title: An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation.
abstract: Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked severe combined immunodeficiency (X-SCID) has prompted safety concerns. We used an adeno-associated virus vector to achieve targeted insertion of a gamma-retroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the patients with X-SCID. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette that failed to activate the LMO2 gene in lymphoid cells. A gamma-retroviral vector with 2 intact LTRs resulted in activation of the LMO2 gene when inserted into the first intron, but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements for their potential for proto-oncogene activation.
authors.affiliation: Department of Hematology, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: Byoung Y
authors.initials: BY
authors.lastname: Ryu
authors.forename: Marguerite V
authors.initials: MV
authors.lastname: Evans-Galea
authors.forename: John T
authors.initials: JT
authors.lastname: Gray
authors.forename: David M
authors.initials: DM
authors.lastname: Bodine
authors.forename: Derek A
authors.initials: DA
authors.lastname: Persons
authors.forename: Arthur W
authors.initials: AW
authors.lastname: Nienhuis
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D041321:Chromosomes, Human, X; D004273:DNA, Neoplasm / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D007438:Introns; D019169:Jurkat Cells; D060588:LIM Domain Proteins; D008667:Metalloproteins / Q000235:genetics*; D010957:Plasmids; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011519:Proto-Oncogenes; D012190:Retroviridae / Q000235:genetics*; D016511:Severe Combined Immunodeficiency / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D060588:LIM Domain Proteins; C085117:LMO2 protein, human; C000715470:MAS1 protein, human; D008667:Metalloproteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins