Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.1.1.69 (BMT)
 2,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously demonstrated that severe graft-versus-host disease (GvHD), associated with the therapeutic infusion of donor lymphocytes after allogeneic marrow transplantation (BMT), can be efficiently controlled by the SFCMM-2-mediated expression of the herpes simplex virus thymidine kinase (HSV-tk) suicide gene into the allogeneic lymphocytes. This was achieved by selective elimination of transduced lymphocytes by ganciclovir (GCV) infusion. Despite the positive results of the pilot clinical trial, two vector-related limitations were observed. The induction of a strong immune response against genetically modified cells was observed in two patients. In addition, the only patient who developed chronic GvHD showed only partial response to ganciclovir treatment. In an attempt to overcome these limitations, we developed a new generation of vectors. The neomycin resistance (neoR) gene was removed from the SFCMM-3 and SFCMM-4 retroviral vectors. These vectors are less immunogenic and able to confer higher ganciclovir sensitivity to transduced human lymphocytes. All the vectors carry a modified form of the low-affinity nerve growth factor receptor cDNA, as cell surface selectable marker (deltaLNGFR). The vectors were compared in terms of gene transfer efficiency, and ability to confer high and specific sensitivity to ganciclovir. The SFCMM-3 vector, carrying the entire HSV-tk gene driven by the LTR promoter, allows efficient transduction of human T lymphocytes and confers the highest GCV sensitivity to transduced lymphocytes with a high and a low proliferation index. The expression of the deltaLNGFR marker allows an easier in vitro manipulation and a faster immune selection of transduced cells compared with neoR selection. Finally, the elimination of the neoR gene removes a potent immunogen from transduced lymphocytes.
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PMID:Herpes simplex virus thymidine kinase gene transfer for controlled graft-versus-host disease and graft-versus-leukemia: clinical follow-up and improved new vectors. 979 8

Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.
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PMID:Expansion and fibronectin-enhanced retroviral transduction of primary human T lymphocytes for adoptive immunotherapy. 1063 78