Transient gene expression mediated by integrase-defective retroviral vectors

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.     

Mutations in p53 cDNA sequence introduced by retroviral vector

The high mutation rates of retroviruses are a potential problem with retroviral vectors. We studied the mutation rates and spectra of p53 sequences transduced with a retroviral vector in a cancer gene therapy model. When p53-deficient H358 non-small cell lung cancer cells were treated with a retroviral vector carrying normal p53 cDNA, most of transduced cells were killed by apoptosis. However, a small number of clones escaped p53-mediated apoptosis. We examined the p53 cDNA structure in these resistant clones. PCR-based analysis showed that 88/102 clones had detectable mutations in p53, including gross rearrangements, deletions/insertions, and base substitutions. To study the mutation rate of the p53 sequence in all transduced clones, the retroviral vector containing the non-functional p53 gene and the Neo-resistant marker gene was introduced into H358 cells. Only one of 95 isolated clones showed a base substitution. These results indicate that the mutation rate of p53 is not particularly high, but there is a significant risk that cancer cells will resist p53 gene therapy as a result of retroviral replication errors.     

Expression of the extracellular domain of OB-cadherin as an Fc fusion protein using bicistronic retroviral expression vector

Osteoblast cadherin (OB-cadherin, also known as cadherin-11) is a Ca²⁺-dependent homophilic cell adhesion molecule that is expressed mainly in osteoblasts. OB-cadherin is expressed in prostate cancer and may be involved in the homing of metastatic prostate cancer cells to bone. The extracellular domain of OB-cadherin may be used to inhibit the adhesion between prostate cancer cells and osteoblasts. In this report, we describe the expression of the extracellular domain of OB-cadherin as an Fc fusion protein (OB-CAD-Fc) in human embryonic kidney 293FT cells using a bicistronic retroviral vector. Coexpression of GFP and OB-CAD-Fc through the bicistronic vector permitted enrichment of OB-CAD-Fc-expressing cells by fluorescence-activated cell sorting. Recombinant OB-CAD-Fc proteins were secreted into cell medium, and about 0.85 mg of purified OB-CAD-Fc protein was purified from 1 l of the conditioned medium using immobilized protein A-affinity chromatography. The purified OB-CAD-Fc was biologically active because it supported the adhesion of PC3 cells and L cells transduced with OB-cadherin. The availability of OB-CAD-Fc offers opportunities to test whether OB-CAD-Fc can be used to inhibit OB-cadherin-mediated prostate cancer bone metastasis in vivo or to generate antibodies for inhibiting the adhesion between prostate cancer cells and osteoblasts.     

p120 serine and threonine phosphorylation is controlled by multiple ligand-receptor pathways but not cadherin ligation

p120-catenin (p120) regulates cadherin turnover and is required for cadherin stability. Extensive and dynamic phosphorylation on tyrosine, serine and threonine residues in the N-terminal regulatory domain has been postulated to regulate p120 function, possibly through modulation of the efficiency of p120/cadherin interaction. Here we have utilized novel phospho-specific monoclonal antibodies to four major p120 serine and threonine phosphorylation sites to monitor individual phosphorylation events and their consequences. Surprisingly, membrane-localization and not cadherin interaction is the main determinant in p120 serine and threonine phosphorylation and dephosphorylation. Furthermore, the phospho-status of these four residues had no obvious effect on p120's role in cadherin complex stabilization or cell-cell adhesion. Interestingly, dephosphorylation was dramatically induced by PKC activation, but PKC-independent pathways were also evident. The data suggest that p120 dephosphorylation at these sites is modulated by multiple cell surface receptors primarily through PKC-dependent pathways, but these changes do not seem to reduce p120/cadherin affinity.     

Factors affecting the performance of different long terminal repeats in the retroviral vector

The long terminal repeat (LTR) of retrovirus contains the nucleotide sequences that control gene expression. Although several different LTRs have been used in the context of retroviral vector, the activity of the various LTRs has not yet been systematically compared for their level of gene expression. We evaluated the effect of four different LTRs on gene expression using luciferase, stem cell factor, and enhanced green fluorescence protein as reporter genes. LTRs tested in this study were derived from Moloney murine leukemia virus, myeloproliferative sarcoma virus, murine stem cell virus, and spleen focus-forming virus. It was found that the level of gene expression is affected by not only LTRs but also the transgenes and the cell types in which gene expression occurs. Furthermore, the presence of other nucleotide sequences such as the internal ribosome entry site (IRES)-neo cassette could also significantly affect gene expression. Our results suggested that the LTR should be chosen carefully, more or less on an empirical basis.     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.     

