CNS Gene Therapy for Mucopolysaccharidosis IIIB w/ AAV

描述(由申请人提供)： 粘多糖病III B(Sanfilippo type 13，MPS IIIB)是一种由于α-N-乙酰氨基葡萄糖苷酶(NaGlu)遗传性缺陷引起的溶酶体储存障碍。该病的特点是轻度躯体疾病和严重的神经变性，大多数患者在6-10岁之前，社会和适应能力迅速和进行性恶化，导致过早死亡。目前还没有治疗MPS III B患者中枢神经系统(CNS)障碍的方法，这种疾病通常是过早死亡的原因。腺相关病毒(AAV)可感染多种组织器官，且致病机制未知，是一种很有前途的基因递送系统。在这个项目中，AAV介导的对MPS III B中枢神经系统疾病的基因治疗将使用基因敲除小鼠模型进行研究。构建了由Cw启动子和内源性脑内启动子神经元特异性烯醇化酶(NSE)启动子驱动的人NaGlu基因的AAV载体，并在MPS IIIB细胞培养和小鼠脑内证实了NaGlu的高效表达和重组NaGlu对溶酶体储存的纠正作用。这些载体将通过直接显微注射的方式进入MPS III B小鼠的多个脑区，以研究rNaGlu在体内更有效的表达和分布，以及注射后溶酶体在脑内储存的纠正。此外，由于基因治疗载体在大脑中的有限分布是中枢神经系统治疗的最大障碍之一，因此将进行研究以开发更有效的脑内基因输送方式。通过直接显微注射将含有增强型绿色荧光蛋白基因的AAV载体导入小鼠多个脑区，以a)比较不同AAV血清型(1、2和5)载体介导的基因表达的分布；b)研究不同注射途径，特别是外周注射途径携带的基因表达在小鼠脑内的扩散；以及c)通过改变AAV病毒载体的传递介质来研究AAV载体的传播；d)。单次注射进行多点输注，增加单次注射后AAV载体的分布。本课题的目的是利用小鼠模型研究AAV介导的基因治疗MPS III B儿童中枢神经系统疾病的可行性，并实现AAV载体在中枢神经系统的广泛分布。该项目的长期目标是开发治疗MPS IIIB患者中枢神经系统疾病的新疗法。

DESCRIPTION (provided by applicant): Mucopolysaccharidosis III B (Sanfilippo type 13, MPS IIIB) is a lysosomal storage disorder due to the inherited deficiency of a-N-acetylglucosaminidase (NaGlu). The disease is characterized by mild somatic disease with severe neurological degeneration in most of patients by 6-10 years of age with rapid and progressive deterioration of social and adaptive abilities leading to premature death. No treatment is currently available for the central nervous system (CNS) disorder of MPS III B patients, which is usually the cause of premature death. Adeno-associated virus (AAV) has been shown to provide a promising gene delivery system for its ability of infecting wide range of tissues/organs and with no known pathogenesis in human. In this project, AAV-mediated gene therapy for the CNS disease of MPS III B is to be studied using a knock-out mouse model. Two AAV-vectors, containing human NaGlu cDNA driven by a CW promoter or the endogenous brain promoter, neuron specific enolase (NSE) promoter, have been constructed and have shown efficient expression of functional NaGlu and the correction of lysosomal storage by recombinant NaGlu in vitro in MPS IIIB cell cultures and in vivo in mouse brain. The vectors are to be delivered into multiple brain areas of MPS III B mice by direct microinjection, to study more efficient in vivo expression and distribution of rNaGlu and the correction of lysosomal storage in the brain after the injection. In addition, studies will be conducted to develop more efficient means of gene delivery into brain because limited distribution of gene therapy vectors in brain is one of the biggest obstacles for CNS therapies. It will be achieved by delivering AAV vectors containing an enhanced green fluorescent protein gene into multiple mouse brain areas by direct microinjection, to a) compare the distribution of gene expression mediated by different AAV serotype (1, 2 and 5) vectors; b) study the dispersion of gene expression delivered into mouse brain through different injection route, especially peripheral delivery; and c) study the spread of AAV vectors by modifying the delivery media of AAV viral vectors; d). Conduct multiple site infusion by a single injection, to increase the distribution of AAV vectors after a single injection. The goal of this project is to study the feasibility of using AAV-mediated gene therapy to treat the CNS disease in MPS III B children and to achieve broad distribution of AAV vectors in CNS, using mouse model. The long-term goal of this project is to develop novel therapy for CNS disease in MPS IIIB patients.