Cloning and characterization of an adenoviral vector for highly efficient and doxycycline-suppressible expression of bioactive human single-chain interleukin 12 in colon cancer.
BACKGROUND
Interleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12.


RESULTS
High gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue.


CONCLUSIONS
VP16 transactivator-mediated and doxycycline-regulated expression of the human interleukin-12 gene enables highly efficient and tightly controlled cytokine expression in human cancer. These data illustrate the potential of the described adenoviral vector system for the safe and superior expression of therapeutic genes in the treatment of colorectal cancer and other malignancies.
Wulff
Holger
H
Department of Medicine, University of Hamburg, University Hospital Hamburg-Eppendorf, Hamburg, Germany. hwulff@web.de <hwulff@web.de>
Krieger
Thorsten
T
Krüger
Karen
K
Stahmer
Ingrid
I
Thaiss
Friedrich
F
Schäfer
Hansjörg
H
Block
Andreas
A
D000256:Adenoviridae / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000008:administration & dosage*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D019073:HT29 Cells; D006801:Humans; D018664:Interleukin-12 / Q000235:genetics* / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*
D018664:Interleukin-12; D004318:Doxycycline