Retroviral transfer of the hENT2 nucleoside transporter cDNA confers broad-spectrum antifolate resistance in murine bone marrow cells.

Antifolate drugs such as methotrexate are commonly used in cancer chemotherapy. It may be possible to increase the antitumor activity of antifolates by the coadministration of drugs that inhibit nucleoside transport, thereby blocking the capacity of tumor cells to salvage nucleotide precursors. An important limitation of this approach is severe myelosuppression caused by many of these drug combinations. For this reason, we have developed a gene therapy strategy to protect bone marrow cells against combined treatment with antifolates and nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of the es nucleoside transporter. A retroviral vector (MeiIRG) was constructed that expressed the NBMPR-insensitive ei transporter, hypothesizing that transduced bone marrow cells would survive drug treatment because of the preservation of nucleoside salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing this system in vivo, decreased myelosuppression was observed in mice transplanted with MeiIRG-transduced bone marrow cells and subsequently treated with trimetrexate and NBMPR-P. In these mice, significant increases were noted in absolute neutrophil count nadirs, reticulocyte indices, and the numbers of myeloid progenitors in the bone marrow. Furthermore, a survival advantage was associated with transfer of the MeiIRG vector, indicating that significant dose intensification was possible with this approach. In summary, the MeiIRG vector can decrease the toxicity associated with the combined use of antifolates and NBMPR-P and thereby may provide a strategy for simultaneously sensitizing tumor cells while protecting hematopoietic cells.

High levels of lymphoid expression of enhanced green fluorescent protein in nonhuman primates transplanted with cytokine-mobilized peripheral blood CD34(+) cells.

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)

Coding region-specific destabilization of mRNA transcripts attenuates expression from retroviral vectors containing class 1 aldehyde dehydrogenase cDNAs.

Class 1 aldehyde dehydrogenases (ALDH-1) function as drug resistance gene products by catalyzing the irreversible conversion of aldophosphamide, an active metabolite of cyclophosphamide, to an inert compound. Because the dose-limiting toxicity of cyclophosphamide is myelosuppression, retrovirus-mediated transfer of ALDH-1 to bone marrow cells has been proposed as a protective strategy. Here we show that expression of ALDH-1 vectors was problematic due to low levels of ALDH-1 mRNA accumulation. A number of vectors containing several different ALDH-1 cDNAs were introduced into a variety of different cell lines either by transfection or transduction. Detectable ALDH-1 protein and enzyme activity was only seen in one transfected cell clone. Cells transduced with ALDH-1 retroviral vectors had no detectable protein expression and very low levels of ALDH-1 mRNA. Analogous vectors containing other drug resistance cDNAs led to much higher levels of steady-state mRNA. The mRNA half-life from ALDH-1 vectors was less than 2 hr suggesting that vector-derived mRNAs were destabilized by ALDH-1 coding sequences. These results suggest that methods which increase the stability of ALDH-1 mRNAs will be important for increased drug resistance in retrovirally transduced hematopoietic cells.

A gene transfer strategy for making bone marrow cells resistant to trimetrexate.

Trimetrexate (TMTX) is an anticancer drug with potential advantages over the more commonly used antifolate, methotrexate (MTX); however, its use has been limited by severe myelosuppression. Retroviral vectors containing mutant dihydrofolate reductase (DHFR) genes have been used to protect bone marrow cells from MTX, suggesting a similar approach could be used for TMTX. We first screened six variants of human DHFR to determine which allowed maximal TMTX resistance in fibroblasts. A variant enzyme containing a Leu-to-Tyr mutation in the 22nd codon (L22Y) was best, allowing a 100-fold increase in resistance over controls. Murine hematopoietic progenitor cells transduced with an L22Y-containing retroviral vector also showed high-level TMTX resistance in vitro. Mice reconstituted with L22Y-transduced bone marrow cells were challenged with a 5-day course of TMTX to determine whether hematopoiesis could be protected in vivo. Transfer of the L22Y vector resulted in consistent protection from TMTX-induced neutropenia and reticulocytopenia at levels that correlated with the proviral copy number in circulating leukocytes. We conclude that the L22Y vector is highly effective in protecting hematopoiesis from TMTX toxicity and may provide a means for increasing the therapeutic utility of TMTX in certain cancers.

Mutations in the gene for human dihydrofolate reductase: an unlikely cause of clinical relapse in pediatric leukemia after therapy with methotrexate.

Resistance to methotrexate (MTX) in some sublines of mammalian cells is reported to be due to one of the following amino acid substitutions in dihydrofolate reductase (DHFR) that lower inhibition by MTX: Gly15 to Trp, Leu22 to Arg or Phe or Phe31 to Trp or Ser. We have produced variants of human DHFR (hDHFR) with these substitutions by directed mutagenesis. Recombinant hDHFR variants expressed in Escherichia coli have greatly decreased inhibition by MTX, but decreased catalytic efficiency, and in one case decreased stability. When a retroviral vector encoding wild-type (wt) hDHFR or one of these variants was introduced into murine fibroblasts or bone marrow progenitors, modest protection from MTX was conferred, even by wt. Relapsed pediatric patients with acute lymphoblastic leukemia who have received multiple courses of high-dose MTX seem most likely to develop such MTX resistance. cDNA was reverse transcribed from blast mRNA from 17 of these patients. However, upon amplification and sequencing of DHFR cDNA, no resistance mutation was found. The explanation for this probably lies in the need for considerable gene amplification to offset lowered catalytic efficiency, and the need for two-base changes for most substitutions, both of which are probably infrequent events.