Protein-induced human iPS cells for personalized cell therapy of PD

蛋白质诱导的人类 iPS 细胞用于 PD 个性化细胞治疗

• 批准号: 8079724
• 负责人: Kwang-Soo Kim
• 金额: $ 33.87万
• 依托单位: MCLEAN HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079724
• 关键词: Animal Model; Behavioral; Biological; Biomedical Research; Calculi; Cell Line; Cell Therapy; Cell Transplantation; Cells; Clinical Data; Clinical Research; Collaborations; Controlled Clinical Trials; Copy Number Polymorphism; Corpus striatum structure; Custom; DNA; Data; Defect; Development; Disease; Disease model; Double-Blind Method; Embryo; Epigenetic Process; Exhibits; Family; Fibroblasts; Functional disorder; Gene Expression; Generations; Genes; Genetic; Genomics; Goals; Human; In Vitro; Infant; Karyotype; Laboratories; Lead; Lesion; Letters; Location; Methods; Midbrain structure; Molecular; Morphology; Motor; Neurodegenerative Disorders; Neurons; Newborn Infant; Oligonucleotides; Outcome; Oxidopamine; Parkinson Disease; Pathology; Patients; Peptides; Physiological; Placebo Control; Pluripotent Stem Cells; Property; Proteins; Publishing; Rattus; Recovery; Regimen; Resolution; Retroviridae; Rodent Model; Somatic Cell; Source; Southern Blotting; Staging; Stem Cell Research; Stem cells; Substantia nigra structure; Techniques; Testing; Therapeutic; Tissues; Transgenes; Transplantation; Validation; Viral; Virus; Work; aphakia mice; base; c-myc Genes; cell type; clinical application; disease mechanisms study; dopaminergic neuron; embryonic stem cell; fetal; human disease; human embryonic stem cell; human embryonic stem cell line; improved; in vivo; induced pluripotent stem cell; insight; male; nerve stem cell; neurotransmission; novel; open label; preclinical study; public health relevance; research study; retroviral transduction; stem cell technology; tumor; vector; viral DNA

DESCRIPTION (provided by applicant): Parkinson's disease (PD) is the second most common neurodegenerative disorder in which the main pathology is selective degeneration of midbrain dopaminergic (mDA) neurons in the substantia nigra. Because of this specific cell loss, PD is considered to be a prime target disease for cell-based therapy. Indeed, numerous clinical and preclinical studies demonstrated the proof-of-principle that cell transplantation is a viable therapeutic regimen for PD treatment once an ideal and unlimited cell source can be established (reviewed in (Redmond, 2002; Li et al., 2008a; Lindvall and Kokaia, 2009). In 2006, Shinya Yamanaka and his colleagues published their groundbreaking work showing that pluripotent stem cells, so called "induced pluripotent stem cells (iPSCs)", can be generated from somatic cells by retroviral transduction of four reprogramming factors (i.e., Oct4, Sox2, Klf4 and c-Myc)(Takahashi and Yamanaka, 2006). Subsequent successful generation of human iPSCs by similar methods (Takahashi et al., 2007; Yu et al., 2007; Park et al., 2008a) offered the possibility to generate disease- or patient-specific stem cells without destruction of embryos. Indeed, these iPSCs offer unprecedented potentials for biomedical research, disease mechanism study, and personalized cell-based therapies. However, current iPSCs suffer from major drawbacks including multiple viral integrations and remaining transgenes at various chromosomal locations, any of which may cause unpredictable genetic dysfunction and/or tumor formation, making these cells unsuitable for clinical applications (Yamanaka, 2009a). With the long-term goal of developing a personalized cell-based therapy of PD using iPSCs, we propose the following three specific aims. First, based on our preliminary results (Kim et al., 2009a), we will establish iPSC lines from healthy and sporadic PD subjects by a novel, DNA-free reprogramming method (i.e., direct delivery of reprogramming proteins). We will extensively evaluate morphological, gene expression, epigenetic, and in vitro and in vivo differentiation properties to establish iPSC lines exhibiting properties similar to human embryonic stem cells (hESCs). We will compare their properties with those of hESC lines and iPSCs generated by conventional retroviral methods. In addtion, we will investigate their chromosomal integrity by state of the art copy number variation analysis. Second, once authentic iPSC lines are established, we will fully characterize and compare their in vitro differentiation properties into neural progenitors and midbrain DA neurons. We will optimize their differentiation into A9 DA neurons and evaluate the molecular, cellular, and electrophysiological characters of DA neurons from these iPS cells. Third, we will initiate to evaluate the potential functional benefits of these iPSC-derived DA neurons in two rodent models of PD; aphakia mice and 6-OHDA lesioned rats, both of which are well established in our laboratory. Biological and behavioral outcomes of these transplantation studies will be systematically investigated. Overall, our proposed experiments will provide invaluable insights and stepping stones, leading to the development of safe, realistic, and ideal cell source for personalized cell-based therapy of PD. PUBLIC HEALTH RELEVANCE: Although in its infant stage, the "induced pluripotent stem cell (iPSC)" technology is a tantalizing new method that can revolutionize biomedical research, disease mechanism studies, and customized cell-based therapies. To explore the potential of iPSCs for personalized cell-based therapy for PD, we propose to establish and characterize clinically viable iPSC lines from healthy and sporadic PD subjects by direct delivery of reprogramming proteins without any virus or DNA vectors. This proposal will provide important stepping-stones for realistic development of a personalized cell-based therapy of PD using DNA- or transgene-free iPSCs, which could be applicable to many other human diseases.

