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”人胚胎干（huES）细胞的治疗潜力得到广泛认可。然而，伦理和技术原因阻碍了常规使用体细胞核移植（SCNT）从患者获得遗传匹配的huES细胞。最近的进展，在确定参与维持的huES细胞表型的关键分子的球员，导致了突破性的报告，描述了体细胞的重编程，迫使表达的转录因子的数量令人惊讶的管理。虽然这些诱导多能干细胞（iPS）细胞似乎在功能上与来自胚泡的huES细胞相同，但使用逆转录病毒载体递送关键基因在人类治疗的背景下仍然是不安全的。因此，除非设计出逆转录病毒递送的替代方案，否则iPS细胞将不适用于临床。我们的建议的具体目标是通过蛋白质转导的方式开发iPS细胞，通过该技术，通过简单地将它们添加到培养基中，使用短细胞穿透肽工程化的重组蛋白在细胞内可用。我们假设蛋白转导非常适合以安全、有效和瞬时的方式递送已知的重编程因子。因此，我们的方法旨在打破阻止iPS细胞临床应用的现有障碍。公共卫生相关性：人类胚胎干细胞（huES）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，huES细胞技术也为产生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于在人类中得到证明。然而，最近几个月，一种基于逆转录病毒递送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的固有风险，所得细胞无法用于临床目的。我们的项目将探索蛋白转导作为病毒递送的替代方案，目标是获得可用于人类治疗目的的iPS细胞。在这方面，该提案既响应了NIH的一般使命，也响应了目前的方案公告（“利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“重编程人类成年体细胞，使其去分化为多能干细胞”。