Transfection with a cDNA encoding a Ser31 or Ser35 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance

Banerjee, Debabrata; Schweitzer, Barry; Volkenandt, Matthias; Li, Ming-Xia; Waltham, Mark; Mineishi, Shin; Zhao, Shi Cheng; Bertino, Joseph R.

doi:10.1016/0378-1119(94)90768-4

Cited by 26 publications

(21 citation statements)

References 14 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This approach facilitates enrichment of populations of progenitor cells transfected with mutant enzyme on exposure to antifolate in vitro. The strategy has been utilized to achieve resistance to MTX with mutated DHFR in murine progenitor cells (Banerjee et al, 1994;Flasshove et al, 1995a;Takebe et al, 2000) and human CD34 þ peripheral human cells in vitro and in vivo (Flasshove et al, 1995b;Sorrentino et al, 1999). Bicistronic retroviral vectors have been used to expand the spectrum of resistance in progenitors, as with the transduction of both mutated DHFR and Pgp into murine bone marrow progenitors (Galipeau et al, 1997), or with DHFR and cytidine deaminase conferring resistance to both MTX and cytosine arabinoside (Sauerbrey et al, 1999), or with cotransfection of mutated DHFR and wild-type aldehyde dehydrogenase to achieve resistance to both MTX and cyclophosphamide (Takebe et al, 2001).…”

Section: Novel Gene Therapy Approaches That Exploit Mutations In Targmentioning

confidence: 99%

Resistance to antifolates

2003

View full text Add to dashboard Cite

Section: Novel Gene Therapy Approaches That Exploit Mutations In Targmentioning

confidence: 99%

Resistance to antifolates

2003

View full text Add to dashboard Cite

“…The doubly substituted F31S/Q35E conferred MTX resistance in the same range as the benchmarking variant L22Y, while variants F31R/F34T/ Q35S, F31R/Q35E and F31A/F34V/Q35H conferred higher MTX resistance (>1 order of magnitude higher). Previous reports have shown that the degree of chemoprotection conferred by a given MTX-resistant variant in adherent cells correlated well with that obtained in BM cells (Banerjee et al, 1994a;Banerjee et al, 1994b;Spencer et al, 1996). The results obtained for the GP þ E-86 cells suggested that variants F31R/Q35E, F31R/F34T/Q35S and F31A/ F34V/Q35H could confer greater myeloprotection at high MTX concentrations than variant L22Y.…”

Section: Mtx Selection Of Infected Gp R E-86 Virus Producer Cells Andmentioning

confidence: 60%

“…Prior ex vivo selection studies on transduced BM cells were performed with various hDHFR variants (Banerjee et al, 1994a;Li et al, 1994;Spencer et al, 1996;Patel et al, 1997;Sauerbrey et al, 1999). The doubly-substituted L22F/F31S (Ercikan-Abali et al, 1996) displayed a greater decrease in MTX affinity than variant L22Y (Lewis et al, 1995), while maintaining sufficient catalytic activity to ensure cell proliferation.…”

Section: Introductionmentioning

confidence: 99%

Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells

Volpato

Mayotte

Fossati

et al. 2011

J of Molecular Recognition

View full text Add to dashboard Cite

Ex vivo selection of transduced hematopoietic stem cells (HSC) with drug-resistance genes offers the possibility to enrich transduced cells prior to engraftment, toward increased reconstitution in transplant recipients. We evaluated the potential of highly methotrexate (MTX)-resistant variants of human dihydrofolate reductase (hDHFR) for this application. Two subsets of hDHFR variants with reduced affinity for MTX that had been previously identified in a bacterial system were considered: those with substitutions at positions 31, 34, and/or 35, and those with substitutions at position 115. The variants were characterized for their resistance to pemetrexed (PMTX), an antifolate that is related to MTX. We observed a strong correlation between decreased binding to both antifolates, although the identity of specific sequence variations modulated the correlation. We chose a subset of hDHFR variants for tests of ex vivo MTX resistance, taking into consideration their residual specific activity and their decrease in affinity for the related antifolates. Murine myeloid progenitors and other differentiated hematopoietic cells were transduced and exposed to MTX in a nucleotide-free medium. Bone marrow (BM) cells including 15% cells infected with F31R/Q35E were enriched to 98% transduced cells within 6 days of ex vivo selection. hDHFR variant F31R/Q35E allowed a strong ex vivo enrichment upon a short exposure to MTX relative to a less resistant variant of hDHFR, L22Y. We have thus demonstrated that bacterial selection of highly antifolate-resistant hDHFR variants can provide selectable markers for rapid ex vivo enrichment of hematopoietic cells.

show abstract

“…Expression of these gene products has been demonstrated to reduce the effects of cytotoxic drugs on bone marrow function. This approach has been successful in protecting against the hematopoietic toxicity of methotrexate with dihydrofolate reductase 2,3 ; against placitaxel, doxorubicin, and vinblastine toxicity using the multidrug resistance gene-1 (MDR1) 4,5 ; and toxicity associated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) with O6-methylguanine DNA methyltransferase (MGMT). [6][7][8][9] Retroviral transduction of mobilized peripheral blood progenitor cells has been used in human clinical trials to transfer heterologous genes into hematopoietic cells.…”

Section: Introductionmentioning

confidence: 99%

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

et al. 2006

View full text Add to dashboard Cite

Administration of chemotherapy is often limited by myelosuppression. Expression of drug-resistance genes in hematopoietic cells has been proposed as a means to decrease the toxicity of cytotoxic agents. In this pilot study, we utilized a retroviral vector expressing methylguanine DNA methyltransferase (MGMT) to transduce hematopoietic progenitors, which were subsequently used in the setting of alkylator therapy (procarbazine, CCNU, vincristine (PCV)) for poor prognosis brain tumors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells were collected by apheresis and enriched for CD34 þ expression. Nine subjects were infused with CD34 þ -enriched cells treated in a transduction procedure involving a 4-day exposure to cytokines with vector exposure on days 3 and 4. No major adverse event was related to the gene therapy procedure. Importantly, the engraftment kinetics of the treated product was similar to unmanipulated peripheral blood stem cells, suggesting that the ex vivo manipulation did not significantly reduce engrafting progenitor cell function. Gene-transduced cells were detected in all subjects. Although the level and duration was limited, patients receiving cells transduced using fibronectin 'preloaded' with virus supernatant appeared to show improved in vivo marking frequency. These findings demonstrate the feasibility and safety of utilizing MGMT-transduced CD34 þ peripheral blood progenitor cells in the setting of chemotherapy.

show abstract

Transfection with a cDNA encoding a Ser31 or Ser35 mutant human dihydrofolate reductase into Chinese hamster ovary and mouse marrow progenitor cells confers methotrexate resistance

Cited by 26 publications

References 14 publications

Resistance to antifolates

Resistance to antifolates

Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase

Contact Info

Product

Resources

About