Blocking Huntingtin Toxicity by Intrabody

通过体内阻断亨廷顿蛋白毒性

• 批准号: 6957329
• 负责人: XIAO-JIANG LI
• 金额: $ 19.13万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-03 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6957329
• 关键词: Huntington' s disease; antigen antibody reaction; blocking antibody; cell line; gene expression; genetically modified animals; green fluorescent proteins; homopeptide; immunocytochemistry; immunoprecipitation; laboratory mouse; monoclonal antibody; nerve /myelin protein; neuropathology; neuroprotectants; protein binding; protein folding; protein protein interaction; terminal nick end labeling; transfection /expression vector

DESCRIPTION (provided by applicant): Huntington disease (HD) belongs to a neurodegeneration disease family in which selective neuronal loss is caused by misfolded proteins. In HD, a polyglutamine (polyQ) expansion (>37 glutamines) in the N-terminal region of Huntington (htt) causes N-terminal htt fragments to misfold or aggregate, conferring neuropathology. Consistently, N-terminal htt fragments containing an expanded polyQ are able to interact with a number of proteins to mediate multiple pathological pathways. Thus, an important therapy approach is to inhibit the expression of mutant Huntington (htt) or its activity by blocking its abnormal interactions with other important proteins. Intracellular antibody (intrabody) provides a promising approach to achieve this goal, as intrabody is able to interact with an antigen intracellular to block its activity ortoxicity. However, normal htt is required for cell survival, and deletion of htt causes cell degeneration. Thus, a challenge for using the intrabody therapy for HD is to identify an intrabody that can selectively inhibit the toxicity of mutant htt but not interfere with the pivotal function of normal htt. Using the same antigen for an antibody (EM48) that reacts preferentially with misfolded and aggregated htt, we have established a hybridoma cell line that generates a monoclonal antibody (mEM48), which also preferentially reacts with mutant htt. By isolating the gene encoding mEM48 from this cell line, we have generated an intrabody that selectively binds mutant htt. We propose to characterize the protective effect of this intrabody on htt's toxicity in HD cellular models. We will also modify this intrabody to increase its expression level and stability. Finally, we will deliver this intrabody to HD mouse brain via viral vectors and to examine its protection against the neuropathology in HD brain. This proof of principle study will have a broad implication for the treatment of other neurodegeneration diseases that are also caused by misfolded proteins.

描述（由申请人提供）： 亨廷顿疾病（HD）属于神经变性疾病家族，其中选择性神经元丧失是由错误折叠的蛋白质引起的。在HD中，亨廷顿（HTT）的N末端区域中的聚谷氨酰胺（PolyQ）膨胀（> 37个谷氨酰胺）导致N末端HTT片段错误地折叠或聚集，从而赋予神经病理学。一致地，包含扩展的PolyQ的N末端HTT片段能够与许多蛋白质相互作用，以介导多种病理途径。因此，一种重要的疗法方法是通过阻止其与其他重要蛋白质的异常相互作用来抑制突变亨廷顿突变体（HTT）的表达。细胞内抗体（内部）提供了实现此目标的有前途的方法，因为内部象征能够与抗原细胞内相互作用以阻断其活性。但是，细胞存活需要正常的HTT，HTT的缺失会导致细胞变性。因此，对HD使用内部疗法的挑战是确定可以选择性地抑制突变体HTT毒性但不会干扰正常HTT的关键功能的内部象征。我们使用相同的抗原（EM48）与错误折叠和聚集的HTT反应，我们建立了产生单克隆抗体（MEM48）的杂交瘤细胞系，该杂交瘤细胞系也优先与突变体HTT反应。通过从该细胞系中隔离编码MEM48的基因，我们产生了一种选择性结合突变体HTT的内部函数。我们建议表征该内部机构对HTT毒性在HD细胞模型中的毒性的保护作用。我们还将修改此内在以提高其表达水平和稳定性。最后，我们将通过病毒载体向HD小鼠脑输送此内在，并检查其对HD脑中神经病理学的保护。这项原则研究证明将对其他神经退行性疾病的治疗具有广泛的影响，这些疾病也是由错误折叠蛋白引起的。