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

项目总结/摘要 该项目的目标是开发一种适用于干细胞治疗的组织工程技术 阿尔茨海默病（AD）。AD是最常见的痴呆症，其特征是突触功能障碍， 神经元缺失目前尚无有效的治疗方法。人神经干细胞移植 用人神经干细胞（hNSCs）替代丢失的神经元是一种很有前途的策略。然而，移植到AD大脑中的细胞 将面临淀粉样β（Aβ）肽水平升高的有毒环境，这将诱导 突触损伤和细胞死亡。我们如何克服这个问题？ 我们先前鉴定了突触蛋白takusan的活性（Tu等人，2007年）。过表达 Takusan在大鼠原代神经元培养物中导致各种突触性质的增强，包括 树突棘密度和AMPA和NMDA介导的谷氨酸受体活性。有趣的是， Aβ暴露会减弱突触的特性。然后，我们在培养的大鼠中过量表达takusan， 用可溶性Aβ寡聚体攻击神经元。值得注意的是，takusan表达减轻了突触 失去这些神经元。通过缺失作图和结构域交换，我们发明了合成的69-aa长的 蛋白hD 2b，其中我们融合了来自discs-large-homolog 5（DLG 5）的人D2 takusan结构域 和人PDZ结合C末端序列。hD 2b在培养的大鼠神经元中的过表达，类似于 全长takusan变体，赋予针对oAβ诱导的突触毒性的保护。 在目前的提议中，我们将生产表达hD 2b的hNSCs，并测试这种遗传修饰是否 有利于开发新的抗AD的细胞替代疗法。这一建议使我们 利用Allele Biotech Inc.在干细胞技术方面的专业知识，知识和发明 hD 2b蛋白的研究。具体目标如下： 目标1.体外培养表达hD 2b的人神经元。 目标二。为了测试表达hD 2b的hNs是否对oAβ诱导的突触毒性更有抵抗力， 与对照hN相比。 目标3.目的：探讨表达hD 2b基因的hNSCs移植到人淀粉样蛋白脑内是否能有效抑制淀粉样蛋白的表达， 前体蛋白（hAPP）转基因小鼠，hAPP-J20，将拯救他们的行为和组织学， 表型。 本提案包括已进行的实验、检测的试剂和结论 发表了一系列高质量的论文，使用大鼠原代神经元。由于拟议的工作是合乎逻辑的 扩展到人类神经元，我们有很高的信心，积极的结果将获得通过1年 第一阶段SBIR工作。在第一阶段完成后，我们预计将继续执行第二阶段的项目 更多的动物试验旨在不久的将来IND和最终与制药公司的合作伙伴关系。

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.