iPSC Generation using Peptide Injection and Site-Selective HDAC Inhibition

描述(申请人提供)：诱导多能干细胞(IPSCs)是再生医学最有希望的细胞来源，具有巨大的治疗潜力。许多研究小组已经证明，通过病毒或质粒载体介导的少量重编程因子的传递，成年成纤维细胞和上皮细胞可以重编程到多潜能的ESC样状态。然而，使用病毒载体或质粒DNA会通过潜在的DNA整合带来肿瘤发生的危险。因此，有必要开发新的递送方法来瞬时表达相关的重编程因子。在这里，我们建议开发不使用病毒或质粒DNA的替代方法来产生IPSC，从而使其在医学应用中更安全。在第一个目标中，我们将测试是否可以使用细菌介导的蛋白质注射来产生IPSCs。我们将利用一种名为III型分泌系统(T3SS)的高效蛋白质输送机制，该系统由一种非细胞毒性铜绿假单胞菌编码。在第二个目标中，我们将测试诱导位点特异性HDAC抑制的化学物质是否可以触发内源性重编程因子的瞬时表达，从而促进IPSC的产生。在这里，我们利用一类被称为吡咯咪唑聚酰胺(PIP)的小分子来探索这种可能性，这种小分子可以以序列特异性的方式与DNA结合。这种更安全的IPSC诱导方法的开发将使IPSCs的临床应用更接近现实。佛罗里达大学为当地和地区经济做出了巨大贡献。2008年，UF创造了2525个就业机会，最近的研究表明，UF每年为佛罗里达州的经济贡献近60亿美元。该大学直接在主校区雇佣了约3.4万人，并通过UF组织，如食品和农业科学研究所，负责在全州范围内创造74,894个就业机会。目前的提案将创造2个新的就业机会，并保留4个现有的就业机会。 公共卫生相关性：诱导多能干细胞(IPSCs)是一种备受期待的再生医学细胞来源，具有巨大的治疗潜力。目前，IPSCs的医学应用受到肿瘤形成危险的阻碍。目前使用病毒或质粒基因传递方法的方案有可能永久损害细胞基因组，并构成癌症启动的风险。因此，有必要开发新的递送方法来瞬时表达适当的重编程因子。在这里，我们将尝试使用(1)高效的蛋白质注射系统和(2)诱导序列特异性基因重新激活的化学物质来克服目前的限制。这种更安全的IPSC诱导方法的开发将使IPSCs的临床应用更接近现实。

DESCRIPTION (provided by applicant): Induced pluripotent stem cells (iPSCs) are the most expected cellular sources for regenerative medicine and hold tremendous therapeutic potential. Many groups have shown that adult fibroblasts and epithelial cells can be reprogrammed to a pluripotent, ESC-like state by viral or plasmid vector-mediated delivery of a small number of reprogramming factors. However, the use of viral vectors or plasmid DNA poses the danger of tumorigenesis through potential DNA integration. Therefore, it is necessary to develop new delivery methods for the transient expression of relevant reprogramming factors. Here, we propose to develop alternative methods for iPSC generation without using virus or plasmid DNA, thus making it safer for medical application. In the first aim, we will test whether we can generate iPSCs using bacteria-mediated protein injection. We will utilize a highly efficient protein delivery machinery, called type III secretion system (T3SS), encoded by a non-cytotoxic strain of Pseudomonas aeruginosa. In the second aim, we will test whether chemicals inducing site-specific HDAC inhibition can trigger transient expression of endogenous reprogramming factors and thus facilitate iPSC generation. Here we explore this possibility by utilizing a class of small molecules called Pyrrole imidazole polyamides (PIPs), which can bind to DNA in a sequence specific manner. Development of such safer iPSC induction methods will make the clinical application of iPSCs one step closer to reality. The University of Florida contributes substantially to the local and regional economy. In 2008, UF created 2,525 jobs and recent studies have shown that UF contributes nearly $6 billion annually to Florida's economy. The university employs about 34,000 people directly on its main campus and via UF organizations, such as the Institute of Food and Agricultural Sciences, is responsible for the creation of 74,894 jobs statewide. The current proposal will create 2 new jobs and retain 4 existing jobs. PUBLIC HEALTH RELEVANCE: Induced pluripotent stem cells (iPSCs) are a highly anticipated cellular source for regenerative medicine and hold tremendous therapeutic potential. Presently, medical application of iPSCs is hampered by the danger of tumor formation. The current protocols utilizing viral or plasmid gene delivery methods, have a potential to damage the cellular genome permanently, and pose a risk for cancer initiation. Therefore, it is necessary to develop new delivery methods for the transient expression of appropriate reprogramming factors. Here we will attempt to overcome current limitations using (1) a highly efficient protein injection system and (2) chemicals to induce sequence specific gene reactivation. Development of such safer iPSC induction methods will make the clinical application of iPSCs one step closer to reality.