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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.

Hepatocellular carcinoma (HCC), the most prevalent histological form of primary liver cancer is one of the most frequent cancer worldwide. This pathology still requires the development of new therapeutical approaches. Gene therapy strategies focusing on the genetic manipulation of accessory cells involved in the immune reaction against cancer cells, or on the direct transduction of tumor cells with transgenes able to "suicide" cancer cells have been largely developed for more than ten years.

Suicide gene therapy combining herpes simplex virus thymidine kinase gene transfer and ganciclovir administration can be envisioned as a powerful therapeutical approach in the treatment of hepatocellular carcinoma; however, safety issues regarding transgene expression in parenchyma cells have to be addressed. In this study, we constructed LATKW, a lentiviral vector expressing the HSV-TkEGFP gene placed under the control of the promoter elements that control the expression of the rat alpha-fetoprotein, and assayed its specific expression in vitro in hepatocarcinoma and nonhepatocarcinoma human cell lines, and in epidermal growth factor stimulated human primary hepatocytes. Using LATKW, a strong expression of the transgene was found in transduced hepatocarcinoma cells compared to a very low expression in nonhepatocarcinoma human cell lines, as assessed by Northern blot, RT-PCR, FACS analysis and ganciclovir-mediated toxicity assay, and no expression was found in lentivirally transduced normal human hepatocytes. Altogether, these results demonstrate the possibility to use a lentivirally transduced expression unit containing the rat alpha-fetoprotein promoter to restrict the HSV-TK-mediated induced GCV sensitivity to human hepatocarcinoma cells.

In a preclinical model for prostate cancer gene therapy, we have tested lentiviral vectors as a practical possibility for the transfer and long-term expression of the EGFP gene both in vitro and in vivo. The human prostate cancer cell lines DU145 and PC3 were transduced using experimental conditions which permitted analysis of the expression from a single proviral vector per cell. The transduced cells stably expressed the EGFP transgene for 4 months. After injection of the transduced cell populations into Nod-SCID mice a decrease in EGFP was only observed in a minority of cases, while the majority of tumors maintained transgene expression at in vitro levels. In vivo injection of viral vector preparations directly into pre-established subcutaneous or orthotopic tumor masses, obtained by implantation of untransduced PC3 and DU145 cells led to a high transduction efficiency. While the efficiency of direct intratumoral transduction was proportional to the dose of virus injected, the results indicated some technical limitations inherent in these approaches to prostate cancer gene therapy.

Gene delivery to dendritic cells (DCs) could represent a powerful method of inducing potent, long-lasting immunity. Although recent studies underline the intense interest in lentiviral vector-mediated monocyte-derived DC transduction, efficient gene transfer methods currently require high multiplicities of infection and are not compatible with clinical constraints. We have designed a strategy to optimize the efficiency and clinical relevance of this approach. Initially, using a third generation lentiviral vector expressing green fluorescent protein, we found that modifying the vector design, the DC precursor cell type, and the DC differentiation stage for transduction results in sustained transgene expression in 75-85% of immature DCs (transduction at a multiplicity of infection of 8). This high efficiency was reproducible among different donors irrespective of whether DCs were expanded from fresh or cryopreserved CD14(+) precursors. We then developed procedures that bypass the need for highly concentrated lentiviral preparations and the addition of polybrene to achieve efficient transduction. DCs transduced under these conditions retain their immature phenotype and immunostimulatory potential in both autologous and allogeneic settings. Furthermore, genetically modified DCs maintain their ability to respond to maturation signals and secrete bioactive IL-12, indicating that they are fully functional. Finally, the level of transgene expression is preserved in the therapeutically relevant mature DCs, demonstrating that there is neither promoter-silencing nor loss of transduced cells during maturation. The novel approach described should advance lentiviral-mediated monocyte-derived DC transduction towards a clinical reality.

Prostate cancer is the second cause of cancer mortality in men in Western countries. To study new therapeutic approaches such as gene therapy, animal models of human prostate cancer with metastatic behavior are mandatory. We used the Nod Scid mouse strain to develop an orthotopic animal model. Two androgen-independent cell lines (PC-3 and DU 145) were used. Local tumor growth and metastases were analyzed. The tumor take rates were close to those reported in the literature. However, a high frequency of various metastatic sites has been observed (liver, lung, spleen, adrenal, kidney, lymph node, and diaphragm). It can be concluded that the Nod Scid mouse is a relevant preclinical animal model to study human prostate cancer. Metastatic sites seem more numerous in comparison to other orthotopic mice models described.

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.

