REDUCIBLE POLYAMIDO ETHYLENIMINE FOR GLP-1 PLASMID DELIVERY

描述(申请人提供)：尽管病毒携带者在基因治疗中显示出有希望的结果，但潜在的免疫原性和宿主染色体整合仍然存在，这导致了人类应用的缺陷。我们提出了一种新的聚合物载体--含有多个二硫键的聚酰胺乙二亚胺(SS-PAEI)。这种特性良好的聚合物表现出更高水平的报告基因表达，与传统的高分子量聚乙烯亚胺相比，细胞毒性显著降低。有报道称，给予胰升糖素样肽-1(GLP-1)可使2型糖尿病患者的血糖完全正常化。然而，GLP-1的血浆半衰期太短，需要连续多次输注才能治疗。在这一应用中，我们建议构建GLP-1质粒，并将其与SS-PAEI一起用于治疗2型糖尿病。GLP-1将通过非病毒SS-PAEI载体输送，以延长GLP-1基因输送的生产时间。这种SS-PAEI载体提高了转染效率，不具免疫原性，使用起来不那么困难。它的高转染率是由于胞体不稳定和快速降解在细胞内释放DNA所致。在这一应用中，我们将通过选择最佳的启动子/增强子和转录因子结合位点来开发有效的GLP-1质粒。构建的GLP-1表达载体具有强启动子/增强子。为了增加核进口，核因子kappaB(NFkB)结合位点将被整合到质粒中。由于这种核进口主要存在于细胞质中，并具有核定位信号，因此它将增强DNA的核进口。此外，还将使用两步转录扩增(TSTA)来增强转录活性。所设计的递送系统的有效性将通过体外转染法和体内2型糖尿病动物模型实验进行评估。

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.