Expression of HSV-TK suicide gene in primary T lymphocytes: the dog as a preclinical model.
Expression of suicide genes (e.g. herpes simplex virus thymidine kinase,HSV-TK) in T cells is an appealing approach to regulate graft-versus-host disease in adoptive immunotherapy. Here we report the optimization of retroviral infection of canine T cells. Canine T cells were stimulated either with phytohemagglutinin (PHA, 2 microg/ml) for 24-72 hours or with 100 U/ml interleukin-2 for seven days. Stimulated cells were co-cultivated with irradiated virus-producing cells. Transduction efficiencies ranged from 4% to 45% using PG13, a gibbon ape leukemia virus envelope (env) pseudotyped packaging cell line. Infection of cells with GPenvAM12, expressing the amphotropic Moloney murine leukemia virus env, did not yield a satisfactory percentage of transduced cells. Enrichment of transduced cells was performed using immunoselection, and gave a purity of up to 98%. Transfusion of 1 x 10(6) transduced cells per kilogram body weight showed that transduced cells could convert mixed chimerism to 100% and transfer immunity to a specific antigen. Transduced cells were repeatedly detected in peripheral blood and bone marrow by polymerase chain reaction with primers specific for the HSV-TK gene. We have demonstrated the feasibility of using the canine model to study gene therapy as a preclinical model.
Weissinger
E M
EM
MHH, Hematology/Oncology, Hannover, Germany. weissinger.eva@mh-hannover.de
Franz
M
M
Voss
C
C
Bonini
C
C
Kremmer
E
E
Kolb
H J
HJ
D000818:Animals; D016026:Bone Marrow Transplantation / Q000276:immunology; D018920:Coculture Techniques; D017931:DNA Primers / Q000737:chemistry; D004285:Dogs; D015774:Ganciclovir / Q000494:pharmacology; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods; D006086:Graft vs Host Disease / Q000276:immunology / Q000628:therapy*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D007376:Interleukin-2 / Q000494:pharmacology; D017708:Leukocyte Transfusion; D008213:Lymphocyte Activation / Q000187:drug effects / Q000276:immunology; D010835:Phytohemagglutinins / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018139:Simplexvirus / Q000201:enzymology*; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology / Q000821:virology; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D007376:Interleukin-2; D010835:Phytohemagglutinins; D012333:RNA, Messenger; D013937:Thymidine Kinase; D015774:Ganciclovir