TET regulated vectors for Parkinson's disease

该项目的长期目标是开发可用作基因治疗载体的病毒载体，以治疗帕金森病（PD）以及其他慢性神经退行性疾病。这样的载体需要掺入启动子元件，该启动子元件允许通过外周给药有效和安全地调节治疗基因。所提出的实验将集中在四环素（泰特）调节的启动子系统的背景下，重组腺相关病毒载体（AAV）和tentiviral载体， 基于人类免疫缺陷病毒（HIV）。将制备一组AV和HIV病毒载体，其中包含由tet调节的启动子驱动的细胞标记物和治疗基因，这些启动子可以通过给予泰特类似物强力霉素（Dox）来打开或关闭。包括人源化绿色荧光蛋白（hrGFP）和非免疫原性基因大鼠碱性磷酸酶（rAP）的细胞标记基因将用于评估载体设计的效率和适用性。将采用定量分析（包括流式细胞术、实时定量逆转录酶-聚合酶链反应（QRT-PCR）、ELISA和计算机形态测定）评估和比较细胞培养物和大鼠黑质纹状体系统中的载体。体内研究将在正常完整大鼠脑和6-OHDA进行性损伤的PD大鼠模型中克隆，以确定与PD相关的损伤是否会影响疗效 含有受调控启动子的载体。这些研究还将确定胶质细胞源性神经营养因子（GDNF）基因治疗在这种PD大鼠模型中的治疗效果在多大程度上是可逆的。使用tet调节载体的GDNF基因递送对多巴胺神经元的影响将使用定量形态测量、分子和行为评价来评价。所有研究都将涉及与该联盟中的Lowenstein和Federoff实验室合作评估宿主免疫反应和载体的染色体效应。成功产生病毒载体，满足大鼠CNS中严格调控、长期表达和可调控性的要求，并具有最小的宿主免疫应答 将推进到PD的非人灵长类临床前试验。

The long-term goal of this project is to develop viral vectors that can be used as gene therapy vehicles to treat Parkinson's disease (PD), as well as other chronic neurodegenerative diseases. Such vectors require the incorporation of a promoter element that permits effective and safe regulation of the therapeutic gene through peripheral drug administration. The proposed experiments will focus on tetracycline(tet)-regulated promoter systems in the context of recombinant adeno-associated viral vectors (AAV) and tentiviral vectors which are based on the human immunodeficiency virus (HIV). A set of AAV and HIV viral vectors will be made that incorporate cellular marker and therapeutic genes driven by tet-regulated promoters that can be turned-on or turned-off by administration of the tet analog doxycycline (Dox). Cellular marker genes including humanized green fluorescent protein (hrGFP) and an non-immunogenic gene, rat alkaline phosphatase (rAP), will be used to assess the efficiency and suitability of the vector designs. Quantitative assays including flow cytometry, real-time, quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR), ELISA and computerized morphometry will be applied to assess and compare vectors in cell culture and in the rat nigrostriatal system. In vivo studies will be clone in both normal intact rat brain and in the 6-OHDA progressively lesioned rat model of PD to determine whether damage related to PD will affect the efficiency of vectors containing regulated promoters. These studies will also determine to what extent the therapeutic effects of glial cell line-derived neurotrophic factor (GDNF) gene therapy in this rat model of PD are reversible. Effects of GDNF gene delivery using tet-regulated vectors on dopamine neurons will be evaluated using quantitative morphometric, molecular and behavioral evaluations. All studies will involve assessment of host immune reactions and chromosomal effects of the vectors in collaboration with the Lowenstein and Federoff labs in this consortium. The successful generation of a viral vector that fulfills the requirements of tight regulation, long-term expression and regulatability with minimal host immune responses in the rat CNS will be advanced to non-human primate preclinical trials for PD.