Long-term transgene expression and survival of transgene-expressing grafts following lentivirus transduction of bone marrow side population cells.
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.
Zhang
Jai-lin
JL
Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska 68198-3285, USA.
Cai
Jin
J
Jackson
John D
JD
Kuszynski
Charles A
CA
Walls
Shannon
S
McIvor
R Scott
RS
Fox
Ira J
IJ
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*
D016207:Cytokines; D015789:HLA-A2 Antigen