Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retroviral gene transfer into hematopoietic cells has many experimental as well as clinical applications. Although transduction efficiency of retroviral vectors is higher than with conventional methods, selection of successfully transduced cells may become mandatory for efficient in vivo transfer. We have been evaluating a retroviral construct that meets the criteria for a clinically acceptable selection system. The Ser31 dihydrofolate reductase (DHFR) mutant confers resistance to methotrexate (MTX) due to decreased binding of the drug. while its enzymatic activity remains adequate for normal folate metabolism. Transduction of this vector into murine hematopoietic cells has been recently described and increase in MTX resistance could be observed. We investigated transduction of CD34-antigen positive subpopulations of hematopoietic progenitor cells and CD34-positive/CD38-negative subpopulations enriched for stem cells. We focused on two sources of primary hematopoietic cells, umbilical cord blood (UCB) and peripheral blood (PB) harvested from patients after mobilization with chemotherapy and/or cytokines. Both contain a large number of lineage restricted and pluripotent progenitor cells and can be expanded extensively ex vivo. Potential clinical applications of gene therapy in such cell populations include correction of inborn enzymatic diseases and support of high-dose chemotherapy by transplanting ex vivo transduced progenitor cells rendered more resistant to cytotoxic drugs. The feasibility and efficiency of retroviral transduction into UCB and PB has been reported recently.
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PMID:Increased resistance to methotrexate in human hematopoietic cells after gene transfer of the Ser31 DHFR mutant. 747 10

Retroviral gene transfer into human myeloid precursor cells allows introduction of marker genes as well as genes conferring resistance to chemotherapeutic drugs. We transduced a human mutant dihydrofolate reductase (DHFR) cDNA into CD34 antigen-positive peripheral blood cells from patients with breast or ovarian cancer obtained after treatment with chemotherapy and granulocyte colony-stimulating factor (G-CSF). This mutant DHFR has been shown to confer resistance to methotrexate (MTX) in murine bone marrow. We established a transduction protocol that permitted ex vivo expansion and selection of transduced early progenitor cells. The number of progenitor cells from transduced CD34-positive cells increased 50-fold after cytokine prestimulation with interleukin-1 (IL-1), c-kit ligand (KL; stem cell factor), and IL-3 and 2 weeks in liquid culture. Transduced colony-forming unit-granulocyte-macrophage (CFU-GM), assayed directly after the transduction procedure, were protected completely against 2 x 10(-8) mol/L MTX, a concentration that significantly reduced the CFU-GM detected in the control population. Gene transfer of the mutant DHFR led to a twofold selective advantage for a pre-CFU population after exposure to MTX in liquid culture (P < .001). Polybrene, in contrast with protamine, significantly inhibited the expansion of progenitors. The presence of proviral DNA was monitored by polymerase chain reaction (PCR) and was detected in greater than 80% of CFU-GM and ex vivo expanded pre-CFU. We have demonstrated that human hematopoietic precursor cells can be expanded extensively after retroviral gene transfer. The same population of early progenitors can be selected ex vivo with low-dose MTX. As long-term expression of transduced genes in human hematopoietic cells remains a problem in vivo, these results may have implications for future clinical trials, especially for the introduction of nonselectable genes.
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PMID:Ex vivo expansion and selection of human CD34+ peripheral blood progenitor cells after introduction of a mutated dihydrofolate reductase cDNA via retroviral gene transfer. 752 65

