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.
References
[1]
Afzelius BA (1976) A human syndrome caused by immotile cilia. Science 193: 317–319.
[2]
Blouin JL, Meeks M, Radhakrishna U, Sainsbury A, Gehring C, et al. (2000) Primary ciliary dyskinesia: a genome-wide linkage analysis reveals extensive locus heterogeneity. Eur J Hum Genet 8: 109–118.
[3]
El Zein L, Omran H, Bouvagnet P (2003) Lateralization defects and ciliary dyskinesia: lessons from algae. Trends Genet 19: 162–167.
[4]
Nonaka S, Tanaka Y, Okada Y, Takeda S, Harada A, et al. (1998) Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95: 829–837.
[5]
Kartagener M, Stucki P (1962) Bronchiectasis with situs inversus. Arch Pediatr 79: 193–207.
[6]
Pedersen H, Rebbe H (1975) Absence of arms in the axoneme of immobile human spermatozoa. Biol Reprod 12: 541–544.
[7]
Douek E, Bannister LH, Dodson HC (1975) Recent advances in the pathology of olfaction. Proc R Soc Med 68: 467–470.
[8]
Ibanez-Tallon I, Gorokhova S, Heintz N (2002) Loss of function of axonemal dynein Mdnah5 causes primary ciliary dyskinesia and hydrocephalus. Hum Mol Genet 11: 715–721.
[9]
Kennedy MP, Omran H, Leigh MW, Dell S, Morgan L, et al. (2007) Congenital heart disease and other heterotaxic defects in a large cohort of patients with primary ciliary dyskinesia. Circulation 115: 2814–2821.
[10]
Moore A, Escudier E, Roger G, Tamalet A, Pelosse B, et al. (2006) RPGR is mutated in patients with a complex X linked phenotype combining primary ciliary dyskinesia and retinitis pigmentosa. J Med Genet 43: 326–333.
[11]
Narayan D, Krishnan SN, Upender M, Ravikumar TS, Mahoney MJ, et al. (1994) Unusual inheritance of primary ciliary dyskinesia (Kartagener's syndrome). J Med Genet 31: 493–496.
[12]
Bartoloni L, Blouin JL, Pan Y, Gehrig C, Maiti AK, et al. (2002) Mutations in the DNAH11 (axonemal heavy chain dynein type 11) gene cause one form of situs inversus totalis and most likely primary ciliary dyskinesia. Proc Natl Acad Sci U S A 99: 10282–10286.
[13]
Budny B, Chen W, Omran H, Fliegauf M, Tzschach A, et al. (2006) A novel X-linked recessive mental retardation syndrome comprising macrocephaly and ciliary dysfunction is allelic to oral-facial-digital type I syndrome. Hum Genet 120: 171–178.
[14]
Duriez B, Duquesnoy P, Escudier E, Bridoux AM, Escalier D, et al. (2007) A common variant in combination with a nonsense mutation in a member of the thioredoxin family causes primary ciliary dyskinesia. Proc Natl Acad Sci U S A 104: 3336–3341.
[15]
Loges NT, Olbrich H, Fenske L, Mussaffi H, Horvath J, et al. (2008) DNAI2 mutations cause primary ciliary dyskinesia with defects in the outer dynein arm. Am J Hum Genet 83: 547–558.
[16]
Olbrich H, Haffner K, Kispert A, Volkel A, Volz A, et al. (2002) Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left-right asymmetry. Nat Genet 30: 143–144.
[17]
Omran H, Kobayashi D, Olbrich H, Tsukahara T, Loges NT, et al. (2008) Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins. Nature 456: 611–616.
[18]
Pennarun G, Escudier E, Chapelin C, Bridoux AM, Cacheux V, et al. (1999) Loss-of-function mutations in a human gene related to Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia. Am J Hum Genet 65: 1508–1519.
[19]
Schwabe GC, Hoffmann K, Loges NT, Birker D, Rossier C, et al. (2008) Primary ciliary dyskinesia associated with normal axoneme ultrastructure is caused by DNAH11 mutations. Hum Mutat 29: 289–298.
