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）：广泛认可“定制”人胚胎茎（色调）细胞的治疗潜力。然而，伦理和技术原因都阻碍了常规使用体细胞核转移（SCNT）来从患者中得出遗传匹配的色调细胞。识别维持色调细胞表型的关键分子参与者的最新进展导致了突破性的报告，描述了细胞的重编程，通过强制表达出令人惊讶的可管理数量的转录因子。尽管这些诱导的多能茎（IPS）细胞似乎在功能上与源自胚泡的色细胞相同，但在人类治疗的背景下，使用逆转录病毒载体来传递关键基因仍然不安全。因此，除非设计逆转录病毒递送的替代方法，否则IPS细胞将不适用。我们建议的具体目的是通过蛋白质转导开发IPS细胞，该技术通过简单地将它们添加到培养基中，通过将其在细胞内提供用短细胞穿透肽设计的重组蛋白。我们假设蛋白质转导非常适合以安全，高效和瞬时的方式提供已知的重编程因子。因此，我们的方法旨在打破现有的障碍，以防止IPS细胞的临床使用。公共卫生相关性：人类胚胎干细胞（HUE）细胞可能代表无限的再生医学组织供应。与体细胞核转移（SCNT）结合在一起，色调细胞技术也为产生与供体的组织相同的可能性打开了大门，但是该应用的原理在人类中仍有待证明。然而，近几个月来，基于关键转录因子逆转录的方法导致人体细胞成功地重编程为诱导的多能茎（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，所得细胞无法使用临床目的。我们的项目将探索蛋白质转导作为病毒输送的替代方案，目的是获得可容易用于人类治疗目的的IPS细胞。在这种情况下，该提案既对NIH的总体任务又回应了本计划的公告（“使用非胚胎来源的人类多能干细胞研究”），因为它专门旨在“重新编程人类成年人体细胞以将人类的成年细胞分解为多能干细胞，以将其重新分化为多能干细胞”。