Lecerf J M, Shirley T L, Zhu Q, Kazantsev A, Amersdorfer P, Housman D E, Messer A, Huston J S
IntraImmune Therapies, Inc., Lexington, MA 02215, USA.
Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4764-9. doi: 10.1073/pnas.071058398.
This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein-protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 N-terminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias.
本研究旨在测试细胞内抗体（intrabodies）作为阻断亨廷顿舞蹈病（HD）发病机制手段的用途。HD的特征是亨廷顿蛋白N端附近的多聚谷氨酰胺异常延长，这会诱导亨廷顿蛋白或其含第1外显子的片段发生病理性蛋白质-蛋白质相互作用并形成聚集体。从一个大型人噬菌体展示文库中筛选出一种对亨廷顿蛋白的17个N端残基具有特异性的单链Fv（sFv）抗体，这些残基与HD第1外显子中的多聚谷氨酰胺相邻。在该疾病的细胞模型中对这种抗亨廷顿蛋白sFv细胞内抗体进行了测试，在该模型中亨廷顿蛋白第1外显子已与绿色荧光蛋白（GFP）融合。转染细胞中扩增重复的HD-多聚谷氨酰胺-GFP的表达显示出与人类HD病理学相似的核周聚集，随着多聚谷氨酰胺长度增加而恶化；这些转染细胞中的聚集体数量为该研究提供了一个可量化的HD模型。与缺乏细胞内抗体的对照相比，抗亨廷顿蛋白sFv细胞内抗体与异常的亨廷顿蛋白-GFP融合蛋白共表达显著减少了聚集体的数量。与核定位信号融合的抗亨廷顿蛋白sFv将亨廷顿蛋白类似物重新靶向到细胞核，这进一步证明了抗亨廷顿蛋白sFv的特异性及其重定向第1外显子亚细胞定位的能力。这项研究表明，细胞内抗体介导的异常神经元蛋白调节可能有助于治疗诸如HD、阿尔茨海默病、帕金森病、朊病毒病和脊髓小脑共济失调等神经退行性疾病。
