Related Experiment Videos
Expression of HSV-TK suicide gene in primary T lymphocytes: the dog as a preclinical model
E M Weissinger1, M Franz, C Voss
1MHH, Hematology/Oncology, Hannover, Germany. weissinger.eva@mh-hannover.de
Abstract:
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.
Insights
Gene therapy using suicide genes like herpes simplex virus thymidine kinase (HSV-TK) in T cells offers a way to control graft-versus-host disease. This study optimized canine T cell gene transfer, showing its preclinical feasibility.
Area of Science:
- * Immunology
- * Gene Therapy
- * Oncology
Background:
- * Adoptive immunotherapy aims to regulate graft-versus-host disease using suicide gene-modified T cells.
- * Herpes simplex virus thymidine kinase (HSV-TK) is a suicide gene utilized for T cell regulation.
Purpose of the Study:
- * To optimize retroviral infection of canine T cells for gene therapy applications.
- * To evaluate the efficacy of gene-modified T cells in a preclinical canine model.
Main Methods:
- * Canine T cells were stimulated with phytohemagglutinin (PHA) or interleukin-2.
- * Cells were co-cultivated with irradiated virus-producing cells (PG13 packaging cell line).
- * Transduction efficiency was assessed, and transduced cells were enriched via immunoselection.
Main Results:
- * Transduction efficiencies ranged from 4% to 45% using the PG13 packaging cell line.
- * Immunoselection achieved up to 98% purity of transduced cells.
- * Transfused cells converted mixed chimerism to 100% and transferred antigen-specific immunity, detected via PCR for HSV-TK.
Conclusions:
- * Retroviral gene transfer into canine T cells was successfully optimized.
- * Gene-modified T cells demonstrated therapeutic potential in a preclinical setting.
- * The canine model is feasible for studying gene therapy strategies for graft-versus-host disease.