Human Neural Stem Cells Engineered against oAβ-Induced Toxicity for Cell Therapy

项目摘要/摘要 该项目的目标是开发一种适用于干细胞疗法的组织工程技术。 治疗阿尔茨海默氏症(AD)。AD是最常见的痴呆症，其特点是突触功能障碍和 神经元丢失。目前还没有治愈这种疾病的有效方法。人神经干细胞移植 (HNSCs)替代丢失的神经元是一种很有前途的策略。然而，移植到阿尔茨海默病大脑中的细胞 将面临淀粉样蛋白-β(A-β)肽水平升高的有毒环境，这将导致 植入神经元的突触损伤和细胞死亡。我们怎样才能克服这个问题呢？ 我们之前发现了突触蛋白Takusan的活性(Tu等人，2007年)。过度表达 大鼠原代神经元培养中的Takusan导致多种突触特性的增强，包括 树突棘密度，以及AMPA和NMDA介导的谷氨酸受体活性。有趣的是，这些是 因接触β而减弱的突触特性。然后我们在培养的大鼠中过表达了竹节散 并用可溶性A-β寡聚体攻击神经元。值得注意的是，Takusan的表达缓解了突触 这些神经元的缺失。通过缺失作图和结构域交换，我们发明了人工合成的69-AA Long 蛋白hD2b，在其中我们融合了来自Diss-Large-Homolog 5(DLG5)的人D2 Takusan结构域 和人PDZ结合的C-末端序列。HD2b在培养的大鼠神经元中的过表达 全长Takusan变体，对OAβ诱导的突触毒性具有保护作用。 在目前的方案中，我们将培养表达hD2b的hNSCs，并测试这种基因改造 有利于开发一种新的治疗AD的细胞替代疗法。这项提议使我们能够 利用我们在Allele Biotech Inc.的干细胞技术专业知识和发明 我们的学术合作者的hD2b蛋白。具体目标如下： 目的1.体外培养表达hD2b的人神经元。 目的2.检测表达hD2b的HN是否对OAβ诱导的突触毒性更具抵抗力 与对照HNS相比.. 目的3.评价表达hD2b的hNSCs能否移植到人淀粉样蛋白的脑内 前体蛋白(HAPP)转基因小鼠HAPP-J20将挽救它们的行为和组织学 表型。 这项建议包括已经进行的实验、试剂测试和结论。 发表在一系列高质量的论文中，使用的是大鼠的原代神经元。由于提议的工作是合乎逻辑的 推广到人类神经元，我们有很高的信心，通过一年的时间将取得积极的结果 第一阶段SBIR工作。在第一阶段完成后，我们预计将继续进行第二阶段的项目执行 旨在不久的将来进行额外的动物试验，并最终与制药公司建立合作伙伴关系。

Project Summary/Abstract The goal of this project is to develop a tissue engineering technology applicable to stem cell-based therapies for Alzheimer's disease (AD). AD is the most common dementia and characterized by synaptic dysfunction and neuronal loss. There is currently no effective cure for the disease. Transplantation of human neural stem cells (hNSCs) to replace lost neurons is a promising strategy. However, the cells transplanted into the AD brain would face a toxic environment with elevated levels of the amyloid-β (Aβ) peptides, which would induce synaptic damage and cell death for implanted neurons. How can we overcome this problem? We previously identified the activity of synaptic protein takusan (Tu et al., 2007). Overexpression of takusan in rat primary neuronal cultures leads to enhancement of various synaptic properties including dendritic-spine density, and AMPA- and NMDA-mediated glutamate receptor activity. Interestingly, these are the synaptic properties that are attenuated by Aβ exposure. We then overexpressed takusan in cultured rat neurons and challenged them by soluble Aβ oligomers. Remarkably, takusan expression mitigated synaptic loss in these neurons. By deletion mapping and domain swapping, we invented the synthetic 69-aa long protein hD2b, in which we fused the human D2 takusan domain derived from discs-large-homolog 5 (DLG5) and a human PDZ-binding C-terminal sequence. Overexpression of hD2b in cultured rat neurons, like that of full-length takusan variants, conferred protection against oAβ-induced synaptic toxicity. In the current proposal, we will produce hNSCs expressing hD2b and test whether this genetic modification is beneficial toward the development of a novel cell-replacement therapy against AD. This proposal allows us to leverage our expertise in stem cell technology at Allele Biotech Inc. with the knowledge and invention of hD2b protein by our academic collaborators. Specific Aims are as follows: Aim 1. To produce human neurons (hNs) that express hD2b in vitro. Aim 2. To test whether hNs expressing hD2b are more resistant against oAβ-induced synaptic toxicity compared to control hNs.. Aim 3. To evaluate whether transplantation of hNSCs expressing hD2b into the brain of human amyloid precursor protein (hAPP) transgenic mice, hAPP-J20, will rescue their behavioral and histological phenotypes. This proposal consists of experiments that have been performed, reagents tested, and conclusions published in series of high quality papers, using rat primary neurons. Since the proposed work is a logical extension to human neurons, we have high confidence that positive results will be obtained through a 1 year phase 1 SBIR effort. Upon completion of phase 1, we expect to continue to phase 2 projects to perform additional animal testing aimed at near-future IND and eventual partnership with pharmaceutical companies.