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.

The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase.

The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

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.

Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes.

Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain. In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity. When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR). The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK. TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.

Transgenic mice expressing the Sh ble bleomycin resistance gene are protected against bleomycin-induced pulmonary fibrosis.

Despite the high efficiency of bleomycin (BLM) as a chemotherapeutic agent against various carcinomas, the potentially lethal and chronic fibrotic response of the lung is a major dose-limiting side effect. Here, we explore the possibility of a direct inhibition of lung tissue injury by in vivo expression of the actinomycetes BLM resistance protein Sh ble. Transgenic mice expressing the Sh ble gene under the control of a composite viral promoter were produced after introduction of the transgene into D3 ES cells. The protein was detected at high level in lungs, spleen, and kidney. We then assessed its ability to modulate the BLM-induced fibrotic response in the transgenic mice in comparison with C57BL/6 and 129/Sv parental mice. Cumulative doses of 300, 400, or 500 mg/kg BLM were administered either by i.p. or s.c. repeated injections in the different strains. Transgenic mice were shown to be clearly less sensitive to BLM toxicity, as assessed by lung histology. The pulmonary hydroxyproline content in the treated transgenic mice was close to its baseline level, whereas it was up to 50% higher than the control level in C57BL/6 and 129/Sv parental mice. These observations are consistent with the hypothesis that a resistance gene specifically expressed in lungs may prevent the BLM-induced inflammation.

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.

Bystander killing of cancer cells by herpes simplex virus thymidine kinase gene is mediated by connexins.

In gene therapy to treat cancer, typically only a fraction of the tumor cells can be successfully transfected with a gene. However, in the case of brain tumor therapy with the thymidine kinase gene from herpes simplex virus (HSV-tk), not only the cells transfected with the gene but also neighboring others can be killed in the presence of ganciclovir. Such a "bystander" effect is reminiscent of our previous observation that the effect of certain therapeutic agents may be enhanced by their diffusion through gap junctional intercellular communication (GJIC). Herein, we present the evidence, from in vitro studies, that gap junctions could indeed be responsible for such a gene therapy bystander effect. We used HeLa cells for this purpose, since they show very little, if any, ability to communicate through gap junctions. When HeLa cells were transfected with HSV-tk gene and cocultured with nontransfected cells, only HSV-tk-transfected HeLa cells (tk+) were killed by ganciclovir. However, when HeLa cells transfected with a gene encoding for the gap junction protein, connexin 43 (Cx43), were used, not only tk+ cells, but also tk- cells were killed, presumably due to the transfer, via Cx43-mediated GJIC, of toxic ganciclovir molecules phosphorylated by HSV-tk to the tk- cells. Such bystander effect was not observed when tk+ and tk- cells were cocultured without direct cell-cell contact between those two types of cells. Thus, our results give strong evidence that the bystander effect seen in HSV-tk gene therapy may be due to Cx-mediated GJIC.

Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle.

The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.

CL307-24, a new antibiotic complex from Saccharopolyspora aurantiaca sp. nov. II. Physico-chemical and biological properties.

CL307-24I, the main component of the CL307-24 complex produced by Saccharopolyspora aurantiaca sp. nov., was found to be a potent inhibitor of yeast mitochondrial ATPase. CL307-24I displayed a high degree of activity towards some coryneform bacteria and also has been shown to possess an insecticidal activity. Its biological and physico-chemical properties clearly distinguish it from previously known ATPase inhibitors.

CL307-24, a new antibiotic complex from Saccharopolyspora aurantiaca sp. nov. I. Taxonomy, fermentation and purification.

CL307-24, a complex of new antibiotics has been isolated from the fermentation broth of Saccharopolyspora aurantiaca sp. nov. The complex was purified by cation-exchange and hydrophobic interaction chromatographies. It was then resolved as one major and three minor components by silica gel chromatography and HPLC.

Genetic improvements of an industrial strain of Aspergillus flavus for urate oxidase production.

Urate oxidase, an enzyme used in human therapy, is currently produced industrially by a strain of Aspergillus flavus. Two strategies of strain improvement were tested in order to obtain higher yields of urate oxidase. The first one, based on a classical mutation-selection protocol, led to the isolation of a mutant strain that overproduced uricase two-fold as compared to the industrial strain. The second one consisted in the construction of transformed strains that had integrated multiple copies of a urate oxidase-expression vector. A twenty-fold improvement in urate oxidase was obtained by this method.

