Generation of Human iPS Cells via Non-integrating Vectors

描述(申请人提供)：干细胞在治疗各种疾病方面具有巨大的治疗潜力，需要替代产生多能细胞的方法。产生患者特异性(或同基因)多能干细胞的一种特别令人兴奋的方法是体细胞重新编程。已经研究了三种重编程方法：i)体细胞核移植(SCNT)方法；ii)基于细胞融合的方法；iii)通过定义的转录因子(Oct3/4和Sox2与Klf4和c-Myc或Nanog和Lin28)重编程体细胞来产生诱导多能干细胞(IPS)。前两种方法的效率极低，需要新鲜的人卵母细胞或形成体细胞/胚胎干细胞，从而限制了其进一步的临床应用。最后一种方法不需要胚胎或卵母细胞，并提供了一种潜在的替代目前来源的ES细胞，从而提供了产生患者和疾病特异性多能干细胞的可能性。这些细胞不仅对无免疫排斥的个体化细胞移植治疗有价值，而且对了解疾病机制、药物筛选、组织工程和毒理学也有价值。IPS细胞主要来源于小鼠体细胞，这些细胞在基因表达谱、DNA甲基化以及可能最显著的自我更新和分化潜能方面与ES细胞相似。然而，这些iPS细胞是通过基因组中的许多突变进行显著的基因修饰的，来自iPS细胞的小鼠发展成肿瘤，主要是通过c-Myc的重新激活和病毒插入突变导致的肿瘤抑制基因的失活。最近，通过腺病毒介导的四种转录因子的基因传递，成功地从成纤维细胞和肝细胞中获得了小鼠iPS细胞。然而，效率极低，从不到0.0001%到0.001%不等。“基因清洁”的人类iPS细胞尚未产生，可能是因为重编程效率不足。最近，我们通过强制表达四个重编程因子来改善iPS细胞的生成，这些重编程因子来自一个通过自切割的2A序列连接的单一开放阅读框架(ORF)。这一改进的系统显著提高了重新编程效率(高达100倍)，并使多个重新编程因素能够在单个矢量中表达。因此，我们打算测试通过非整合载体强制表达来自单个ORF的重编程因子是否能促进从体细胞产生基因清洁的人iPS细胞。为了解决这个问题，我们提出了以下两个具体目标：目的1：通过自复制异体载体从单个ORF诱导表达重编程因子，从体细胞中产生人iPS细胞；目的2：通过腺病毒介导的单个ORF中重编程因子的基因转移，从体细胞中产生人iPS细胞。与公共卫生相关：该项目将通过体细胞重新编程的方法产生“基因清洁的”人类ES样多能干细胞。因此，这是使用人类胚胎干细胞进行再生医学应用的一种替代方案。

DESCRIPTION (provided by applicant): Stem cells have great therapeutic potential for treatment of various diseases, and alternative methods for generation of pluripotent cells are needed. A particularly exciting method for the generation of patient-specific (or isogenic) pluripotent stem cells is somatic cell reprogramming. Three reprogramming approaches have been investigated: i) the somatic cell nuclear transfer (SCNT) approach; ii) cell fusion-based approach; iii) generation of induced pluripotent stem (iPS) cells by reprogramming of somatic cells via the defined transcription factors (Oct3/4 and Sox2 with Klf4 and c-Myc or Nanog and Lin28). The first two approaches are limited by extremely low efficiency and the requirement for fresh human oocytes or formation of somatic/embryonic stem (ES) hybrid cells, thereby limiting their further clinical applications. The last approach does not require embryos or oocytes, and provides a potential alternative to the current source of ES cells, thereby offering the possibility of generating patient- and disease-specific pluripotent stem cells. These cells may be valuable for not only personalized cell transplantation therapy without immune rejection but also for understanding disease mechanisms, drug screening, tissue engineering, and toxicology. iPS cells were generated mainly from mouse somatic cells, and these cells are similar to ES cells in terms of gene expression profile, DNA methylation, and perhaps most significantly self-renewal and differentiation potential. However, these iPS cells are significantly genetically modified with many mutations in the genome, and mice derived from iPS cells develop tumors, mainly through reactivation of c-Myc and inactivation of tumor suppressor genes resulting from viral insertional mutagenesis. Recently, mouse iPS cells were successfully generated from fibroblasts and liver cells via adenovirus-mediated gene delivery of the four transcription factors. However, the efficiency is extremely low, ranging from less than 0.0001% to 0.001%. 'Genetically clean' human iPS cells have not been generated, maybe because of inadequate reprogramming efficiency. We recently improved the generation of iPS cells by forced expression of four reprogramming factors from one single open reading frame (ORF) that was linked by self-cleaving 2A sequences. This improved system significantly increased reprogramming efficiency (up to 100 times) and enabled multiple reprogramming factors to be expressed in a single vector. Therefore, we intend to test whether forced expression of the reprogramming factors from a single ORF via non-integrating vectors enhances generation of 'genetically clean' human iPS cells from somatic cells. To address this question, we propose the following two Specific Aims: Aim 1: To generate human iPS cells from somatic cells by inducible expression of the reprogramming factors from a single ORF via a self-replication episomal vector; Aim 2: To generate human iPS cells from somatic cells by adenovirus-mediated gene transfer of the reprogramming factors in a single ORF. PUBLIC HEALTH RELEVANCE: This project will generate 'genetically clean' human ES-like pluripotent stem cells by somatic cell reprogramming approaches. Therefore, this is an alternative to the use of human embryonic stem cells for regenerative medicine applications.