Intrabodies as novel neurological therapeutics

这项建议的目标是将工程化的细胞内抗体(体内)优化为新的 用于治疗亨廷顿病(HD)的临床试剂和药物发现工具， 与错误折叠的蛋白质引起的其他神经退行性疾病具有广泛、长期的相关性。 体内抗体利用抗体的靶向性与细胞内蛋白形成复合体，并 已经在治疗癌症和艾滋病的临床试验中。研究设计始于 体内试验与单链抗体抗亨廷顿蛋白(HTT)体内(ScFv C4)已显示 在细胞系、器官切片培养和果蝇HD中显著挽救HD表型 模型；加上更新的单域体内(VL 12.3)，显示出更强的抗HTT 原位聚集特性。体内基因的传递将利用非灵长类慢病毒， 马传染性贫血病毒(EIAV)，具有VSVG或狂犬病-G包膜作为一个基因 治疗载体，并通过一些实验与使用由 合作者。核内HTT异常聚集和聚集的定量分析 32级，并使用野外活动行为来评估体内的疗效 外显子1转基因(R6/1)和HDH基因敲入(Q111)小鼠的脑内注射 相同的近亲交配遗传背景。同时，筛选和测试一小部分较新的 体内将使用神经细胞中的抗聚集、保护和毒性测试。 台词。然后，最成功的新体内将进行如上所述的测试。如果修正是 对于不完整的个体体内，联合治疗将在细胞和体内进行测试。在… 在这些研究的结束时，我们将建立最佳的体内特征 最终的HD疗法和进一步的药物发现。这些方法通常也应该是 适用于其他因蛋白质折叠异常而引起的神经退行性疾病 积聚，包括阿尔茨海默氏症、帕金森氏症和普恩病毒疾病。

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.