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（iPSCs）是再生医学中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，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被重编程为多能的、类似于esc的状态。然而，使用病毒载体或质粒DNA通过潜在的DNA整合带来肿瘤发生的危险。因此，有必要为相关重编程因子的瞬时表达开发新的递送方法。在此，我们建议开发不使用病毒或质粒DNA的iPSC生成替代方法，从而使其更安全地用于医学应用。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注射来生成iPSCs。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（T3SS），由一种无细胞毒性的铜绿假单胞菌菌株编码。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的生成。在这里，我们通过利用一类被称为吡咯咪唑聚酰胺（PIPs）的小分子来探索这种可能性，它可以以特定的序列方式与DNA结合。这种更安全的诱导多能干细胞方法的发展将使多能干细胞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，佛罗里达大学创造了2525个工作岗位，最近的研究表明，佛罗里达大学每年为佛罗里达经济贡献近60亿美元。该大学在其主校区直接雇用了约34,000人，并通过佛罗里达大学组织，如食品和农业科学研究所，负责在全州创造74,894个工作岗位。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。

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.