Constant and steady transgene expression of interferon-γ by optimization of plasmid construct for safe and effective interferon-γ gene therapy

Background Hydrodynamic injection of pmCMV(enh)-hEF-1(prom)-muIFN-gamma, a plasmid DNA (pDNA) expressing murine interferon (IFN)-gamma 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-gamma 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-gamma gene transfer, we have developed plasmid vectors encoding murine IFN-gamma under the control of different combinations of promoter and enhancer sequences. Methods The promoter and enhancer sequence of pmCMV(enh)-hEF-1(prom)-huIFN-gamma, a prototype plasmid expressing human IFN-gamma, 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-gamma concentration was measured periodically. On the basis of the results obtained, murine IFN-gamma 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-gamma showed a high but declining concentration of IFN-gamma. 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-gamma. Copyright (C) 2012 John Wiley & Sons, Ltd.