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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: 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.

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

IFNγ-HBD fusion proteins are localized on the cell surface. (a) COS-7 cells transfected with IFNγ or IFNγ-HBD fusion proteins expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ, and the cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (blue). The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. Green signals represent IFNγ. (b) COS-7 cells transfected with IFNγ (closed area) or IFNγ-HBD fusion proteins (open area) expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ. The amounts of IFNγ both inside and outside the cells were measured by flow cytometry. The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. The result of IFNγ is shown in all the graphs. HBD, heparin-binding domain; IFN, interferon.
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fig2: IFNγ-HBD fusion proteins are localized on the cell surface. (a) COS-7 cells transfected with IFNγ or IFNγ-HBD fusion proteins expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ, and the cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (blue). The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. Green signals represent IFNγ. (b) COS-7 cells transfected with IFNγ (closed area) or IFNγ-HBD fusion proteins (open area) expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ. The amounts of IFNγ both inside and outside the cells were measured by flow cytometry. The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. The result of IFNγ is shown in all the graphs. HBD, heparin-binding domain; IFN, interferon.

Mentions: To visualize the cellular localization of IFNγ and IFNγ-HBD fusion proteins, immunofluorescent staining of COS-7 cells was performed using anti-IFNγ antibody (Figure 2a). To detect IFNγ and IFNγ-HBD fusion proteins both inside and outside the cells, cells were treated with 0.1% Triton X-100 to permeabilize the cell membrane. Strong IFNγ signals were observed in all the cells transfected with any plasmid. When the cells were not treated with 0.1% Triton X-100, no significant signals were detected in the cells transfected with pCMV-IFNγ, whereas positive signals were detected in the cells transfected with pCMV-IFNγ-HBD1, pCMV-IFNγ-HBD2, or pCMV-IFNγ-HBD3. In addition, these localization of IFNγ-HBDs on cell surface were observed in other two types of cells, mouse embryonic fibroblast cell line NIH 3T3 and human hepatocellular carcinoma cell line HepG2 (data not shown).


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)

IFNγ-HBD fusion proteins are localized on the cell surface. (a) COS-7 cells transfected with IFNγ or IFNγ-HBD fusion proteins expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ, and the cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (blue). The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. Green signals represent IFNγ. (b) COS-7 cells transfected with IFNγ (closed area) or IFNγ-HBD fusion proteins (open area) expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ. The amounts of IFNγ both inside and outside the cells were measured by flow cytometry. The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. The result of IFNγ is shown in all the graphs. HBD, heparin-binding domain; IFN, interferon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: IFNγ-HBD fusion proteins are localized on the cell surface. (a) COS-7 cells transfected with IFNγ or IFNγ-HBD fusion proteins expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ, and the cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (blue). The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. Green signals represent IFNγ. (b) COS-7 cells transfected with IFNγ (closed area) or IFNγ-HBD fusion proteins (open area) expressing pDNA were fixed and stained with monoclonal antibodies against IFNγ. The amounts of IFNγ both inside and outside the cells were measured by flow cytometry. The cells were additionally permeabilized with 0.1% TritonX-100 (top) or not (bottom) before staining. The result of IFNγ is shown in all the graphs. HBD, heparin-binding domain; IFN, interferon.
Mentions: To visualize the cellular localization of IFNγ and IFNγ-HBD fusion proteins, immunofluorescent staining of COS-7 cells was performed using anti-IFNγ antibody (Figure 2a). To detect IFNγ and IFNγ-HBD fusion proteins both inside and outside the cells, cells were treated with 0.1% Triton X-100 to permeabilize the cell membrane. Strong IFNγ signals were observed in all the cells transfected with any plasmid. When the cells were not treated with 0.1% Triton X-100, no significant signals were detected in the cells transfected with pCMV-IFNγ, whereas positive signals were detected in the cells transfected with pCMV-IFNγ-HBD1, pCMV-IFNγ-HBD2, or pCMV-IFNγ-HBD3. In addition, these localization of IFNγ-HBDs on cell surface were observed in other two types of cells, mouse embryonic fibroblast cell line NIH 3T3 and human hepatocellular carcinoma cell line HepG2 (data not shown).

Bottom Line: 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.

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