Generation of an efficient graft-versus-leukemia (GVL) effect in patients with hematological malignancies who relapse after allogeneic bone marrow transplantation depends in part upon the number of infused T lymphocytes. Currently, a GVL reaction cannot be achieved without inducing concomitant graft-versus-host disease (GVHD); thus, one strategy is to try to modulate this GVL/GVHD ratio. We engineered human T lymphocytes with herpes simplex virus-thymidine kinase and neomycin resistance genes, with an LXSN-derived vector that confers a ganciclovir-specific sensitivity to the transduced T cells. We analyzed proliferation, interleukin-2 production, alloreactivity in a mixed lymphocyte culture, and clonogenicity during the different stages of retroviral infection and G418 selection. Our results confirm that a sufficient number of transduced T lymphocytes can be obtained after selection for clinical studies. Their proliferative activity, alloresponsiveness, and ability to produce and respond to interleukin-2 were retained. Compared with control populations, their clonogenicity, as assessed by limiting dilution assays, was reduced after retroviral infection and G418 selection by 1.6 and 2.9 logs, respectively, with both viral supernatant incubation and coculture procedures. This study shows that infection and selection with the thymidine kinase-neomycin resistance gene retroviral vector significantly reduces the number of functional T lymphocytes. This finding should be taken into account when establishing the dose of T lymphocytes necessary to trigger a modulated GVL/GVHD effect.

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

Relative transduction efficiency with retroviral vector-producing clones was assayed by cocultivating TF-1, a human CD34+ hematopoietic cell line and YR-2, a sheep B-lymphoid cell line, with LacZ containing vector-producing cells, and then by scoring the percentage of X-Gal+ cells. At the same time, viral titer was estimated by titration assay with murine fibroblasts. Results clearly demonstrated a lack of correlation between viral titer and efficiency of transduction into hematopoietic cells, which depends neither on the type of packaging cell line, PG-13 and GP-envAM12 in this study, nor on the type of LacZ containing retroviral vector. These results strongly favor consideration of interactions between producers and target cells of the study for the screening of producing cell lines.

T lymphocytes are a promising cell vehicle for gene therapy purposes. By cocultivating retroviral vector producing cells and target cells, highly efficient gene transfer was achieved with activated human T lymphocytes derived from peripheral blood with vectors carrying different forms of the bacterial beta-galactosidase gene including the regular LacZ gene, the Sh-ble::LacZ gene and the nlsLacZ gene. Infection kinetics of T cells activated by a combination of monoclonal antibodies directed against CD2 and CD28 indicated that the highest efficiencies of transduction were obtained when the cocultivation began 4 days after stimulation. In fact, with the FLac vector, a new retroviral vector which expresses the Sh-ble::LacZ gene, we observed up to 78% transduction efficiency assessed by X-gal staining performed 2 days after the end of the cocultivation. Expression of the transduced genes was observed throughout the period of culture. Neither the cocultivation step nor the expression of the transduced Sh-ble::LacZ gene altered cell culture proliferation or the expression of selected cell surface antigens. In addition, we showed that CD4+ and CD8+ T cells were equally transduced.

Recombinant granulocyte colony-stimulating factor (rhG-CSF) has been shown to hasten granulocyte recovery after autologous BMT. In current protocols, rhG-CSF treatment starts 1 day after BM reinfusion. Our study retrospectively examined the effects on haematological recovery of a day 6 delayed administration. Seventy-eight patients receiving autologous BMT for malignant lymphoma (21 non-Hodgkin's lymphoma and 9 Hodgkin's disease) or solid tumors (33 breast carcinoma and 5 ovarian carcinoma) were split up into three study groups. Two groups receiving a 5 micrograms/kg/day of rhG-CSF starting either 1 day (day +1 group, n = 25 patients) or 6 days (day +6 group, n = 24 patients) after BM reinfusion were compared with 29 historical control patients. Granulocyte recovery to 0.5 x 10(9)/l was 12 days in day +6 and day +1 groups versus 16 days in control group (p < 0.005) without any difference in other hematological parameters, infectious complications or length of hospitalisation between the three groups. The day +6 administration allows elimination of a median of 7 days rhG-CSF. It has been concluded that the day +6 administration gives the same clinical benefit as day +1 administration with consequent cost reductions.

