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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

Characteristics of IFNγ-HBD fusion proteins. (a) Schematic representation of wild-type and cell surface-interacted interferonγ (IFNγ) genes encoding IFNγ and IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3. (b) The concentration of IFNγ in the culture medium of COS7 cells (open column) and cell lysates (solid column) 24 hours after transfection of pCpG-IFNγ and pCpG-IFNγ-HBDn (0.3 μg/well). Results are expressed as mean ± SEM (n = 3). (c) Western blotting analysis of IFNγ-HBD fusion proteins. Lane 1, IFNγ; lane 2, IFNγ-HBD1; lane 3, IFNγ-HBD2; and lane 4, IFNγ-HBD3. (d) Heparan sulfate-coating plate was incubated with serial dilutions of IFNγ (solid circles), IFNγ-HBD1 (open circles), IFNγ-HBD2 (open triangles), and IFNγ-HBD3 (open squares) for 2 hours at room temperature. Each protein bound to heparan sulfate was detected by ELISA. Results are expressed as mean ± SEM (n = 3). Asterisk (*) indicates t-test statistically different (P < 0.05) from the IFNγ group at the same concentration. ELISA, enzyme-linked immunosorbent assay; HBD, heparin-binding domain; IFN, interferon.
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fig1: Characteristics of IFNγ-HBD fusion proteins. (a) Schematic representation of wild-type and cell surface-interacted interferonγ (IFNγ) genes encoding IFNγ and IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3. (b) The concentration of IFNγ in the culture medium of COS7 cells (open column) and cell lysates (solid column) 24 hours after transfection of pCpG-IFNγ and pCpG-IFNγ-HBDn (0.3 μg/well). Results are expressed as mean ± SEM (n = 3). (c) Western blotting analysis of IFNγ-HBD fusion proteins. Lane 1, IFNγ; lane 2, IFNγ-HBD1; lane 3, IFNγ-HBD2; and lane 4, IFNγ-HBD3. (d) Heparan sulfate-coating plate was incubated with serial dilutions of IFNγ (solid circles), IFNγ-HBD1 (open circles), IFNγ-HBD2 (open triangles), and IFNγ-HBD3 (open squares) for 2 hours at room temperature. Each protein bound to heparan sulfate was detected by ELISA. Results are expressed as mean ± SEM (n = 3). Asterisk (*) indicates t-test statistically different (P < 0.05) from the IFNγ group at the same concentration. ELISA, enzyme-linked immunosorbent assay; HBD, heparin-binding domain; IFN, interferon.

Mentions: One, two, or three repeats of the HBD of EC-SOD (RKKRRR) were genetically fused to the C-terminal of IFNγ to obtain IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, respectively. We used the pcDNA3 plasmid backbone to construct plasmid vectors expressing IFNγ-HBDn (n = 1, 2, 3) for in vitro studies. Figure 1a shows the schematic images of plasmids encoding IFNγ or IFNγ-HBDs. African green monkey kidney COS-7 cells were transfected with each plasmid, and the amounts of IFNγ and IFNγ-HBDs in culture media and cell lysates were measured 24 hours after transfection. The concentration of IFNγ-HBD fusion proteins in the culture media of the cells transfected with pCMV-IFNγ-HBD1, pCMV-IFNγ-HBD2, or pCMV-IFNγ-HBD3 was much lower than the concentration of IFNγ in the media of the cells transfected with pCMV-IFNγ (Figure 1b). Western blot analysis of the culture media of the cells transfected with pCMV-IFNγ showed a single band of around 35 kDa, which is consistent with the molecular weight of IFNγ homodimer (Figure 1c, lane 1). The culture media of cells transfected with pCMV-IFNγ-HBD1, pCMV-IFNγ-HBD2, or pCMV-IFNγ-HBD3 showed a band with a molecular weight slightly higher than 35 kDa (Figure 1c, lanes 2–4), suggesting that the fusion proteins designed are expressed in the cells after transfection.


