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

描述(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能(MDA)神经元的选择性变性。由于这种特殊的细胞丢失，帕金森病被认为是以细胞为基础的治疗的首要目标疾病。事实上，大量的临床和临床前研究证明，一旦建立了理想和无限的细胞来源，细胞移植是治疗帕金森病的一种可行的治疗方案(见Redmond，2002；Li等人，2008a；Lindvall和Kokaia，2009)。2006年，Shinya Yamanaka和他的同事发表了他们的开创性工作，表明可以通过逆转录病毒转导四种重编程因子(即Oct4、Sox2、Klf4和c-Myc)从体细胞中产生多能干细胞，即所谓的“诱导多能干细胞(IPSCs)”(Takahashi和Yamanaka，2006)。随后通过类似方法成功地产生了人类IPSCs(Takahashi等人，2007年；Yu等人，2007；Park等人，2008a)，提供了在不破坏胚胎的情况下产生针对疾病或患者的干细胞的可能性。事实上，这些IPSCs在生物医学研究、疾病机制研究和个性化细胞疗法方面提供了前所未有的潜力。然而，目前IPSCs的主要缺陷包括多个病毒整合和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，2009a)。我们的长期目标是利用IPSCs开发一种个性化的基于细胞的帕金森病治疗方法，我们提出了以下三个具体目标。首先，基于我们的初步结果(Kim等人，2009a)，我们将通过一种新的无DNA重编程方法(即直接交付重编程蛋白)从健康和散发性帕金森病患者中建立IPSC系。我们将广泛评估形态、基因表达、表观遗传学以及体外和体内分化特性，以建立具有类似人类胚胎干细胞(HESCs)特性的IPSC系。我们将把它们的特性与传统逆转录病毒方法产生的hESC和IPSCs的特性进行比较。此外，我们还将通过最新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向神经前体细胞和中脑DA神经元分化的特性。我们将优化其向A9 DA神经元的分化，并评估从这些iPS细胞分化出的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损毁大鼠，这两种模型都在我们实验室得到了很好的证实。这些移植研究的生物学和行为结果将被系统地调查。总体而言，我们提出的实验将提供宝贵的见解和垫脚石，导致开发安全、现实和理想的个体化细胞治疗帕金森病的细胞源。 与公共卫生相关：尽管“诱导多能干细胞(IPSC)”技术还处于初级阶段，但它是一种诱人的新方法，可以彻底改变生物医学研究、疾病机制研究和基于细胞的定制化疗法。为了探索IPSCs用于个体化细胞治疗帕金森病的可能性，我们建议建立和鉴定来自健康和散发性帕金森病患者的临床可行的IPSC系，方法是在没有任何病毒或DNA载体的情况下直接传递重编程蛋白。这一提议将为使用DNA或无转基因IPSCs进行基于细胞的个性化帕金森病治疗的现实发展提供重要的垫脚石，该疗法可能适用于许多其他人类疾病。

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.