Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

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.

SuperScript II Reverse Transcriptase, as recommended by the manufacturer (Invitrogen, Grand Island, NY). The P4 (forward: 5'-TTGTGTGTGGTCAAGGAGACGG-3') and P5 specific primers were designed to amplify the full-length double-stranded cDNA (2319 bp) with the Expand High Fidelity PCR System kit, according to manufacturer's instructions (Roche Applied Science, France). Six Supplementary sets of specific primers were used to amplify (products from 371 to 594 bp) and to sequence DNAI1 cDNA (primer sequences available upon request). Purified PCR products were sequenced using the Big Dye ® sequencing chemistry (Applied Biosystems, Foster City, CA). Full-length DNAI1 cDNA PCR product was cloned into pCR ® II-TOPO ® vector and used to transform DH5α One Shot competent cells, according to the manufacturer's protocol (Invitrogen). Cloned products were sequenced as previously described.
Addition of enzyme restriction sites in 5' and 3' DNAI1 cDNA sequence was performed using two sets of primers. The BAMHIDNAI1_for and NCOIDNAI1_for primers (forward) added BamH I and Nco I restriction sites upstream DNAI1 cDNA sequence, respectively. The XHOIDNAI1_rev primer (reverse) added a Xho I restriction site downstream DNAI1 cDNA sequence.
Addition of hemagglutinin (HA) tag downstream DNAI1 cDNA sequence was performed by PCR using lentiviral vectors containing DNAI1 cDNA (pK+DNAI1 or pK-DNAI1) as template and upDNAI1_for (forward)/lowHA_rev (reverse) primers with the Expand High Fidelity PCR System kit, following manufacturer's instructions (Roche Applied Science).

RT-PCR Analysis
Tagged DNAI1 gene expression was revealed by reverse transcription PCR (RT-PCR) on infected HAECs. Non-ciliated cells were collected the day of collagen digestion (J') and ciliated cells were collected when they were fully covered by cilia (J'+28).
Poly(A) + mRNA was isolated by the Dynabeads Oligo(dT) 25 purification kit, according to the manufacturer's protocol (Dynal Biotech, Norway). cDNA was synthesized by tag specific priming, using HART_rev (reverse: 5'-GGCATAGTCGGGGACGTCGTA-3') or P5 DNAI1 specific primer, and SuperScript II Reverse Transcriptase, following manufacturer's instructions (Invitrogen). A specific pair of primers located on DNAI1 cDNA sequence (DNAI1seq5.2F/DNAI1seq5.2R, located in exons 13-14 and exon 19, respectively) was used to amplify a 545 bp PCR product under standard conditions. Absence of genomic DNA contamination was confirmed by PCR with alpha-tubulin primers which could amplify either a 320 bp fragment on cDNA or a 468 bp fragment on genomic DNA (protocol is available upon request).

Viral Vector Production and Titration
The lentiviral vector was produced and titrated as described by Negre et al. (2,3). The About 2.5×10 5 cells were plated one day before transfection. The day of transfection, 1 µg of each plasmid was diluted in 25 µL 150 mM NaCl and 5 µL of ExGen500 (Euromedex, France) was added to 20 µL of 150 Mm NaCl. This last mix was added to diluted plasmids, incubated ten minutes at room temperature before transfer on 293Bosc cells. Cells were incubated for three hours with plasmids at 37°C, 5% CO 2 , and then supplementary medium was added for 12-hours incubation before complete changing. Forty-eight hours posttransfection, medium was removed; cells were rinsed with cooled 1X PBS, detached from the dish using a scraper and centrifuged at 70 g for 5 minutes. The supernatant was removed and the pellet was stored at -80°C or was resuspended with an extraction buffer (1X TE: 25 mM   Tris, 2  At day J'+31 (31-days post-collagen-digestion), ciliated cells were fixed in 3% glutaraldehyde in 0,1 M sodium cacodylate buffer (pH 7.4) for two to three hours, followed by an additional OsO 4 0,1 M phosphate buffer (pH 7.4) fixation for one hour. The material was dehydrated in a graded ethanol series and then embedded in epoxy resin. One micron sections were prepared for TEM and axoneme ultrastructure analysis.