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.

Hyperthermia enhances CTL cross-priming.

Dendritic cells (DCs) loaded with killed allogeneic melanoma cells can cross-prime naive CD8(+) T cells to differentiate into melanoma-specific CTLs in 3-wk cultures. In this study we show that DCs loaded with killed melanoma cells that were heated to 42 degrees C before killing are more efficient in cross-priming of naive CD8(+) T cells than DCs loaded with unheated killed melanoma cells. The enhanced cross-priming was demonstrated by several parameters: 1) induction of naive CD8(+) T cell differentiation in 2-wk cultures, 2) enhanced killing of melanoma peptide-pulsed T2 cells, 3) enhanced killing of HLA-A*0201(+) melanoma cells in a standard 4-h chromium release assay, and 4) enhanced capacity to prevent tumor growth in vitro in a tumor regression assay.

Interleukin 7-engineered stromal cells: a new approach for hastening naive T cell recruitment.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.

Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro.

Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines.

Comparison of murine leukemia virus, human immunodeficiency virus, and adeno-associated virus vectors for gene transfer in multiple myeloma: lentiviral vectors demonstrate a striking capacity to transduce low-proliferating primary tumor cells.

Genetic modification of primary tumor cells by gene transfer is of major interest to study the role of specific genes in the biology of a given malignancy and to modify tumor cells for therapeutic use. Multiple myeloma (MM) is a low-proliferating cancer, with often less than 1% of the cells in the S phase of the cell cycle. As primary myeloma cells are notoriously difficult to transduce, we conducted a comparison of various viral vectors, known to integrate the transgene of interest into the target genome, for their ability to stably promote the expression of an enhanced green fluorescent protein (EGFP) transgene.

Expression of a model gene in prostate cancer cells lentivirally transduced in vitro and in vivo.

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.

A Nod Scid mouse model to study human prostate cancer.

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.

Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: optimized design and procedures for highly efficient transduction compatible with clinical constraints.

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.

Transient detection of beta-galactosidase activity in hematopoietic cells, following reinjection of retrovirally marked autologous blood progenitors in patients with breast or ovarian cancer receiving high-dose chemotherapy.

Objective: The aim of this report is to demonstrate the feasibility and safety of genetically modifying autologous human blood CD34(+) cells in vitro, with a retroviral vector that encodes a marker gene. The fate of genetically modified cells and their progeny was followed in vivo, after reinfusion in patients treated with high-dose chemotherapy for poor-prognosis breast or ovarian carcinomas.

Patients And Methods: Six patients received genetically modified autologous peripheral blood progenitors, together with unmanipulated aphereses, following high-dose chemotherapy.

Retrovirus-mediated gene transfer of the cytokine genes interleukin-1beta and tumor necrosis factor-alpha into human neuroblastoma cells: consequences for cell line behavior and immunomodulatory properties.

We have investigated the value of a gene therapy approach for neuroblastoma (NB), based on retroviral transduction of the IL-1beta or TNF-alpha cytokine genes into human NB lines. Secretion of the corresponding cytokine, was demonstrated in all lines, although with considerable quantitative variations. Cytokine gene expression significantly reduced the proliferation index (p = 0.

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.

Peripheral stem cells in bone marrow transplantation. Peripheral blood stem cell and gene therapy.

Mobilized peripheral blood stem cells characterized by sustained re-populating ability could be optimal target cells for ex-vivo gene transfer. In spite of very attractive preliminary results obtained in the murine studies, therapeutically efficient gene transfer and expression in human targeted cells must be proven. In recent years, effort has been spent on the identification of factors limiting gene transfer efficiency of haematopoietic stem cells.

T-lymphocyte function after retroviral-mediated thymidine kinase gene transfer and G418 selection.

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.

T lymphocyte transduction with herpes simplex virus-thymidine kinase (HSV-tk) gene: comparison of four different infection protocols.

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping.

Expression of the nlsLacz gene in dendritic cells derived from retrovirally transduced peripheral blood CD34+ cells.

Background And Objective: Gene transfer and expression of exogenous genetic information coding for an immunogenic protein in antigen presenting cells (APCs) can promote an immune response. This was investigated by retroviral transfer of a marker gene into CD34+ derived APCs.

Design And Methods: To achieve long term expression of a specific transgene in APCs, G-CSF mobilized peripheral blood CD34+ cell populations were retrovirally transduced with the bacterial nlsLacZ, a marker gene used here as a model, in the presence of IL-3, IL-6, GM-CSF and SCF prior to being induced to differentiate into dendritic and macrophage cells by GM-CSF and TNF-a.

Ex vivo manipulations alter the reconstitution potential of mobilized human CD34+ peripheral blood progenitors.

The phenotype and functions of CD34+ cells isolated from peripheral blood (PB) of steady-state healthy volunteers (ssPB-CD34), and of patients or healthy volunteers after mobilization (mPB-CD34) were investigated. ssPB-CD34+ cells contain a lymphoid cell population that co-express T or B cell markers, while mPB-CD34+ cells lack this population. After 5-day culture, significantly higher levels of expansion in cell, CD34+ cell, and HPP-CFC numbers were induced in ssPB-CD34+ cells, as compared to mPB-CD34+ cells.

In vivo rescue of a silent tax-deficient bovine leukemia virus from a tumor-derived ovine B-cell line by recombination with a retrovirally transduced wild-type tax gene.

The lack of bovine leukemia virus (BLV) expression is a consistent finding in freshly isolated ovine tumor cells and in the B-cell lines derived from these tumors. In order to gain further insight into the mechanisms of BLV silencing in these tumors, we have used the YR2 B-cell line, which was derived from the leukemic cells of a BLV-infected sheep. This cell line contains a single, monoclonally integrated, silent provirus, which cannot be reactivated either by stimulation in vitro or by in vivo injection of the tumor cells or cloned proviral DNA in sheep.

Anti-GM-CSF monoclonal antibody therapy for refractory acute leukemia.

Several phase I trials and pilot studies using Monoclonal Antibody (MoAb) have been performed in B-cell neoplasms, but this approach has not until now been extensively tested in myeloid leukemias. Recently, we evaluated the use of anti-Granulocyte-Macrophage Colony-Stimulating Factor MoAb (Anti-GM-CSF MoAb) in acute myeloid leukemia (AML). Eight patients fulfilled inclusion criteria and received a single course of Anti-GM-CSF MoAb infusion during 5 to 15 days.

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

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.

Preferential photoinactivation of leukemia cells by aluminum phthalocyanine.

The efficacy of chloroaluminum phthalocyanine (AlPc) for photodynamic therapy (PDT) has been evaluated in vitro on acute myeloid leukemia (AML) cells, normal peripheral blood leukocytes (PBL) and mobilized peripheral blood stem cells (mPBSC). The selectivity of the treatment has been evaluated by mixing PBL and TF-1, an erythroleukemic cell line. Upon photoradiation, this photosensitizer leads to a significant and preferential photokilling of leukemia cells in comparison to normal cells.