Pilot Studies of Gene Therapy for Primary Ciliary Dyskinesia

描述（由申请人提供）：本申请涉及广泛的挑战领域（15）转化科学和特定的挑战主题，15-OD（ORR）-101：预防、早期发现和治疗罕见疾病的试点项目。具体而言，该申请专注于开发基因疗法作为罕见疾病原发性纤毛运动障碍（PCD）的治疗。PCD主要作为常染色体隐性遗传疾病遗传，其由排列在呼吸道上的运动纤毛的正常功能所需的基因突变引起。在气道中，正常纤毛活动的破坏导致粘膜纤毛清除的缺乏，并导致呼吸道感染的发病率增加。目前，没有针对PCD的特异性治疗方法，受影响的个体患有终生复发性呼吸道感染，最终导致支气管扩张。最近，已在少数纤毛蛋白（包括动力蛋白中间链1）中鉴定出导致显著部分PCD的突变。在PCD病例中突变的特定基因的鉴定提供了使用基因转移技术来纠正遗传缺陷并恢复这些患者正常纤毛功能和肺部健康的机会。为了验证基因治疗可以有效治疗PCD的总体假设，这项工作将使用新开发的PCD小鼠模型。该小鼠模型在动力蛋白中间链1基因中具有突变，并表现出人类疾病的许多特征，包括呼吸纤毛的结构缺陷、纤毛活性受损、完全缺乏粘液纤毛清除以及呼吸系统疾病的自发发展。一种新开发的基因转移载体，是基于马传染性贫血病毒和假型与禽斑流感病毒血凝素蛋白将被用来表达正常的动力蛋白中间链1蛋白在小鼠模型的PCD。将在体外和体内测量基因转移载体校正纤毛活性和粘膜纤毛清除的能力。此外，将测试相同病毒载体在体外纠正人PCD细胞中纤毛活性和粘膜纤毛功能的能力。这些研究将促进基因治疗作为PCD和其他疾病的治疗方法的发展。该提案的具体目标是：1.验证表达正常Dnaic 1 cDNA的HA假型慢病毒载体可以在体外恢复PCD小鼠气管气道上皮细胞纤毛活性的假设。2.为了检验以下假设：表达正常Dnaic 1 cDNA的HA假型慢病毒载体可以提供纤毛活性和粘膜纤毛清除的稳定、长期校正，并在体内预防PCD小鼠的疾病。3.检验HA假型慢病毒载体可恢复纤毛活性和粘膜纤毛转运至人PCD细胞的假设。 公共卫生相关性：该项目将测试一种新的治疗方法，用于治疗因基因突变引起的疾病，这些基因突变对气道细胞的正常功能至关重要，并患有频繁的肺部感染。将测试一种新的基因治疗载体，以确定它是否可以将突变基因的正常拷贝转移到受影响的细胞中并纠正它们的功能。从这些研究中获得的信息将推动基因治疗领域的发展，并可能对许多遗传性疾病的治疗有用。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic, 15-OD(ORDR)-101: Pilot projects for prevention, early detection and treatment of rare diseases. Specifically, this application is focused on developing gene therapy as a treatment for the rare disease Primary Ciliary Dyskinesia (PCD). PCD is predominantly inherited as an autosomal recessive disease caused by mutations in genes that are required for the normal function of the motile cilia that line the respiratory tract. In the airways, disruption of normal ciliary activity results in an absence of mucociliary clearance and leads to an increased incidence of respiratory infections. Currently, there is no specific treatment for PCD, and affected individuals suffer from a life-long pattern of recurring respiratory infections, ultimately leading to bronchiectasis. Recently, mutations that cause a significant fraction of PCD have been identified in a small number of ciliary proteins, including dynein intermediate chain 1. The identification of the specific genes that are mutated in cases of PCD provides the opportunity to use gene transfer techniques to correct the genetic defect and restore normal ciliary function and pulmonary health to these patients. To test the overall hypothesis that gene therapy can be an effective treatment for PCD, this work will use a newly developed mouse model of PCD. This mouse model has a mutation in the dynein intermediate chain 1 gene and exhibits many of the features of the human disease, including structural defects of respiratory cilia, impaired ciliary activity, a complete lack of mucociliary clearance, and the spontaneous development of respiratory disease. A newly developed gene transfer vector that is based on the equine infectious anemia virus and is pseudotyped with fowl plaque influenza virus hemagglutinin protein will be used to express the normal dynein intermediate chain 1 protein in the mouse model of PCD. The ability of the gene transfer vector to correct ciliary activity and mucociliary clearance will be measured both in vitro and in vivo. In addition, the same viral vector will be tested for its ability to correct ciliary activity and mucociliary function in human PCD cells in vitro. These studies will advance the development of gene therapy as a treatment for PCD and other diseases. The specific aims of this proposal are: 1. To test the hypothesis that an HA-pseudotyped lentiviral vector expressing the normal Dnaic1 cDNA can restore ciliary activity to tracheal airway epithelial cells from PCD mice in vitro. 2. To test the hypothesis that an HA-pseudotyped lentiviral vector expressing the normal Dnaic1 cDNA can provide stable, long-term correction of ciliary activity and mucociliary clearance and prevent disease in PCD mice in vivo. 3. To test the hypothesis that HA-pseudotyped lentiviral vectors can restore ciliary activity and mucociliary transport to human PCD cells. PUBLIC HEALTH RELEVANCE: This project will test a new therapy for people who have diseases caused by mutations in genes that are important for the proper function of the cells that line their airways and suffer from frequent lung infections. A new gene therapy vector will be tested to determine if it can transfer a normal copy of the mutated gene into the affected cells and correct their function. The information gained from these studies will advance the field of gene therapy and may be useful to the treatment of many genetic diseases.