Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression.

Gene transfer into muscle by electroporation with low-voltage and long-pulse (LV/LP, 100 V/50 msec) currents was shown to be more efficient than simple intramuscular DNA injection. Nevertheless, transgene expression declined from day 7 and only reached 10% of the maximum 3 weeks after electroporation. We have optimized electroporation conditions including voltage, pulse number, and the amount of injected luciferase-encoding plasmid DNA in the tibialis anterior muscle. Using high-voltage and short-pulse (HV/SP, 900 V/100 microsec) currents, we observed an average 500-fold increase in luciferase expression, in comparison with nonelectroporated muscle. Moreover, sustained and long-lasting gene expression was observed for at least 6 months. When we compared HV/SP currents with LV/LP currents, luciferase expression was similar 24 hr after electroporation. One month later, whereas luciferase expression was stable in muscle electroporated with HV/SP currents, it decreased 600-fold in muscle electroporated with LV/LP currents. In conclusion, electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression in muscle.

Increased bax expression is associated with cell death induced by ganciclovir in a herpes thymidine kinase gene-expressing glioma cell line.

The herpes simplex virus thymidine kinase gene (HSV-tk) was stably transfected into rat C6 glioma cells (C6tk) in order to characterize the mechanisms underlying cell toxicity induced in vitro by the guanosine analog ganciclovir (GCV). The results demonstrate the efficiency of the HSV-tk/GCV system in ablating most of the tumoral cells within 7 to 8 days of treatment with 20 mivroM GCV; however, a few cells still survive. C6tk cells arrest in the S phase of the cell cycle after 2 days of drug treatment before undergoing cell death. Microscopic analysis reveals dying cells with ultrastructural characteristics consistent with apoptosis; we cannot rule out, however, that necrotic cell death may also be occurring. The cytotoxicity induced by GCV is not associated with changes in the expression of p53 protein, suggesting that cell cycle arrest and cell death may occur through a p53-independent pathway. C6tk cells constitutively express Bcl-xL and Bax proteins; when exposed to GCV, Bcl-xL levels do not change but Bax accumulation is rapidly induced. These findings suggest that the balance between Bcl-xL and Bax proteins may be of importance in determining the sensitivity of tumoral cells to GCV.

Mycoplasma: a new potential vector for gene therapy?

The introduction of foreign genetic material in living cells is the basis of the current protocols of gene therapy. The concept that the de novo synthesis of a foreign therapeutic protein requires the entrance of the corresponding gene into target cells via virus or synthetic vectors is directly inherited from experiments on bacterial transduction or transformation. However, the difficulties inherent in the penetration and the expression of foreign DNA into eucaryotic cells are probably responsible for the low efficiency of this therapeutic approach. In this paper, we explore the possibility of avoiding these limiting critical steps by expressing the foreign gene on the surface rather than inside the target cells by the use of mycoplasma, the smallest reported living cell. The absence of transfer of genetic information between this vector and eucaryotic target cells, the sensibility of mycoplasmas to antibiotics and their cytadherance are among the interesting features of this potential vector. The interest of this new vector in the case, e.g. of the gene-directed enzyme prodrug therapy of solid tumors, is discussed.

[The biology of nasopharyngeal carcinoma in 2001: update and perspective]. / La biologie des carcinomes nasopharyngés en 2001: mise à jour et perspectives.

The phenotype of malignant epithelial cells in nasopharyngeal carcinomas (NPC) results from latent infection by the Epstein-Barr virus (EBV) combined to cell gene alterations, especially those affecting the p16/Ink 4 gene. At the site of the primary tumor, NPC are strongly infiltrated by non-malignant EBV-negative T-lymphocytes. There are experimental clues suggesting that these lymphocytes are involved in tumor development, for example by providing anti-apoptotic signals to malignant epithelial cells. The amazing geographic distribution of NPC is accounted for by the conjunction of several risk factors in endemic regions. These risk factors are related to the diffusion of one or several susceptibility genes, the probable existence of viral strains with high oncogenic potential and non-viral environmental factors, especially dietary factors. The perspectives of immunotherapy in NPC are still unclear since viral proteins detected in tumors are poorly immunogenic (EBNA1, LMP1). Targeted molecules designed to interfere with viral and cellular oncoprotein signals will probably have interesting applications for the treatment of NPC.

Bacterial DNA methylation and gene transfer efficiency.

The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of 5-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, but the possible consequences of the presence of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+), dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and C(m)C(A/T)GG sequences in eukaryotic cells or organisms.

Therapeutic efficacy of the thymidine kinase/ganciclovir system on large experimental gliomas: a nuclear magnetic resonance imaging study.

Contradictory experimental results and human trials have questioned the clinical relevance of the HSVtk/ganciclovir system. To bypass the problem of transfection efficiency, we used a glioma cell line stably expressing the HSVtk gene, which was also fully characterized from gene to protein. We also designed a more clinically relevant experimental protocol, consisting of late GCV delivery on large tumor formations. In short-term studies, histological examination revealed a significant decrease in tumor volume in GCV-treated animals from day 1 or from day 10 after cell inoculation. We observed that late GCV delivery is as efficient as early delivery, probably because GCV can reach tumor cells more easily when neoangiogenesis occurs. In long-term experiments, the survival of treated rats bearing 15-day tumors was improved by 60% compared with C6 control animals. Surprisingly, a 30% survival rate was observed in C6TK control animals. Nuclear magnetic resonance imaging demonstrated, in all surviving animals, a complete regression of tumors without mass effect. These results clearly demonstrate that the HSVtk/GCV system remains a potent therapeutic strategy, even when tested in large tumors, in contrast with the microscopic tumor formations previously reported.