New Cell Reprogramming Technology to Produce Dopaminergic Neurons

 描述(由申请人提供)：细胞重编程和治疗有限责任公司将开发新的细胞重编程技术，以生产用于治疗帕金森氏病(PD)的细胞治疗产品。细胞重新编程技术的进步，以产生所需类型的患者特定细胞，将使再生医学领域发生革命性变化。在过去的几十年里，已经发展了几种细胞重编程方法，如核移植、细胞融合和带有多潜能因子的转基因或转导。然而，这些技术中的大多数需要通过转基因、转导、细胞融合或核移植使细胞核暴露于重新编程的大分子。这引发了几个技术、安全和道德问题。化学遗传学是细胞重新编程的另一种方法，它使用小的细胞膜穿透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、可逆和 剂量依赖，允许通过微调其浓度和组合对特定结果进行精确控制。最近，利用化学遗传学的方法(参与染色质结构和功能调节的小分子和特定的细胞信号通路的组合)，我们已经能够从表达成熟的多巴胺(DA)标记的人骨髓间充质干细胞(HMSCs)中产生神经元细胞，释放多巴胺，表现出成熟神经元的电生理特性，并形成突触。拟议的第一阶段研究的目标是优化这种细胞重编程技术，以增加可植入(FOX2A/TH/Nurr1)中脑DA神经元的产量，并阐明这些专门细胞在帕金森病动物模型中的治疗效果。第二阶段的研究将集中于这些DA细胞的临床分级制造，并在几种帕金森病的临床前致病和病因动物模型上测试它们的治疗效果。从I/II阶段工作中产生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期后产品将是临床相关的DA神经元的生产，用于帕金森病的治疗。为了实现这些目标，第一阶段的目标包括：特定目标1将检验这样一个假设，即通过在我们最近开发的神经诱导协议中以特定的时间和特定的顺序添加特定的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将被用来评估未成熟和成熟的多巴胺能神经元特异性标志物的表达。多巴胺的释放将通过酶联免疫吸附试验进行评估。特定目的2将测试DA细胞移植到6-羟基多巴胺(6-OHDA)小鼠和帕金森病大鼠模型中的假设，即DA细胞将存活、整合并将促进苯丙胺诱导的旋转行为的恢复，并将在前肢使用和动作障碍测试中显示出改善。

 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.