Generation of gene therapeutic method for diabetic neuropathy by AAV vector

噬菌体展示是一项强大的技术，可以识别针对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结合，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小鼠背根神经节（DRG）的肽，这些肽表达与m13噬菌体的次要外壳蛋白（pIII）融合的随机7聚肽。在体外生物筛选过程中，通过与分离的DRG神经元孵育5个周期的富集和暴露于无关细胞系的2个周期的减去，获得了113个噬菌体斑块。对该集合序列的分析确定了在生物筛选过程中反复发生的三个肽克隆。噬菌体抗体染色显示，这三种肽与不同大小的DRG神经元结合。为了确定GST-肽融合蛋白在体内是否能识别神经元细胞，我们将个体GST-肽融合蛋白注射到小鼠蛛网膜下腔，观察到DRG神经元胞浆中免疫反应性GST的出现，其大小分布与体外观察到的相似，表明GST-肽融合蛋白在体内被不同的DRG神经元识别和吸收。归巢肽序列的鉴定为进一步研究DRG神经元在体内和体外的功能提供了有力的工具，并为DRG神经元疾病的神经元特异性药物和基因传递治疗开辟了可能性。

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.