title: Long-term transgene expression and survival of transgene-expressing grafts following lentivirus transduction of bone marrow side population cells.
abstract: BACKGROUND
  Successful transduction of hematopoietic stem cells is essential if gene therapy is to be used clinically to induce immunologic tolerance.
  
  
  METHODS
  Hoechst 33342 staining was used to isolate a population of bone marrow cells enriched for stem cells, termed side population (SP) cells. Murine bone marrow SP cells were transduced with HLA-A2.1-expressing VSV-G-pseudotyped lentivirus or retrovirus vectors under identical conditions.
  
  
  RESULTS
  After transduction without prestimulating cytokines, which minimizes cell cycling and helps maintain stem cell pluripotency, the HLA-A2.1 gene was found in the DNA of 56% of CFU-GM colonies derived from lentivirus-transduced SP cells, but in only 4% of colonies derived from retrovirus-transduced SP cells. Lentivirus and retrovirus transduction including cytokine prestimulation produced the same degree of integration as that following lentivirus-transduction of non-prestimulated cells. Transplantation of 5,000 lentivirus-transduced SP cells into lethally irradiated mice resulted in long-term expression of the HLA-A2.1 transgene in peripheral blood progeny of bone marrow SP cells and prolonged skin graft survival across this class I MHC barrier until the time of animal sacrifice.
  
  
  CONCLUSIONS
  Recombinant lentivirus, but not retrovirus vectors, effectively transduced SP cells that were not prestimulated with cytokines and lentivirus-transduced SP cells successfully repopulated lethally irradiated C57BL/6 mice, animals where there is no selective advantage to repopulation with transduced cells. Transplantation of a relatively small number of transduced SP cells led to prolonged transgene mRNA expression and antigen-specific survival of grafts expressing the foreign MHC transgene.
authors.affiliation: Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska 68198-3285, USA.
authors.forename: Jai-lin
authors.initials: JL
authors.lastname: Zhang
authors.forename: Jin
authors.initials: J
authors.lastname: Cai
authors.forename: John D
authors.initials: JD
authors.lastname: Jackson
authors.forename: Charles A
authors.initials: CA
authors.lastname: Kuszynski
authors.forename: Shannon
authors.initials: S
authors.lastname: Walls
authors.forename: R Scott
authors.initials: RS
authors.lastname: McIvor
authors.forename: Ira J
authors.initials: IJ
authors.lastname: Fox
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D016207:Cytokines / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics*; D006085:Graft Survival; D015789:HLA-A2 Antigen / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D051379:Mice; D016038:Skin Transplantation; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes / Q000235:genetics*
chemical_list: D016207:Cytokines; D015789:HLA-A2 Antigen