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

描述(申请人提供)：大多数衰弱的神经退行性疾病缺乏有效的治疗选择。细胞替代疗法已被认为是一种很有前途的长期解决方案。然而，获取用于治疗的适当细胞一直是一个重大挑战。胎儿组织来源的神经元、胚胎干细胞来源的神经元和诱导的多能干细胞来源的神经元都受到伦理问题和/或畸胎瘤形成的影响。转分化，即一种成体细胞类型可以被重新编程为另一种不同谱系的成体细胞类型，而不需要经历完全去分化到多能性状态，这是一种有吸引力的方法来产生摆脱这些缺点的细胞。Wernig等人最近证实了神经元转分化(NTD)，将Brn2、Ascl2和Myt1转录因子(TF)导入鼠和人成纤维细胞，将它们转化为功能神经元，效率高达20%，称为诱导神经元(IN)细胞。这些INS是有丝分裂后的，不会经历增殖的祖细胞状态，这使得它们成为基于细胞的治疗中枢神经系统疾病(如癫痫、中风、亨廷顿病和帕金森病)的有吸引力的细胞来源。然而，由于这些INS是由慢病毒产生的，因此必须开发一种替代的衍生策略用于临床应用。在我们的初步研究中，我们已经成功地通过非病毒传递已报道的TF来产生表达泛神经元标记Tuj1和Map2的鼠和人成纤维细胞。转分化效率很低，但我们认为还有很大的改进空间。这项建议的目的是开发有效的策略，使用非病毒方法从成人细胞中产生功能神经元。我们提出了以下具体目标：(1)优化非病毒载体对成人细胞转分化效率的影响；(2)研究底物性质对神经细胞转分化效率的影响。

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.