Prevention of diabetes by pDNA encoded with IL-10

通过编码 IL-10 的 pDNA 预防糖尿病

• 批准号: 7915665
• 负责人: Jagdish Singh
• 金额: $ 7.18万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7915665
• 关键词: 18 year old; 20 year old; Affect; Amines; Animal Model; Autoimmune Process; Biocompatible; Biological Assay; Blood Glucose; Buffers; Cell Nucleus; Cells; Characteristics; Charge; Chromatography; Confocal Microscopy; Coumarins; DNA delivery; Development; Diabetes Mellitus; Diabetes prevention; Electron Microscopy; Embryo; Emulsions; Ensure; Enzyme-Linked Immunosorbent Assay; Gel; Gel Chromatography; Gene Delivery; Glycolic-Lactic Acid Polyester; Goals; Histology; Human; IL10 gene; In Vitro; Individual; Insulin; Insulin-Dependent Diabetes Mellitus; Interferons; Interleukin-10; Kidney; Measurement; Mediating; Methacrylates; Methylmethacrylate; Molecular Weight; Mus; Newly Diagnosed; Nuclear; Pancreas; Plasmids; Play; Polymers; Population; Prevention; Proteins; Research; Role; Serum; Shapes; Solvents; Surface; System; T-Lymphocyte; Techniques; Testing; Th1 Cells; Therapeutic; Tissues; Toxic effect; Transfection; United States; Weight; alpha benzopyrone; ammonium bromide; biomaterial compatibility; cell mediated immune response; chronic autoimmune disease; copolymer; cytokine; density; evaporation; extracellular; gel electrophoresis; gene delivery system; gene therapy; in vivo; light microscopy; light scattering; monomer; nanoparticle; nanoparticulate; non-viral gene delivery; plasmid DNA; poly(DL-lactide); prevent; protective effect; public health relevance; surfactant; tertiary amine; vector; zeta potential

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.

描述（由申请人提供）：在美国，2080 万人（占人口的 7%）患有糖尿病。在所有新诊断的 I 型糖尿病病例中，约 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，影响着世界 0.3% 的人口。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介素 10 (IL-10) 的质粒，从而预防 1 型糖尿病。我们假设，在阳离子表面活性剂存在下，阳离子聚合物和聚丙交酯乙交酯 (PLGA) 的组合将产生具有高正 zeta 电位的纳米颗粒，这将有助于将编码 IL-10 基因的带负电荷的质粒 DNA 有效负载到表面；负载有质粒DNA的带正电荷的纳米颗粒具有生物相容性，可以在体外和体内高效转染细胞并表达蛋白质。为了检验我们的假设，我们计划研究以下具体目标：（1）。以甲基丙烯酸2-二甲基氨基乙酯(DMAEMA)和甲基丙烯酸甲酯(MMA)为单体，随着DMAEMA摩尔比的增加，合成甲基丙烯酸酯共聚物。将通过凝胶渗透1色谱法表征共聚物的重均分子量，并通过1H NMR表征共聚物的数均分子量。 （2）。以十六烷基三甲基溴化铵为阳离子表面活性剂，采用双乳液溶剂蒸发技术，将阳离子聚合物与PLGA共混物制备成阳离子纳米粒子。将分别通过动态光散射、电子显微镜、zeta 电位测量、紫外分光光度计、滴定法和凝胶电泳来表征纳米颗粒的尺寸、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。 （3）。使用负载香豆素 6 的阳离子纳米粒子，通过共聚焦显微镜研究人胚肾 (HEK 293) 细胞的细胞内化。将使用编码 IL-10 的治疗性质粒研究 HEK 293 细胞中阳离子纳米粒子的体外转染效率。 IL-10和IL-10的表达将通过酶联免疫吸附测定进行定量。 （4）。分别使用 MTT 测定和光学显微镜评估阳离子纳米粒子在小鼠体内的体外和体内生物相容性。 （5）。研究阳离子纳米颗粒在小鼠体内递送编码白细胞介素 10 的质粒的效率及其预防 1 型糖尿病发病的能力。拟议的研究将有助于开发高效、低毒的非病毒基因传递载体，以传递编码 IL-10 基因的质粒来预防 1 型糖尿病。 公共卫生相关性：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 细胞选择性破坏引起的，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，并确保有效地将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粒子的正zeta电位及其转染效率。该研究将在动物模型的体外和体内进行。拟议的研究将有助于开发高效、低毒的非病毒基因传递载体，以传递编码 IL-10 基因的质粒来预防 1 型糖尿病。