We have administered a recombinant adenovirus vector (AdCFTR) containing the normal human CFTR cDNA to the nasal and bronchial epithelium of four individuals with cystic fibrosis (CF). We show that this vector can express the CFTR cDNA in the CF respiratory epithelium in vivo. With doses up to 2 x 10(9) pfu, there was no recombination/complementation or shedding of the vector or rise of neutralizing antibody titres. At 2 x 10(9) pfu, a transient systemic and pulmonary syndrome was observed, possibly mediated by interleukin-6. Follow-up at 6-12 months demonstrated no long term adverse effects. Thus, it is feasible to use an adenovirus vector to transfer and express the CFTR cDNA in the respiratory epithelium of individuals with CF. Correction of the CF phenotype of the airway epithelium might be achieved with this strategy.

Variable in-frame skipping of exon 9 in cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts (exon 9-) occurs in the respiratory epithelium. To explore the genetic basis of this event, we evaluated respiratory epithelial cells and blood leukocytes from 124 individuals (38 with cystic fibrosis (CF), 86 without CF). We found an inverse relationship between the length of the polythymidine tract at the exon 9 splice branch/acceptor site and the proportion of exon 9- CFTR mRNA transcripts. These results strongly indicate a genetic basis in vivo modulating post-transcriptional processing of CFTR mRNA transcripts.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene but the association between mutation (genotype) and disease presentation (phenotype) is not straightforward. We have been investigating whether variants in the CFTR gene that alter splicing efficiency of exon 9 can affect the phenotype produced by a mutation. A missense mutation, R117H, which has been observed in three phenotypes, was found to occur on two chromosome backgrounds with intron 8 variants that have profoundly different effects upon splicing efficiency. A close association is shown between chromosome background of the R117H mutation and phenotype. These findings demonstrate that the genetic context in which a mutation occurs can play a significant role in determining the type of illness produced.

The most common mutation of the cystic fibrosis transmembrane conductance regulator gene, CFTR, associated with the clinical disorder cystic fibrosis (CF) is called "APhe5N," a triple-base deletion resulting in loss of phenylalanine at residue 508 of the predicted 1480-amino acid CFTR CFTR gene expression quantified by limited polymerase chain reaction amplification showed that in normal individuals, CFTR mRNA transcripts are expressed in nasal, tracheal, and bronchial epithelial cells at -1-2 copies per cell, more than 100-fold greater than in pharyngeal epithelium. Importantly, allele-specific hybridization studies demonstrated that the normal and APhe5" CFTR alleles are expressed in the respiratory epithelium in similar amounts.Cystic fibrosis (CF) is a fatal recessive disorder caused by mutations of the CF transmembrane conductance regulator gene, CFTR, whose postulated protein product is a 1480-amino acid membrane protein thought to modulate Clsecretion across the apical membrane of epithelial cells (1-4). The most common mutation of the CFTR gene is called "Afe 508 ," a triple-base deletion resulting in loss of the codon for phenylalanine at residue 508 of the predicted protein sequence found on ;'"70% ofchromosomes containing an abnormal CFTR gene (3, 4). All organs with exocrine glands are affected by abnormalities of the CFTR gene, but the major clinical manifestations are in the respiratory tract, with impacted mucus, chronic infection, and inflammation with subsequent derangements of airways and lung parenchyma (1). It is not known how mutations in the CFTR gene cause the respiratory abnormalities, but it is assumed to be directly related to abnormalities in expression of the CFTR gene in the respiratory epithelium. As an approach to this question, the present study is directed toward quantifying in vivo CFTR gene expression at the mRNA level in respiratory epithelium of normals and individuals with CF. METHODS