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell caused by the BCR/ABL gene rearrangement. The only curative therapy is allogeneic transplantation. Although autologous transplants may prolong survival, most patients relapse because of disease persisting in the host and in the graft. Continued administration of chemotherapy after transplant could reduce the incidence of relapse provided that the autograft can be protected by transfer of a drug-resistance gene. However, CML autografts will almost certainly contain malignant stem cells that will also be rendered drug-resistant. The presence of the BCR/ABL oncoprotein is necessary and sufficient for malignant transformation seen in CML. We thus hypothesized that transfer of a vector that combines a drug-resistance gene with anti-BCR/ABL antisense (AS) sequences may allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduced CML stem and progenitor cells functionally normal. We constructed a retroviral vector, LasBD, that combines the methotrexate (MTX)-resistant tyrosine-22 dihydrofolate-reductase (tyr22-DHFR) gene and AS sequences directed at the b3a2 BCR/ABL breakpoint. b3a2 BCR/ABL containing 32D and MO7e cells were transduced with LasBD and selected in MTX for 14 days. Expression of the AS sequences reduced BCR/ABL mRNA and p210(BCR/ABL) protein levels by 6- to 10-fold in most cells. This subsequently led to the restoration of normal function of BCR/ABL cDNA+ cells: they grew significantly slower in the presence of interleukin-3 (IL-3); they underwent apoptotic cell death when cultured without IL-3; and they had restored expression and function of adhesion receptors. These effects were specific, because LasBD-containing AS sequences directed at the b3a2 BCR/ABL breakpoint did not affect p190(BCR/ABL)-containing cells. LasBD also rendered 20% to 30% of primary Ph- and Ph+ CD34(+) cells MTX-resistant and decreased BCR/ABL mRNA levels in MTX resistant Ph+ CD34(+) cells by 10-fold. Expression of the MTX-resistant DHFR gene and the AS sequences has been stable for at least 1 year in vitro and for more than 70 days in vivo. Finally, LasBD decreased tumorigenicity of 32DBCR/ABL cells in vivo by 3 to 4 logs. In conclusion, the tyr22-DHFR gene in the LasBD vector can protect normal hematopoietic cells from MTX-mediated toxicity, whereas the AS sequences in LasBD can suppress expression of the BCR/ABL gene and restore normal function of BCR/ABL cDNA-containing cells. The LasBD vector may therefore prove to be an extremely useful adjunct in autologous transplantation for CML.
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PMID:Gene therapy for chronic myelogenous leukemia (CML): a retroviral vector that renders hematopoietic progenitors methotrexate-resistant and CML progenitors functionally normal and nontumorigenic in vivo. 938 83

Umbilical cord blood cells (UCB) have become a major target population for experimental and clinical studies using transfer of genes involved in inborn enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high frequency, and expanding these cells ex vivo generates sufficient numbers of hematopoietic precursors for transplantation into adults, e.g., as supportive treatment. As clinical reports about retroviral transduction into UCB cells have not been as encouraging as the first preclinical data, we have established a retroviral transduction system that allows expansion and selection of hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that confers MTX resistance. We observed increased resistance to MTX in transduced granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ UCB cells with the virus-producing cell line, or after incubation with virus-containing supernatant. The supernatant-based transduction protocol included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold selective advantage of transduced versus nontransduced progenitor cells after exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction analysis revealed integration of proviral DNA into the majority of transduced colonies before and after ex vivo expansion. The retroviral vector and transduction protocol reported here provides an experimental system for selection and expansion of retrovirally transduced progenitor/stem cells from UCB that may help improve the efficiency of current clinical gene therapy strategies.
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PMID:Retroviral transduction of human CD34+ umbilical cord blood progenitor cells with a mutated dihydrofolate reductase cDNA. 945 43

We investigated whether transduction of human cord blood progenitor cells can be increased by spinoculation in fibronectin fragment CH-296 (FN)-coated tubes. Bicistronic vectors PA317/LgEIN, containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (neo) genes, and PG13/LgDIN, containing the dihydrofolate reductase and neo genes, were used to transduce CD34-enriched human cord blood cells. Transduction by spinoculation in FN-coated tubes (spin/FN+) was compared with spinoculation in noncoated tubes (spin/FN-) and transduction in plates coated with FN (plate/FN+). Antibody to TGF-beta was added to spin/FN+ to evaluate its impact on transduction. Using producer cell line PA317/LgEIN for transduction of CD34+ cord blood cells, FACS analysis for expression of EGFP revealed mean transduction of 30.6+/-4.3, 9.1+/-1.6, and 21.1+/-6.5% of CD34+ cells in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. Transduction of CD+CD38low cells was also higher in the spin/FN+ arm as compared with transduction in the spin/FN- arm. These results were corroborated by colony-forming assays. Antibody to TGF-beta did not further increase transduction. Using a different producer cell line, PG13/pLgDIN, a higher number of G418-resistant CFU-GM was observed in the spin/FN+ as compared with the plate/FN+ and spin/FN-arms. NOD/SCID mice were transplanted with transduced, CD34-enriched human cord blood cells, and persistence of transduced human cells was analyzed in the mice marrows after 6-8 weeks: 32.8, 6.0, and 23.9% human G418-resistant CFU-GM colonies were observed in the spin/FN+, spin/FN-, and plate/FN+ arms, respectively. These results suggest that spinoculation in FN-coated tubes increases transduction of early human cord blood progenitor cells as compared with spinoculation in noncoated tubes.
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PMID:Increased gene transfer into human cord blood cells by centrifugation-enhanced transduction in fibronectin fragment-coated tubes. 1058 31

