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specific, activation-induced transgene expression. Our objective was to devel-
op a retroviral vector for stable and activation-induced transgene expression
in T-lymphocytes.
Results
First, we compared the transcriptional potency of the full-length IL-2 promot-
er with that of a synthetic promoter composed of 3 repeats of the Nuclear Fac-
tor of Activated T-Cells (NFAT) element following activation of transfected
Jurkat T-cells expressing the large SV40 T antigen (Jurkat TAg). Although the
NFAT3 promoter resulted in a stronger induction of luciferase reporter ex-
pression post stimulation, the basal levels of the IL-2 promoter-driven reporter
expression were much lower indicating that the IL-2 promoter can serve as a
more stringent activation-dependent promoter in T-cells. Based on this data,
we generated a self-inactivating retroviral vector with the full-length human
IL-2 promoter, namely SINIL-2pr that incorporated the enhanced green flu-
orescent protein (EGFP) fused to herpes simplex virus thymidine kinase as a
reporter/suicide “bifunctional” gene. Subsequently, Vesicular Stomatitis Vi-
rus-G Protein pseudotyped retroparticles were generated for SINIL-2pr and
used to transduce the Jurkat T-cell line and the ZAP-70-deficient P116 cell
line. Flow cytometry analysis showed that EGFP expression was markedly en-
hanced post co-stimulation of the gene-modified cells with 1 µ M ionomycin
and 10 ng/ml phorbol 12-myristate 13-acetate (PMA). This activation-in-
duced expression was abrogated when the cells were pretreated with 300 nM
cyclosporin A.
Conclusion
These results demonstrate that the SINIL-2pr retrovector leads to activation-
inducible transgene expression in Jurkat T-cell lines. We propose that this de-
sign can be potentially exploited in several cellular immunotherapy applica-
tions.
background
T-cells have several features that render them attractive target cells for cell and
gene therapy applications. In addition to their diverse and critical role in the im-
mune system, T-cells are readily available from human peripheral blood, they can
be easily isolated, activated, expanded to large numbers in vitro, and reinfused
with a potential to circulate for a long time in vivo. Indeed, T-cells have been
successfully utilized for adoptive immunotherapy of cancer, viral infections, and
autoimmune diseases.
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