title: Methotrexate selection of long-term culture initiating cells following transduction of CD34(+) cells with a retrovirus containing a mutated human dihydrofolate reductase gene.
abstract: 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.
authors.affiliation: Program of Molecular Pharmacology and Experimental Therapeutics, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. ntakebe@som.umaryland.edu
authors.forename: Naoko
authors.initials: N
authors.lastname: Takebe
authors.forename: Li-Cheng
authors.initials: LC
authors.lastname: Xu
authors.forename: Karen L
authors.initials: KL
authors.lastname: MacKenzie
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Bertino
authors.forename: Malcolm A S
authors.initials: MA
authors.lastname: Moore
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D000964:Antimetabolites, Antineoplastic / Q000009:adverse effects / Q000494:pharmacology*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000737:chemistry; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000502:physiology; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008164:Luminescent Proteins; D008727:Methotrexate / Q000009:adverse effects / Q000494:pharmacology*; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D012190:Retroviridae / Q000235:genetics*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013995:Time; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D018952:Antigens, CD34; D000964:Antimetabolites, Antineoplastic; D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate