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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

基本信息

批准号：
7806438
负责人：
PETER E NEWBURGER
金额：
$ 24.42万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-04-10 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7806438
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

项目摘要

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(申请人提供)：慢性肉芽肿病(CGD)是一种遗传性的原发免疫缺陷疾病，其特征是由过氧化氢酶阳性细菌和真菌反复严重感染。引起CGD的分子缺陷导致吞噬细胞NADPH氧化酶组分之一的缺失、低表达或功能障碍，该组分负责产生杀菌活性氧物种。X连锁CGD是由编码细胞色素b重链糖蛋白gp91-Phox的细胞色素b基因突变引起的。这种新型的酵母细胞壁颗粒(YCWP)DNA递送技术以阳离子聚合物纳米复合体的形式将DNA捕获在多孔细胞壁的“幽灵”中，使DNA在颗粒内化后在巨噬细胞中得到控制释放。微米大小的YCWP可以口服，以便将治疗性基因传递到腹膜和肠壁巨噬细胞，然后这些巨噬细胞迁移到全身各个位置。我们推测，这种创新的方法将导致编码正常gp91-Phox的野生型基因的表达，并纠正CGD吞噬细胞的功能缺陷。具体地说，我们建议：1.利用Cybb基因敲除小鼠的腹膜吞噬细胞，在体外系统中检测YCWP的传递和cDNAs的表达，并将编码gp91-Phox的基因转入体外系统，以进行基因功能的瞬时替换和CGD表型的纠正。测量将包括颗粒摄取的荧光显微镜；基因表达和过氧化氢产生的分子、组织化学和流式细胞术分析；以及评估杀菌功能的细菌杀灭分析。2.采用小鼠CGD Cybb基因敲除模型，在体内系统中检测YCWP腹腔和口服给药对编码gp91-Phox基因的c DNA功能表达的影响。检测将包括AIM 1中使用的检测，以及体内宿主防御检测。这些研究的结果，如果成功，将证明这种新的CGD基因治疗系统的可行性，并将为大型动物研究和翻译研究提供强有力的基础，以弥合动物和人类基因治疗的差距。我们希望拟议的探索性/发展性研究最终将为CGD的基因治疗带来一种安全、有效的替代方法。公共卫生相关性：这项拟议的研究如果成功，将证明一种新的口服基因治疗系统的可行性，用于治疗慢性肉芽肿疾病，一种重要的原发免疫缺陷疾病。这一发现将为转化研究提供强有力的基础，最终达到人类基因治疗该病的临床试验。