Somatic cell reprogramming by protein transduction

通过蛋白质转导进行体细胞重编程

• 批准号: 7761208
• 负责人: Juan Dominguez-Bendala
• 金额: $ 22.72万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7761208
• 关键词: Address; Adult; Area; Basic Science; Biological; Biological Process; Cell Culture Techniques; Cell Line; Cell Therapy; Cells; Clinical; Coupled; Culture Media; Custom; Derivation procedure; Diabetes Mellitus; Embryo; Environment; Ethics; Fibroblasts; Funding Mechanisms; Genes; Genetic Transcription; Germ Layers; Goals; Human; Human Resources; In Vitro; Insertional Mutagenesis; Investigation; Maintenance; Medical; Membrane; Methods; Mission; Molecular; Mus; NIH Program Announcements; Outcome; Patients; Peptides; Phenotype; Pluripotent Stem Cells; Procedures; Protein Engineering; Proteins; Protocols documentation; Qualifying; Recombinant Proteins; Regenerative Medicine; Reporting; Research; Research Institute; Retroviral Vector; Risk; Series; Somatic Cell; Source; Stem Cell Research; System; Technology; Tertiary Protein Structure; Testing; Therapeutic; Time; Tissues; Training; Translating; Undifferentiated; United States National Institutes of Health; Viral; Viral Vector; base; blastocyst; cellular transduction; design; embryonic stem cell; experience; human embryonic stem cell; human stem cells; in vivo; induced pluripotent stem cell; innovation; novel; potency testing; prevent; public health relevance; somatic cell nuclear transfer; stem; stem cell technology; success; transcription factor

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".

描述（由申请人提供）：“定制”人类胚胎干（huES）细胞的治疗潜力已得到广泛认可。然而，伦理和技术原因阻碍了常规使用体细胞核移植 (SCNT) 从患者体内获取基因匹配的 huES 细胞。最近在鉴定参与维持 huES 细胞表型的关键分子方面取得的进展带来了突破性的报告，描述了通过强制表达数量惊人且易于管理的转录因子来对体细胞进行重编程。虽然这些诱导多能干 (iPS) 细胞在功能上似乎与源自囊胚的 huES 细胞相同，但使用逆转录病毒载体传递关键基因在人类治疗中仍然不安全。因此，除非设计出逆转录病毒递送的替代方案，否则 iPS 细胞将无法应用于临床。我们提案的具体目标是通过蛋白质转导来开发 iPS 细胞，通过这种技术，只需将短细胞穿透肽工程化的重组蛋白添加到培养基中，即可在细胞内使用它们。我们假设蛋白质转导非常适合以安全、有效和瞬时的方式传递已知的重编程因子。因此，我们的方法旨在打破阻碍 iPS 细胞临床使用的现有障碍。公共健康相关性：人类胚胎干 (huES) 细胞可能代表着再生医学组织的无限供应。与体细胞核移植 (SCNT) 相结合，huES 细胞技术也为生成与供体基因相同的组织的可能性打开了大门，但这种应用的原理仍有待在人类中得到证实。然而，最近几个月，一种基于关键转录因子的逆转录病毒传递的方法成功地将人类体细胞重编程为诱导多能干（iPS）细胞。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固有的风险，所得细胞无法用于临床目的。我们的项目将探索蛋白质转导作为病毒传递的替代方案，目标是获得可轻松用于人类治疗目的的 iPS 细胞。在这种背景下，该提案既响应了 NIH 的总体使命，也响应了当前的计划公告（“使用非胚胎来源的人类多能干细胞研究”），因为它专门针对“对人类成体细胞进行重新编程以去分化”转化为多能干细胞”。