Intrabodies as novel neurological therapeutics

描述(由申请人提供)：这项建议的目标是优化工程细胞内抗体(体内)作为治疗亨廷顿病(HD)的新型临床试剂和药物发现工具，与由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，并已在治疗癌症和艾滋病的临床试验中。研究设计从体内测试开始，用单链抗体抗亨廷顿素(HTT)体内(ScFv C4)，它已经显示出在细胞系、器官型切片培养和果蝇HD模型中HD表型的显著挽救；加上较新的单域体内(VL 12.3)，它在原位显示出更强的抗HTT聚集特性。体内基因的传递将利用具有VSVG或狂犬病-g包膜的非灵长类慢病毒-马传染性贫血病毒(EIAV)作为基因治疗载体，并进行一些实验，以与合作者提供的AAV载体传递进行比较。核内HTT异常聚集和聚集、DARPP-32水平和旷场活动行为的定量分析将被用于评估在相同近交遗传背景下向外显子1转基因(R6/1)和HDH敲入(Q111)小鼠模型脑内注射HTT的效果。同时，将在神经元细胞系中使用抗聚集、保护和毒性测试来筛选和测试较新的体内。然后，最成功的新体内将进行如上所述的测试。如果个别体内的矫正不完整，联合治疗将在细胞和体内进行测试。在这些研究结束时，我们将为最终的HD治疗和进一步的药物发现建立最佳的体内特征。这些方法也应该普遍适用于其他由蛋白质折叠和堆积异常引起的神经退行性疾病，包括阿尔茨海默氏症、帕金森氏症和普里恩病。

DESCRIPTION (provided by applicant): 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.