New Cell Reprogramming Technology to Produce Dopaminergic Neurons

产生多巴胺能神经元的新细胞重编程技术

• 批准号: 8904832
• 负责人: Arshak R Alexanian
• 金额: $ 15万
• 依托单位: CELL REPROGRAMMING AND THERAPEUTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8904832
• 关键词: Adverse effects; Amphetamines; Animal Model; Animals; Behavior; Behavioral Assay; Biological; Bone Marrow; Brain-Derived Neurotrophic Factor; Cell Differentiation process; Cell Nucleus; Cell Separation; Cell Transplants; Cell fusion; Cell membrane; Cell physiology; Cell surface; Cells; Chemicals; Chromatin Structure; Clinical; Coculture Techniques; Corpus striatum structure; Data; Dopamine; Dopaminergic Cell; Dose; Engraftment; Enzyme-Linked Immunosorbent Assay; Ethical Issues; Exhibits; FGF8 gene; Failure; Family Felidae; Floor; Forelimb; GDNF gene; Gene Expression Profile; Generations; Genome; Goals; Growth Factor; Human; In Vitro; Ketamine; Legal patent; Lesion; Mesenchymal Stem Cells; Methods; Midbrain structure; Modeling; Modification; Mus; Neurons; Outcome; Oxidopamine; Parkinson Disease; Patients; Phase; Phenotype; Production; Property; Protocols documentation; Rattus; Regenerative Medicine; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Rotation; SHH gene; Safety; Signal Pathway; Signal Transduction; Synapses; Technology; Testing; Therapeutic; Therapeutic Effect; Time; Transfection; Transplantation; Work; Xylazine; chemical genetics; clinical application; clinically relevant; cost effective; dopaminergic differentiation; dopaminergic neuron; embryonic stem cell; genetic approach; immunocytochemistry; improved; in vivo; induced pluripotent stem cell; inhibitor/antagonist; killings; morphogens; nuclear transfer; phase 1 study; phase 2 study; pre-clinical; progenitor; public health relevance; purmorphamine; relating to nervous system; restoration; secretase; small molecule

 DESCRIPTION (provided by applicant): Cell Reprogramming & Therapeutics LLC will develop new cellular reprogramming technology to produce cell therapeutic product for the treatment of Parkinson's disease (PD). Advances in cell reprogramming technologies to generate patient-specific cells of a desired type will revolutionize the field of regenerative medicine. Over the lat decades several cell reprogramming methods such as nuclear transfer, cell fusion and transfection or transduction with pluripotent factors have been developed. However, the majority of these technologies require the exposure of cell nuclei to reprogramming large molecules via transfection, transduction, cell fusion or nuclear transfer. This raises several technical, safety and ethical issues. Chemical genetics is an alternative approach for cell reprogramming that uses small, cell membrane penetrable substances to regulate multiple cellular processes including cell plasticity. The main advantages of this approach over the above-mentioned technologies are that the biological effects of small molecules are typically rapid, reversible and dose-dependent, allowing precise control over specific outcomes by fine-tuning their concentrations and combinations. Recently, using chemical genetics approach (the combination of small molecules that are involved in the regulation of chromatin structure and function and specific cell signaling pathways), we have been able to generate neuronal cells from human mesenchymal stem cells (hMSCs) that expressed mature dopaminergic (DA) markers, released dopamine, exhibited electrophysiological properties of maturing neurons, and formed synapses. The goal of the proposed Phase I studies is the optimization of this cell reprogramming technology to increase the production of engraftable (FOX2A+/TH+/Nurr1+) midbrain DA neurons and elucidation of the therapeutic effects of these specialized cells in an animal model of PD. Phase II studies will focus on clinical grade manufacturing of these DA cells and testing their therapeutic effect in several preclinical pathogenic and etiologic animal models of PD. Commercial and clinically compatible research products emerging from Phase I/II work include a DA neuron differentiation kit and a technology for large-scale clinical grade production of DA neurons. The ultimate post-Phase II product will be the production of clinically relevant DA neurons for PD therapy. To achieve these goals, the Phase I aims include: Specific Aim 1 will test the hypothesis that the efficiency of DA specification and maturation can be further improved by addition of specific cell signaling modulators and growth factors to our recently developed neural induction protocol at a certain time and in a specific order. Immunocytochemistry and RT-PCR will be used to evaluate the expression of markers specific to immature and mature dopaminergic neurons. Dopamine release will be assessed by ELISA. Specific Aim 2 will test the hypothesis that DA cells transplanted into the 6- hydroxydopamine (6-OHDA) mice and rat models of PD, will survive, integrate and will promote restoration of amphetamine-induced rotation behavior and will show improvements in tests of forelimb use and akinesia.

 描述（由申请人提供）：Cell Reprogramming & Therapeutics LLC 将开发新的细胞重编程技术来生产用于治疗帕金森病（PD）的细胞治疗产品。细胞重编程技术的进步可产生所需类型的患者特异性细胞，这将彻底改变再生医学领域。近几十年来，已经开发了几种细胞重编程方法，例如核移植、细胞融合和多能因子转染或转导。然而，大多数这些技术需要通过转染、转导、细胞融合或核移植将细胞核暴露于重编程大分子。这引发了一些技术、安全和道德问题。化学遗传学是细胞重编程的另一种方法，它使用小的、细胞膜可穿透的物质来调节包括细胞可塑性在内的多种细胞过程。与上述技术相比，这种方法的主要优点是小分子的生物效应通常是快速的、可逆的和 剂量依赖性，可以通过微调其浓度和组合来精确控制特定结果。最近，利用化学遗传学方法（参与染色质结构和功能以及特定细胞信号通路调节的小分子的组合），我们已经能够从人类间充质干细胞（hMSC）中产生神经元细胞，这些细胞表达成熟的多巴胺能（DA）标记，释放多巴胺，表现出成熟神经元的电生理特性，并形成突触。拟议的 I 期研究的目标是优化这种细胞重编程技术，以增加可移植 (FOX2A+/TH+/Nurr1+) 中脑 DA 神经元的产生，并阐明这些特殊细胞在 PD 动物模型中的治疗效果。 II 期研究将重点关注这些 DA 细胞的临床级制造，并在几种 PD 临床前致病和病因动物模型中测试其治疗效果。 I/II 期工作中出现的商业和临床兼容的研究产品包括 DA 神经元分化试剂盒和用于大规模临床级生产 DA 神经元的技术。最终的 II 期后产品将是生产用于 PD 治疗的临床相关 DA 神经元。为了实现这些目标，第一阶段的目标包括： 具体目标 1 将测试以下假设：通过在特定时间和特定顺序向我们最近开发的神经诱导方案中添加特定细胞信号调节剂和生长因子，可以进一步提高 DA 规范和成熟的效率。免疫细胞化学和 RT-PCR 将用于评估未成熟和成熟多巴胺能神经元特异标记的表达。多巴胺释放将通过 ELISA 进行评估。具体目标 2 将测试以下假设：移植到 6-羟基多巴胺 (6-OHDA) 小鼠和 PD 大鼠模型中的 DA 细胞将存活、整合并促进安非他明诱导的旋转行为的恢复，并将在前肢使用和运动不能测试中显示出改善。