Derivation of Functional Neurons from Human Adult Cells by Nonviral Gene Delivery

描述（由申请人提供）：大多数衰弱性神经退行性疾病缺乏有效的治疗选择。细胞替代疗法已被提出作为一种有前途的长期解决方案。然而，获得用于治疗的适当细胞一直是一个重大挑战。胎儿组织衍生的神经元、胚胎干细胞衍生的神经元和诱导多能干细胞衍生的神经元都遭受伦理问题和/或畸胎瘤形成的担忧。转分化，其中成体细胞类型可以重编程为不同谱系的另一种成体细胞类型，而不经历完全去分化为多能状态，代表了产生摆脱这些缺点的细胞的有吸引力的方法。神经元转分化（NTD）最近由Wernig等人证实，其中，将Brn 2、Ascl 2和Myt 11转录因子（TF）递送至小鼠和人成纤维细胞以高达20%的效率将它们转化为功能性神经元，称为诱导神经元（iN）细胞。这些iN是有丝分裂后的，并且不经过增殖祖细胞状态，这使得它们成为用于CNS疾病（例如癫痫、中风、亨廷顿病和帕金森病）的基于细胞的治疗的有吸引力的细胞来源。然而，由于这些iN已经由慢病毒产生，因此必须开发用于临床应用的替代衍生策略。在我们的初步研究中，我们已经成功地产生小鼠和人类成纤维细胞表达的泛神经元标记Tuj 1和MAP 2的非病毒传递的报告TF。转分化效率较低，但我们相信有足够的改进空间。该提案的目的是开发有效的策略，使用非病毒方法从成人细胞产生功能性神经元。我们的目标是：（1）优化非病毒载体诱导人成体细胞向神经元转分化的效率;（2）研究底物性质对神经元转分化效率的影响。 公共卫生相关性：大多数使人衰弱的神经退行性疾病缺乏有效的治疗选择。细胞替代疗法已被提出作为一种有前途的长期解决方案。该提案旨在开发使用非病毒方法从成人细胞产生功能性神经元的有效策略，使其成为基于细胞的CNS疾病（如癫痫、中风、亨廷顿病和帕金森氏病）治疗的有吸引力的细胞来源。

DESCRIPTION (provided by applicant): Most debilitating neurodegenerative diseases lack effective treatment options. Cell replacement therapy has been proposed as a promising long-term solution. However, procurement of the appropriate cells for therapy has been a significant challenge. Fetal tissue-derived neurons, embryonic stem cell-derived neurons, and induced pluripotent stem cell-derived neurons all suffer from concerns of ethical issues and/or teratoma formation. Transdifferentiation, where an adult cell type can be reprogrammed to another adult cell type of distinct lineage without going through complete de-differentiation to a pluripotent state, represents an attractive approach to generate cells rid of these shortcomings. Neuronal transdifferentiation (NTD) was most recently demonstrated by Wernig et al., where a delivery of Brn2, Ascl2, and Myt1l transcription factors (TFs) to mouse and human fibroblasts converted them into functional neurons with up to 20% efficiency, referred to as induced neuronal (iN) cells. These iNs are postmitotic and do not pass through a proliferative progenitor state, attributes making them an attractive cell source for cell-based therapy of CNS disorders such as epilepsy, stroke, Huntington disease, and Parkinson's disease. However, since these iNs have been generated by lentiviruses, an alternative derivation strategy must be developed for clinical applications. In our preliminary studies we have succeeded in generating mouse and human fibroblasts expressing the pan-neuronal markers Tuj1 and MAP2 by nonviral delivery of the reported TFs. The transdifferentiation efficiency is low, but we believe there is ample room for improvement. The objective of this proposal is to develop effective strategies to generate functional neurons from adult human cells using nonviral methods. We propose to pursue the following specific aims: (1) Optimize efficiency of neuronal transdifferentiation from human adult cells by nonviral vectors; (2) Investigate effects of substrate properties on efficiency of neurona transdifferentiation. PUBLIC HEALTH RELEVANCE: Most debilitating neurodegenerative diseases lack effective treatment options. Cell replacement therapy has been proposed as a promising long-term solution. This proposal aims to develop effective strategies to generate functional neurons from adult human cells using nonviral methods, making them an attractive cell source for cell-based therapy of CNS disorders such as epilepsy, stroke, Huntington disease, and Parkinson's disease.