Effect of the content of unmethylated CpG dinucleotides in plasmid DNA on the sustainability of transgene expression.
BACKGROUND
Nonviral gene transfer generally suffers from short-term expression of transgenes. We have previously demonstrated that plasmids with reduced CpG content exhibited a more prolonged expression of murine interferon (IFN)-beta or IFN-gamma, which was effective in inhibiting metastatic tumor growth. A further extension of the duration of transgene expression could be achieved by controlling the number and location of CpG motifs in plasmid DNA.


METHODS
Luciferase-expressing plasmids with differing CpG content were injected into the tail vein of mice by the hydrodynamic injection method. The effects of CpG content on the duration of transgene expression were examined, focusing on cytosine methylation and pro-inflammatory cytokines. Based on the findings, IFN-gamma-expressing plasmids were constructed and their transgene expression and inhibitory effect on pulmonary metastasis were evaluated.


RESULTS
Plasmids with a few CpG motifs showed a prolonged luciferase activity in the liver. Methylation of CpG motifs in plasmids reduced the expression and the extent of this reduction was greater for plasmids with a high CpG content. Pro-inflammatory cytokines hardly affected the expression. pCpG-Mu gamma, the IFN-gamma-expressing plasmid, which contains 20 CpG motifs only in the cDNA region, exhibited a sustained IFN-gamma concentration at therapeutic levels, and had a great inhibitory effect on the pulmonary metastasis of tumor cells.


CONCLUSIONS
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.
Mitsui
Masaru
M
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
Nishikawa
Makiya
M
Zang
Lei
L
Ando
Mitsuru
M
Hattori
Kayoko
K
Takahashi
Yuki
Y
Watanabe
Yoshihiko
Y
Takakura
Yoshinobu
Y
D000818:Animals; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics*; D016207:Cytokines / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D019175:DNA Methylation; D015226:Dinucleoside Phosphates / Q000235:genetics*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D018836:Inflammation Mediators / Q000378:metabolism; D007267:Injections; D007371:Interferon-gamma / Q000097:blood / Q000235:genetics; D008156:Luciferases / Q000378:metabolism; D008175:Lung Neoplasms / Q000556:secondary; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D013997:Time Factors; D019076:Transgenes / Q000235:genetics*
D016207:Cytokines; D015226:Dinucleoside Phosphates; D018836:Inflammation Mediators; D007371:Interferon-gamma; D003596:Cytosine; D004247:DNA; D008156:Luciferases