Intrabodies as novel neurological therapeutics

该提议的目标是优化工程化的细胞内抗体（胞内抗体）作为新的免疫调节剂。 用于治疗亨廷顿病（HD）的临床试剂和药物发现工具， 与其他由错误折叠蛋白引起的神经退行性疾病具有广泛的长期相关性。 胞内抗体利用抗体的靶特异性与胞内蛋白形成复合物， 已经在进行治疗癌症和艾滋病的临床试验。研究设计始于 用单链Fv抗亨廷顿蛋白（htt）胞内抗体（scFv C4）进行的体内测试显示， 在细胞系、器官型切片培养物和果蝇HD中显著拯救HD表型 模型;加上一个较新的单域胞内抗体（VL 12.3），显示更强的抗htt 原位聚集特性。胞内抗体基因的递送将利用非灵长类慢病毒， 马传染性贫血病毒（EIAV），具有VSVG或Rabies-g包膜，作为一个基因 治疗载体，一些实验与使用AAV载体的递送进行比较，所述AAV载体由 合作者异常核htt积累和聚集的定量测定，DARPP- 32个水平和旷场活动行为将用于评估胞内抗体的疗效 递送到外显子1转基因（R6/1）和Hdh敲入（Q111）小鼠模型的脑中。 相同的近亲遗传背景与此同时，对一小部分较新的病毒进行筛选和测试 胞内抗体将在神经元细胞中使用抗聚集、保护和毒性测定进行 线最成功的新胞内抗体将如上所述进行测试。如果纠正是 由于单个胞内抗体的不完全性，将在细胞和体内测试组合疗法。在 在这些研究结束时，我们将建立胞内抗体的最佳特性， 最终的HD治疗和进一步的药物发现。这些方法一般还应 适用于由异常蛋白质折叠引起的其他神经退行性疾病， 积累，包括阿尔茨海默氏症，帕金森氏症和朊病毒疾病。

The goal of this proposal is to optimize engineered intracellular antibodies (intrabodies) as novel clinical reagents and drug discovery tools for the treatment of Huntington's Disease (HD), with broad, long-term relevance to other neurodegenerative disorders caused by misfolded proteins. Intrabodies use the target specificity of antibodies to form complexes with intracellular proteins, and are already in clinical trials for treatment of cancers and AIDS. The research design starts with in vivo testing with a single-chain Fv anti-huntingtin (htt) intrabody (scFv C4) that has shown significant rescue of HD phenotypes in cell lines, organotypic slice cultures and a Drosophila HD model; plus a newer single domain intrabody (VL 12.3)that shows even stronger anti-htt aggregation properties in situ. Delivery of the intrabody genes will utilize a non-primate lentivirus, Equine Infectious Anemia Virus (EIAV), with either a VSVG or Rabies-g envelope, as one gene therapy vector, with some experiments to compare with delivery using AAV vectors provided by a collaborator. Quantitative assays of abnormal nuclear htt accumulation and aggregation, DARPP- 32 levels, and open field activity behavior will be used to assess the efficacy of the intrabodies delivered to the brains of Exon 1 transgenic (R6/1) and Hdh knock-in (Q111) mouse models on the same inbred genetic background. Simultaneously, screening and testing of a small pool of newer intrabodies will be done using anti-aggregation, protection, and toxicity assays in neuronal cell lines. The most successful of the new intrabodies will then be tested as above. If correction is incomplete with individual intrabodies, combination therapies will be tested in cells and in vivo. At the end of these studies, we will have established the optimal characteristics of intrabodies for eventual HD therapeutics and further drug discovery. These approaches should also be generally applicable for other neurodegenerative diseases that result from abnormal protein folding and accumulation, including Alzheimer's, Parkinson's, and prion diseases.