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Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice.

Ando M, Takahashi Y, Yamashita T, Fujimoto M, Nishikawa M, Watanabe Y, Takakura Y - Mol Ther Methods Clin Dev (2014)

Bottom Line: Sustained gene delivery of interferon (IFN) γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight.These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer.Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biopharmaceutics and Drug Metabolism , Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo-ku, Kyoto, Japan.

ABSTRACT
Sustained gene delivery of interferon (IFN) γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD) of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

No MeSH data available.


Related in: MedlinePlus

Biological activities of IFNγ-HBD fusion proteins. B16-BL6 cells transfected with pGAS-Luc and phRL-TK were incubated with serial dilutions of IFNγ (solid circles) and IFNγ-HBD fusion proteins (open circles) for 24 hours. pGAS-Luc expressed firefly luciferase under the control of the interferon gamma activated site was used to evaluate the IFNγ activities. phRL-TK expressed renilla luciferase was used to be normalized of transfection efficiencies and cell numbers. The result of IFNγ is shown in all the graphs. (a) IFNγ-HBD1, (b) IFNγ-HBD2, (c) IFNγ-HBD3. The ratio was normalized to give x-fold values relative to those of the untreated group and the half maximum effective concentration (EC50) of each protein was calculated. Results are expressed as mean ± SEM (n = 3). GAS, interferon gamma activated site; HBD, heparin-binding domain; IFN, interferon.
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fig3: Biological activities of IFNγ-HBD fusion proteins. B16-BL6 cells transfected with pGAS-Luc and phRL-TK were incubated with serial dilutions of IFNγ (solid circles) and IFNγ-HBD fusion proteins (open circles) for 24 hours. pGAS-Luc expressed firefly luciferase under the control of the interferon gamma activated site was used to evaluate the IFNγ activities. phRL-TK expressed renilla luciferase was used to be normalized of transfection efficiencies and cell numbers. The result of IFNγ is shown in all the graphs. (a) IFNγ-HBD1, (b) IFNγ-HBD2, (c) IFNγ-HBD3. The ratio was normalized to give x-fold values relative to those of the untreated group and the half maximum effective concentration (EC50) of each protein was calculated. Results are expressed as mean ± SEM (n = 3). GAS, interferon gamma activated site; HBD, heparin-binding domain; IFN, interferon.

Mentions: The biological activity of the IFNγ-HBD fusion proteins was examined using mouse melanoma B16-BL6 cells transfected with pGAS-Luc, a plasmid-encoding firefly luciferase under the control of the interferon gamma activated site (GAS) (Figure 3). In B16-BL6 cells, the amount of heparan sulfate on the surface of cells is small, because this cell line expresses heparanase, which is a heparan sulfate-degrading enzyme. Therefore, we were simply able to evaluate the biological activities of “free” IFNγ-HBD fusion proteins in vitro. Serially diluted IFNγ or IFNγ-HBD fusion proteins collected from culture media of COS-7 cells were added to each well of B16-BL6 cells transfected with pGAS-Luc. The addition of IFNγ increased the luciferase activity in a concentration-dependent manner. The fusion proteins also increased the activity, but the increase was a little smaller than that by IFNγ. The half maximal effective concentration (EC50) values calculated from the dose–response curves were 66.0, 80.5, 81.7, and 83.6 pg/ml for IFNγ, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, respectively.


Prevention of adverse events of interferon γ gene therapy by gene delivery of interferon γ-heparin-binding domain fusion protein in mice.