We have used a murine retrovirus vector containing an enhanced green fluorescent protein complimentary DNA (EGFP cDNA) to dynamically follow vector-expressing cells in the peripheral blood (PB) of transplanted rhesus macaques. Cytokine mobilized CD34(+) cells were transduced with an amphotropic vector that expressed EGFP and a dihydrofolate reductase cDNA under control of the murine stem cell virus promoter. The transduction protocol used the CH-296 recombinant human fibronectin fragment and relatively high concentrations of the flt-3 ligand and stem cell factor. Following transplantation of the transduced cells, up to 55% EGFP-expressing granulocytes were obtained in the peripheral circulation during the early posttransplant period. This level of myeloid marking, however, decreased to 0.1% or lower within 2 weeks. In contrast, EGFP expression in PB lymphocytes rose from 2%-5% shortly following transplantation to 10% or greater by week 5. After 10 weeks, the level of expression in PB lymphocytes continued to remain at 3%-5% as measured by both flow cytometry and Southern blot analysis, and EGFP expression was observed in CD4(+), CD8(+), CD20(+), and CD16/56(+) lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets soon after transplantation. Such sustained levels of lymphocyte marking may be therapeutic in a number of human gene therapy applications that require targeting of the lymphoid compartment. The transient appearance of EGFP(+) myeloid cells suggests that transduction of a lineage-restricted myeloid progenitor capable of short-term engraftment was obtained with this protocol. (Blood. 2000;95:445-452)
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PMID:High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells. 1062 48

Autologous transplantation of bone marrow cells (BMCs) transduced with the multidrug resistance 1 (MDR1) gene or dihydrofolate reductase (DHFR) gene has already been applied in clinical chemoprotection trials. However, anticancer drugs frequently used in high-dose chemotherapy (HDC), such as alkylating agents, are not relevant to MDR1 or DHFR gene products. In this context, we have previously reported that glutathione S-transferase-pi (GST-pi) gene-transduced human CD34(+) cells showed resistance in vitro against 4-hydroperoxicyclophosphamide, an active form of cyclophosphamide (CY). In the present study, a subsequent attempt was made in a murine model to evaluate the effectiveness of transplantation of GST-pi-transduced BMCs to protect bone marrow against high-dose CY. The gene transfection was carried out retrovirally, employing a recombinant fibronectin fragment. Transfection efficiency into CFU-GM was 30%. After the transplantation, recipient mice (GST-pi mice) received three sequential courses of high-dose CY. As the chemotherapy courses advanced, both shortening of recovery period from WBC nadir and shallowing of WBC nadir were observed. In contrast to the fact that three of seven control mice died, possibly due to chemotoxicity, all seven GST-pi mice were alive after the third course, at which point the vector GST-pi gene was detected in 50% of CFU-GM derived from their BMCs and peripheral blood mononuclear cells. When BMCs obtained from these seven mice were retransplanted into secondary recipient mice, 20% of CFU-GM from BMCs showed positive signals for vector GST-pi DNA after 6 months. These data indicate that the GST-pi gene can confer resistance to bone marrow against CY by being transduced into long-term repopulating cells.
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PMID:GST-pi gene-transduced hematopoietic progenitor cell transplantation overcomes the bone marrow toxicity of cyclophosphamide in mice. 1095 1