Colony stimulating factors and especially rHuG-GSF, the first available neutrophil growth factor, have led to considerable interest in the field of stem cell transplantation because of their ability to induce stem cell peripheralization either alone or in association with high-dose chemotherapy. Few data exist, however, on the impact of rHuG-CSF on large scale bone marrow collection and autologous bone marrow transplantation (ABMT). This phase I, non-randomized, dose escalation study of rHu-G-CSF (lenograstim) administered to 30 patients at doses ranging from 1 to 40 micrograms/kg/day for 5 days before bone marrow harvesting showed that priming with rHu-G-CSF in vivo increased the number of bone marrow cells and D14 myeloid restricted progenitors (CFU-GM) and led to a better neutrophil recovery after ABMT compared with a contemporary unprimed control population. Otherwise, this study established that 5 days of rHuG-CSF therapy, as a sole stimulus, induced a tenfold increase in the circulating CFU-GM amongst which immature progenitors, estimated by the Delta assay (secondary CFU-GM grown after 7 days of liquid culture/primary CFU-GM), are detected. These conclusions were valid for doses as low as 2 micrograms/kg/day which induced only mild neutrophilia up to the highest dose (40 micrograms/kg/day) and suggest that a short course of rHuG-CSF is beneficial in increasing the stem cell collection.

We recently cloned an additional member of the receptor type tyrosine kinase class III. This new gene, called Flt3 by our group [Rosnet, O., Matteï, M.G., Marchetto, S. & Birnbaum, D. (1991). Genomics, 9, 380-385; Rosnet, O., Marchetto, S., deLapeyriere, O. & Birnbaum, D. (1991). Oncogene, 6, 1641-1650] and Flk2 by others [Matthews, W., Jordan, C.T., Wieg, G.W., Pardoll, D. & Lemischka, I.R. (1991). Cell, 65, 1143-1152] is strongly related to the important developmental genes Kit, Fms and Pdgfr. The murine 3.2-kb full-length cDNA, when introduced into COS-1 cells, shows the expression of two polypeptides with apparent molecular weights of 155 kDa and 132 kDa. Treatment of cells with N-linked glycosylation inhibitors results in the expression of a 110-kDa protein. We have shown that FLT3 contains an intrinsic tyrosine kinase activity. A point mutation in a highly conserved residue within the phosphoryltransferase domain inactivates the catalytic function of this receptor, whereas activation by way of a chimeric molecule between the ligand-binding domain of colony-stimulating factor type 1 (CSF-1) receptor (CSF-1R) and the kinase domain of FLT3 results, in the presence of CSF-1, in the development of the transforming activity of this receptor as shown by anchorage-independent cell growth. Finally, expression analysis of the FLT3 protein shows that, in addition to the hematopoietic system, FLT3 is strongly expressed in neural, gonadal, hepatic and placental tissues in the mouse.

We report here 16 autologous bone marrow transplantations (ABMT) for poor prognosis B or pre-B malignancies (16 acute lymphoblastic leukemias (ALL), three Burkitt lymphomas, one multiple myeloma) in 11 adults and five children where in vitro purging was accomplished by means of floating immunobeads. This method was developed to avoid non-specific killing by complement or toxin or batch-to-batch variability and provides a 3 log reduction of tumor in a model of B lymphoid malignancies. Low density bone marrow mononuclear cells were incubated for 30 min at 4 degrees C with anti CD10 (ALB2 Immunotech) and/or anti CD19 (Bg4) monoclonal antibodies (MoAb) and then mixed with low density polypropylene beads precoated with a rat antimouse MoAb. After 1 h at 4 degrees C the beads with target cells were decanted; the depleted marrow was collected through a microfilter and cryoperserved. After immunodepletion the recovery of nucleated cells was 75% with a median of 0.75 x 10(8) cells/kg (range 0.3-3.6) and the recovery of hematopoietic progenitors was 83% with a median of 2.9 x 10(4) CFU-GM/kg. The conditioning regimen consisted of busulfan 16 mg/kg and melphalan 140 mg/m2 for three patients, fractionated total body irradiation (TBI) following melphalan 140 mg/m2 for nine patients, TBI and cyclophosphamide 120 mg/m2 for two patients and TBI associated with melphalan and cyclophosphamide for two patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-cell adhesion is essential for many immunological functions. The LFA-1 molecule, a member of a superfamily of adhesion molecules, participates in adhesion which is critical to the function of each of the three major subsets of leukocytes: lymphocytes, monocytes and granulocytes. Putative LFA-1 ligands have been identified functionally in different laboratories using three different monoclonal antibodies that inhibit LFA-1-mediated leukocyte adhesion in particular model systems; however, there may be more than one LFA-1 ligand. We have directly compared the three relevant monoclonal antibodies, and show that each binds to the same molecule, intercellular-adhesion molecule-1 (ICAM-1). Most important, B, T and myeloid cells adhere specifically to purified ICAM-1-coated surfaces; such adhesion has distinctive requirements for Mg2+ and Ca2+. This constitutes biochemical evidence that ICAM-1 functions as a ligand for LFA-1-dependent adhesion by a variety of leukocytes.