Electrotransfer of CpG free plasmids enhances gene expression in skin.

Skin is a very suitable target for gene therapy and DNA vaccination due to its accessibility, its surface and its ability to produce transgenes. Gene electrotransfer (GET) to the skin is under development for clinical applications for DNA vaccine or local treatment such as wound healing. Local treatments are effective if the expression of the plasmid affects only the local environment (skin) by inducing an efficient concentration over a prolonged period. In this study, we evaluate the control of expression in the skin of a plasmid coding a fluorescent protein by its CpG (cytosine-phosphate-guanine motif) content. Two fluorescent reporter genes are evaluated: tdTomato and GFP. The expression is followed on the long term by in vivo fluorescence imaging. Our results show that GET mediated expression in the skin can be controlled by the CpG content of the plasmid. Long term expression (>120 days) can be obtained at high level with CpG-free constructs associated with a proper design of the electrodes where the field distribution mediating the gene electrotransfer is present deep in the skin.

Concomitant expression of E. coli cytosine deaminase and uracil phosphoribosyltransferase improves the cytotoxicity of 5-fluorocytosine.

The prodrug activation system formed by the E. coli codA gene encoding cytosine deaminase (CD) and 5-fluorocytosine (5-FC) developed for selective cancer chemotherapy suffers from a sensitivity limitation in many tumour cells. In an attempt to improve the CD/5-FC suicide association, we combined the E. coli upp gene encoding uracil phosphoribosyltransferase (UPRT) with codA gene to create the situation prevailing in E. coli, a bacterium very efficient in metabolising 5-FC. The constitutive expression of the two genes cloned on an E. coli-animal cell shuttle plasmid either in a linked or in a fused configuration was evaluated in E. coli strains selected and engineered to mimic the 5-FC metabolism encountered in mammalian cells. The simultaneous expression of codA and upp genes generated a cooperative effect resulting in a dramatic increase in 5-FC sensitivity of cells compared to the expression of codA alone. Furthermore, it was shown that the association of UPRT with CD facilitated the uptake of 5-FC, in the situation where the drug penetrates cells by passive diffusion as in mammalian cells, by directly channeling 5-fluorouracil, the product of CD, to 5-fluoroUMP, the product of UPRT.

Prevention of bleomycin-induced pulmonary fibrosis after adenovirus-mediated transfer of the bacterial bleomycin resistance gene.

A serious limitation in the use of the DNA-cleaving, antitumoral-antibiotic, bleomycin during chemotherapy is pulmonary toxicity. Lung injury induced by bleomycin is characterized by an increased deposition of interstitial extracellular matrix proteins in the alveolar wall that compromises respiratory function. Several drugs have been tested in animal models to prevent the pulmonary toxicity of bleomycin, but have not led to a useful clinical treatment because of their adverse effects on other tissues. We have shown that transgenic mice expressing Streptoalloteichus hindustanus (Sh) ble bleomycin resistance protein in pulmonary epithelial cells in the lungs are protected against bleomycin-induced toxicity in lungs. In the present study, we used intranasal administration by adenovirus-mediated gene transfer of the bleomycin resistance Sh ble gene to mouse lung for prevention of bleomycin-induced pulmonary fibrosis. We constructed recombinant adenoviruses Ad.CMVble and Ad.RSVble harboring the bleomycin resistance Sh ble gene under the control of the cytomegalovirus early promoter and the Rous sarcoma virus early promoter, respectively. Transgene expression was detected in epithelia of conducting airways and alveolar septa by immunostaining with a rabbit polyclonal antibody directed against the bleomycin resistance protein and persisted for the duration of drug treatment; i.e., up to 17 d. No toxic effect was seen in adenovirus-treated mice. Pretreatment of mice with Ad.CMVble or Ad.RSVble completely prevented collagen deposition 42-133 d after bleomycin treatment, as measured by lung OH-proline content. Histologic studies indicated that there was little or no lung injury in the adenovirus/bleomycin-treated mice compared with the bleomycin-treated mice. These observations may lead to new approaches for the prevention of bleomycin-induced pulmonary fibrosis.

