Novel Approach to Oral Gene Therapy for Chronic Granulomatous Disease

描述（由申请人提供）：慢性肉芽肿病（CGD）是一种遗传性原发性免疫缺陷疾病，其特征是过氧化氢酶阳性细菌和真菌的严重复发性感染。导致CGD的分子缺陷导致吞噬细胞中负责产生杀微生物活性氧的NADPH氧化酶成分之一的缺失、低表达或功能障碍。x连锁CGD是由编码细胞色素b重链糖蛋白gp91-phox的CYBB基因突变引起的。新的酵母细胞壁颗粒（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“幽灵”中，为巨噬细胞内化颗粒时DNA的受控释放提供了条件。微米大小的YCWP可以口服，以便将治疗基因传递给腹膜和肠壁巨噬细胞，然后这些巨噬细胞迁移到全身各处。我们假设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。具体而言，我们建议：1。利用Cybb敲除小鼠腹膜吞噬细胞，转染编码gp91-phox的cDNA，在体外系统中检测YCWP的传递和cDNA的表达，以短暂替代基因功能和纠正CGD表型。测量将包括用于颗粒摄取的荧光显微镜；基因表达和过氧化产物的分子、组织化学和流式细胞术检测；以及用于评价杀菌功能的杀菌试验。2. 使用小鼠Cybb敲除CGD模型，在体内系统中测试腹腔和口服YCWP对编码gp91-phox的cDNA的功能表达。检测将包括Aim 1中使用的检测，以及宿主防御的体内检测。这些研究的结果，如果成功，应该证明这种新型基因治疗系统的可行性，并将为大型动物研究和转化研究提供坚实的基础，以弥合从动物到人类基因治疗的差距。我们希望所提出的探索性/发展性研究将最终导致一种安全、有效的基因治疗CGD的替代方法。公共卫生相关性：拟议的研究如果成功，将证明一种新的口服基因治疗系统治疗慢性肉芽肿病的可行性，这是一种重要的原发性免疫缺陷疾病。这些发现将为转化研究提供坚实的基础，最终为人类基因治疗该疾病的临床试验奠定基础。

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.