Readministration of helper-dependent adenovirus to mouse lung.

Adenovirus vectors (Ad) are widely used in gene therapy studies, including those aimed at treating cystic fibrosis lung disease. Various approaches have been investigated to blunt the host immune response to Ad, including development of helper-dependent (HD) Ad. The host cytotoxic T-cell response to HD-Ad is generally lower than to earlier-generation Ad. However, antibodies are formed which could inhibit the efficacy of HD-Ad readministration. In this first study of HD-Ad readministration to the lung, we found that a second administration of HD-Ad to mice was possible with minimal loss of transgene expression. In contrast, when first-generation (FG) Ad was administered initially, followed by HD-Ad or FG-Ad, transgene expression was reduced. Significantly lower concentrations of antibodies against Ad were found in lung lavage fluid and serum from mice that received two doses of HD-Ad (when the initial HD-Ad lacked a transgene), compared to mice that received FG-Ad followed by HD-Ad. These data suggest that readministration of HD-Ad for lung gene therapy may be feasible.

Gene delivery to human sweat glands: a model for cystic fibrosis gene therapy.

Gene therapy vectors are mostly studied in cultured cells, rodents, and sometimes in non-human primates, but it is useful to test them in human tissue prior to clinical trials. In this study, we investigated the possibility of using human sweat glands as a model for testing cystic fibrosis (CF) gene therapy vectors. Human sweat glands are relatively easy to obtain from skin biopsy, and can be tested for CFTR function. Using patients' sweat glands could provide a safe model to study the efficacy of CF gene therapy. As the first step to explore using sweat glands as a model for CF gene therapy, we examined various ex vivo gene delivery methods for a helper-dependent adenovirus (HD-Ad) vector. Gene delivery to sweat glands in skin organ culture was studied by topical application, intradermal injection or submerged culture. We found that transduction efficiency can be enhanced by pretreating isolated sweat glands with dispase, which suggests that the basement membrane is a critical barrier to gene delivery by adenoviral vectors. Using this approach, we showed that Cftr could be efficiently delivered to and expressed by the epithelial cells of sweat glands with our helper-dependent adenoviral vector containing cytokeratin 18 regulatory elements. Based on this study we propose that sweat glands might be used as an alternative model to study CF gene therapy in humans.

Challenges and strategies for cystic fibrosis lung gene therapy.

Gene replacement therapy represents an interesting new approach for the treatment of cystic fibrosis (CF) lung disease. Basic research suggests that CF gene therapy is feasible, but major technological challenges must be addressed before clinical applications are likely to succeed. Therapeutic genes can be delivered to and expressed in human airways, but the number of cells expressing the transgene is relatively low. The inefficiency of gene delivery is largely attributable to the remarkable defenses of human airways. Maintaining long-term transgene expression in airway cells is also a significant obstacle. Recent advances have been made in the development of vectors, expression cassettes, and delivery techniques for enhancing airway gene transfer and expression. These advances have the potential to improve the efficiency of lung gene therapy and to achieve clinical benefits for CF patients in the future.

Targeting transgene expression for cystic fibrosis gene therapy.

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.

A human epithelium-specific vector optimized in rat pneumocytes for lung gene therapy.

Gene therapy vectors based on mammalian promoters offer the potential for increased cell specificity and may be less susceptible than viral promoters to transcription attenuation by host cytokines. The human cytokeratin 18 (K18) gene is naturally expressed in the lung epithelia, a target site for gene therapies to treat certain genetic pediatric lung diseases. Our original vector based on the promoter and 5' control elements of K18 offered excellent epithelial cell specificity but relatively low expression levels compared with viral promoters. In the present study, we found that adding a stronger SV40 poly(A) signal boosted primary rat lung epithelial cell expression but greatly reduced cell specificity. Addition of a 3' portion of the K18 gene to our vector as a 3' untranslated region (UTR) improved epithelial cell-specific expression by reducing expression in lung fibroblasts. The effect of the 3' UTR was not related to gross differences in cell-specific splicing. A deletion variant of this UTR further increased lung epithelial cell expression while retaining some cell specificity. These data illustrate the possibilities for using 3' UTR to regulate cell-specific transgene expression. Our improved K18 vector should prove useful for pediatric lung gene therapy applications.

