Intrabodies as novel neurological therapeutics

描述（由申请人提供）：本提案的目标是优化工程化细胞内抗体（胞内抗体），作为治疗亨廷顿病（HD）的新型临床试剂和药物发现工具，与错误折叠蛋白引起的其他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，并已在临床试验中用于治疗癌症和艾滋病。该研究设计从使用单链Fv抗亨廷顿蛋白（htt）胞内抗体（scFv C4）的体内测试开始，该抗体已在细胞系、器官型切片培养物和果蝇HD模型中显示出对HD表型的显著拯救;加上一种较新的单结构域胞内抗体（VL 12.3），该抗体在原位显示出更强的抗htt聚集特性。胞内抗体基因的递送将利用具有VSVG或Rabies-g包膜的非灵长类慢病毒，马传染性贫血病毒（EIAV）作为一种基因治疗载体，其中一些实验与使用合作者提供的AAV载体的递送进行比较。将使用异常核htt积累和聚集、DARPP- 32水平和旷场活动行为的定量测定来评估递送至相同近交遗传背景的外显子1转基因（R6/1）和Hdh敲入（Q111）小鼠模型的脑的胞内抗体的功效。同时，将在神经元细胞系中使用抗聚集、保护和毒性测定来筛选和测试一小部分较新的胞内抗体。最成功的新胞内抗体将如上所述进行测试。如果单个胞内抗体的校正不完全，则将在细胞和体内测试组合疗法。在这些研究结束时，我们将建立胞内抗体的最佳特征，用于最终的HD治疗和进一步的药物发现。这些方法也应该普遍适用于由异常蛋白质折叠和积累引起的其他神经退行性疾病，包括阿尔茨海默病、帕金森病和朊病毒病。

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.