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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

Suppression of the leaky expression of Ad genes in culture cells and mouse organs by insertion of the miRNA-targeted sequences. (a) HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour and harvested at 12 hours after transduction. (b) Restoration of the leaky expression of Ad genes in HuH-7 cells by LNA-modified ASO complementary to miR-122a. HuH-7 cells were transfection with LNA-modified ASO complementary to miR-122a or an LNA control at 10 nmol/l. Twenty-four hours after transduction, HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour, and harvested at 12 hours after transduction. The Ad gene expression levels in the cells transduced with Ad vectors were determined by real-time RT-PCR. (c,d) C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Two days after administration, (c) the livers and (d) the spleens were harvested. The Ad gene expression levels in the cells and mice transduced with Ad vectors were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (a,b: n = 4; c,d: n = 5–6). *P < 0.05 in comparison with (a,c,d) Ad-L2 or the (b) LNA control.
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fig2: Suppression of the leaky expression of Ad genes in culture cells and mouse organs by insertion of the miRNA-targeted sequences. (a) HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour and harvested at 12 hours after transduction. (b) Restoration of the leaky expression of Ad genes in HuH-7 cells by LNA-modified ASO complementary to miR-122a. HuH-7 cells were transfection with LNA-modified ASO complementary to miR-122a or an LNA control at 10 nmol/l. Twenty-four hours after transduction, HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour, and harvested at 12 hours after transduction. The Ad gene expression levels in the cells transduced with Ad vectors were determined by real-time RT-PCR. (c,d) C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Two days after administration, (c) the livers and (d) the spleens were harvested. The Ad gene expression levels in the cells and mice transduced with Ad vectors were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (a,b: n = 4; c,d: n = 5–6). *P < 0.05 in comparison with (a,c,d) Ad-L2 or the (b) LNA control.

Mentions: In order to examine whether leaky expression of the Ad genes was suppressed by insertion of the miRNA-targeted sequences in an miRNA-dependent manner, HuH-7 cells, which highly express miR-122a,23 were transduced with the luciferase-expressing Ad vectors, and the Ad gene expression levels were determined 12 hours after transduction. All the Ad vectors examined exhibited similar levels of luciferase production in HuH-7 cells (data not shown). Ad-E2A-122aT-L2 mediated 2.2-fold lower E2A gene expression than did Ad-L2 (Figure 2a). Ad-E4-122aT-L2 and Ad-pIX-122aT-L2 exhibited 4- and 6.5-fold lower E4 and pIX gene expression levels, respectively, than did Ad-L2. Insertion of the miR-142-3p–targeted sequences in the 3′-UTR also reduced the expression levels of the E4 and pIX genes in spite of the undetectable levels of miR-142-3p expression in HuH-7 cells, probably due to non-specific suppression via the insertion of miRNA-targeted sequences; however, the levels of suppression of the E4 and pIX genes realized by insertion of the miR-142-3p–targeted sequences were significantly lower than those by insertion of the miR-122a–targeted sequences. A reduction in E4 gene expression was also found for Ad-pIX-122aT-L2. It was unclear why the E4 gene expression was reduced for Ad-pIX-122aT-L2. The expression levels of Ad genes other than those described above were not significantly reduced in HuH-7 cells. The E2A, E4, and pIX gene expressions were also significantly suppressed in K562 cells, which is a human chronic myelogenous leukemia cell line highly expressing miR-142-3p,24 by insertion of the miR-142-3p–targeted sequences in the 3′-UTR of these Ad genes (Supplementary Figure S1). Note that all the E4 ORFs can be detected by the primers for the E4 gene used in this study. The E4 mRNA levels in the graph represent the sum of each E4 ORF mRNA level.


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)

Suppression of the leaky expression of Ad genes in culture cells and mouse organs by insertion of the miRNA-targeted sequences. (a) HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour and harvested at 12 hours after transduction. (b) Restoration of the leaky expression of Ad genes in HuH-7 cells by LNA-modified ASO complementary to miR-122a. HuH-7 cells were transfection with LNA-modified ASO complementary to miR-122a or an LNA control at 10 nmol/l. Twenty-four hours after transduction, HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour, and harvested at 12 hours after transduction. The Ad gene expression levels in the cells transduced with Ad vectors were determined by real-time RT-PCR. (c,d) C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Two days after administration, (c) the livers and (d) the spleens were harvested. The Ad gene expression levels in the cells and mice transduced with Ad vectors were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (a,b: n = 4; c,d: n = 5–6). *P < 0.05 in comparison with (a,c,d) Ad-L2 or the (b) LNA control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: Suppression of the leaky expression of Ad genes in culture cells and mouse organs by insertion of the miRNA-targeted sequences. (a) HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour and harvested at 12 hours after transduction. (b) Restoration of the leaky expression of Ad genes in HuH-7 cells by LNA-modified ASO complementary to miR-122a. HuH-7 cells were transfection with LNA-modified ASO complementary to miR-122a or an LNA control at 10 nmol/l. Twenty-four hours after transduction, HuH-7 cells were transduced with Ad vectors at an MOI of 10 for 1 hour, and harvested at 12 hours after transduction. The Ad gene expression levels in the cells transduced with Ad vectors were determined by real-time RT-PCR. (c,d) C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Two days after administration, (c) the livers and (d) the spleens were harvested. The Ad gene expression levels in the cells and mice transduced with Ad vectors were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (a,b: n = 4; c,d: n = 5–6). *P < 0.05 in comparison with (a,c,d) Ad-L2 or the (b) LNA control.
Mentions: In order to examine whether leaky expression of the Ad genes was suppressed by insertion of the miRNA-targeted sequences in an miRNA-dependent manner, HuH-7 cells, which highly express miR-122a,23 were transduced with the luciferase-expressing Ad vectors, and the Ad gene expression levels were determined 12 hours after transduction. All the Ad vectors examined exhibited similar levels of luciferase production in HuH-7 cells (data not shown). Ad-E2A-122aT-L2 mediated 2.2-fold lower E2A gene expression than did Ad-L2 (Figure 2a). Ad-E4-122aT-L2 and Ad-pIX-122aT-L2 exhibited 4- and 6.5-fold lower E4 and pIX gene expression levels, respectively, than did Ad-L2. Insertion of the miR-142-3p–targeted sequences in the 3′-UTR also reduced the expression levels of the E4 and pIX genes in spite of the undetectable levels of miR-142-3p expression in HuH-7 cells, probably due to non-specific suppression via the insertion of miRNA-targeted sequences; however, the levels of suppression of the E4 and pIX genes realized by insertion of the miR-142-3p–targeted sequences were significantly lower than those by insertion of the miR-122a–targeted sequences. A reduction in E4 gene expression was also found for Ad-pIX-122aT-L2. It was unclear why the E4 gene expression was reduced for Ad-pIX-122aT-L2. The expression levels of Ad genes other than those described above were not significantly reduced in HuH-7 cells. The E2A, E4, and pIX gene expressions were also significantly suppressed in K562 cells, which is a human chronic myelogenous leukemia cell line highly expressing miR-142-3p,24 by insertion of the miR-142-3p–targeted sequences in the 3′-UTR of these Ad genes (Supplementary Figure S1). Note that all the E4 ORFs can be detected by the primers for the E4 gene used in this study. The E4 mRNA levels in the graph represent the sum of each E4 ORF mRNA level.

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