[20]
Guichard C, Harricane MC, Lafitte JJ, Godard P, Zaegel M, et al. (2001) Axonemal dynein intermediate-chain gene (DNAI1) mutations result in situs inversus and primary ciliary dyskinesia (Kartagener syndrome). Am J Hum Genet 68: 1030–1035.
[21]
Zariwala MA, Leigh MW, Ceppa F, Kennedy MP, Noone PG, et al. (2006) Mutations of DNAI1 in primary ciliary dyskinesia: evidence of founder effect in a common mutation. Am J Respir Crit Care Med 174: 858–866.
[22]
Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, et al. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272: 263–267.
[23]
Harvey BG, Leopold PL, Hackett NR, Grasso TM, Williams PM, et al. (1999) Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive administration of a recombinant adenovirus. J Clin Invest 104: 1245–1255.
[24]
Limberis M, Anson DS, Fuller M, Parsons DW (2002) Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer. Hum Gene Ther 13: 1961–1970.
[25]
Negre D, Duisit G, Mangeot PE, Moullier P, Darlix JL, et al. (2002) Lentiviral vectors derived from simian immunodeficiency virus. Curr Top Microbiol Immunol 261: 53–74.
[26]
Negre D, Mangeot PE, Duisit G, Blanchard S, Vidalain PO, et al. (2000) Characterization of novel safe lentiviral vectors derived from simian immunodeficiency virus (SIVmac251) that efficiently transduce mature human dendritic cells. Gene Ther 7: 1613–1623.
[27]
Jorissen M, Willems T, Van der Schueren B, Verbeken E, De Boeck K (2000) Ultrastructural expression of primary ciliary dyskinesia after ciliogenesis in culture. Acta Otorhinolaryngol Belg 54: 343–356.
[28]
Wang G, Davidson BL, Melchert P, Slepushkin VA, van Es HH, et al. (1998) Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia. J Virol 72: 9818–9826.
[29]
Wang G, Slepushkin V, Zabner J, Keshavjee S, Johnston JC, et al. (1999) Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect. J Clin Invest 104: R55–62.
[30]
Jeffery PK, Li D (1997) Airway mucosa: secretory cells, mucus and mucin genes. Eur Respir J 10: 1655–1662.
[31]
Chilvers MA, McKean M, Rutman A, Myint BS, Silverman M, et al. (2001) The effects of coronavirus on human nasal ciliated respiratory epithelium. Eur Respir J 18: 965–970.
[32]
Ibricevic A, Pekosz A, Walter MJ, Newby C, Battaile JT, et al. (2006) Influenza virus receptor specificity and cell tropism in mouse and human airway epithelial cells. J Virol 80: 7469–7480.
[33]
Szecsi J, Boson B, Johnsson P, Dupeyrot-Lacas P, Matrosovich M, et al. (2006) Induction of neutralising antibodies by virus-like particles harbouring surface proteins from highly pathogenic H5N1 and H7N1 influenza viruses. Virol J 3: 70.
[34]
Evans MJ, Johnson LV, Stephens RJ, Freeman G (1976) Renewal of the terminal bronchiolar epithelium in the rat following exposure to NO2 or O3. Lab Invest 35: 246–257.
[35]
Park KS, Wells JM, Zorn AM, Wert SE, Laubach VE, et al. (2006) Transdifferentiation of ciliated cells during repair of the respiratory epithelium. Am J Respir Cell Mol Biol 34: 151–157.
[36]
Hajj R, Baranek T, Le Naour R, Lesimple P, Puchelle E, et al. (2007) Basal cells of the human adult airway surface epithelium retain transit-amplifying cell properties. Stem Cells 25: 139–148.
[37]
Szecsi J, Drury R, Josserand V, Grange MP, Boson B, et al. (2006) Targeted retroviral vectors displaying a cleavage site-engineered hemagglutinin (HA) through HA-protease interactions. Mol Ther 14: 735–744.
[38]
Lufino MM, Edser PA, Wade-Martins R (2008) Advances in high-capacity extrachromosomal vector technology: episomal maintenance, vector delivery, and transgene expression. Mol Ther 16: 1525–1538.
