Pilot Studies of Gene Therapy for Primary Ciliary Dyskinesia

描述(申请人提供)：本申请涉及广泛的挑战领域(15)翻译科学和具体的挑战主题，15-OD(ORDR)-101：预防、早期发现和治疗罕见疾病的试点项目。具体地说，这项应用的重点是开发基因疗法作为治疗罕见疾病原发性睫状体运动障碍(PCD)的方法。PCD主要是一种常染色体隐性遗传病，由呼吸道活动纤毛正常功能所需的基因突变引起。在呼吸道中，正常纤毛活动的中断会导致粘液纤毛清除不足，并导致呼吸道感染的发生率增加。目前，没有针对PCD的特效治疗方法，受影响的人一生都会反复发生呼吸道感染，最终导致支气管扩张。最近，在包括动力蛋白中间链1在内的一小部分纤毛蛋白中发现了导致PCD的很大一部分的突变。PCD患者中突变的特定基因的鉴定为利用基因转移技术纠正遗传缺陷和恢复这些患者的正常睫状功能和肺健康提供了机会。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。这种小鼠模型具有动力蛋白中间链1基因的突变，并显示出许多人类疾病的特征，包括呼吸道纤毛结构缺陷、纤毛活动受损、完全缺乏粘液纤毛清除，以及呼吸系统疾病的自发发展。以马传染性贫血病毒为基础，以鸡斑块流感病毒血凝素蛋白为假型的基因转移载体，将在PCD小鼠模型中表达正常的动力蛋白中间链1蛋白。基因转移载体纠正纤毛活性和粘液纤毛清除的能力将在体外和体内进行测量。此外，同样的病毒载体将在体外测试其在人PCD细胞中纠正纤毛活动和粘液纤毛功能的能力。这些研究将推动基因治疗作为PCD和其他疾病的治疗方法的发展。本研究的具体目的是：1.验证表达正常DNAIC1基因的HA伪型慢病毒载体能够在体外恢复PCD小鼠气管上皮细胞纤毛活动的假说。2.验证HA伪型慢病毒载体表达正常DNAIC1基因能够稳定、长期纠正PCD小鼠体内纤毛活动和粘液纤毛清除，预防疾病的假说。3.验证HA伪型慢病毒载体可恢复人PCD细胞纤毛活动和粘液纤毛转运的假说。 与公共卫生相关：该项目将测试一种新的治疗方法，用于治疗因基因突变而导致疾病的人，这些突变对呼吸道细胞的正常功能非常重要，并患有频繁的肺部感染。一种新的基因治疗载体将进行测试，以确定它是否可以将突变基因的正常副本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.