Bacterial mediated generation of iPS cells

描述(申请人提供)：胚胎干细胞(ES)是胚胎来源的多能细胞系，可以在体内产生每一种细胞类型。因此，这些细胞是研究组织形成和疾病发展机制的宝贵工具，并为组织修复提供了一个有前途的“替代细胞”来源。然而，胚胎来源的ES细胞的研究，特别是在将其用作疾病模型或可移植的替代细胞方面，一直受到阻碍新系产生的调控障碍和获得“患者特有的”、组织相容的细胞的困难。令人兴奋的最新发现使成年体细胞能够直接“重新编程”成类似ES的“诱导多能(IPS)细胞”，似乎降低了这些障碍，为生产用于研究和治疗的患者特有的多能细胞提供了一种简便的机制。IPS技术的革命性发展打开了许多新的机会，也许最令人兴奋的潜在应用是使用患者特定的组织进行移植。然而，目前对体细胞重新编程的最有效方法是通过逆转录病毒载体将编码四种哺乳动物转录因子的cDNA导入体细胞。考虑到病毒介导的插入事件可能激活癌基因或无效的肿瘤抑制因子，因此人们非常担心将这些操纵细胞返回给患者的安全性，从而引起对重新编程细胞的致瘤性的担忧。因此，为了响应尤里卡的倡议，我们建议开发一种全新的系统来规避与病毒产生的iPS细胞相关的问题，方法是重新设计专门的细菌跨王国分泌系统，将iPS转化蛋白而不是毒力蛋白直接输送到哺乳动物细胞。这是一种创新的高风险方法，如果成功，将是体细胞重新编程的重大进步，将对加速向患者提供基于iPS的移植疗法产生巨大影响，并提供一种潜在地提高iPS细胞生成效率的新方法。 与公共卫生相关：干细胞生物学方面出现了一项重大突破，证明了通过将cDNA导入细胞，分化的体细胞可以转化为多能细胞。这些方法为针对患者的细胞替代疗法提供了巨大的潜力，然而目前将替代细胞引入患者体内的一个主要障碍是DNA整合到这些细胞的染色体中可能会导致肿瘤的形成。为了绕过这一主要障碍，走向针对患者的治疗，我们建议开发一种新的技术，使用重新设计的跨王国细菌分泌系统，直接引入重新编程的蛋白质，而不是编码它们到分化细胞的cDNA。

DESCRIPTION (provided by applicant): Embryonic stem (ES) cells are embryo-derived pluripotent cell lines that can give rise to each and every cell type in the body. As such, these cells are invaluable tools for research into the mechanisms of tissue formation and the development of disease, and provide a promising source of "replacement cells" for tissue repair. However, research with embryo-derived ES cells, particularly with respect to their use as disease models or transplantable replacement cells, has been hampered by regulatory hurdles impeding the derivation of new lines and by difficulties in obtaining "patient-specific", histocompatible cells. Exciting recent discoveries enabling direct "reprogramming" of adult somatic cells to ES-like "induced pluripotent (iPS) cells" appear to have lowered these hurdles, providing a facile mechanism for the production of patient-specific pluripotent cells for research and therapy. The revolutionarily development of iPS technology has opened many new opportunities perhaps the most exciting potential application being the use of patient-specific tissues for transplantation. However, currently the most efficient ways to reprogram somatic cells involve the introduction of cDNAs that encode four mammalian transcription factors into somatic cells via retroviral vectors. There is great concern regarding the safety of returning these manipulate cells to patients given the risk that virus- mediated insertional events can activate oncogenes or inactive tumor suppressors thus raising concern for the tumorigenicity of the reprogrammed cells. Thus, in response to the EUREKA initiative, we propose to develop a completely novel system to circumvent the issues associated with virally-generated iPS cells by reengineering specialized bacterial transkingdom secretion systems to deliver the iPS transforming proteins rather than virulence proteins directly into mammalian cells. This is an innovative and high risk approach that if successful would be a major advance in somatic reprogramming and would have tremendous impact on accelerating the delivery of iPS-based transplant therapies to patients as well as provide a new means of potentially increasing the efficiency of the generation of iPS cells. PUBLIC HEALTH RELEVANCE: A major breakthrough in stem cell biology has emerged with the demonstrated that differentiated somatic cells can be converted to pluripotent cells through the introduction of cDNAs into cells. These approaches hold tremendous potential for the patient-specific replacement based cell therapies, however currently a major roadblock in introducing replacement cells into patients is the possibility that the integration of DNA into the chromosomes of these cells can induce tumor formation. To circumvent this major roadblock in moving towards patient-specific therapies, we propose to develop a novel technology using re-engineered transkingdom bacterial secretion systems to directly introduce reprogramming proteins rather than the cDNAs that encode them into the differentiated cells.