Use of lipoplex-induced nuclear factor-kappaB activation to enhance transgene expression by lipoplex in mouse lung.
BACKGROUND
Although lipofection-induced TNF-alpha can activate nuclear factor kappaB (NF-kappaB), which, in turn, increases the transgene expression from plasmid DNA in which any NF-kappaB responsive element is incorporated, no attempts have been made to use such biological responses as NF-kappaB activation against a vector to enhance vector-mediated gene transfer.


METHODS
A lipoplex composed of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium and cholesterol liposome and plasmid DNA encoding firefly luciferase under the control of the cytomegalovirus immediate early promoter (pCMV-Luc) was intravenously injected into mice. Luciferase activity as well as NF-kappaB activation in the lung were evaluated. Then, a novel plasmid DNA, pCMV-kappaB-Luc, was constructed by inserting 5 repeats of NF-kappaB-binding sequences into the pCMV-Luc.


RESULTS
NF-kappaB in the lung was activated by injection of the lipoplex and its nuclear localization was observed. An injection of lipopolysaccharide 30 min prior to the lipofection further activated NF-kappaB. At the same time, the treatment significantly increased the transgene expression by lipoplex, suggesting a positive correlation between expression and NF-kappaB activity. Based on these findings, we tried to enhance the lipoplex-based transgene expression by using NF-kappaB activation. The lipoplex consisting of pCMV-kappaB-Luc showed a 4.7-fold increase in transgene expression in the lung compared with that with pCMV-Luc.


CONCLUSIONS
We demonstrated that NF-kappaB activation by lipoplex can be used to enhance lipoplex-mediated transgene expression by inserting NF-kappaB-binding sequences into plasmid DNA. These findings offer a novel method for designing a vector for gene transfer in conjunction with biological responses to it.
Kuramoto
Takeshi
T
Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Nishikawa
Makiya
M
Thanaketpaisarn
Oranuch
O
Okabe
Takayuki
T
Yamashita
Fumiyoshi
F
Hashida
Mitsuru
M
D000818:Animals; D002784:Cholesterol / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008081:Liposomes / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology; D008168:Lung / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000378:metabolism / Q000494:pharmacology; D014018:Tissue Distribution; D019076:Transgenes / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D008081:Liposomes; D016328:NF-kappa B; D000644:Quaternary Ammonium Compounds; D014409:Tumor Necrosis Factor-alpha; C054005:N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium; D002784:Cholesterol