Use of the PSA enhancer core element to modulate the expression of prostate- and non-prostate-specific basal promoters in a lentiviral vector context.

Composite promoters combining the prostate-specific antigen (PSA) enhancer core element with promoter elements derived from gene coding for human prostate-specific transglutaminase gene, prostate-specific membrane antigen gene, prostate-specific antigen, rat probasin or phosphoglycerate kinase were characterized for their ability to specifically express the enhanced green fluorescent protein (EGFP) gene in prostate versus non-prostate cancer cell lines when transferred with a human immunodeficiency virus-1-based lentiviral vector. By themselves minimal proximal promoter elements were found to inefficiently promote relevant tissue-specific expression; in all the vectors tested, addition of the PSA enhancer core element markedly improved EGFP expression in LnCaP, a cancer prostate cell line used as a model for prostate cancer. The composite promoter was inactive in HuH7, a hepatocarcinoma cell line used as a model of neighboring non-prostate cancer cells. Among the promoters tested, the combination of the PSA enhancer and the rat probasin promoter showed both high specificity and a strong EGFP expression. Neither a high viral input nor the presence of the cPPT/CTS sequence affected composite promoter behavior. Our data suggest that composite prostate-specific promoters constructed by combining key elements from various promoters can improve and/or confer tissue specific expression in a lentiviral vector context.

Death of motoneurons induced by trophic deprivation or by excitotoxicity is not prevented by overexpression of SMN.

The telomeric copy of the survival motor neuron gene (SMN1) is deleted or mutated in all spinal muscular atrophy (SMA) patients and these patients present mainly a loss in spinal motoneurons. Although studies performed in HeLa cells suggest that SMN may be involved in the biogenesis and possibly in recycling of spliceosomal small nuclear ribonucleoproteins (snRNPs), no link has been established between this function and the consequence of the absence of SMN in the specific loss of motoneurons. We attempted to answer the question of whether SMN plays a direct role in motoneuron survival by transducing cultured motoneurons with lentiviral vectors coding either for an antisense Smn mRNA or for full-length or truncated forms of SMN. We studied their effect on survival under different anti- or proapoptotic culture conditions. Our results show that increased levels of SMN are unable to protect motoneurons from death induced by trophic deprivation or by excitotoxicity. These results suggest that SMN is not a survival factor per se for motoneurons. In addition, overexpression of a truncated form of SMN shown to induce a modified subcellular localization and to exert a dominant-negative effect on snRNP biogenesis and RNA splicing in HeLa cells was ineffective in modifying both localization and survival in motoneurons.

Gene transfer to hepatocellular carcinoma: transduction efficacy and transgene expression kinetics by using retroviral and lentiviral vectors.

Gene therapy is an attractive therapy for hepatocarcinoma, and several approaches have been studied using murine leukemia virus-derived retroviruses. We compared gene transfer efficacy and transgene expression kinetics after transduction of hepatocarcinoma cell lines using enhanced green fluorescent protein (EGFP)-expressing murine leukemia virus-derived retroviral vectors and HIV-derived lentiviral vectors. First, we showed that both retroviral and lentiviral vectors efficiently transduce cycling hepatocarcinoma cell lines in vitro. However, after cell cycle arrest, transduction efficacy remained the same for lentiviral vectors but it decreased by 80% for retroviral vectors. Second, we studied EGFP expression kinetics using lentiviral vectors expressing EGFP under the control of cytomegalovirus (CMV) or phosphoglycerolkinase (PGK) promoter. We show that the CMV promoter allows a stronger EGFP expression than the PGK promoter. However, in contrast to PGK-driven EGFP expression, which persists up to 2 months after transduction, CMV-driven EGFP expression rapidly decreased with time. This phenomenon is due to promoter silencing, and EGFP expression can be restored in transduced cells by using transcription activators such as interleukin-6 or phorbol myristate acetate/ionomycin and, to a lesser extent, the demethylating agent 5'-azacytidine. Altogether, our results suggest that lentiviral vectors, which allow efficient transduction of hepatocarcinoma cell lines with a strong and a sustained expression according to the promoter used, are promising tools for gene therapy of hepatocarcinomas.

The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule.

We have recently described Poliovirus Receptor Related 2 (PRR2), a new cell surface molecule homologous to the poliovirus receptor (PVR/CD155). Both molecules are transmembrane glycoproteins belonging to the Ig superfamily (IgSF). They contain 3 Ig domains of V, C2, and C2 types in their extracellular regions that share 51% aa identity. The PRR2 gene encodes two mRNA isoforms of 3.0 kb (hPRR2 [short form]) and 4.4 kb (hPRR2delta [long form]), both widely expressed in human tissues, including hematopoietic cells. To further characterize PRR2 expression during hematopoiesis and to analyze its function, we have developed a monoclonal antibody (MoAb) directed against its extracellular region (R2.477). PRR2 was expressed in 96% of the CD34(+), 88% of the CD33(+), and 95% of the CD14(+) hematopoietic lineages and faintly in the CD41 compartment. Ectopic expression of both PRR2 cDNAs induced marked cell aggregation. A soluble chimeric receptor construct with the Fc fragment of human IgG1 (PRR2-Fc) as well as a fab fragment of the anti-PRR2 MoAb (R2.477) inhibit aggregation. PRR2-Fc binds specifically to PRR2-expressing cells. These results suggest that PRR2 is a homophilic adhesion receptor. PRR2 was also expressed at the surface of endothelial cells at the intercellular junctions of adjacent cells but not at the free cellular edges. Homophilic interactions are associated with dimerization of isoforms of PRR2 and lead to the tyrosine phosphorylation of PRR2delta. Altogether, these results suggest that homophilic properties of PRR2 could participate to the regulation of hematopoietic/endothelial cell functions.