Consistent with the fact that the clinical disorder cystic fibrosis (CF) is manifested on epithelial surfaces, active transcription of the CF transmembrane conductance regulator (CFTR) gene and CFTR mRNA transcripts are detectable in a variety of epithelial cells, suggesting CFTR gene expression might be epithelial cell-specific. However, analysis of the CFTR gene promoter suggests it is a housekeeping gene, implying more widespread expression than only in epithelial cells. To evaluate the latter hypothesis, various human cells of non-epithelial origin, including lung fibroblasts, U-937 histiocytic lymphoma cells, K-562 erythroleukemia cells, HL-60 promyelocytic leukemia cells as well as freshly isolated blood lymphocytes, neutrophils, monocytes, and alveolar macrophages were examined for CFTR gene expression. Although Northern analysis failed to show CFTR mRNA transcripts in these cells, amplification of mRNA (after conversion to cDNA) by polymerase chain reaction combined with Southern analysis demonstrated the presence of CFTR mRNA transcripts at low levels in all cells evaluated except HL-60 cells. Comparative quantitative analysis showed fibroblasts contained 200-400 fold less CFTR mRNA transcripts than the T84 and HT-29 colon carcinoma epithelial cell lines, but had similar levels of CFTR transcripts to those of other epithelial cell lines. Nuclear transcription run-on analyses demonstrated very low level CFTR gene transcription in fibroblasts and U-937 cells, similar to that of other epithelial cells, but lower than the T84 and HT-29 colon carcinoma cell lines. Interestingly, while chromatin DNA of fibroblasts had no DNase I hypersensitivity sites in the 5' flanking region of the CFTR gene, HT-29 chromatin DNA exhibited four DNase I accessible sites in the same region, suggesting that these sites may be related to more active transcription of the CFTR gene in the intestinal epithelial cells than in fibroblasts.

73, 89, 90, and 92% CFTR transcripts with inframe deletion of exon 9, respectively, despite normal sweat Cl-and no clinical manifestation of CF. In the context that only 8% or less of bronchial CFTR transcripts need exon 9 to maintain normal airway function, these observations strongly suggest that either exon 9 is not necessary for CFTR structure and /or function or that only a very small fraction of bronchial epithelial cells need to express normal CFIR mRNA transcripts with exon 9 to perform the function of CFITR sufficient to maintain a normal phenotype in vivo. (J. Clin. Invest. 1992.

The predicted protein domains coded by exons 9–12 and 19–23 of the 27 exon cystic fibrosis transmembrane conductance regulator (CFTR) gene contain two putative nucleotide‐binding fold regions. Analysis of CFTR mRNA transcripts in freshly isolated bronchial epithelium from 12 normal adult individuals demonstrated that all had some CFTR mRNA transcripts with exon 9 completely deleted (exon 9‐ mRNA transcripts). In most (9 of 12), the exon 9‐ transcripts represented less than or equal to 25% of the total CFTR transcripts. However, in three individuals, the exon 9‐ transcripts were more abundant, comprising 39, 62 and 66% of all CFTR transcripts. Re‐evaluation of the same individuals 2–4 months later showed the same proportions of exon 9‐ transcripts. Of the 24 CFTR alleles in the 12 individuals, the sequences of the exon‐intron junctions relevant to exon 9 deletion (exon 8‐intron 8, intron 8‐exon 9, exon 9‐intron 9, and intron 9‐exon 10) were identical except for the intron 8‐exon 9 region sequences. Several individuals had varying lengths of a TG repeat in the region between splice branch and splice acceptor consensus sites. Interestingly, one allele in each of the two individuals with 62 and 66% exon 9‐ transcripts had a TT deletion in the splice acceptor site for exon 9. These observations suggest either the unlikely possibility that sequences in exon 9 are not critical for the functioning of the CFTR or that only a minority of the CFTR mRNA transcripts need to contain exon 9 sequences to produce sufficient amounts of a normal CFTR to maintain a normal clinical phenotype.

Human bronchial epithelium is exquisitely sensitive to high O2 levels, with tracheobronchitis usually developing after 12 h of exposure to 100% O2. To evaluate whether this vulnerability results from inability of the bronchial epithelium to provide adequate antioxidant protection, we quantified antioxidant gene expression in bronchial epithelium of normal volunteers at baseline and after exposure to 100% O2 in vivo. After 14.8 +/- 0.2 h of 100% O2, 24 of 33 individuals had evidence of tracheobronchitis. Baseline gene expression of CuZn superoxide dismutase (SOD), MnSOD, and catalase in bronchial epithelium was very low (CuZnSOD 4.1 +/- 0.8 transcripts/cell, MnSOD 5.1 +/- 0.9, catalase 1.3 +/- 0.2), with control gamma-actin expression relatively abundant (50 +/- 6 transcripts/cell). Importantly, despite 100% O2 exposure sufficient to cause tracheobronchitis in most individuals, antioxidant mRNA transcripts/cell in bronchial epithelium did not increase (P > 0.5). Catalase activity in bronchial epithelium did not change after exposure to hyperoxia (P > 0.05). Total SOD activity increased mildly (P < 0.01) but not sufficiently to protect the epithelium. Together, the very low levels of expression of intracellular antioxidant enzymes and the inability to upregulate expression at the mRNA level with oxidant stress likely have a role in human airway epithelium susceptibility to hyperoxia.