GENE THERAPHY FOR PARKINSON'S DISEASE

描述：（申请人摘要中的逐字记录） 该项目旨在为神经退行性疾病开发新的基因疗法。 在之前的支持期间，我们专注于腺病毒（Ad）载体， 递送编码GDNF（胶质细胞系衍生的神经营养因子）的基因。 将Ad-GDNF注射到进行性黑质或纹状体中， 帕金森病变性模型对多巴胺能神经元的保护作用 抗神经毒素6-OHDA诱导的细胞死亡。Ad-GDNF注射入 纹状体也阻止了行为和分子变化的获得， 在DA缺乏的年轻和老年大鼠中发生。该提案的重点是 假设抗凋亡基因传递也将保护DA神经元， 并与神经营养素的递送具有协同作用 因子基因携带阻断特异性凋亡性死亡的基因的病毒载体 包括XIAP、显性负性caspase-9、bcl-2和bclxl在内的信号通路， 研究对单独或单独的DA神经元的存活和功能的影响。 与神经营养因子、GDNF或neurturin组合。基因会 使用无辅助的HSV：AAV递送至培养物和大鼠脑中的DA神经元 杂交扩增子载体。这些载体将包含双向 驱动治疗基因和细胞表达的表达盒 标记基因，绿色荧光蛋白，以允许特异性评估 转导的细胞。将使用四环素控制表达 在一些实施方案中，转基因表达是“开启”的， 四环素激活剂（TA）和不存在多西环素（Dox）。向量 其中TA由DA细胞的病毒启动子驱动， 启动子，酪氨酸羟化酶（TH）。“治疗性”基因的效果将是 研究使用非神经元细胞，DA细胞系，MN 9D，和原代胎儿DA 用神经毒素、MPP+或6-OHDA或其它细胞损伤处理的神经元。 将在以下动物中研究治疗基因的体内作用：1）患有以下疾病的大鼠： 接受胚胎DA神经元移植，和2）大鼠已经接受了一个 黑质纹状体投射的进行性6-OHDA病变。可逆性 将通过施用Dox来研究效果。对DA神经元的影响将是 使用定量形态测定和分子技术进行评价， 行为评估该项目还旨在继续评价 新一代病毒载体，包括E2 b缺失的Ad、完全消化的Ad，以及 HSV：AAV扩增子，用于黑质纹状体中的稳定性和表达水平 系统这些研究涉及儿童研究所的研究人员之间的合作。 纪念医院和西北大学医学院，并与 神经退行性疾病和损伤的新疗法的开发， CNS。

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.