Recruitment of damaged DNA to the nuclear matrix in hamster cells following ultraviolet irradiation.

We examined the relationship between the nuclear matrix and DNA in the dihydrofolate reductase domain following irradiation of Chinese hamster cells with UV light. The fraction of matrix-bound DNA increased in transcribed and non-transcribed regions during a 3 h period after irradiation. However, no increase was observed with excision repair-deficient cells mutant for the ERCC1 gene. The major UV-induced lesion, the cyclobutane pyrimidine dimer, increased in frequency in the matrix-bound DNA 1 h after irradiation, in both transcribed and non-transcribed regions, but decreased subsequently. This phenomenon was also lacking in excision repair-deficient cells. These data demonstrate that recruitment of lesion-containing DNA to the nuclear matrix occurs following UV irradiation and suggest that this recruitment is dependent upon nucleotide excision repair. This is consistent with the concept of a 'repair factory' residing on the nuclear matrix at which excision repair occurs.

Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli.

We compared the removal of pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] and cyclobutane pyrimidine dimers (CPDs) from the genome of repair-proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion. We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2). The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs.

Digestion of damaged DNA by the T7 DNA polymerase-exonuclease.

We have investigated the 3'-5'-exonuclease activity of phage T7 DNA polymerase for its usefulness as an approach for the detection of lesions in DNA. Unlike the T4 DNA polymerase-exonuclease, which is commonly used to map the position and frequency of lesions in very small DNA fragments, T7 DNA polymerase-exonuclease is able to hydrolyse almost completely the large fragments from KpnI-restricted mammalian DNA. However, we found that the exonuclease was also able to hydrolyse DNA containing several kinds of lesions: cyclobutane pyrimidine dimers, thymine glycols, and mono-adducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen and 5'-methyl-isopsoralen. Modifications of the reaction conditions did not significantly alter the extent of hydrolysis. These properties distinguish the T7 DNA polymerase-exonuclease from the T4 DNA polymerase-exonuclease and make the T7 DNA polymerase-exonuclease unsuitable for detecting several types of lesions in DNA.

Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli.

An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli. The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI (Schaaper, R.M., Dunn, R.L., and Glickman, B.W. (1987) J. Mol. Biol. 198, 187-202). On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed. A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand. This bias is reduced in an excision repair deficient (UvrB-) strain. In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand. This bias is also markedly reduced in the UvrB- strain. In light of recent work Mellon and Hanawalt (Mellon, I., and Hanawalt, P.C. (1989) Nature 342, 95-98) describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E. coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis.

Uracil-DNA glycosylase activity affects the mutagenicity of ethyl methanesulfonate: evidence for an alternative pathway of alkylation mutagenesis.

Mutagenesis induced by the alkylating agent ethyl methanesulfonate (EMS) is thought to occur primarily via mechanisms that involve direct mispairing at alkylated guanines, in particular, O6-ethyl guanine. Recent evidence indicates that alkylation of guanine at the O-6 position might enhance the deamination of cytosine residues in the complementary strand. To determine whether such deamination of cytosine could play a role in the production of mutations by EMS, the efficacy of this agent was tested in uracil-DNA glycosylase deficient (Ung) strains of Escherichia coli. The Ung- strains showed a linear response with increasing doses of EMS. This response was independent of the umuC gene product. In contrast, the Ung+ strains yielded a dose-squared response that became linear at higher doses of EMS when the cells were defective for the umuC gene product. These results support a model for mutagenesis involving the deamination of cytosines opposite O6-alkylated guanines followed by an error-prone repair event.