Human CD34(+) cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous hematopoietic disorders. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human hematopoietic cells, we investigated a dominant selection strategy using a variant of the DHFR gene (DHFR(L22Y)). For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFR(L22Y). Here we demonstrate efficient in vitro selection and enrichment of lentivirus vector-transduced human CD34(+) hematopoietic cells from fetal liver, umbilical cord blood, bone marrow, and peripheral blood after cytokine mobilization. Growth of transduced human CD34(+) cells in semisolid culture under selective pressure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14). Selection for DHFR(L22Y)(+) cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting that development of such strategies holds promise for future in vivo application.
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PMID:In vitro selection of lentivirus vector-transduced human CD34+ cells. 1098 66

The genetic transfer of drug resistance to hematopoietic cells is an attractive approach to overcoming myelosuppression caused by high-dose chemotherapy. Because cyclophosphamide (CTX) and methotrexate (MTX) are commonly used non-cross-resistant drugs, generation of dual drug resistance in hematopoietic cells that allows dose intensification may increase anti-tumor effects and circumvent the emergence of drug-resistant tumors. We constructed a retroviral vector containing both a human cytosolic ALDH-1 cDNA and a human doubly mutated DHFR cDNA (Phe22/Ser31; termed F/S in the description of constructs) to generate increased resistance to both CTX and MTX. Infection of NIH3T3 cells resulted in increased resistance to both 4-hydroperoxycyclophosphamide (4HC) (1.9 +/- 0.1-fold) and MTX (73 +/- 2.8-fold). Transduced human CD34(+) enriched hematopoietic progenitor cells were also resistant to both 4HC and MTX by CFU-GM readout. Lethally irradiated mice transplanted with SFG-ALDH-IRES-F/S or mock-transduced bone marrow cells were treated with high-dose pulse CTX or high-dose CTX/MTX. Animals receiving marrow not transduced with ALDH-1 or mutated DHFR cDNA died from CTX or CTX/MTX toxicity, whereas mice transduced with ALDH-1 and mutated DHFR cDNA-containing marrow were able to tolerate the same doses of CTX or CTX/MTX treatment posttransplant. These data taken together indicate that ALDH-1 overexpression and mutant DHFR increased both 4HC and MTX resistance in vitro and in the in vivo mouse model. This construct may be useful for protecting patients from high-dose CTX- and MTX-induced myelosuppression.
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PMID:Generation of dual resistance to 4-hydroperoxycyclophosphamide and methotrexate by retroviral transfer of the human aldehyde dehydrogenase class 1 gene and a mutated dihydrofolate reductase gene. 1116 15

A limitation of successful stem cell gene transfer to hematopoietic stem cells is low transduction efficiency. To overcome this hurdle and develop a gene transfer strategy that might be clinically feasible, retroviral vectors containing a drug resistance gene were utilized to transduce human CD34(+)-enriched cells and select gene-modified cells by drug administration. We constructed a high-titer retroviral vector containing a fusion gene (F/S-EGFP) consisting of a mutated dihydrofolate reductase (DHFR) (Leu22-->Phe22, Phe31-->Ser31; F/S) gene and enhanced green fluorescent protein (EGFP) cDNA. To test whether the fusion gene could function as a selectable marker, transduced CD34(+) cells were assayed in long-term stromal co-cultures with and without addition of methotrexate (MTX). Without MTX exposure, the vector-transduced CD34(+) cells generated 22-50% EGFP(+) cobblestone area forming cells (CAFC) at week 5. By contrast, the vector-transduced cells cultured with MTX produced 96-100% EGFP(+) CAFC in four separate experiments. These are the first investigations to demonstrate selection for transduced long-term culture initiating cells using MTX. The DHFR/MTX system holds promise for improving selection of gene-transduced hematopoietic progenitor cells in vivo.
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PMID:Methotrexate selection of long-term culture initiating cells following transduction of CD34(+) cells with a retrovirus containing a mutated human dihydrofolate reductase gene. 1189 48


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