Non-viral Delivery of Neurotrophic Factors to the CNS

描述（由申请人提供）：治疗中枢神经系统（CNS）疾病的新疗法的开发通常受到候选药物（例如小分子、肽、蛋白质或病毒）对脑和/或脊髓内靶细胞的生物利用度差的阻碍。特别地，重组胶质细胞系衍生的神经营养因子（GDNF）已显示在体内神经疾病和CNS损伤的多种动物模型中具有强大的神经保护和神经恢复作用，然而，其在治疗人类神经病症中的成功受到其向大得多的人脑的不良递送的限制。本项目的总体目标是评估一种新型重组融合蛋白的治疗潜力，该融合蛋白由GDNF连接到破伤风毒素的神经元结合片段（破伤风毒素片段C，或TTC）组成。我们的研究有四个具体目标：（1）设计，表达和纯化GDNF：TTC融合蛋白;（2）表征这些融合蛋白的功能活性;（3）确定脑实质内，脑室内和肌内给药后GDNF：TTC融合蛋白在CNS中的分布，持久性和组织水平;（4）使用帕金森病小鼠模型研究融合蛋白的神经保护活性。目标1中的实验将涉及使用cDNA构建体（目前都在进行中）和重组蛋白纯化技术。目标2中的研究将采用原代神经元细胞培养结合免疫细胞化学、酶免疫测定和形态测定技术来评估GDNF：TTC融合蛋白的体外功能特性。目标3中的研究将类似地使用免疫细胞化学和酶免疫测定方法来研究融合蛋白向小鼠和大鼠CNS的体内递送。最后，目的4中的实验将使用神经化学方法来研究GDNF：TTC在小鼠体内的神经保护作用。虽然目前的工作可能会产生一种新的治疗帕金森氏病，从我们的GDNF：TTC的表征获得的信息可能会导致其在其他神经系统疾病，如肌萎缩侧索硬化症的使用。（ALS）。

DESCRIPTION (provided by applicant): The development of new therapies to treat disorders of the central nervous system (CNS) is often hindered by the poor bioavailability of candidate drugs (e.g. small molecules, peptides, proteins, or viruses) to target cells within the brain and/or spinal cord. In particular, recombinant glial cell line derived neurotrophic factor (GDNF) has been shown to have robust neuroprotective and neurorestorative effects in a variety of animal models for neurological disease and CNS injury in vivo, however, its success in treating human neurological disorders has been limited by its poor delivery to the much larger human brain. The overall goal of this project is to evaluate the therapeutic potential of a novel recombinant fusion protein composed of GDNF linked to the neuronal binding fragment of tetanus toxin (tetanus toxin fragment C, or TTC). Our study has four specific aims: (1) design, express, and purify GDNF:TTC fusion proteins; (2) characterize the functional activity of these fusion proteins; (3) determine the distribution, persistence, and tissue levels of GDNF:TTC fusion proteins in the CNS following intraparenchymal, intraventricular, and intramuscular administration; and (4) investigate the neuroprotective activity of the fusion proteins using a mouse model of Parkinson's disease. The experiments in Aim 1 will involve the use of cDNA constructs (all currently in hand) and recombinant protein purification techniques. The studies in Aim 2 will employ primary neuron cell culture in conjunction with immunocytochemical, enzyme immunoassay, and morphometric techniques to assess the functional properties of the GDNF:TTC fusion proteins in vitro. The studies in Aim 3 will similarly use immunocytochemical and enzyme immunoassay methods to investigate the delivery of fusions proteins to mouse and rat CNS in vivo. Finally, the experiments in Aim 4 will use a neurochemical approach to investigate the neuroprotective effects of GDNF:TTC in mice in vivo. While the present work may give rise to a new treatment for Parkinson's Disease, the information obtained from our characterization of GDNF:TTC may lead to its use in other neurological disorders such as amyotrophic lateral sclerosis.(ALS).