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

基本信息

批准号：
9444809
负责人：
Arshak R Alexanian
金额：
$ 5万
依托单位：
CELL REPROGRAMMING AND THERAPEUTICS, LLC
依托单位国家：
美国
项目类别：
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/9444809
关键词：
Adverse effects Alpha Cell 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 Electrophysiology (science)Engraftment Enzyme-Linked Immunosorbent Assay Ethical Issues Etiology Exhibits FGF8 gene Failure Family Felidae Floor Forelimb GDNF gene Gene Expression Profile Generations Genome Goals Growth Factor Human Hydroxydopamines In Vitro Injectable Ketamine Legal patent Lesion Mesenchymal Stem Cells Methods Midbrain structure Modeling Modification Mus Neurons Outcome Parkinson Disease Pathogenicity 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

项目摘要

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，以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将产生患者所需类型的特异性细胞。在过去的几十年里，再生医学领域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。这些技术需要通过转染、转导、细胞融合或核转移将细胞核暴露于重编程大分子，这引发了一些技术、安全和问题。化学遗传学是使用小的、细胞膜可穿透物质进行细胞重编程的另一种伦理方法。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、可逆和可逆的。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，我们使用化学遗传学方法（参与染色质结构和功能以及特定细胞信号传导途径调节的小分子的组合）。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。第一阶段研究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，第二阶段研究将重点阐明这些特殊细胞在 PD 动物模型中的治疗效果。这些 DA 细胞的临床级制造，并在几种 PD 临床前致病性和病因学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。分化试剂盒和大规模临床级 DA 神经元生产技术最终的 II 期产品将是生产用于 PD 治疗的临床相关 DA 神经元。将测试以下假设：通过在特定时间以特定顺序向我们最近开发的神经诱导方案中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。用于评估未成熟和成熟多巴胺能神经元特异性标记物的表达将通过 ELISA 进行评估具体目标 2 将测试 DA 细胞移植到 PD 的 6-羟基多巴胺 (6-OHDA) 小鼠和大鼠模型中的假设。，将存活、整合并促进安非他明引起的旋转行为的恢复，并将在前肢使用和运动不能的测试中显示出改善。