Development of a transformation system for the thermophilic fungus Talaromyces sp. CL240 based on the use of phleomycin resistance as a dominant selectable marker.

A transformation system for the thermophilic cellulolytic fungus Talaromyces sp. CL240 has been developed, using the phleomycin resistance gene from Streptoalloteichus hindustanus (Sh ble) as a dominant selectable marker. The plasmids (pAN8-1 and pUT720) carrying the Sh ble gene under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter, allowed selection of phleomycin-resistant transformants. A new promoter sequence cloned from chromosomal DNA of Trichoderma reesei (pUT737) was also able to drive efficient expression of the Sh ble gene in Talaromyces sp. CL240, resulting in the selection of transformants that were highly resistant to phleomycin.

Efficient secretion of human lysozyme fused to the Sh ble phleomycin resistance protein by the fungus Tolypocladium geodes.

Tolypocladium geodes strain NC50 was transformed by different integrating vectors bearing both a synthetic gene encoding human lysozyme (HLz) and the Sh ble phleomycin resistance marker, either in separate expression cassettes or in transcriptional or translational fusion configurations. Clones derived from all vectors were able to secrete HLz. The highest productivities in shake flasks (up to 150 mg l-1 in 5 days) were obtained when HLz was fused at the C-terminal end of the Sh ble protein. The fusion protein is efficiently secreted and release of active lysozyme occurs by extracellular proteolytic cleavage in the junction peptide.

Transformation of Aspergillus flavus: construction of urate oxidase-deficient mutants by gene disruption.

A transformation procedure based on the complementation of a genetic defect was developed using a nitrate reductase-deficient mutant of Aspergillus flavus. The initial transformation efficiency was improved 40-fold by combining factors in a planned experimental program. Although low, this transformation rate was sufficient to obtain transformants in which the urate oxidase-encoding gene (uaZ) was disrupted in a gene replacement experiment. These new uaZ- strains were unable to utilize uric acid as the unique nitrogen source and could be reversed directly to the wild-type phenotype in second order transformation experiments using a urate oxidase-expressing vector.

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.

A screening method to identify antibiotics of the aminoglycoside family.

A simple and selective screening method was developed for detecting new aminoglycoside (AMG) antibiotics from actinomycetes strains propagated on solid culture media. The first phase of the screening program is designed to isolate AMG-type activities using: 1) two Serratia marcescens strains, one susceptible and one resistant to AMGs, 2) the high tolerance of AMGs to heat in acidic solutions and 3) the specific resistance of a streptothricin producing strain of Streptomyces lavendulae to streptothricin antibiotics. The second phase of the screening program identifies already known AMG antibiotics through the characteristic spectrum of action which each AMG shows toward a group of bacterial strains synthesizing various AMG-inactivating enzymes.

High efficiency transformation of Tolypocladium geodes conidiospores to phleomycin resistance.

A convenient and efficient transformation system has been developed for the filamentous fungus Tolypocladium geodes. In contrast to most of the commonly described techniques requiring prior preparation of protoplasts or spheroplasts, this method leads to high efficiency transformation of T. geodes conidiospores following moderate lytic enzyme treatment. Competent cells so obtained are still resistant to osmotic pressure and can be stored frozen without loss of viability. The highest transformation frequency (3-5 x 10(3) transformants per microgram of DNA) was obtained with plasmid pUT737 containing the Sh ble gene conferring phleomycin resistance under the control of a strong promoter isolated from Trichoderma reesei. Southern hybridization revealed multiple integration sites of plasmid DNA into the T. geodes nuclear DNA despite the absence of homology between the transforming DNA and the recipient genome. Instability could not be detected for the phleomycin phenotype during more than five generations of mitotic growth under non-selective conditions.

Cloning of an aminoglycoside-resistance-encoding gene, kamC, from Saccharopolyspora hirsuta: comparison with kamB from Streptomyces tenebrarius.

An aminoglycoside-resistance-encoding gene (kamC) has been isolated from the sporaricin producer, Saccharopolyspora (Sac.) hirsuta, and expressed both in Streptomyces lividans and Escherichia coli. The pattern of resistance conferred by this gene was identical to that given by another gene (kamB) previously isolated from Streptomyces tenebrarius. In accordance with the known action of the kamB product, the Sac, hirsuta determinant also encodes a methyltransferase that modifies 16S rRNA, thereby rendering ribosomes refractory to certain aminoglycosides. The nucleotide sequences of both genes have been determined and comparison of the deduced amino acid sequences reveals a high degree of similarity.