Tolerance by selective in vivo expansion of foreign major histocompatibility complex-transduced autologous bone marrow.
BACKGROUND
Application of gene therapy to induce antigen-specific immune tolerance could be important for transplantation or treatment of autoimmune diseases. Hematopoietic stem cell-based gene therapy has been hampered by relatively weak gene expression in vivo and loss of transduced cells over time. Selective expansion of transduced hematopoietic stem cells has been accomplished by incorporating the dihydrofolate reductase (DHFR) gene into the gene transfer vector.


METHODS
To assess whether this strategy could be applied to transplantation, we constructed a retroviral vector plasmid (KA274) containing the cDNA encoding human leukocyte antigen (HLA)-A2.1 and a tyr22 mutant DHFR and generated vesicular stomatitis virus-G-pseudotyped recombinant retrovirus by transfection into 293GPG cells. Bone marrow cells from C57BL/6 mice were infected with KA274 at a multiplicity of infection of 100, and transplanted into lethally irradiated syngeneic mice.


RESULTS
After transplantation with transduced bone marrow, the proportion of peripheral blood cells expressing HLA-A2 ranged from 3.2% to 38% and increased 2- to 4.9-fold after selection for DHFR-expressing cells using trimetrexate and nitrobenzylmercaptpurine riboside 5' monophosphate. HLA-A2 expression remained above pretreatment levels throughout the study. Cytotoxic spleen cells from reconstituted mice lysed third-party HLA-B7-expressing targets but were unable to lyse HLA-A2-expressing targets. All KA274 reconstituted C57BL/6 mice accepted skin grafts from HLA-A2.1 transgenic mice for more than 245 days but rejected third-party Balb/c skin grafts in 12 days.


CONCLUSIONS
Long-term transgene expression and immunologic tolerance to retrovirus-encoded HLA-A2, equivalent to that obtained by donor bone marrow transplantation, was accomplished, and selective expansion of transduced bone marrow cells was induced using DHFR as a selectable marker.
Zhang
Jia Lin
JL
Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska 68198-3285, USA.
Cai
Jin
J
Walls
Shannon
S
Jackson
John D
JD
Kuszynski
Charles A
CA
Zhao
Yong
Y
Pawliuk
Robert
R
Leboulch
Philippe
P
Fox
Ira J
IJ
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology; D016026:Bone Marrow Transplantation / Q000379:methods*; D017690:Cell Transplantation; D018076:DNA, Complementary / Q000378:metabolism; D005434:Flow Cytometry; D006084:Graft Rejection; D006085:Graft Survival; D015789:HLA-A2 Antigen / Q000737:chemistry; D006412:Hematopoietic Stem Cells / Q000166:cytology; D008285:Major Histocompatibility Complex; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009154:Mutation; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D016038:Skin Transplantation; D013997:Time Factors
D018076:DNA, Complementary; D015789:HLA-A2 Antigen