Generation of gene therapeutic method for diabetic neuropathy by AAV vector

AAV载体产生糖尿病神经病变基因治疗方法

• 批准号: 18590934
• 负责人: YASUDA Hitoshi
• 金额: $ 2.49万
• 依托单位: Shiga University of Medical Science
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590934/
• 关键词: diabetic neuropathy; dorsal root ganglion; phage vector; RT-PCR; cDNA; erythropoietin; neuronal growth factor; AVVベクター; 遺伝子治療; ファージ; RNAi

Phage display is a powerful technology to identify peptide sequence motifs that target a particular tissue or cell type in the body. Coupling such peptides to drugs and genes would enable their targeted delivery to specific cells and tissues in vitro and in vivo. In this research, we isolated peptides that home to mouse dorsal root ganglion (DRG) from a phage library expressing random7-mer peptides fused to a minor coat protein (pIII) of the M13phage. An in vitro biopanning procedure yielded 113phage plaques after 5 cycles of enrichment by incubation with isolated DRG neurons and two cycles of subtraction by exposure to irrelevant cell lines. Analyses of the sequences of this collection identified three peptide clones that occurred repeatedly during the biopanning procedure. Phage-antibody staining revealed that the three peptides bound to DRG neurons of different sizes. To determine if the peptides would recognize neuronal cells in vivo, we injected individual GST-peptide-fusion proteins into the subarachnoid space of mice and observed the appearance of immunoreactive GST in the cytosol of DRG neurons with a similar size distribution as that observed in vitro, indicating that the GST-peptide-fusion proteins were recognized and taken up by different DRG neurons in vivo. The identification of homing peptide sequences provides a powerful tool for future studies on DRG neuronal function in vitro and in vivo, and opens up the possibility of neuron-specific drug and gene delivery in the treatment of diseases affecting DRG neurons.

噬菌体展示是一种强大的技术，用于鉴定靶向体内特定组织或细胞类型的肽序列基序。将这些肽与药物和基因偶联将使它们能够在体外和体内靶向递送到特定的细胞和组织。在这项研究中，我们从噬菌体文库中分离出了与M13噬菌体的次要外壳蛋白（pIII）融合的随机7聚体肽，这些肽归巢于小鼠背根神经节（DRG）。体外生物淘选程序产生113噬菌体噬斑后，5个循环的富集与分离的DRG神经元和两个循环的扣除暴露于无关的细胞系。对该集合的序列的分析鉴定了在生物淘选过程中重复出现的三个肽克隆。噬菌体抗体染色显示，这三个肽结合到不同大小的DRG神经元。为了确定肽是否会在体内识别神经元细胞，我们将单个GST-肽融合蛋白注射到小鼠的蛛网膜下腔中，并观察到在DRG神经元的胞质溶胶中具有与体外观察到的相似的尺寸分布的免疫反应性GST的出现，这表明GST-肽融合蛋白被体内不同的DRG神经元识别和摄取。归巢肽序列的鉴定为未来体外和体内研究背根节神经元功能提供了有力的工具，并开辟了神经元特异性药物和基因递送治疗影响背根节神经元的疾病的可能性。

项目成果

Isolation of specific peptides that home to dorsal root ganglion neurons in mice

• DOI: 10.1016/j.neulet.2008.01.062
• 发表时间: 2008-04
• 期刊: Neuroscience Letters
• 影响因子: 2.5
• 作者: J. Oi;T. Terashima;Hideto Kojima;M. Fujimiya;Kengo Maeda;R. Arai;L. Chan;H. Yasuda;Atsunori Kashiwagi;H. Kimura