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首页> 外文期刊>PLoS Genetics >Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy
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Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

机译：慢病毒前体基因治疗后人气道缺乏上皮细胞的睫状跳动恢复

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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.
机译：原发性睫状运动障碍是一种异质性遗传疾病，其特征在于位于呼吸道的上皮细胞的纤毛功能障碍，导致反复的呼吸道感染。尽管进行了终生的生理疗法和抗生素治疗，但受影响患者的肺部逐渐被破坏，导致呼吸功能不全。已在10％的原发性睫状运动障碍病例中描述了Dynein轴突中间链1型（DNAI1）基因的隐性突变。我们的目标是在缺乏DNAI1的人气道上皮细胞中恢复正常的纤毛搏动。使用以水泡性口腔炎病毒糖蛋白假型化的猿猴免疫缺陷病毒为基础的慢病毒载体，可通过由延伸因子1启动子驱动的DNAI1 cDNA转导培养的人气道上皮细胞。分别通过RT-PCR和Western blot检测转导基因的转录和翻译。慢病毒转导了由于复合杂合突变而导致DNAI1缺陷的人气道上皮细胞，因此具有不能运动的纤毛且没有外部动力蛋白臂。记录纤毛的跳动并进行纤毛的电子显微镜检查。在用慢病毒处理的人细胞中检测到转导的DNAI1基因的转录和翻译。此外，运动不正常的纤毛恢复了正常的搏动，并重新出现了外部的动力蛋白。我们证明有可能通过使用慢病毒转导具有治疗基因的细胞来获得缺陷的人气道上皮细胞的纤毛搏动频率的正常化。该初步步骤构成了从原发性睫状运动障碍的体内基因治疗的角度来看必不可少的概念证明。这是该疾病首次证明纤毛跳动的恢复。

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