Non-viral Delivery of Neurotrophic Factors to the CNS

描述（由申请人提供）：治疗肌萎缩性侧索硬化症（ALS）的新疗法的开发经常受到候选药物对中枢神经系统（CNS）内受影响运动神经元的生物利用度差的阻碍。因此，尽管胶质细胞系衍生的神经营养因子（GDNF）和胰岛素样生长因子-1 （IGF-1）在实验动物中对损伤的运动神经元具有强大的促进生存的作用，但它们在治疗ALS患者方面的成功似乎受到了更大的人类中枢神经系统中运动神经元访问不足的阻碍。我们假设破伤风毒素片段C （TTC）与GDNF或IGF-1的遗传或化学融合将通过一种或多种与TTC的神经细胞结合特性相关的机制改善生长因子向运动神经元的传递。因此，本项目的主要目的是评估IGF-1:TTC和GDNF:TTC在ALS实验动物模型中的神经保护活性是否优于各自单独使用的生长因子。我们的研究有四个具体目标：(1)优化IGF-1:TTC和GDNF:TTC的设计、表达和纯化；(2)鉴定这些融合蛋白的纯度、稳定性和基本功能活性；(3)检测脑室内或肌肉内给药后融合蛋白在啮齿动物中枢神经系统中的生物利用度和功能活性；(4)研究融合蛋白在肌萎缩侧索硬化症（ALS）转基因大鼠模型中的神经保护活性。Aims 1的实验将使用重组蛋白表达和纯化技术来产生融合蛋白，Aims 2和Aims 3的研究将使用免疫细胞化学、Western blot、酶免疫测定和形态计量技术来评估GDNF:TTC和IGF-1:TTC在培养细胞和全动物中的功能特性。最后，Aim 4的实验将采用转基因动物进行生存和行为实验，评估GDNF:TTC和/或IGF-1:TTC在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ALS的方法，但从我们对IGF-1:TTC和GDNF:TTC的表征中获得的信息也可能导致在其他神经系统疾病中使用这些融合蛋白。

DESCRIPTION (provided by applicant): The development of new therapies to treat amyotrophic lateral sclerosis (ALS) is often hindered by the poor bioavailability of candidate drugs to affected motor neurons within the central nervous system (CNS). Thus, while both glial cell line-derived neurotrophic factor (GDNF) and insulin-like growth factor-1 (IGF-1) have had robust survival promoting effects on injured motor neurons in experimental animals, their success in treating ALS patients appears to have been thwarted by insufficient access to motor neurons in the much larger human CNS. We hypothesize that genetic or chemical fusion of tetanus toxin fragment C (TTC) to either GDNF or IGF-1 will improve growth factor delivery to motor neurons through one or more mechanisms related to the nerve cell binding properties of TTC. The primary goal of this project, then, is to assess whether the neuroprotective activity of IGF-1:TTC and GDNF:TTC in an experimental animal model of ALS is superior to that of the respective growth factor alone. Our study has four specific aims: (1) optimize the design, expression, and purification of IGF-1:TTC and GDNF:TTC; (2) characterize the purity, stability, and basic functional activity of these fusion proteins; (3) examine the bioavailability and functional activity of the fusion proteins in rodent CNS following intracerebroventricular or intramuscular administration; and (4) investigate the neuroprotective activity of the fusion proteins in a transgenic rat model of amyotrophic lateral sclerosis (ALS). The experiments in Aim 1 will use recombinant protein expression and purification techniques to generate the fusion proteins, while the studies in Aims 2 and 3 will employ immunocytochemical, Western blot, enzyme immunoassay, and morphometric techniques to assess the functional properties of GDNF:TTC and IGF-1:TTC in cultured ceils and whole animals. Finally, the experiments in Aim 4 will employ transgenic animals in survival and behavioral experiments to assess the neuroprotective effects of GDNF:TTC and/or IGF-1 :TTC in vivo. While the present work may give rise to a new treatment for ALS, the information obtained from our characterization of IGF-1:TTC and GDNF:TTC may lead to the use of these fusion proteins in other neurological disorders as well.