Prevention of diabetes by pDNA encoded with IL-10

描述（由申请人提供）：在美国，2080万人（占人口的7%）患有糖尿病。所有新诊断的I型糖尿病病例中约有75%发生在18岁以下的个体中。1型糖尿病是一种慢性自身免疫性疾病，影响世界人口的0.3%。它是选择性破坏胰腺2-细胞的结果。该研究的主要目标是开发一种纳米颗粒基因递送载体，用于高效核递送编码白细胞介素-10（IL-10）的质粒，以预防1型糖尿病。我们假设阳离子聚合物和聚（丙交酯-共-乙交酯）（PLGA）在阳离子表面活性剂存在下的共沉淀将产生具有高正ζ电位的纳米颗粒，这将促进编码IL-10基因的带负电荷的质粒DNA在表面上的有效装载;负载质粒DNA的带正电荷的纳米颗粒具有生物相容性，能够有效地转染细胞并表达蛋白质，无论是在体外还是在体内。为了验证我们的假设，我们计划研究以下具体目标：（1）。以甲基丙烯酸二甲氨基乙酯（DMAEMA）和甲基丙烯酸甲酯（MMA）为单体，通过增加DMAEMA的摩尔比合成甲基丙烯酸酯共聚物。将通过凝胶渗透色谱法表征共聚物的重均分子量，并通过1H NMR表征数均分子量。（二）、以十六烷基三甲基溴化铵为阳离子表面活性剂，将阳离子聚合物与PLGA共混，采用复乳溶剂蒸发法制备阳离子纳米粒。将分别通过动态光散射、电子显微镜、zeta电位测量、UV分光光度计、滴定法和凝胶电泳表征纳米颗粒的大小、形状、电荷密度、质粒加载效率、缓冲能力和质粒DNA的结构完整性。（三）、利用共聚焦显微镜研究香豆素6阳离子纳米粒对人胚肾细胞（HEK 293）的细胞内化作用。将使用编码IL-10的治疗性质粒研究阳离子纳米颗粒在HEK 293细胞中的体外转染效率。将通过酶联免疫吸附试验定量IL-10的表达。（四）、分别采用MTT法和光学显微镜法评价阳离子纳米粒的体外和小鼠体内生物相容性。（五）、目的研究阳离子纳米粒介导白细胞介素10（IL-10）质粒在小鼠体内的有效性及其对1型糖尿病的预防作用。该研究将有助于开发一种高效、低毒的非病毒基因载体，以递送编码IL-10基因的质粒用于预防1型糖尿病。 1型糖尿病是一种慢性自身免疫性疾病，影响世界人口的0.3%。它是由T淋巴细胞介导的胰腺2-细胞的选择性破坏引起的，这导致身体产生胰岛素的能力逐渐降低。基因递送载体需要特殊的功能来克服细胞外和细胞内的障碍，并确保有效的DNA递送到细胞核。阳离子聚合物和阳离子表面活性剂的使用将协同增强纳米颗粒的正ζ电位和它们的转染效率。研究将在体外和动物模型中进行。该研究将有助于开发一种高效、低毒的非病毒基因载体，以递送编码IL-10基因的质粒用于预防1型糖尿病。

DESCRIPTION (provided by applicant): In the United States, 20.8 million people (7% of the population) suffer from diabetes. About 75% of all newly diagnosed cases of type I diabetes occurs in individuals younger than 18 years of age. Type 1 diabetes is a chronic autoimmune disease affecting 0.3% of world's population. It results from selective destruction of pancreatic 2-cells. The major goal of the proposed research is to develop a nanoparticulate gene delivery vector for highly efficient nuclear delivery of plasmid encoding interleukin-10 (IL-10) for prevention of type 1 diabetes. We hypothesize that the combination of cationic polymer and poly (lactide-co-glycolide) (PLGA) in the presence of a cationic surfactant will produce nanoparticles with high positive zeta potential that will facilitate efficient loading of negatively charged plasmid DNA encoding IL-10 gene on the surface; and the positively charged nanoparticles loaded with plasmid DNA are biocompatible and can efficiently transfect the cells and express the protein both in vitro and in vivo. To test our hypotheses, we plan to study the following specific aims: (1). To synthesize methacrylate copolymers using monomers, 2- dimethyl amino ethyl methacrylate (DMAEMA) and methylmethacrylate (MMA) with increasing molar ratio of DMAEMA. The copolymers will be characterized for weight average molecular weight by gel permeation 1 chromatography and number average molecular weight by H NMR. (2). To prepare cationic nanoparticles using a blend of cationic polymer and PLGA by double emulsion solvent evaporation technique, using cetyl trimethyl ammonium bromide as a cationic surfactant. The nanoparticles will be characterized for size, shape, charge density, plasmid loading efficiency, buffering ability, and structural integrity of plasmid DNA by dynamic light scattering, electron microscopy, zeta potential measurement, UV spectrophotometer, titrimetric, and gel electrophoresis, respectively. (3). To study the cellular internalization in Human Embryonic Kidney (HEK 293) cells by confocal microscopy, using cationic nanoparticles loaded with coumarin 6. In vitro transfection efficiency of cationic nanoparticles in HEK 293 cells will be studied, using a therapeutic plasmid encoding IL-10. The expression of and IL-10 will be quantified by enzyme-linked immunosorbent assay. (4). To evaluate in vitro and in vivo in mice biocompatibility of cationic nanoparticles, using an MTT assay and light microscopy, respectively. (5). To study the efficiency of cationic nanoparticles to deliver plasmid encoding Interleukin-10 in vivo in mice and its ability to prevent the onset of type 1diabetes. The proposed study will contribute towards the development of a high efficiency and low toxicity non-viral gene delivery vehicle in order to deliver plasmid encoding IL-10 gene for prevention of type 1 diabetes. PUBLIC HEALTH RELEVANCE: Type 1 diabetes is a chronic autoimmune disease affecting 0.3% of world's population. It results from selective destruction of pancreatic 2-cells mediated by T lymphocytes which leads to gradual reduction in body's ability to produce insulin. The gene delivery vectors need special features to overcome extracellular and intracellular barriers, and ensure efficient DNA delivery to the nucleus. The use of cationic polymer and cationic surfactant will synergistically enhance the positive zeta potential of nanoparticles and their transfection efficiency. The study would be conducted in vitro and in vivo in animal model. The proposed study will contribute towards the development of a high efficiency and low toxicity non-viral gene delivery vehicle in order to deliver plasmid encoding IL-10 gene for prevention of type 1 diabetes.