Bacterial mediated generation of iPS cells

描述（由申请人提供）：胚胎干（ES）细胞是胚胎来源的多能细胞系，可以产生体内的每种细胞类型。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，特别是关于它们作为疾病模型或可移植替代细胞的用途，受到阻碍新细胞系衍生的监管障碍和获得“患者特异性”组织相容性细胞的困难的阻碍。最近令人兴奋的发现使得能够将成体体细胞直接“重编程”为ES样“诱导多能（iPS）细胞”似乎已经降低了这些障碍，为生产用于研究和治疗的患者特异性多能细胞提供了一种简便的机制。iPS技术的革命性发展开辟了许多新的机会，也许最令人兴奋的潜在应用是使用患者特异性组织进行移植。然而，目前重编程体细胞的最有效方法涉及通过逆转录病毒载体将编码四种哺乳动物转录因子的cDNA引入体细胞。考虑到病毒介导的插入事件可能激活癌基因或失活的肿瘤抑制因子，因此引起了对重编程细胞致瘤性的关注，因此对将这些操作细胞返回患者的安全性存在极大的担忧。因此，为了响应尤里卡倡议，我们建议开发一种全新的系统，通过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递送到哺乳动物细胞中，来规避与病毒产生的iPS细胞相关的问题。这是一种创新且高风险的方法，如果成功，将是体细胞重编程的重大进展，并将对加速向患者提供基于iPS的移植疗法产生巨大影响，并提供可能提高iPS细胞生成效率的新方法。 公共卫生相关性：干细胞生物学的一个重大突破已经出现，证明分化的体细胞可以通过将cDNA导入细胞中转化为多能细胞。这些方法对于基于患者特异性置换的细胞疗法具有巨大的潜力，然而目前将置换细胞引入患者的主要障碍是DNA整合到这些细胞的染色体中可能诱导肿瘤形成。为了绕过这一主要障碍，在走向患者特异性治疗，我们建议开发一种新的技术，使用重新设计的transkingdom细菌分泌系统直接引入重编程蛋白质，而不是编码它们的cDNA到分化的细胞。

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.