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(申请人提供)：在美国，有2080万人(占总人口的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岁以下的人群中。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。本研究的主要目标是开发一种纳米颗粒基因载体，用于高效核传递编码白介素10(IL-10)的质粒，以预防1型糖尿病。我们假设阳离子聚合物与聚丙交酯-乙交酯共聚物(PLGA)在阳离子表面活性剂的存在下将产生具有高正Zeta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有效地负载在表面；而负载有正电荷的纳米颗粒具有生物相容性，能够有效地在体外和体内转染细胞和表达蛋白质。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-2-二甲氨基乙酯(DMAEMA)和甲基丙烯酸甲酯(MMA)为单体，随DMAEMA摩尔比的增加，合成了甲基丙烯酸酯共聚物。共聚物的重均相对分子质量将通过凝胶渗透层析进行表征，数均相对分子质量将通过氢核磁共振进行表征。(2)。以十六烷基三甲基溴化铵为阳离子表面活性剂，采用复乳化溶剂挥发法制备了阳离子聚合物/PLGA共混物。通过动态光散射、电子显微镜、Zeta电位测量、紫外光谱、滴定和凝胶电泳法分别对纳米粒子的大小、形状、电荷密度、载质粒效率、缓冲能力和质粒DNA的结构完整性进行表征。(3)。用共聚焦显微镜研究阳离子纳米粒负载香豆素6对人胚胎肾(HEK 293)细胞的细胞内化作用。体外研究阳离子纳米粒对HEK 293细胞的转染效率。用酶联免疫吸附试验检测IL-10和IL-10的表达。(4)。分别采用四甲基偶氮唑盐比色法和光学显微镜评价阳离子纳米粒在体外和体内的生物相容性。(5)。目的：研究阳离子纳米粒体内携带IL-10基因的效率及其对1型糖尿病的预防作用。这项研究将有助于开发一种高效、低毒的非病毒基因递送载体，以便递送编码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0.3%的人口。它是由T淋巴细胞介导的选择性破坏胰腺2细胞造成的，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提高纳米粒子的正Zeta电位和转染率。这项研究将在体外和体内进行动物模型。这项研究将有助于开发一种高效、低毒的非病毒基因递送载体，以便递送编码IL-10基因的质粒来预防1型糖尿病。