title: Enhanced in vivo selection of bone marrow cells by retroviral-mediated coexpression of mutant O6-methylguanine-DNA-methyltransferase and HOXB4.
abstract: To attain therapeutic levels of gene-modified hematopoietic stem cells, it may be necessary in the majority of disorders to provide an in vivo selective advantage that facilitates the expansion of their numbers. A popular strategy to achieve in vivo selection has been to employ drug selection while coexpressing a transgene that conveys chemoresistance, such as O6-methylguanine-DNA-methyltransferase (MGMT). An alternate approach is to confer an enhanced proliferative potential upon gene-modified hematopoietic stem cells through the delivery of the homeobox transcription factor HOXB4. By developing a novel tricistronic retroviral vector, we have facilitated the simultaneous coexpression of a mutant version of MGMT and HOXB4 in retrovirally transduced bone marrow. Using an in vivo competitive repopulation assay, we demonstrate that primary bone marrow cells containing this construct show enhanced reconstitution following transplant and improved selection subsequent to chemotherapeutic challenge in comparison to cells expressing either HOXB4 or MGMT alone. This selection advantage was evident even when HOXB4/MGMT-coexpressing cells were infused along with a large excess of unmodified cells. We propose that this selection cassette may facilitate the in vivo expansion of gene-modified hematopoietic stem cells at a level in excess of previous strategies.
authors.affiliation: Gene Therapy, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Milsom
authors.forename: Lorna B
authors.initials: LB
authors.lastname: Woolford
authors.forename: Geoffrey P
authors.initials: GP
authors.lastname: Margison
authors.forename: R Keith
authors.initials: RK
authors.lastname: Humphries
authors.forename: Leslie J
authors.initials: LJ
authors.lastname: Fairbairn
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D003114:Colony-Forming Units Assay; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D018398:Homeodomain Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009154:Mutation; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics* / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D000077204:Temozolomide; D014157:Transcription Factors
chemical_list: D018398:Homeodomain Proteins; C513262:Hoxb4 protein, mouse; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; C088636:O(6)-benzyl-2'-deoxyguanosine; D049452:Green Fluorescent Proteins; D003606:Dacarbazine; D019853:O(6)-Methylguanine-DNA Methyltransferase; D003849:Deoxyguanosine; D000077204:Temozolomide