title: Efficiency of transduction of highly purified murine hematopoietic stem cells by lentiviral and oncoretroviral vectors under conditions of minimal in vitro manipulation.
abstract: The development of leukemias in several children with severe combined immunodeficiency disease who were transplanted with retroviral vector-transduced bone marrow cells has renewed concerns about the risks associated with the random integration of proviral sequences into chromosomal DNA. One theoretical way to reduce the risks of insertional mutagenesis would be to employ transduction/transplantation protocols that minimize the total number of genetically modified cells and associated proviral integration "events" introduced into recipients. Toward this end, we have developed a transduction protocol that involves the short-term incubation of highly purified murine stem cells with high-titer recombinant lentivirus vectors in the presence of serum-free medium and the cytokines SCF and TPO. Competitive repopulation studies showed that stem cells transduced in this way possessed the same reconstitutive ability as fresh, unmanipulated cells. Animals transplanted with only 200-2000 transduced cells were efficiently reconstituted with the genetically modified cells, and most hematopoietic cells in the recipients expressed the transgene. In contrast, the use of high-titer oncoretroviral vectors in conjunction with the same transduction/transplantation protocol resulted in only low levels of gene marking in vivo. The use of a similar transduction/transplantation strategy in future clinical studies may offer distinct advantages over current protocols.
authors.affiliation: Department of Genetics, Harvard Medical School and Molecular Medicine, Children's Hospital, Boston, MA 02115, USA.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Mostoslavsky
authors.forename: Darrell N
authors.initials: DN
authors.lastname: Kotton
authors.forename: Attila J
authors.initials: AJ
authors.lastname: Fabian
authors.forename: John T
authors.initials: JT
authors.lastname: Gray
authors.forename: Jeng-Shin
authors.initials: JS
authors.lastname: Lee
authors.forename: Richard C
authors.initials: RC
authors.lastname: Mulligan
mesh_terms: D000818:Animals; D016207:Cytokines / Q000494:pharmacology; D005819:Genetic Markers; D015316:Genetic Therapy / Q000009:adverse effects; D005822:Genetic Vectors / Q000235:genetics*; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D011533:Proviruses / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D016207:Cytokines; D005819:Genetic Markers