The duration of transgene expression of IFN-gamma was successfully increased by reducing the CpG content of IFN-expressing plasmid vector, which resulted in an increased anticancer activity of IFN gene transfer.

Plasmid DNA (pDNA) expressing mouse interferon (IFN)-b or IFN-c (pCMV-Mub and pCMV-Muc, respectively) has been shown to be effective in inhibiting the growth of colon carcinoma CT-26 cells in the liver (Kobayashi et al., Molecular Therapy 2002;6:737-44). The therapeutic effect of such IFN gene transfer could be significantly increased by the sustained expression of IFNs. In the present study, CpG-reduced pDNA encoding IFN-b or IFN-c (pGZB-Mub and pGZB-Muc, respectively) was constructed. pCMV-Mub and pCMV-Muc were used as conventional CpG-replete pDNAs. Each pDNA was injected into the tail vein of mice by the hydrodynamics-based procedure. An injection of pGZB-Mub resulted in very high IFN-b activities in the serum for at least 24 hr after injection, whereas the IFN-b activity after pCMV-Mub injection declined quickly. About a 14-fold greater amount of IFN-b was produced from pGZB-Mub than from pCMV-Mub. pGZB-Mub markedly inhibited the pulmonary metastasis of CT-26 cells. Similar, but more marked results were obtained with pGZB-Muc: it increased the area under the concentration-time curve by more than a 60-fold and the mean residence time of IFN-c 4-fold compared with pCMV-Muc. The survival time of the pGZB-Muc-treated mice was significantly (p < 0.05) longer than that of the saline-or pCMV-Muc-treated mice. These results indicate that long-term expression of IFN can be achieved by CpG-reduced pDNA and sustained IFN gene expression results in enhanced therapeutic effects of IFN gene transfer against tumor metastasis. ' 2007 Wiley-Liss, Inc.