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)

Characteristics of IFNγ-HBD fusion proteins. (a) Schematic representation of wild-type and cell surface-interacted interferonγ (IFNγ) genes encoding IFNγ and IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3. (b) The concentration of IFNγ in the culture medium of COS7 cells (open column) and cell lysates (solid column) 24 hours after transfection of pCpG-IFNγ and pCpG-IFNγ-HBDn (0.3 μg/well). Results are expressed as mean ± SEM (n = 3). (c) Western blotting analysis of IFNγ-HBD fusion proteins. Lane 1, IFNγ; lane 2, IFNγ-HBD1; lane 3, IFNγ-HBD2; and lane 4, IFNγ-HBD3. (d) Heparan sulfate-coating plate was incubated with serial dilutions of IFNγ (solid circles), IFNγ-HBD1 (open circles), IFNγ-HBD2 (open triangles), and IFNγ-HBD3 (open squares) for 2 hours at room temperature. Each protein bound to heparan sulfate was detected by ELISA. Results are expressed as mean ± SEM (n = 3). Asterisk (*) indicates t-test statistically different (P < 0.05) from the IFNγ group at the same concentration. ELISA, enzyme-linked immunosorbent assay; HBD, heparin-binding domain; IFN, interferon.
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fig1: Characteristics of IFNγ-HBD fusion proteins. (a) Schematic representation of wild-type and cell surface-interacted interferonγ (IFNγ) genes encoding IFNγ and IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3. (b) The concentration of IFNγ in the culture medium of COS7 cells (open column) and cell lysates (solid column) 24 hours after transfection of pCpG-IFNγ and pCpG-IFNγ-HBDn (0.3 μg/well). Results are expressed as mean ± SEM (n = 3). (c) Western blotting analysis of IFNγ-HBD fusion proteins. Lane 1, IFNγ; lane 2, IFNγ-HBD1; lane 3, IFNγ-HBD2; and lane 4, IFNγ-HBD3. (d) Heparan sulfate-coating plate was incubated with serial dilutions of IFNγ (solid circles), IFNγ-HBD1 (open circles), IFNγ-HBD2 (open triangles), and IFNγ-HBD3 (open squares) for 2 hours at room temperature. Each protein bound to heparan sulfate was detected by ELISA. Results are expressed as mean ± SEM (n = 3). Asterisk (*) indicates t-test statistically different (P < 0.05) from the IFNγ group at the same concentration. ELISA, enzyme-linked immunosorbent assay; HBD, heparin-binding domain; IFN, interferon.
Mentions: One, two, or three repeats of the HBD of EC-SOD (RKKRRR) were genetically fused to the C-terminal of IFNγ to obtain IFNγ-HBD1, IFNγ-HBD2, and IFNγ-HBD3, respectively. We used the pcDNA3 plasmid backbone to construct plasmid vectors expressing IFNγ-HBDn (n = 1, 2, 3) for in vitro studies. Figure 1a shows the schematic images of plasmids encoding IFNγ or IFNγ-HBDs. African green monkey kidney COS-7 cells were transfected with each plasmid, and the amounts of IFNγ and IFNγ-HBDs in culture media and cell lysates were measured 24 hours after transfection. The concentration of IFNγ-HBD fusion proteins in the culture media of the cells transfected with pCMV-IFNγ-HBD1, pCMV-IFNγ-HBD2, or pCMV-IFNγ-HBD3 was much lower than the concentration of IFNγ in the media of the cells transfected with pCMV-IFNγ (Figure 1b). Western blot analysis of the culture media of the cells transfected with pCMV-IFNγ showed a single band of around 35 kDa, which is consistent with the molecular weight of IFNγ homodimer (Figure 1c, lane 1). The culture media of cells transfected with pCMV-IFNγ-HBD1, pCMV-IFNγ-HBD2, or pCMV-IFNγ-HBD3 showed a band with a molecular weight slightly higher than 35 kDa (Figure 1c, lanes 2–4), suggesting that the fusion proteins designed are expressed in the cells after transfection.

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