To determine the effect of different promoters on the expression of an altered dihydrofolate reductase (DHFR) gene conferring methotrexate (MTX) resistance in different cell types, double-copy retroviral vectors were constructed carrying a murine mutant DHFR under the control of five different promoters, i.e., human adenosine deaminase (ADA), simian virus 40 (SV40), thymidine kinase (TK), human beta-actin, and cytomegalovirus (CMV). Their expression was compared in NIH-3T3 cells, three human leukemia cell lines, and mouse bone marrow. The variant DHFR is readily expressed from these various promoters in retroviral vectors at a selectable level. In 3T3 cells, the DHFR constructs containing the SV40 promoter conferred the highest levels of resistance to MTX. In K562 and Raji cells, the construct with the TK promoter produced the highest level of resistance. However granulocyte-macrophage colony-forming unit (CFU-GM) colonies from mouse marrow were more resistant to MTX when infected with vectors containing the SV40 promoter and ADA promoter as compared to the other promoter constructs. These studies show that mouse fibroblast cell lines such as NIH-3T3 do not predict the effectiveness of retroviral-mediated gene transfer for marrow progenitor cells, and that the activity of retroviral vector-encoded promoters vary in an unpredictable manner from cell type to cell type. Possible implications for basic gene transfer studies and clinical applications are discussed.

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.

No abstract

A double-copy Moloney leukemia virus-based retroviral construct containing both the NeoR gene and a mutant human dihydrofolate reductase (DHFR) cDNA (Ser31 mutant) was used to transduce NIH 3T3 and mouse bone marrow (BM) progenitor cells. This resulted in increased resistance of these cells to methotrexate (MTX). The transduced BM progenitor cells were returned to lethally irradiated mice. The recipients transplanted with marrow cells infected with the recombinant virus showed protection from lethal MTX toxicity as compared with mock- infected animals. Evidence for integration of the proviral DNA was obtained by amplification of proviral DNA by polymerase chain reaction (PCR) and Southern analysis. Sequencing a portion of the PCR-amplified human DHFR cDNA showed the presence of the mutation. These studies with the human Ser31 mutant DHFR cDNA gave results comparable with those obtained with the mutant murine DHFR cDNA (Leu to Arg22) in developing MTX-resistant BM. The Ser31 mutant human DHFR cDNA is currently being tested for infection of human CD34+ human BM and peripheral blood stem cells in vitro.

No abstract