Gene Therapy for Neurodegenerative Lysosomal Storage Diseases

描述（由申请人提供）：溶酶体贮积病（lsd）是一种单基因缺陷，导致分解代谢溶酶体酶的缺乏和溶酶体中一种或多种大分子底物的积累。GM1神经节脂质沉积症是由溶酶体酶-半乳糖苷酶（betagal）缺乏引起的，导致进行性和致命的神经变性。人类GM1神经节脂质沉积症根据疾病严重程度和发病年龄分为三种类型，分别为婴儿、青少年和成人发病形式。基于敲除小鼠模型的非凡初步结果，腺相关病毒载体的基因治疗是一种非常有前途的治疗GM1神经节脂质病的策略。然而，老鼠的大脑比人类婴儿的大脑小1000 - 2000倍，复杂程度也低得多，这就要求在老鼠身上获得的结果要在大脑大小和复杂程度更接近人类的动物身上复制。具有良好特征的猫GM1神经节脂质病模型，其大脑大小仅为人类婴儿的15倍，将用于测试AAV载体在更大更复杂的大脑中的治疗效果。此外，小鼠初步研究采用小鼠β -半乳糖苷酶cDNA，尽管用于人类临床试验的AAV载体将表达人β -半乳糖苷。因此，在开始人体临床试验之前，有必要进行生物等效性研究，以确保人β -基因cDNA的功能。本应用程序中的实验旨在通过以下具体目标将AAV基因治疗推进到人类临床试验。在Aim 1中，将在实质内或csf介导的AAV载体递送到猫GM1脑后进行短期治疗研究。此外，将在GM1小鼠的短期实验中直接比较人和小鼠的betagal。在Aim 2中，将通过临床、行为和疾病进展的生化分析对GM1猫和小鼠的治疗效果进行长期研究。最后，Aim 3将测试AAV衣壳文库的体内选择原理（分子进化），以鉴定血管内（iv）输注后对GM1小鼠中枢神经系统（CNS）具有趋向性的AAV载体，并测试其在这些小鼠中的治疗效果。验证这种方法在静脉输注后产生具有中枢神经系统取向的AAV载体，将为开发此类载体用于人类应用铺平道路。

DESCRIPTION (Provided by Applicant): Lysosomal storage diseases (LSDs) are single gene defects that result in the deficiency of a catabolic lysosomal enzyme and accumulation of one or more of its macromolecular substrates in the lysosome. GM1 gangliosidosis results from deficiency of the lysosomal enzyme beta-galactosidase (betagal), leading to progressive and fatal neurodegeneration. Human GM1 gangliosidosis occurs as three types based on disease severity and age of onset, classified as infantile, juvenile, and adult-onset forms. Gene therapy with adeno-associated viral vectors is a very promising strategy for treatment of GM1 gangliosidosis based on extraordinary preliminary results in the knockout mouse model. However, the mouse brain is 1,000-2,000 times smaller and much less complex than the brain of a human infant, requiring that results obtained in mice be reproduced in an animal with a brain size and complexity more similar to humans. The well-characterized feline model of GM1 gangliosidosis, with a brain size only 15 times smaller than a human infant, will be utilized to test therapeutic efficacy of AAV vectors in a larger and more complex brain. In addition, preliminary mouse studies employed a mouse beta-galactosidase cDNA, although AAV vectors for human clinical trials will express human betagal. Therefore, it is necessary to perform bio-equivalency studies to ensure the functionality of the human betagal cDNA before initiating human clinical trials. The experiments in this application are designed to advance AAV gene therapy toward human clinical trials through the following specific aims. In Aim 1, short-term therapeutic studies will be performed after intraparenchymal or CSF-mediated delivery of AAV vectors to the feline GM1 brain. Also, a direct comparison of human and mouse betagal will be conducted in short-term experiments in GM1 mice. In Aim 2 long-term studies of therapeutic efficacy in GM1 cats and mice will be conducted using clinical, behavioral, and biochemical assays of disease progression. Finally, Aim 3 will test the principle of in vivo selection of an AAV capsid library (molecular evolution) to identify AAV vectors with tropism for the GM1 mouse central nervous system (CNS) after intravascular (iv) infusion, and then test their therapeutic efficacy in these mice. Validation of this approach to generate AAV vectors with CNS-tropism after iv infusion will pave the way for development of such vectors for human application.