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Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome.

Shimizu K, Sakurai F, Tomita K, Nagamoto Y, Nakamura S, Katayama K, Tachibana M, Kawabata K, Mizuguchi H - Mol Ther Methods Clin Dev (2014)

Bottom Line: These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells.The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences.Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a-mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan.

ABSTRACT
Leaky expression of adenovirus (Ad) genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3'-untranslated region (UTR) of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a-mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

No MeSH data available.


Related in: MedlinePlus

Ad vector-mediated transgene expression in mice. (a) Luciferase production in the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the luciferase gene under the control of a CMV promoter at 1 × 1010 IFU/mouse, and the livers were harvested and subjected to luciferase expression analysis 2 days after administration. Luciferase production in the liver was determined by luminescence assay. The data are expressed as the mean values ± SD (n = 5–6). *P < 0.05 in comparison with Ad-L2. (b) mSEAP production in the serum following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the mSEAP gene under the control of an AHA promoter at 1 × 1010 IFU/mouse. Blood samples were collected via retro-orbital bleeding on the indicated days after injection. mSEAP production in the serum was determined by SEAP chemiluminescence assay. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-AHASEAP. mSEAP expression in the PBS-treated mice was below the detectable level.
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fig6: Ad vector-mediated transgene expression in mice. (a) Luciferase production in the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the luciferase gene under the control of a CMV promoter at 1 × 1010 IFU/mouse, and the livers were harvested and subjected to luciferase expression analysis 2 days after administration. Luciferase production in the liver was determined by luminescence assay. The data are expressed as the mean values ± SD (n = 5–6). *P < 0.05 in comparison with Ad-L2. (b) mSEAP production in the serum following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the mSEAP gene under the control of an AHA promoter at 1 × 1010 IFU/mouse. Blood samples were collected via retro-orbital bleeding on the indicated days after injection. mSEAP production in the serum was determined by SEAP chemiluminescence assay. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-AHASEAP. mSEAP expression in the PBS-treated mice was below the detectable level.

Mentions: In order to evaluate the in vivo transgene expression levels induced by the Ad vectors carrying miRNA-targeted sequences, luciferase expression in the liver was examined 2 days following administration (Figure 6a). The luciferase expression levels in the liver induced by the Ad vectors carrying miRNA-targeted sequences, with the exceptions of Ad-E2A-122aT-L2 and Ad-E4-122aT-L2, were comparable to those by Ad-L2. Ad-E4-122aT-L2–mediated luciferase expression in the liver was 15-fold lower than that mediated by Ad-L2. The lower luciferase expression levels of Ad-E4-122aT-L2 in the liver were probably due to the significant suppression of E4 gene expression. Previous studies demonstrated that the E4 gene products, especially the E4 ORF3 gene product, enhanced the transcriptional activity of a CMV promoter.9,10,32


Suppression of leaky expression of adenovirus genes by insertion of microRNA-targeted sequences in the replication-incompetent adenovirus vector genome.

Shimizu K, Sakurai F, Tomita K, Nagamoto Y, Nakamura S, Katayama K, Tachibana M, Kawabata K, Mizuguchi H - Mol Ther Methods Clin Dev (2014)

Ad vector-mediated transgene expression in mice. (a) Luciferase production in the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the luciferase gene under the control of a CMV promoter at 1 × 1010 IFU/mouse, and the livers were harvested and subjected to luciferase expression analysis 2 days after administration. Luciferase production in the liver was determined by luminescence assay. The data are expressed as the mean values ± SD (n = 5–6). *P < 0.05 in comparison with Ad-L2. (b) mSEAP production in the serum following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the mSEAP gene under the control of an AHA promoter at 1 × 1010 IFU/mouse. Blood samples were collected via retro-orbital bleeding on the indicated days after injection. mSEAP production in the serum was determined by SEAP chemiluminescence assay. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-AHASEAP. mSEAP expression in the PBS-treated mice was below the detectable level.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362365&req=5

fig6: Ad vector-mediated transgene expression in mice. (a) Luciferase production in the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the luciferase gene under the control of a CMV promoter at 1 × 1010 IFU/mouse, and the livers were harvested and subjected to luciferase expression analysis 2 days after administration. Luciferase production in the liver was determined by luminescence assay. The data are expressed as the mean values ± SD (n = 5–6). *P < 0.05 in comparison with Ad-L2. (b) mSEAP production in the serum following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors expressing the mSEAP gene under the control of an AHA promoter at 1 × 1010 IFU/mouse. Blood samples were collected via retro-orbital bleeding on the indicated days after injection. mSEAP production in the serum was determined by SEAP chemiluminescence assay. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-AHASEAP. mSEAP expression in the PBS-treated mice was below the detectable level.
Mentions: In order to evaluate the in vivo transgene expression levels induced by the Ad vectors carrying miRNA-targeted sequences, luciferase expression in the liver was examined 2 days following administration (Figure 6a). The luciferase expression levels in the liver induced by the Ad vectors carrying miRNA-targeted sequences, with the exceptions of Ad-E2A-122aT-L2 and Ad-E4-122aT-L2, were comparable to those by Ad-L2. Ad-E4-122aT-L2–mediated luciferase expression in the liver was 15-fold lower than that mediated by Ad-L2. The lower luciferase expression levels of Ad-E4-122aT-L2 in the liver were probably due to the significant suppression of E4 gene expression. Previous studies demonstrated that the E4 gene products, especially the E4 ORF3 gene product, enhanced the transcriptional activity of a CMV promoter.9,10,32

Bottom Line: These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells.The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences.Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a-mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan.

ABSTRACT
Leaky expression of adenovirus (Ad) genes occurs following transduction with a conventional replication-incompetent Ad vector, leading to an induction of cellular immunity against Ad proteins and Ad protein-induced toxicity, especially in the late phase following administration. To suppress the leaky expression of Ad genes, we developed novel Ad vectors by incorporating four tandem copies of sequences with perfect complementarity to miR-122a or miR-142-3p into the 3'-untranslated region (UTR) of the E2A, E4, or pIX gene, which were mainly expressed from the Ad vector genome after transduction. These Ad vectors easily grew to high titers comparable to those of a conventional Ad vector in conventional 293 cells. The leaky expression of these Ad genes in mouse organs was significantly suppressed by 2- to 100-fold, compared with a conventional Ad vector, by insertion of the miRNA-targeted sequences. Notably, the Ad vector carrying the miR-122a-targeted sequences into the 3'-UTR of the E4 gene expressed higher and longer-term transgene expression and more than 20-fold lower levels of all the Ad early and late genes examined in the liver than a conventional Ad vector. miR-122a-mediated suppression of the E4 gene expression in the liver significantly reduced the hepatotoxicity which an Ad vector causes via both adaptive and non-adaptive immune responses.

No MeSH data available.


Related in: MedlinePlus