Gene Therapy for Neurodegenerative Lysosomal Storage Diseases

描述(申请人提供)：溶酶体储存疾病(LSD)是单基因缺陷，导致分解代谢溶酶体酶缺乏，并在溶酶体中积聚其一个或多个大分子底物。GM1神经节苷脂沉积症是由于溶酶体酶β-半乳糖苷酶(β-Galactoside ase，β-Gal)缺乏所致，导致进行性和致命性神经变性。人类GM1神经节苷脂沉积症根据疾病严重程度和发病年龄分为三种类型，分为婴儿型、青少年型和成年型。基于基因敲除小鼠模型的非凡初步结果，用腺相关病毒载体进行基因治疗是治疗GM1神经节苷脂增多症的一种非常有前途的策略。然而，小鼠的大脑比人类婴儿的大脑小1000到2000倍，复杂程度也低得多，这就要求在小鼠身上获得的结果必须在大脑大小和复杂程度更接近人类的动物身上复制。GM1神经节苷脂增多症的猫科动物模型，其大脑大小只有人类婴儿的15倍，将被用来测试AAV载体在更大、更复杂的大脑中的治疗效果。此外，尽管用于人类临床试验的AAV载体将表达人β-半乳糖苷酶，但初步的小鼠研究使用了小鼠β-半乳糖苷酶基因。因此，在启动人体临床试验之前，有必要进行生物等效性研究，以确保人Betagal基因的功能。本申请中的实验旨在通过以下特定目标将AAV基因治疗推向人类临床试验。在目标1中，将在实质内或脑脊液介导的AAV载体向猫GM1脑内传递AAV载体后进行短期治疗研究。此外，还将在GM1小鼠身上进行短期实验，直接比较人和小鼠的Betagal。在Aim中，将使用疾病进展的临床、行为和生化分析，对GM1猫和小鼠的治疗效果进行2项长期研究。最后，Aim 3将测试体内选择AAV衣壳文库(分子进化)的原理，以确定血管内(Iv)注射后对GM1小鼠中枢神经系统(CNS)具有趋向性的AAV载体，然后在这些小鼠身上测试其治疗效果。将该方法用于静脉输注后产生具有中枢神经系统趋向性的AAV载体的验证，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.