Pathogenesis and possible therapies modelled in ovine Batten disease

描述（由申请人提供）：神经元蜡样脂褐质沉积症（NCL，Batten病）是儿童的毁灭性遗传性致命性神经退行性疾病。目前没有有效的治疗方法，姑息治疗很困难。在新西兰绵羊中对CLN6形式的研究已经证明在理解生物化学和临床前病理学方面是非常宝贵的。这些绵羊具有类似人类的大脑解剖学、生理学和遗传组织，并显示出以皮质萎缩为主的类似神经病理学，以及类似的临床疾病发展。本项目的总体目标是完成对病毒载体基因疗法体内试验的理解，以便开始。分子生物学、生物化学和免疫组织学实验将确定校正所需的基因产物及其亚细胞驻留位点。将用慢病毒和腺相关病毒衍生的载体测试神经元培养物，以确定将基因转导到绵羊神经元和神经胶质细胞中的适用性。产前和临床前神经病理学研究表明，胶质细胞活化在这种疾病的发病机制中起主要作用。进一步的研究将确定神经胶质细胞活化和炎症的级联反应，以确定关键步骤，从而确定最有效的干预点。将在体内测试用抗炎药物长期治疗对绵羊疾病发展的有效性。病理学研究还表明，在受影响绵羊的大脑中，神经发生和细胞从脑室下区迁移到受影响区域的时间延长。其程度将通过体内新细胞的BrdU标记和抗体检测来确定。最后，通过将携带报道基因的优选载体靶向注射到受影响的羊脑中并检测基因表达的扩散，来确定这种迁移对于将病毒载体中的基因携带到需要它的区域的有用性。这些研究旨在提供直接在绵羊上测试基因治疗的所有要求，并结合抑制炎症。靶细胞将与与其相关的CLN 6基因产物一起被沿着鉴定。治疗的时间窗将与抑制炎症在减缓疾病进程中的作用一起沿着定义。最有效的病毒载体将被确定，并建立注射到SVZ的技术。Revelance：该项目旨在获得知识，以确定在大型动物形式的Batten病中的现实疗法，该疗法可以测试是否可能用于人类。所获得的大部分知识将与其他疾病和其他治疗策略相关，如阿尔茨海默病和干细胞移植。

DESCRIPTION (provided by applicant): The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are devastating inherited fatal neurodegenerative diseases of children. At present there are no effective therapies and palliative care is difficult. Studies on a CLN6 form in New Zealand sheep have proved invaluable in understanding the biochemistry and preclinical pathology. These sheep have a human-like brain anatomy, physiology and genetic organization, and display similar neuropathology dominated by cortical atrophy, and a similar development of clinical disease. The overall aim of this project is to complete understanding to a point where in vivo trials of viral vector gene therapies can begin. Molecular biology, biochemistry and immunohistological experiments will define the gene product required for correction and its subcellular site of residence. Neuron cultures will be be tested with lenti- and adeno-associated virally derived vectors to determine suitability for transduction of the gene into sheep neurons and glial cells. Prenatal and preclinical neuropathology studies indicated that glial activation has a primary role in the pathogenesis of this disease. Further studies will define the cascade of glial cell activation and inflammation, to determine the critical steps and thus determine the most effective point for intervention. The effectiveness of chronic treatment with anti-inflammatory drugs on the development of the disease in sheep will be tested in vivo. Prolonged neurogenesis and migration of cells from the subventricular zone to the affected areas in the brains of affected sheep was also indicated in the pathology studies. The extent of this will be established by BrdU labeling of new cells in vivo and antibody detection. Finally the usefulness of this migration for carrying a gene in viral vectors to the areas where it is required will be determined by targeted injection of the preferred vector carrying a reporter gene into affected sheep brains and detection of the dispersal of gene expression. These studies are aimed at providing all the requirements for directly testing gene therapy on the sheep, in combination with suppression of inflammation. The target cells will have been identified along with the CLN6 gene product relevant to them. The time window for therapy will have been defined along with the role of suppression of inflammation in slowing the course of the disease. The most effective viral vector will have been identified and the techniques for injection into the SVZ established. REVELANCE: This project intends to gain knowledge to define realistic therapies in a large animal form of Batten disease that can be tested for possible human use. Much of the knowledge gained will be relevant to other diseases and other treatment strategies, such as Alzheimer's disease and stem cell transplantation.