Novel Approach to Oral Gene Therapy for Chronic Granulomatous Disease

慢性肉芽肿性疾病口服基因治疗的新方法

• 批准号: 7740349
• 负责人: PETER E NEWBURGER
• 金额: $ 20.49万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-10 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7740349
• 关键词: 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one; Abbreviations; Acetates; Animals; Bacteria; Biological Assay; Cell Wall; Chronic Granulomatous Disease; Clinical Trials; Complementary DNA; Complex; Cytochromes b; DNA; DNA delivery; Defect; Development; Disease; EWS/FLI 1 Type 1 antisense oligonucleotide; Evaluation; Fluorescence Microscopy; Gene Expression; Generations; Genes; Glucans; Glycoproteins; Host Defense; Human; Immunologic Deficiency Syndromes; In Vitro; Infection; Influenza Hemagglutinin; Inherited; Investigation; Knock-out; Knockout Mice; Label; Lead; Link; Location; Long Terminal Repeats; Mannans; Measurement; Methods; Modeling; Molecular; Mus; Mutation; NADPH Oxidase; Nitroblue Tetrazolium; Oral; Peptide Elongation Factor 1; Peritoneal; Peroxides; Phagocytes; Phorbol; Phorbols; Polymers; Reactive Oxygen Species; Recurrence; Research; System; Technology; Testing; Transfection; Translational Research; Yeasts; base; cDNA Expression; catalase; controlled release; dihydrorhodamine 123; disease phenotype; enhanced green fluorescent protein; fungus; gene function; gene therapy; glucosylceramidase; in vivo; innovation; intraperitoneal; killings; macrophage; microbicide; nano; novel; novel strategies; particle; public health relevance; therapeutic gene; uptake

DESCRIPTION (provided by applicant): Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency disorder characterized by severe recurrent infections by catalase positive bacteria and fungi. The molecular defects causing CGD result in the absence, low expression, or malfunction of one of the phagocyte NADPH oxidase components responsible for the generation of microbicidal reactive oxygen species. X-linked CGD results from mutations in the CYBB gene encoding the cytochrome b heavy chain glycoprotein, gp91-phox. The novel yeast cell wall particle (YCWP) DNA delivery technology traps DNA in the form of cationic polymer nano-complexes within porous cell wall "ghosts," providing for controlled release of the DNA upon particle internalization in macrophages. The micron-sized YCWP can be administered orally in order to deliver a therapeutic gene to peritoneal and gut wall macrophages, which then migrate to locations throughout the body. We hypothesize that this innovative approach will result in expression of the wild type gene encoding normal gp91-phox and correction of the functional defect in CGD phagocytes. Specifically, we propose to: 1. Test YCWP delivery and cDNA expression in an in vitro system using elicited peritoneal phagocytes from Cybb knock-out mice and transfection with cDNA encoding gp91-phox for transient replacement of gene function and correction of the CGD phenotype. Measurements will include fluorescence microscopy for particle uptake; molecular, histochemical and flow cytometric assays of gene expression and peroxide generation; and bacterial killing assays for evaluation of microbicidal function. 2. Test intraperitoneal and oral YCWP delivery for functional expression of cDNA encoding gp91-phox in an in vivo system using a murine Cybb knock-out model of CGD. Assays will include those used in Aim 1, as well as in vivo assays of host defense. The results of these studies, if successful, should demonstrate the feasibility of this novel gene therapy system for CGD and would provide a strong basis for large animal studies and translational research to bridge the gap from animal to human gene therapy. We hope that the proposed exploratory/developmental investigations will eventually lead to a safe, effective alternative method for gene therapy of CGD. PUBLIC HEALTH RELEVANCE: The proposed research, if successful, would demonstrate the feasibility of a novel oral gene therapy system for chronic granulomatous disease, an important primary immune deficiency disorder. The findings would provide a strong basis for translational research culminating in clinical trials for human gene therapy for the disease.

描述（由申请方提供）：慢性肉芽肿病（CGD）是一种遗传性原发性免疫缺陷疾病，其特征为过氧化氢酶阳性细菌和真菌引起的严重复发性感染。引起CGD的分子缺陷导致负责产生杀微生物活性氧的吞噬细胞NADPH氧化酶组分之一的缺失、低表达或功能障碍。X连锁CGD由编码细胞色素B重链糖蛋白gp 91-phox的CYBB基因突变引起。新型酵母细胞壁颗粒（YCWP）DNA递送技术将DNA以阳离子聚合物纳米复合物的形式捕获在多孔细胞壁“鬼”内，从而在颗粒内化于巨噬细胞中时提供DNA的受控释放。微米大小的YCWP可以口服给药，以将治疗基因传递到腹膜和肠壁巨噬细胞，然后迁移到全身的位置。我们假设，这种创新的方法将导致野生型基因编码正常gp 91-phox的表达和CGD吞噬细胞的功能缺陷的纠正。具体而言，我们建议：1.在体外系统中使用Cybb敲除小鼠的引发的腹膜吞噬细胞测试YCWP递送和cDNA表达，并用编码gp 91-phox的cDNA转染，用于基因功能的瞬时替换和CGD表型的校正。测量将包括颗粒摄取的荧光显微镜检查;基因表达和过氧化物生成的分子、组织化学和流式细胞术测定;以及用于评价杀微生物功能的细菌杀灭测定。2.使用CGD的鼠Cybb敲除模型测试腹膜内和口服YCWP递送在体内系统中编码gp 91-phox的cDNA的功能性表达。测定将包括在目标1中使用的那些，以及宿主防御的体内测定。这些研究的结果，如果成功的话，应该证明这种新的基因治疗系统的可行性CGD，并将提供一个强大的基础，为大型动物研究和转化研究，以弥合差距，从动物到人类的基因治疗。我们希望所提出的探索性/发展性研究将最终导致一个安全，有效的CGD基因治疗的替代方法。公共卫生相关性：这项研究如果成功，将证明一种新型口服基因治疗系统治疗慢性肉芽肿病的可行性，慢性肉芽肿病是一种重要的原发性免疫缺陷疾病。这一发现将为转化研究提供强有力的基础，最终导致人类基因治疗疾病的临床试验。