IN VIVO EVALUATION OF CNS GENE THERAPY USING ANIMAL MODELS

这个项目的总体目标是使用动物模型 神经病理性溶酶体储存障碍(SDS)的发展和评估 中枢神经系统靶向基因的体内和体内研究进展 送货。为了促进这些研究，我们建立了育种 黏多糖病I型(FMPS I)和GM2猫的菌落 神经节苷脂沉积症(FGM2)和构建的“基因敲除”型小鼠模型 尼曼-皮克病(MNPD)和辛德勒病(MSD)。具体的 本项目的目标是：1)表征生物化学 小鼠模型的异常、神经病理和临床病程。 将研究每种小鼠疾病的自然历史，水平 剩余的酶活性和底物积累将是 记录下来，神经病理将通过以下方式进行评估和量化 形态计量分析。这些研究将提供基本的基准 可用于评估治疗干预效果的数据。 2)比较血细胞的进入、迁移和耐受性。 造血干细胞后中枢神经系统内的衍生细胞 移植(HSCT)。将获得造血干细胞(HSC) 取自正常小鼠和猫，并用逆转录病毒载体标记，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进行研究，以评估 在此期间，是否存在与年龄相关的“机会之窗” 造血干细胞来源的细胞进入中枢神经系统。移植的动物将被牺牲 在植入后的不同时间对他们的大脑进行Betagal分析 用于评估移植的耐受性和迁移性的表达 细胞。3)生化、病理和临床评价。 体外基因治疗对神经病理性LSD动物的影响 模特们。溶酶体过度表达/分泌盒(开发于 项目1和2)将被插入逆转录病毒载体并用于 从神经性LSD动物模型中转导HSCs。自体造血干细胞移植 将在受影响的动物身上使用中枢神经系统的最佳条件 入选及其生化、病理和临床疗效 这一治疗方法将受到监测。我们还将评估 溶酶体过表达/分泌载体的体外导入 使用神经细胞移植策略的受影响动物的中枢神经系统 在项目3.4中开发)评估体内CNS- 利用动物模型系统进行靶向基因传递和治疗。 使用项目1-3中开发的媒介和交付系统， 溶酶体过表达/分泌载体将直接输送 通过短暂的血液中断进入受影响动物的大脑- 经颈动脉注入高渗甘露醇或直接注入脑屏障 脑室内或蛛网膜下腔注射。生化、病理 这些治疗方法的临床效果将是 已评估。

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.