Chhin Brigitte, Negre Didier, Merrot Olivier, Pham Jacqueline, Tourneur Yves, Ressnikoff Denis, Jaspers Martine, Jorissen Mark, Cosset François-Loïc, Bouvagnet Patrice
Université de Lyon, Lyon, France.
PLoS Genet. 2009 Mar;5(3):e1000422. doi: 10.1371/journal.pgen.1000422. Epub 2009 Mar 20.
Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1-deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT-PCR and western blot, respectively. Human airway epithelial cells that were DNAI1-deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease.
原发性纤毛运动障碍是一种异质性遗传病，其特征是呼吸道内衬上皮细胞的纤毛功能障碍，导致反复的呼吸道感染。尽管进行了终身的物理治疗和使用抗生素，但受影响患者的肺部仍会逐渐受损，导致呼吸功能不全。在10%的原发性纤毛运动障碍病例中，已发现动力蛋白轴丝中间链1型（DNAI1）基因存在隐性突变。我们的目标是恢复DNAI1缺陷的人气道上皮细胞的正常纤毛摆动。一种基于猿猴免疫缺陷病毒并假型化有水泡性口炎病毒糖蛋白的慢病毒载体，被用于用由伸长因子1启动子驱动的DNAI1的cDNA转导培养的人气道上皮细胞。分别通过逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测转导基因的转录和翻译情况。因复合杂合突变而缺乏DNAI1、因此纤毛无运动能力且没有外动力蛋白臂的人气道上皮细胞，被该慢病毒转导。记录纤毛摆动情况并对纤毛进行电子显微镜检查。在用慢病毒处理的人细胞中检测到了转导的DNAI1基因的转录和翻译。此外，无运动能力的纤毛恢复了正常摆动，外动力蛋白臂重新出现。我们证明，通过使用慢病毒将治疗性基因转导至细胞，可以使缺陷的人气道上皮细胞的纤毛摆动频率恢复正常。这一初步步骤构成了一个概念验证，对于原发性纤毛运动障碍的体内基因治疗前景而言是不可或缺的。这是首次在这种疾病中证明纤毛摆动得以恢复。
