Generation of Human iPS Cells via Non-integrating Vectors

描述（由申请人提供）：干细胞在治疗多种疾病方面具有巨大的治疗潜力，需要其他方法来产生多能细胞。体细胞重编程是产生患者特异性（或等基因）多能干细胞的一种特别令人兴奋的方法。研究了三种重编程方法：1)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）对体细胞进行重编程，产生诱导多能干细胞（iPS）。前两种方法的效率极低，并且需要新鲜的人类卵母细胞或形成体细胞/胚胎干（ES）杂交细胞，从而限制了它们的进一步临床应用。最后一种方法不需要胚胎或卵母细胞，为目前的胚胎干细胞来源提供了一种潜在的替代方法，从而提供了产生针对患者和疾病的多能干细胞的可能性。这些细胞可能不仅对无免疫排斥的个性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细胞主要由小鼠体细胞产生，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力方面与胚胎干细胞相似。然而，这些iPS细胞在基因组中发生了显著的基因修饰和许多突变，来自iPS细胞的小鼠主要通过病毒插入突变导致的c-Myc的再激活和肿瘤抑制基因的失活而发生肿瘤。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生成了小鼠iPS细胞。然而，效率极低，范围从不到0.0001%到0.001%。“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。我们最近通过从一个开放阅读框（ORF）中强制表达四个重编程因子来改进iPS细胞的生成，该ORF由自裂2A序列连接。这种改进的系统显著提高了重编程效率（高达100倍），并使多个重编程因子能够在单个向量中表达。因此，我们打算测试通过非整合载体从单个ORF中强制表达重编程因子是否能增强体细胞中“基因清洁”的人类iPS细胞的产生。为了解决这个问题，我们提出了以下两个具体目标：目标1：通过一个自我复制的episomal载体，通过单个ORF诱导表达重编程因子，从体细胞中产生人类iPS细胞；目的2：通过腺病毒介导的重编程因子在单个ORF中的基因转移，从体细胞中生成人iPS细胞。公共卫生相关性：该项目将通过体细胞重编程方法产生“基因清洁”的人类es样多能干细胞。因此，这是一种替代人类胚胎干细胞用于再生医学应用的方法。

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.