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. CONCLUSION: 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.

Biliary stent clogging solved by nanotechnology? In vitro study of inorganic-organic sol-gel coatings for teflon stents.

BACKGROUND & AIMS: The major drawback of plastic stents for biliary drainage is the occlusion by sludge. Sludge is accrued because the stent surface allows for the adherence of proteins, glycoproteins, or bacteria and the bile flow is insufficient to clean the surface. In this study, experience from nanotechnology to achieve a clean surface by improved soil-release characteristics is used to optimize biliary stent surface. The aim of this study was to examine sludge accumulation in relation to surface characteristics designed by nanotechnology. METHODS: A variety of inorganic-organic sol-gel-coated stents were incubated in sterilized human bile and enzyme-active Escherichia coli for 35 days. Materials were Teflon (DuPont, Wilmington, DE) coated with hydrophobic Clearcoat (NTC, Tholey, Germany), Teflon with sol-gel coating synthesized of organic epoxides of 190 g/mol or 500 g/mol, and propylaminosilane without or with fluorsilanes for increased hydrophobicity. Scanning electron microscopy and semiquantitative analysis, blinded to the type of coating, were used to determine the amount of sludge accumulated on the surface. RESULTS: Sludge deposition was reduced on the designed surfaces as compared with uncoated Teflon and Clearcoat. The performance of high molecular (500 g/mol) was superior to that of low molecular (190 g/mol) epoxide ligand. However, increasing hydrophobicity by adding fluoraminosilanes resulted in increased adherence of sludge. Less than a micrometer-thin sol-gel coating is inexpensive because very little coating material is required. This is the first published data comparing systematically modified surfaces of biliary stents using nanotechnology. CONCLUSIONS: Optimized soil release by sol-gel nanocoating of plastic stents may prevent biliary plastic stents from clogging.