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（体内抗体）作为治疗亨廷顿病（HD）的新型临床试剂和药物发现工具，与其他由错误折叠蛋白质引起的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目标特异性与细胞内蛋白形成复合物，并已用于治疗癌症和艾滋病的临床试验。研究设计从体内测试开始，使用单链Fv抗亨廷顿蛋白（htt）体内（scFv C4）进行测试，该测试在细胞系、器官型切片培养和果蝇HD模型中显示出对HD表型的显着拯救；加上一个新的单域体内（VL 12.3），在原位显示出更强的抗ht聚集特性。体内基因的递送将利用一种非灵长类慢病毒——马传染性贫血病毒（EIAV）作为一种基因治疗载体，该病毒带有VSVG或狂犬病-g包膜，并进行一些实验，与使用合作者提供的AAV载体进行递送进行比较。在相同的近交遗传背景下，通过定量分析异常核htt积累和聚集、DARPP- 32水平和开放场活动行为来评估外显子1转基因（R6/1）和Hdh敲入（Q111）小鼠模型的体内递送到大脑的效果。同时，将在神经细胞系中使用抗聚集、保护和毒性试验来筛选和测试一小部分新的体内细胞。然后，最成功的新内体将按上述方法进行测试。如果单个体内的校正不完全，联合疗法将在细胞和体内进行试验。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确定体内的最佳特征。这些方法也应该普遍适用于其他由异常蛋白质折叠和积累引起的神经退行性疾病，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.