Somatic cell reprogramming by protein transduction

描述（由申请人提供）：“定制”人类胚胎干细胞（huES）的治疗潜力得到广泛认可。然而，伦理和技术上的原因阻碍了常规使用体细胞核移植（SCNT）从患者身上获得基因匹配的胚胎干细胞。最近在确定参与维持hes细胞表型的关键分子方面取得的进展，导致了突破性的报道，这些报道描述了通过强制表达数量惊人的可管理的转录因子来重编程体细胞。虽然这些诱导多能干细胞（iPS）似乎在功能上与来自囊胚的胚胎干细胞相同，但在人类治疗的背景下，使用逆转录病毒载体递送关键基因仍然是不安全的。因此，除非设计出逆转录病毒的替代方案，否则iPS细胞将无法在临床上应用。我们建议的具体目标是通过蛋白质转导来开发iPS细胞，这是一种技术，通过简单地将重组蛋白与短细胞穿透肽一起添加到培养基中，就可以在细胞内获得重组蛋白。我们假设蛋白质转导非常适合以安全、有效和短暂的方式传递已知的重编程因子。因此，我们的方法旨在打破现有的阻碍iPS细胞临床应用的障碍。公共卫生相关性：人类胚胎干细胞可能代表再生医学组织的无限供应。与体细胞核移植（SCNT）相结合，hes细胞技术也为产生与供体基因相同的组织打开了大门，但这种应用的原理仍有待于在人类身上得到证实。然而，最近几个月，一种基于逆转录病毒传递关键转录因子的方法成功地将人类体细胞重编程为诱导多能干细胞（iPS）。尽管这些发现具有生物学意义，但由于使用逆转录病毒载体所固有的风险，由此产生的细胞不能用于临床目的。我们的项目将探索蛋白质转导作为病毒传递的替代方案，目标是获得可随时用于人类治疗目的的iPS细胞。在这种情况下，该提案既响应了NIH的总体使命，也响应了当前的项目公告（“使用非胚胎来源的人类多能干细胞研究”），因为它的具体目标是“对人类成人体细胞进行重新编程，使其去分化为多能干细胞”。

DESCRIPTION (provided by applicant): The therapeutic potential of "custom-made" human embryonic stem (huES) cells is widely acknowledged. However, both ethical and technical reasons stand in the way of the routine use of somatic cell nuclear transfer (SCNT) to derive genetically matched huES cells from patients. Recent progress at identifying the key molecular players involved in the maintenance of the huES cell phenotype has led to breakthrough reports describing the reprogramming of somatic cells by forcing the expression of a surprisingly manageable number of transcription factors. While these induced pluripotent stem (iPS) cells appear to be functionally identical to huES cells derived from blastocysts, the use of retroviral vectors to deliver the critical genes is still unsafe in the context of human therapies. Thus, unless alternatives to retroviral delivery are devised, iPS cells will not be clinically applicable. The specific aim of our proposal is to develop iPS cells by means of protein transduction, a technology by which recombinant proteins engineered with short cell-penetrating peptides are made available inside the cells by simply adding them to the culture medium. We postulate that protein transduction is well suited to deliver known reprogramming factors in a safe, efficient and transient manner. Our method, therefore, is designed to break existing barriers that prevent the clinical use of iPS cells. PUBLIC HEALTH RELEVANCE: Human embryonic stem (huES) cells might represent an unlimited supply of tissues for regenerative medicine. Coupled to somatic cell nuclear transfer (SCNT), huES cell technologies also opened the door to the possibility of generating tissues genetically identical to those of the donor, but the principle of this application remains to be proven in humans. In recent months, however, an approach based on the retroviral delivery of key transcription factors led to the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells. Despite the biological significance of these findings, the resulting cells are unusable for clinical purposes, due to the risks inherent to the use of retroviral vectors. Our project will explore protein transduction as an alternative to viral delivery, with the goal of obtaining iPS cells that could be readily used for human therapeutic purposes. In this context, the proposal responds both to the general mission of the NIH and to the present Program Announcement ("Human Pluripotent Stem Cell Research Using Non-Embryonic Sources"), as it is specifically aimed at "Reprogramming human adult somatic cells to dedifferentiate into pluripotent stem cells".