疾病。在SBIR第一阶段提案中，我们将1）生成非集成 用共价连接的心脏特异性重编程因子功能化的生物相容性纳米颗粒，2）优化 直接成纤维细胞到心肌细胞重编程效率，和3）表征所得物的功能性质 人心肌细胞我们的建议的结果将允许进一步，更全面的第二阶段研究， 通过心脏动物模型中纤维化瘢痕的直接重编程评价体内直接重编程iCM 损伤，并将提供宝贵的新工具，直接对患者的不同治疗药物的疗效评价- 具有完整基因组的特异性成纤维细胞衍生的心肌细胞。

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.