title: Selectively weakened binding of methotrexate by human dihydrofolate reductase allows rapid ex vivo selection of mammalian cells.
abstract: 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.
authors.affiliation: Département de biochimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada.
authors.forename: Jordan P
authors.initials: JP
authors.lastname: Volpato
authors.forename: Nadine
authors.initials: N
authors.lastname: Mayotte
authors.forename: Elena
authors.initials: E
authors.lastname: Fossati
authors.forename: Vanessa
authors.initials: V
authors.lastname: Guerrero
authors.forename: Guy
authors.initials: G
authors.lastname: Sauvageau
authors.forename: Joelle N
authors.initials: JN
authors.lastname: Pelletier
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D020134:Catalytic Domain; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D004351:Drug Resistance / Q000187:drug effects; D005493:Folic Acid Antagonists / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D005971:Glutamates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008727:Methotrexate / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000378:metabolism; D000068437:Pemetrexed; D012641:Selection, Genetic; D013379:Substrate Specificity / Q000187:drug effects; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic
chemical_list: D005493:Folic Acid Antagonists; D005971:Glutamates; D008024:Ligands; D050505:Mutant Proteins; D000068437:Pemetrexed; D006147:Guanine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate