REDUCIBLE POLYAMIDO ETHYLENIMINE FOR GLP-1 PLASMID DELIVERY

描述（由申请人提供）：虽然病毒载体在基因治疗中已经显示出很好的效果，但潜在的免疫原性和宿主染色体整合仍然存在，这在人类应用中造成了一个缺点。我们提出了一种新的聚合物载体——含多个二硫键的聚胺亚胺（SS-PAEI）。与传统的高分子量聚乙烯亚胺相比，该聚合物具有更高水平的报告基因表达和更低的细胞毒性。据报道，胰高血糖素样肽1 （GLP-1）的使用完全使2型糖尿病患者的血糖正常化。然而，血浆半衰期太短，治疗使用GLP-1需要连续多次注射。在本应用中，提出构建GLP-1质粒并将其与SS-PAEI一起递送用于治疗2型糖尿病。GLP-1质粒将通过非病毒SS-PAEI载体传递，延长基因传递产生GLP-1的时间。这种SS-PAEI载体提高了转染效率，无免疫原性，使用难度小。它的高转染是由于胞体不稳定和快速降解释放细胞内的DNA。在本应用中，我们将通过选择最佳启动子/增强子和转录因子结合位点来开发有效的GLP-1质粒。用强启动子/增强子构建GLP-1质粒。为了增加核输入，核因子κ B （NFkB）结合位点将被纳入质粒。由于这种核输入主要存在于细胞质中，并且具有核定位信号，因此会增强DNA的核输入。此外，将采用两步转录扩增（TSTA）来增强转录活性。将通过体外转染和2型糖尿病动物模型的体内实验来评估所设计的传递系统的有效性。

DESCRIPTION (provided by applicant): Although viral carriers in gene therapy have shown promising results, potential immunogenicity and host chromosomal integration still remain, which causes a drawback in human application. We propose a new polymer carrier, polyamido ethylenimine containing multiple disulfide bonds (SS-PAEI). This well characterized polymer presented higher level of reporter gene expression with a significantly lower cellular toxicity than conventional high molecular weight polyethylenimine. It has been reported that glucagon-like peptide 1 (GLP-1) administration completely normalized blood glucose in type 2 diabetic patients. However, plasma half life is too short and continuous infusion of multiple injections is required for therapeutic use of GLP-1. In this application, construction of GLP-1 plasmid and its delivery with SS-PAEI are proposed for the treatment of type 2 diabetes. GLP-1 plasmid will be delivered by non-viral SS-PAEI carrier for prolonged time of GLP-1 production by gene delivery. This SS-PAEI carrier improves transfection efficiency, non-immunogenic and less difficult to use. Its high transfection is due to cytosomal destabilization and fast degradation to release DNA in the cells. In this application, we will develop an effective GLP-1 plasmid by selecting the optimal promoter/enhancer and transcription factor binding sites. GLP-1 plasmid will be constructed with strong promoter/enhancer. To increase nuclear import, the nuclear factor kappa B (NFkB) binding sites will be incorporated into plasmid. Since this nuclear import is mainly presented in cytoplasm and has nuclear localization signal, it will enhance nuclear import of DNA. In addition, two step transcriptional amplification (TSTA) will be employed to enhance transcriptional activity. The effectiveness of the designed delivery system will be evaluated by both in vitro transfection and in vivo experiment with type 2 diabetic animal model.