title: Ciliary beating recovery in deficient human airway epithelial cells after lentivirus ex vivo gene therapy.
abstract: 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.
authors.affiliation: Université de Lyon, Lyon, France.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Chhin
authors.forename: Didier
authors.initials: D
authors.lastname: Negre
authors.forename: Olivier
authors.initials: O
authors.lastname: Merrot
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Pham
authors.forename: Yves
authors.initials: Y
authors.lastname: Tourneur
authors.forename: Denis
authors.initials: D
authors.lastname: Ressnikoff
authors.forename: Martine
authors.initials: M
authors.lastname: Jaspers
authors.forename: Mark
authors.initials: M
authors.lastname: Jorissen
authors.forename: François-Loïc
authors.initials: FL
authors.lastname: Cosset
authors.forename: Patrice
authors.initials: P
authors.lastname: Bouvagnet
mesh_terms: D056444:Axonemal Dyneins; D002923:Cilia / Q000502:physiology*; D004398:Dyneins / Q000008:administration & dosage* / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism / Q000473:pathology*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007619:Kartagener Syndrome / Q000628:therapy*; D016086:Lentivirus / Q000235:genetics; D012137:Respiratory System / Q000166:cytology*; D014161:Transduction, Genetic
chemical_list: C425071:DNAI1 protein, human; D056444:Axonemal Dyneins; D004398:Dyneins