GENE THERAPHY FOR PARKINSON'S DISEASE

描述：（逐字摘自申请人摘要）长期目标 该项目旨在开发针对神经退行性疾病的新型基因疗法。 在之前的支持期间，我们重点关注腺病毒（Ad）载体 递送编码 GDNF（神经胶质细胞系源性神经营养因子）的基因。 Ad-GDNF 注射到进行性进展的黑质或纹状体中 帕金森病保护多巴胺能（DA）神经元的变性模型 对抗神经毒素 6-OHDA 诱导的细胞死亡。 Ad-GDNF 注射到 纹状体还阻止了行为和分子变化的获得 发生在 DA 缺乏的年轻和老年大鼠中。该提案的重点是 假设抗凋亡基因传递也能保护 DA 神经元 体外和体内，并与神经营养物质的递送具有协同作用 因子基因。含有阻止特定细胞凋亡的基因的病毒载体 途径，包括 XIAP、显性失活 caspase-9、bcl-2 和 bclxl 将 单独或单独研究对 DA 神经元存活和功能的影响 与神经营养因子、GDNF 或神经营养因子联合使用。基因将是 使用无辅助 HSV:AAV 递送至培养物和大鼠大脑中的 DA 神经元 混合扩增子载体。这些向量将包含双向 驱动治疗基因和细胞的表达盒 标记基因，绿色荧光蛋白，以允许具体评估 转导的细胞。使用四环素控制表达 反应元件，使得转基因表达在存在以下物质的情况下“开启” 四环素激活剂 (TA) 和无多西环素 (Dox) 的情况。向量 TA 由 DA 细胞的病毒启动子驱动 启动子，酪氨酸羟化酶（TH）。 “治疗”基因的作用将是 使用非神经元细胞、DA 细胞系、MN9D 和原代胎儿 DA 进行研究 用神经毒素、MPP 或 6-OHDA 或其他细胞损伤处理的神经元。 将在以下对象中研究治疗基因的体内作用：1) 接受了胎儿 DA 神经元的移植物，以及 2) 接受了 黑质纹状体投射的进行性 6-OHDA 损伤。可逆性 将通过施用阿霉素来研究效果。对 DA 神经元的影响将是 使用定量形态测量和分子技术进行评估， 行为评估。该项目还旨在继续评估 新一代病毒载体，包括 E2b 删除的广告、完全去除的广告，以及 HSV：AAV 扩增子，用于黑质纹状体的稳定性和表达水平 系统。这些研究涉及儿童医院的研究人员之间的合作 纪念医院和西北大学。医学。学校及相关 开发针对神经退行性疾病和损伤的新疗法 中枢神经系统。

DESCRIPTION: (Verbatim from the Applicant's Abstract) The long-term goal of this project is to develop novel gene therapies for neurodegenerative diseases. In the previous support period, we focused on adenoviral (Ad) vectors to deliver the gene encoding GDNF (glial cell line-derived neurotrophic factor). Ad-GDNF injected into either the substantia nigra or striatum of a progressive degeneration model of Parkinson's disease protected dopaminergic (DA) neurons against cell death induced by the neurotoxin 6-OHDA. Ad-GDNF injected into the striatum also prevented the acquisition of behaviors and molecular changes that occurred in DA deficient young and aged rats. This proposal focuses on the hypothesis that anti-apoptotic gene delivery will also protect DA neurons in vitro and in vivo and have a synergistic effect with delivery of neurotrophic factor genes. Viral vectors harboring genes that block specific apoptotic death pathways, including XIAP, a dominant-negative caspase-9, bcl-2 and bclxl will be studied for effects on survival and function of DA neurons either alone or in combination with neurotrophic factors, GDNF or neurturin. Genes will be delivered to DA neurons in culture and in rat brain using helper free HSV:AAV hybrid amplicon vectors. These vectors will incorporate bidirectional expression cassettes that drive both the therapeutic gene and the cellular marker gene, green fluorescent protein, to permit specific evaluation of transduced cells. Expression will be controlled using the tetracycline responsive element such that transgene expression is "on" in the presence of tetracycline activator (TA) and in the absence of doxcycline (Dox). Vectors will be made in which TA is driven by a viral promoter of the DA cellular promoter, tyrosine hydroxylase (TH). Effects of the 'therapeutic' genes will be studied using non-neuronal cells, the DA cell line, MN9D, and primary fetal DA neurons treated with the neurotoxins, MPP+ or 6-OHDA or other cellular insults. In vivo effects of therapeutic genes will be studied in: 1) rats that have received grafts of fetal DA neurons, and 2) rats that have received a progressive 6-OHDA lesion of the nigrostriatal projection. Reversibility of effects will be studied by administration of Dox. Effects on DA neurons will be evaluated using quantitative morphometric and molecular techniques and behavioral evaluations. This project also aims to continue its evaluation of new generation viral vectors, including E2b deleted Ad, totally gutted Ad, and HSV:AAV amplicon, for stability and levels of expression in the nigrostriatal system. The studies involve collaborations among investigators at Children's Memorial Hospital and Northwestern Univ. Med. School and are relevant to the development of novel therapies for neurodegenerative diseases and injuries to the CNS.