Ando M, Takahashi Y, Yamashita T, Fujimoto M, Nishikawa M, Watanabe Y, Takakura Y - Mol Ther Methods Clin Dev (2014)

Biological activities of IFNγ-HBD fusion proteins. B16-BL6 cells transfected with pGAS-Luc and phRL-TK were incubated with serial dilutions of IFNγ (solid circles) and IFNγ-HBD fusion proteins (open circles) for 24 hours. pGAS-Luc expressed firefly luciferase under the control of the interferon gamma activated site was used to evaluate the IFNγ activities. phRL-TK expressed renilla luciferase was used to be normalized of transfection efficiencies and cell numbers. The result of IFNγ is shown in all the graphs. (a) IFNγ-HBD1, (b) IFNγ-HBD2, (c) IFNγ-HBD3. The ratio was normalized to give x-fold values relative to those of the untreated group and the half maximum effective concentration (EC50) of each protein was calculated. Results are expressed as mean ± SEM (n = 3). GAS, interferon gamma activated site; HBD, heparin-binding domain; IFN, interferon.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362348&req=5

fig3: Biological activities of IFNγ-HBD fusion proteins. B16-BL6 cells transfected with pGAS-Luc and phRL-TK were incubated with serial dilutions of IFNγ (solid circles) and IFNγ-HBD fusion proteins (open circles) for 24 hours. pGAS-Luc expressed firefly luciferase under the control of the interferon gamma activated site was used to evaluate the IFNγ activities. phRL-TK expressed renilla luciferase was used to be normalized of transfection efficiencies and cell numbers. The result of IFNγ is shown in all the graphs. (a) IFNγ-HBD1, (b) IFNγ-HBD2, (c) IFNγ-HBD3. The ratio was normalized to give x-fold values relative to those of the untreated group and the half maximum effective concentration (EC50) of each protein was calculated. Results are expressed as mean ± SEM (n = 3). GAS, interferon gamma activated site; HBD, heparin-binding domain; IFN, interferon.
Mentions: The biological activity of the IFNγ-HBD fusion proteins was examined using mouse melanoma B16-BL6 cells transfected with pGAS-Luc, a plasmid-encoding firefly luciferase under the control of the interferon gamma activated site (GAS) (Figure 3). In B16-BL6 cells, the amount of heparan sulfate on the surface of cells is small, because this cell line expresses heparanase, which is a heparan sulfate-degrading enzyme. Therefore, we were simply able to evaluate the biological activities of “free” IFNγ-HBD fusion proteins in vitro. Serially diluted IFNγ or IFNγ-HBD fusion proteins collected from culture media of COS-7 cells were added to each well of B16-BL6 cells transfected with pGAS-Luc. The addition of IFNγ increased the luciferase activity in a concentration-dependent manner. The fusion proteins also increased the activity, but the increase was a little smaller than that by IFNγ. The half maximal effective concentration (EC50) values calculated from the dose–response curves were 66.0, 80.5, 81.7, and 83.6 pg/ml for IFNγ, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, respectively.

Bottom Line: Sustained gene delivery of interferon (IFN) γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight.These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer.Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biopharmaceutics and Drug Metabolism , Graduate School of Pharmaceutical Sciences, Kyoto University , Sakyo-ku, Kyoto, Japan.

ABSTRACT
Sustained gene delivery of interferon (IFN) γ can be an effective treatment, but our previous study showed high levels of IFNγ-induced adverse events, including the loss of body weight. These unwanted events could be reduced by target-specific delivery of IFNγ after in vivo gene transfer. To achieve this, we selected the heparin-binding domain (HBD) of extracellular superoxide dismutase as a molecule to anchor IFNγ to the cell surface. We designed three IFNγ derivatives, IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, each of which had 1, 2, or 3 HBDs, respectively. Each plasmid-encoding fusion proteins was delivered to the liver, a model target in this study, by hydrodynamic tail vein injection. The serum concentration of IFNγ-HBD2 and IFNγ-HBD3 after gene delivery was lower than that of IFNγ or IFNγ-HBD1. Gene delivery of IFNγ-HBD2, but not of IFNγ-HBD3, effectively increased the mRNA expression of IFNγ-inducible genes in the liver, suggesting liver-specific distribution of IFNγ-HBD2. Gene delivery of IFNγ-HBD2-suppressed tumor growth in the liver as efficiently as that of IFNγ with much less symptoms of adverse effects. These results indicate that the adverse events of IFNγ gene transfer can be prevented by gene delivery of IFNγ-HBD2, a fusion protein with high cell surface affinity.

No MeSH data available.


Related in: MedlinePlus