A novel protein transduction method to generate induced pluripotent stem cells wi

描述(申请人提供)：成熟的成年细胞可以被重新编程来产生诱导多能干细胞(IPSCs)，就其自我更新和分化能力而言，就像胚胎干细胞一样，这一证明引发了人们对这项技术的极大兴趣。最初的重编程方法集中在使用可能产生突变和/或肿瘤的基因转导方法来传递四个基因(Oct4、Sox2和Klf4+c-Myc或NANOG+Lin 28)。最近，有研究表明，将4种蛋白质输送到成体细胞也可以产生IPSCs。然而，直接用蛋白质重编程有两个主要问题：IPSC生成效率非常低(0.001-0.006%)和极高的蛋白质重编程成本，使其在常规基础上使用不切实际。目前提案的总体目标是应用两项新技术，使IPSC的生成更加实用、简单和负担得起。第一种是QQ-蛋白质转导技术，这是一种简单而高效的方法，将纯化的重编程蛋白特异性地运送到细胞的细胞核中，以启动和维持持续的蛋白质重编程，以生成iPSC。第二种是能够生产克量蛋白质的细菌表达系统，与一种新的体内蛋白质复性技术相结合，可以以非常低的成本生产大量纯化的、功能重新编程的蛋白质。这些新技术的应用有望将产生IPSC的效率大幅提高1000倍。此外，它将使IPSC的创建变得简单得多，并将显著降低成本，因此该技术可以被比当前技术多得多的研究人员使用。这将对IPSC研究和再生医学产生革命性的影响。该领域所依赖的商业产品和服务将拥有一个快速增长的市场。该项目的商业利益将使Techtown Ventures为密歇根州东南部的经济复苏做出贡献。 公共卫生相关性：这项研究的结果将使不使用遗传物质创造诱导多潜能干细胞(IPSC)变得更简单、更安全和负担得起。这些进展将使大多数研究人员能够接触到这项技术，从而刺激创造新的细胞系，用于研究许多不同的疾病。它们还将使从患者的正常细胞中创造出多潜能干细胞成为可能，这些干细胞可以在临床上用于取代患病或受伤的细胞。

DESCRIPTION (provided by applicant): The demonstration that mature adult cells can be reprogrammed to create induced pluripotent stem cells (iPSCs), which are like embryonic stem cells with respect to their self-renewal and differentiation capacities, has sparked a great deal of interest in this technology. The initial methods for reprogramming focused on delivery of four genes (Oct4, Sox2, and either Klf4 plus c-Myc, or NANOG plus Lin 28) using genetic transduction methods that potentially can create mutations and/or tumors. More recently, it was demonstrated that delivery of 4 proteins to adult cells could also generate iPSCs. However, reprogramming directly with proteins suffers from two major problems: very low efficiency of iPSC generation (0.001-0,006%) and extremely high cost of reprogramming proteins, making it not practical for use on a routine basis. The overall objective of the current proposal is to apply 2 novel technologies to make generation of iPSCs more practical, simple and affordable. The first is the QQ-protein transduction technology, which is a simple and highly efficient method to specifically deliver purified reprogramming proteins into the nuclei of cells for initiating and maintaining constant protein reprogramming for iPSC generation. The second is a bacterial expression system capable of producing gram quantities of protein combined with is a novel in vivo protein refolding technique to produce large quantity of purified, functional reprogramming proteins at a very low cost. The application of these new technologies is expected to significantly enhance the efficiency of generating iPSC by 1000-fold. In addition, it will make creation of iPSCs much simpler and will significantly reduce the cost so the technology can be used by many more investigators than current technologies. This will have a revolutionary effect on iPSC research and on regenerative medicine. The commercial products and services upon which the field depends will have a rapidly growing market. The commercial benefits of this project will allow TechTown Ventures to contribute to the economic recovery of Southeast Michigan. PUBLIC HEALTH RELEVANCE: The results of this research will make creation of induced pluripotential stem cells (iPSC) without use of genetic material simpler, safer and affordable. These advances will put the technology in reach of most researchers and thereby stimulate the creation of new cell lines for studies on many different diseases. They will also make it possible to create pluripotential stem cells from a patient's normal cells that can be used clinically to replace the diseased or injured cells.