iPSC Generation using Peptide Injection and Site-Selective HDAC Inhibition

描述（由申请人提供）：诱导多能干细胞（iPSC）是再生医学最受期待的细胞来源，具有巨大的治疗潜力。许多研究小组已经表明，成年成纤维细胞和上皮细胞可以通过病毒或质粒载体介导的少量重编程因子的递送重编程为多能性ESC样状态。然而，病毒载体或质粒DNA的使用通过潜在的DNA整合造成肿瘤发生的危险。因此，有必要开发新的递送方法以瞬时表达相关重编程因子。在这里，我们建议开发不使用病毒或质粒DNA的iPSC生成的替代方法，从而使其在医学应用中更安全。在第一个目标中，我们将测试我们是否可以使用细菌介导的蛋白质注射产生iPSC。我们将利用一种高效的蛋白质递送机制，称为III型分泌系统（T3 SS），由铜绿假单胞菌的非细胞毒性菌株编码。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，我们探索这种可能性，利用一类小分子称为吡咯咪唑聚酰胺（PIP），它可以结合到DNA的序列特异性的方式。这种更安全的iPSC诱导方法的开发将使iPSC的临床应用更接近现实。佛罗里达大学为当地和区域经济做出了巨大贡献。在2008年，UF创造了2,525个工作岗位，最近的研究表明，UF每年为佛罗里达的经济贡献近60亿美元。该大学直接在其主校区雇用约34，000人，并通过用友组织，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个新的工作岗位，并保留4个现有的工作岗位。 公共卫生相关性：诱导多能干细胞（iPSC）是再生医学的一种备受期待的细胞来源，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递送方法的方案具有永久性损伤细胞基因组的可能性，并造成癌症发生的风险。因此，有必要开发新的递送方法用于瞬时表达适当的重编程因子。在这里，我们将试图克服目前的限制，使用（1）一个高效的蛋白质注射系统和（2）化学品诱导序列特异性基因的再激活。这种更安全的iPSC诱导方法的开发将使iPSC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.