title: Constant and steady transgene expression of interferon-γ by optimization of plasmid construct for safe and effective interferon-γ gene therapy.
abstract: BACKGROUND
  Hydrodynamic injection of pmCMV(enh) -hEF-1(prom) -muIFN-γ, a plasmid DNA (pDNA) expressing murine interferon (IFN)-γ with a murine cytomegalovirus (mCMV) enhancer and a human elongation factor (EF)-1 promoter, has been proven effective for the treatment of cancer and atopic dermatitis in mice. However, the initial peak of IFN-γ soon after injection was quite high compared to the steady level for subsequent periods, which could cause unwanted adverse effects. Therefore, in the present study, aiming to optimize the efficacy/side-effect ratio of IFN-γ gene transfer, we have developed plasmid vectors encoding murine IFN-γ under the control of different combinations of promoter and enhancer sequences.
  
  
  METHODS
  The promoter and enhancer sequence of pmCMV(enh) -hEF-1(prom) -huIFN-γ, a prototype plasmid expressing human IFN-γ, was replaced or deleted to obtain various pDNAs. To assess the transgene expression profile, each pDNA was delivered to mice by hydrodynamic injection and the serum IFN-γ concentration was measured periodically. On the basis of the results obtained, murine IFN-γ expressing pDNAs were constructed and the body weight change was monitored as an indicator of adverse effects.
  
  
  RESULTS
  The prototype pmCMV(enh) -hEF-1(prom) -huIFN-γ showed a high but declining concentration of IFN-γ. Those containing hROSA26 promoter expressed the transgene in a more constant manner with no initial high concentrations and scarcely reduced the body weight.
  
  
  CONCLUSIONS
  These results indicate that hROSA26 promoter, irrespective of the presence and type of enhancers, is suitable for achieving constant and steady level of transgene expression and effective in avoiding the body weight loss caused by high concentrations of IFN-γ.
authors.affiliation: Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Science, Kyoto University, Kyoto, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ando
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Makiya
authors.initials: M
authors.lastname: Nishikawa
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Watanabe
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Takakura
mesh_terms: D000818:Animals; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D010957:Plasmids; D011401:Promoter Regions, Genetic; D019076:Transgenes
chemical_list: D007371:Interferon-gamma