Overexpression of DNA polymerase beta sensitizes mammalian cells to 2',3'-deoxycytidine and 3'-azido-3'-deoxythymidine.

Mammalian DNA polymerase beta is a DNA repair enzyme expressed constitutively at a low level. In vitro, purified DNA polymerase (Pol) beta incorporates the nucleotide analogues 2'-3' deoxycytidine (ddC)-triphosphate and 3'-azido-3'-deoxythymidine (AZT)-triphosphate into DNA, causing chain termination. We have tested the possibility of enhancing the cytotoxicity of these chain terminators against mammalian cells by increasing the level of Pol beta. Chinese hamster ovary AA8 and murine melanoma B16 cell lines were stably transfected with rat pol beta cDNA under the control of a viral enhancer/promoter. We found that overexpression of Pol beta sensitized the cells to ddC and AZT. To confirm the role of this polymerase in this process, we prepared cell extracts from the control and Pol beta overexpressing Chinese hamster ovary cell lines and tested in vitro their capacity to incorporate ddC-triphosphate and AZT-triphosphate into DNA. We found that inhibition of DNA replication by both chain terminators was more pronounced when extracts from pol beta-transfected cells were used, providing a direct evidence of the involvement of Pol beta in the sensitization process. In addition, we showed that cotransfection with bacterial or viral thymidine/thymidylate kinase genes enhanced the Pol beta-mediated cytotoxicity of AZT, suggesting that phosphorylation and polymerization activities might be combined to potentiate their respective effects. These observations may be useful for improving therapeutic efficiency of DNA chain terminators.

Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus.

Thymidylate kinase (dTMP kinase; EC 2.7.4.9) catalyzes the phosphorylation of dTMP to form dTDP in both de novo and salvage pathways of dTTP synthesis. The nucleotide sequence of the tmk gene encoding this essential Escherichia coli enzyme is the last one among all the E. coli nucleoside and nucleotide kinase genes which has not yet been reported. By subcloning the 24.0-min region where the tmk gene has been previously mapped from the lambda phage 236 (E9G1) of the Kohara E. coli genomic library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987), we precisely located tmk between acpP and holB genes. Here we report the nucleotide sequence of tmk, including the end portion of an upstream open reading frame (ORF 340) of unknown function that may be cotranscribed with the pabC gene. The tmk gene was located clockwise of and just upstream of the holB gene. Our sequencing data allowed the filling in of the unsequenced gap between the acpP and holB genes within the 24-min region of the E. coli chromosome. Identification of this region as the E. coli tmk gene was confirmed by functional complementation of a yeast dTMP kinase temperature-sensitive mutant and by in vitro enzyme assay of the thymidylate kinase activity in cell extracts of E. coli by use of tmk-overproducing plasmids. The deduced amino acid sequence of the E. coli tmk gene showed significant similarity to the sequences of the thymidylate kinases of vertebrates, yeasts, and viruses as well as two uncharacterized proteins of bacteria belonging to Bacillus and Haemophilus species.

A selectable bifunctional beta-galactosidase::phleomycin-resistance fusion protein as a potential marker for eukaryotic cells.

The Sh ble gene, conferring phleomycin resistance (PhR), was fused in frame to both the 3' and 5' ends of the Escherichia coli lacZ gene. The bifunctionality of the resulting 130-kDa hybrid proteins was demonstrated in E. coli and in the fungus, Tolypocladium geodes. PhR transformants of both organisms could be selected for. All transformants from E. coli and most from T. geodes displayed beta Gal activity. In the fungal host, higher transformation frequencies and greater levels of beta Gal activity were observed in clones harboring the lacZ::Sh ble fusion, as compared to the Sh ble::lacZ configuration. This system appears to be a potentially useful tool for the direct selection of transformants, and the evaluation of gene expression and regulation in a wide variety of prokaryotic and eukaryotic hosts.

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus.

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.