title: Ex vivo expansion of retrovirally transduced primate CD34+ cells results in overrepresentation of clones with MDS1/EVI1 insertion sites in the myeloid lineage after transplantation.
abstract: Activation of proto-oncogenes by retroviral insertion is an important issue delaying clinical development of gene therapy. We have reported the nonrandom persistence of hematopoietic clones with vector insertions within the MDS1/EVI1 locus following transplantation of rhesus macaques. We now ask whether prolonged culture of transduced CD34(+) cells before transplantation selects for clones with insertions in the MDS1/EVI11 or other proto-oncogene loci. CD34(+) cells were transduced with standard retroviral vectors for 4 days and then continued in culture for an additional 6 days before transplantation. A 15% of insertions identified in granulocytes 6 months post-transplant were in MDS1/EVI11, significantly increased compared to the frequency in animals transplanted with cells immediately following transduction. MDS1/EVI1 clones became more dominant over time post-transplantation in one animal that was followed long term, accompanied by an increased overall copy number of vector-containing granulocytes, with one MDS1/EVI1 clone eventually accounting for 100% of transduced granulocytes and marrow colony-forming unit (CFU). This vector insertion increased the expression of Evi1 mRNA. There was no overrepresentation of MDS1/EVI1 insertions contributing to lymphoid lineages. Strategies involving prolonged ex vivo expansion of transduced cells may increase the risk of genotoxicity.
authors.affiliation: Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892-1290, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Sellers
authors.forename: Theotonius J
authors.initials: TJ
authors.lastname: Gomes
authors.forename: Andre
authors.initials: A
authors.lastname: Larochelle
authors.forename: Rebecca
authors.initials: R
authors.lastname: Lopez
authors.forename: Rima
authors.initials: R
authors.lastname: Adler
authors.forename: Allen
authors.initials: A
authors.lastname: Krouse
authors.forename: Robert E
authors.initials: RE
authors.lastname: Donahue
authors.forename: Richard W
authors.initials: RW
authors.lastname: Childs
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism*; D045744:Cell Line, Tumor; D017690:Cell Transplantation / Q000379:methods*; D002478:Cells, Cultured; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008253:Macaca mulatta / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000235:genetics*; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D018952:Antigens, CD34; C000715470:MAS1 protein, human; D015514:Oncogene Proteins, Fusion; D000090063:Proto-Oncogene Mas