PDGF receptor activation induces p120-catenin phosphorylation at serine 879 via a PKCα-dependent pathway

p120-catenin (p120) is required for cadherin stability and is thought to have a central role in modulating cell-cell adhesion. Several lines of evidence suggest that S/T phosphorylation may regulate p120 activity, but the upstream kinases involved have not been established, nor has a discreet measurable function been assigned to an individual site. To approach these issues, we have generated p120 phospho-specific monoclonal antibodies to several individual phosphorylation sites and are using them to pinpoint upstream kinases and signaling pathways that control p120 activity. Protein Kinase C (PKC) has been implicated as a signaling intermediate in several cadherin-associated cellular activities. Signaling events that activate PKC induce rapid phosphorylation at p120 Serine 879 (S879), suggesting that p120 activity is regulated, in part, by one or more PKC isoforms. Here, we find that physiologic activation of a G-protein coupled receptor (i.e., endothelin receptor), as well as several Receptor Tyrosine Kinases, induce rapid and robust p120 phosphorylation at S879, suggesting that these pathways crosstalk to cadherin complexes via p120. Using Va2 cells and PDGF stimulation, we show for the first time that PDGFR-mediated phosphorylation at this site is dependent on PKCα, a conventional PKC isoform implicated previously in disruption of adherens junctions.     

A speedy method to detect inserted DNA fragment in cell lines transfected with retroviral vectors

Cells transfected by retroviral vectors are brought in agene of particular interest and are very useful in avariety of experiments. It is essential to testify that theDNA fragment was successfully introduced into the cellstogether with the retroviral vectors. Polymerase chainreaction is believed to be a fast and convenient method forthis purpose when using primers flanking the cloning siteof the inserted DNA. Unfortunately, a single PCR reactionoften fails to amplify the targeted fragment because of theexistence of endogenous virus DNA in cell genome. However,in this study we conducted a procedure for a single PCR,using vector-specific primers as well as a nested PCR, andsuccessfully detected the DNA fragments cloned in MFGretroviral vectors in 22 transfected cell lines. We alsoproved that real time quantitative PCR in combination withMFG-specific primer is useful to determine copy number ofthe retroviral vector in murine producer cell lines.     

Luminometric method for screening retroviral protease inhibitors

We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni-NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason-Pfizer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors confirmed that this method is sufficiently sensitive and specific for high-throughput screening of retroviral protease inhibitors.     

In vivo diversification and migrations of chick embryo heart muscle cells: a morphometric analysis with ALV- and SNV-based non-replicative vectors

 By means of a reporter gene we previously demonstrated that non-replicative Avian Leukemia Virus- and Spleen Necrosis Virus-based retroviral vectors were preferentially expressed in the heart of avian embryos from different species. Using a computer-assisted approach, we now compare clones tagged by the two types of vectors, for volume, anatomical and subanatomical localisation, number of Hoechst-stained cell nuclei and mean cell division time during the period of heart morphogenesis, i.e. from stages 17–19 to 34 of Hamburger and Hamilton (1951). This analysis demonstrates that clones labelled by the two types of viruses display similar features and bring about new insights on the relationships between mitotic and migratory properties of the myocardial cells and histogenesis of the heart. Since only exteriormost cells were tagged with our inoculation procedure, our analysis shows that: (1) at stages 17–19, the myocardium is composed of cells with diverse potentials; some cells still retain the capacity to divide extensively and participate to different heart muscle layers, whilst most are restricted in their multiplication potential and contribute to single muscle layers; (2) about half of the clones are located deep in the heart wall, revealing extensive cell migrations from the heart surface to the ventricular trabeculae, the first migrating cells tagged being detected 20 h after viral inoculation. The presence of these cells is consistent with the finding of a large number of compact trabecular clones 5 days later suggesting that these cells divide mainly after completing migration. Our approach provides new insights as well as quantitative data on the different processes involved in heart morphogenesis, namely multiplication, migration and localisation of heart muscle cells. Received: 10 March 1996 / Accepted in revised form: 7 July 1996     

Retroviral vectors

The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology.