Highly-efficient gene transfer with retroviral vectors into human T lymphocytes on fibronectin.
Genetically modified lymphocytes have been successfully used for correction of ADA deficiency in children and in controlling graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation. Low transduction efficiencies are, however, limiting for gene therapeutic strategies based on lymphocytes. In this study we compared protocols for highly efficient gene transfer into human T cells using retroviral vector-containing supernatant. We showed that infection of both human primary T cells and CD4+ Jurkat cells is most efficient on the matrix component fibronectin. Transduction was carried out with a retroviral vector encoding both the human intracytoplasmatically truncated low-affinity nerve growth factor receptor (deltaLNGFR) as a gene transfer marker and the Herpes simplex virus thymidine kinase for negative selection. Based on LNGFR expression genetically modified cells were enriched to near purity by magnetic cell sorting (MACS). Enriched cells could be shown to be highly sensitive to ganciclovir.
Bone Marrow Transplantation, University Hospital Eppendorf, Hamburg, Germany.
B
B
Fehse
U M
UM
Schade
Z
Z
Li
A
A
Uhde
S
S
Koch
B
B
Goller
R
R
Rüger
N
N
Fehse
M
M
Stockschläder
A R
AR
Zander
D018926:Anti-Allergic Agents / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism*; D015774:Ganciclovir / Q000494:pharmacology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D039041:Integrin alpha4beta1; D016023:Integrins / Q000378:metabolism; D019169:Jurkat Cells; D017469:Receptors, Fibronectin / Q000378:metabolism; D016201:Receptors, Lymphocyte Homing / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000821:virology*; D013937:Thymidine Kinase / Q000235:genetics
D018926:Anti-Allergic Agents; D005353:Fibronectins; D039041:Integrin alpha4beta1; D016023:Integrins; D017469:Receptors, Fibronectin; D016201:Receptors, Lymphocyte Homing; D017475:Receptors, Nerve Growth Factor; D013937:Thymidine Kinase; D015774:Ganciclovir