IN VIVO EVALUATION OF CNS GENE THERAPY USING ANIMAL MODELS

该项目的总体目标是使用动物模型， 神经病性溶酶体贮积症（SD），以开发和评估前 中枢神经系统（CNS）靶向基因的体内和体内方法 交付. 为了促进这些研究，我们建立了育种 患有I型粘多糖样变性（fMPS I）和GM 2的猫的菌落 神经节苷脂沉积症（fGM 2）和构建的“敲除”小鼠模型 尼曼-匹克病（mNPD）和辛德勒病（mSD）。 具体 该项目的目标是：1）表征生物化学 异常、神经病理学和临床过程。 将研究每种鼠疾病的自然史， 残留酶活性和底物积累的影响将是 记录，并通过以下方式评估和定量神经病理学： 形态定量分析 这些研究将提供必要的基线 可以用来评估治疗干预效果的数据。 2)从造血的角度比较- 造血干细胞移植后， 移植（HSCT） 将获得造血干细胞（HSC） 来自正常小鼠和猫，并用表达 β-半乳糖苷酶（Betagal）活性。 HSCT将在子宫内进行， 在新生儿和发育成熟的动物中， 是否有年龄相关的“机会之窗”， HSC衍生的细胞进入CNS。 将处死移植动物 在移植后的不同时间， 表达来评估移植的细胞的持久性和迁移性。 细胞 3)评估生化、病理和临床 离体基因治疗在神经病性LSD动物中的有效性 模型 溶酶体过表达/分泌盒（在2004年开发） 项目1和2）将被插入逆转录病毒载体，并用于 来自神经病性LSD动物模型的HSCs。 自体HSCT 将使用CNS的最佳条件在受累动物中进行 进入和生化，病理和临床有效性 将对这种治疗方法进行监测。 我们还将评估 离体 将溶酶体过表达/分泌载体递送到 使用神经细胞移植策略的受影响动物的CNS 在项目3中开发。 4)评价体内CNS- 使用动物模型系统的靶向基因递送和治疗。 利用项目1-3中开发的载体和投放系统， 溶酶体过表达/分泌载体将被直接递送 通过短暂的血液破坏进入受影响动物的大脑 脑屏障通过高渗甘露醇或直接注入 脑室内或蛛网膜下注射。 生物化学，病理学 这些治疗方法的临床效果将是 评估。

The overall objective of this project is to use animal models of neuropathic lysosomal storage disorders (SDs) to develop and evaluate ex vivo and in vivo approaches to central nervous system (CNS)-targeted gene delivery. To facilitate these studies, we have established breeding colonies of cats with mucopolysaccharidosis Type I (fMPS I) and GM2 gangliosidosis (fGM2) and constructed 'knock out' mouse models of Type A Niemann-Pick disease (mNPD) and Schindler disease (mSD). The specific aims of this project are to: 1) Characterize the biochemical abnormalities, neural pathology and clinical course of the murine models. The natural history of each murine disease will be studied, the levels of residual enzymatic activity and substrate accumulation will be documented, and the neuropathology will be assessed and quantitated by morphometric analysis. These studies will provide essential baseline data by which one can evaluate the effects of therapeutic intervention. 2) Compare the entrance, migration and persistance of hematopoietically- derived cells in the CNS following hematopoietic stem cell transplantation (HSCT). Hematopoietic stem cells (HSC) will be obtained from normal mice and cats and marked with retroviral vectors expressing Beta-galactosidase (Betagal) activity. HSCT will be performed in utero, in neonates and in developmentally mature animals in order to evaluate whether there are age dependent 'windows of opportunity' during which HSC-derived cells enter the CNS. Transplanted animals will be sacrificed at various times post-engraftment and their brains analyzed for Betagal expression to assess the persistance and migration of the transplanted cells. 3) Evaluate the biochemical, pathological and clinical effectiveness of ex vivo gene therapy in the neuropathic LSD animal models. Lysosomal overexpression/secretion cassettes (developed in Projects 1 and 2) will be inserted into retroviral vectors and used to transduce HSCs from the neuropathic LSD animal models. Autologous HSCT will be performed in affected animals using optimal conditions for CNS entry and the biochemical, pathological and clinical effectiveness of this therapeutic approach will be monitored. We will also evaluate the ex vivo delivery of lysosomal overexpression/secretion vectors into the CNS of affected animals using the neural cell transplantation strategies developed in Project 3. 4) Evaluate the effectiveness of in vivo CNS- targeted gene delivery and therapy using the animal models systems. Using the vectors and delivery systems developed in Projects 1-3, lysosomal overexpression/secretion vectors will be delivered directly into the brains of affected animals by transient disruption of the blood- brain barrier via carotoid infusion of hyperosmolar mannitol or direct intraventricular or subarachnoid injections. The biochemical, pathologic and clinical effectiveness of these therapeutic approaches will be evaluated.