title: Selection with a regulated cell growth switch increases the likelihood of expression for a linked gamma-globin gene.
abstract: Several lines of evidence indicate that in vivo drug selection can be used to overcome the low rates of gene transfer and engraftment encountered in many hematopoietic stem cell gene therapy settings. However, whether selection imposed on one transcription cassette effects the likelihood of expression from a second, independent transcription cassette within the same vector has been less well studied. In order to address this issue, we engineered an oncoretrovirus vector to express two separate transcription units: (i) a bicistronic cassette encoding both GFP and a pharmacologically regulated cell growth switch based on the thrombopoietin receptor Mpl; and (ii) a highly position-dependent second cassette encoding human gamma-globin. Studies in cell cultures and in mice transplanted with transduced marrow indicated that selective expansion increased by more than 9-fold the fraction of erythroid cells expressing the linked but separate expression cassette for gamma-globin. This increase was far greater then that observed for the bicistronic GFP gene, and cannot be explained by a simple increase in the fraction of cells containing provirus. These results suggest that selective expansion favors erythroid stem/progenitor cells with provirus integrated at chromosomal sites which are relatively resistant to silencing position effects.
authors.affiliation: Department of Medicine, Division of Medical Genetics, University of Washington, Box 357720, HSB K236F, 1705 NE Pacific Street, Seattle, WA 98195-7720, USA. demery@u.washington.edu
authors.forename: David W
authors.initials: DW
authors.lastname: Emery
authors.forename: Julie
authors.initials: J
authors.lastname: Tubb
authors.forename: Yumiko
authors.initials: Y
authors.lastname: Nishino
authors.forename: Tamon
authors.initials: T
authors.lastname: Nishino
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Otto
authors.forename: George
authors.initials: G
authors.lastname: Stamatoyannopoulos
authors.forename: C Anthony
authors.initials: CA
authors.lastname: Blau
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D016026:Bone Marrow Transplantation; D048708:Cell Growth Processes / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D041905:Erythroid Cells / Q000166:cytology* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D011518:Proto-Oncogene Proteins / Q000235:genetics; D018121:Receptors, Cytokine / Q000235:genetics; D053628:Receptors, Thrombopoietin; D012190:Retroviridae / Q000235:genetics; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D016662:Virus Integration; D005719:gamma-Globulins / Q000235:genetics*
chemical_list: D011518:Proto-Oncogene Proteins; D018121:Receptors, Cytokine; D053628:Receptors, Thrombopoietin; D005719:gamma-Globulins; C082218:MPL protein, human; D049452:Green Fluorescent Proteins