Non-viral Delivery of Neurotrophic Factors to the CNS

描述(申请人提供)：治疗肌萎缩侧索硬化症(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生物利用度差的阻碍。因此，尽管胶质细胞源性神经营养因子(GDNF)和胰岛素样生长因子-1(IGF-1)都对实验动物受损的运动神经元具有强大的存活促进作用，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获取不足的阻碍。我们推测，破伤风毒素片段C(TTC)与GDNF或IGF-1的基因或化学融合将通过与TTC的神经细胞结合特性相关的一个或多个机制来促进生长因子向运动神经元的输送。因此，该项目的主要目标是评估在ALS的实验动物模型中，IGF-1：TTC和GDNF：TTC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；以及(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。AIM 1中的实验将使用重组蛋白表达和纯化技术来产生融合蛋白，而AIMS 2和3中的研究将使用免疫细胞化学、蛋白质印迹、酶免疫分析和形态计量学技术来评估GDNF：TTC和IGF-1：TTC在培养细胞和整个动物中的功能特性。最后，目标4中的实验将在存活和行为学实验中使用转基因动物，以评估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可能会导致这些融合蛋白在其他神经疾病中的使用。

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.