描述（由申请人提供）：帕金森病（PD）是第二常见的神经退行性疾病，其主要病理是黑质中脑多巴胺（mDA）神经元的选择性变性。由于这种特定的细胞损失，PD被认为是基于细胞的治疗的主要目标疾病。事实上，许多临床和临床前研究证明了原理证明，即一旦可以建立理想的和无限的细胞来源，细胞移植是用于PD治疗的可行的治疗方案（综述于（雷德蒙，2002; Li et al.，2008年a; Lindvall和Kokaia，2009年）。2006年，Shinya Yamanaka和他的同事发表了他们的开创性工作，表明多能干细胞，所谓的“诱导多能干细胞（iPSC）"，可以通过逆转录病毒转导四种重编程因子（即，Oct 4，Sox 2，Klf 4和c-Myc）（Takahashi和Yamanaka，2006）。随后通过类似的方法成功产生人iPSC（Takahashi et al.，2007年; Yu等人，2007年; Park等人，2008年a）提供了在不破坏胚胎的情况下产生疾病或患者特异性干细胞的可能性。事实上，这些iPSCs为生物医学研究、疾病机制研究和个性化细胞治疗提供了前所未有的潜力。然而，目前的iPSC存在主要缺点，包括多个病毒整合和在不同染色体位置的剩余转基因，其中任何一个都可能导致不可预测的遗传功能障碍和/或肿瘤形成，使这些细胞不适合临床应用（Yamanaka，2009 a）。随着开发使用iPSC的个性化基于细胞的PD治疗的长期目标，我们提出了以下三个具体目标。首先，根据我们的初步结果（Kim等人，2009 a），我们将通过一种新的无DNA重编程方法（即，直接递送重编程蛋白）。我们将广泛评估形态学，基因表达，表观遗传，以及在体外和体内分化特性，以建立表现出类似于人类胚胎干细胞（hESC）的特性的iPSC系。我们将比较它们的特性与通过常规逆转录病毒方法产生的hESC系和iPSC的特性。此外，我们将通过最先进的拷贝数变异分析来研究它们的染色体完整性。其次，一旦建立了真正的iPSC系，我们将充分表征和比较它们在体外分化为神经祖细胞和中脑DA神经元的特性。我们将优化其分化为A9 DA神经元，并评估这些iPS细胞的DA神经元的分子，细胞和电生理特性。第三，我们将开始评估这些iPSC衍生的DA神经元在两种PD啮齿动物模型中的潜在功能益处;无晶状体小鼠和6-OHDA损伤大鼠，这两种模型都在我们的实验室中得到了很好的建立。这些移植研究的生物学和行为学结果将被系统地研究。总的来说，我们提出的实验将提供宝贵的见解和垫脚石，从而为PD的个性化细胞治疗开发安全，现实和理想的细胞来源。 公共卫生相关性：虽然在其婴儿阶段，“诱导多能干细胞（iPSC）”技术是一个诱人的新方法，可以彻底改变生物医学研究，疾病机制研究和定制的细胞为基础的治疗。为了探索iPSC用于PD个性化细胞治疗的潜力，我们建议通过直接递送重编程蛋白而不使用任何病毒或DNA载体来建立和表征来自健康和散发性PD受试者的临床上可行的iPSC系。这一提议将为使用无DNA或无转基因iPSC的PD个性化细胞治疗的现实发展提供重要的垫脚石，这可能适用于许多其他人类疾病。