Retroviral-mediated gene transfer of CD34-enriched bone marrow and peripheral blood cells during autologous stem cell transplantation for multiple myeloma.
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M S Dilber
Publications and source records attributed to M S Dilber.
Retroviral-mediated gene transfer into human myeloma cells.
We have studied retroviral-mediated gene transfer into human myeloma cells. Bone marrow cells were obtained from four patients with advanced myeloma, where the marrow was heavily infiltrated with myelomatous plasma cells. Myeloma cells were isolated by immunomagnetic separation, using the high-affinity B-B4 monoclonal antibody. Following separation, cells were transduced with the LN retroviral vector, which carries the gene for neomycin phosphotransferase, by incubation in cell-free supernatant with or without a growth-factor combination of IL-3, IL-6 and SCF. After infection, the cells were cultured for 9 d in RPMI-1640 and 10% FCS, either in the presence or absence of the neomycin analogue G418. Transduction efficiency was 1.5-3.8%, when compared to the number of cells at initiation of the culture, and 5.0-50.0% when compared to the number of surviving infected cells cultured without G418. The gene transfer rate was similar whether or not growth factors were present during the retroviral infection. These preclinical data provide evidence that retroviral-mediated gene transfer into human myeloma cells is feasible, and form part of the basis for current clinical studies of gene marking of bone marrow or peripheral blood progenitor cells before autologous stem cell transplantation in multiple myeloma.
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Basic fibroblast growth factor increases retroviral-mediated gene transfer into human hematopoietic peripheral blood progenitor cells.
Retroviral vector-mediated gene transfer into human hematopoietic stem cells (HSC) may permit gene therapy of certain genetic diseases. Stimulation of HSC with hematopoietic growth factors (GF) has been shown to increase the level of retroviral transduction. We have studied the effect of basic fibroblast growth factor (bFGF), alone and in combination with other GFs, on the efficiency of transfer of the bacterial neomycin phosphotransferase (neoR) gene into human CD34(+)-enriched peripheral blood hematopoietic progenitor cells. The combination of bFGF, interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in a transduction efficiency of 37 and 35% for G418-resistant colony-forming units-granulocyte/macrophage (CFU-GM) and mixed colonies multipotent colony-forming units (CFU-GEMM), respectively, which was significantly higher than the corresponding figures obtained with IL-3, IL-6, and SCF. The optimal concentration of bFGF was between 20 and 200 ng/mL. bFGF alone had no effect on the transduction rate. These results indicated a synergism in the action of bFGF, IL-3, IL-6, and SCF to enhance gene transduction rates into human hematopoietic progenitor cells.
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Intercellular spread of GFP-VP22.
BACKGROUND: The herpes simplex virus type 1 (HSV-1) VP22 polypeptide has been reported to mediate intercellular trafficking of heterologous proteins fused to its C- or N-terminus, a feature making it a useful tool in bystander cell-targeted gene therapy. METHODS: Here we show, by detection of Green Fluorescent Protein (GFP) fused to VP22, its subcellular distribution in living producer (transfected) and recipient (non-transfected) cells as well as in cells after fixation. Four cell lines from different species were used. RESULTS: Different fractions of translocated GFP-VP22 fusion protein could be detected in fixed recipient cells by two different methods of fixation. Functional GFP in live recipient cells could not be detected. CONCLUSIONS: Our study indicates that the VP22-chimeric protein transfer in its present form is suboptimal in terms of protein function. However, after fixation, the GFP signal in 100% of cells in a monolayer can be detected even at moderate transfection efficiency.
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Gene transfer into hematopoietic cells: progress, problems and prospects.
Gene therapy is the introduction of genetic material into somatic cells in order to correct a genetic defect or provide a new therapeutic function. Among the numerous potential cellular targets for gene therapy, hematopoietic stem cells (HSC) may be viewed as one of the best candidates for such genetic modification. More than 250 clinical gene transfer protocols have been reported from around the world, and almost one in three involves manipulation of hematopoietic cells. The introduction of a new gene into the DNA of HSC and expression of the gene product in their progeny are exciting approaches to treatment of congenital and acquired diseases. Although it has been shown that hematopoietic progenitor cells, or even LTC-IC, can be easily transduced with high efficiency by retroviral vectors in vitro, and that long-term expression and high transduction can be obtained in mice after transplantation of the gene manipulated cells, this has not been observed in primate models or in human clinical trials. The reason for this discrepancy is not properly known. The major problems of gene therapy for cancer have been transduction rate, selectivity, and effectiveness due to the heterogeneity of genetic alterations found in human tumors. Overcoming these limitations in gene therapy requires not only improvement in cell biology and immunology, but also development of better delivery systems. Nevertheless, gene therapy is still promising and offers encouragement for future applications in clinical practice.
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