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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-induced immune responses following intravenous administration of Ad vectors into mice. (a) Hexon-specific IFN-γ+ CD8+ T cells in the splenocytes. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Fifteen days after the injection, the splenocytes were harvested. The splenocytes were incubated with hexon peptide for 6 hours. (b) Infiltration of lymphocytes into the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, liver mononuclear cells were isolated and analyzed for cells expressing CD3, CD4, and CD8 by fluorocytometry. The data are expressed as the mean values ± SD (n = 6). N. S., not significant. (c) IFN-γ and chemokine mRNA levels in the liver after administration of Ad-L2 and Ad-E4-122aT-L2. C57BL/6 mice were treated with Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, IFN-γ and chemokine mRNA levels in the liver were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (n = 3–6). (d) Anti-Ad antibody levels in the serum following intravenous administration of Ad vectors. C57BL/6 mice were intravenously administered as described above. Anti-Ad antibody levels in the serum were determined by ELISA 14 days after administration. The data are expressed as the mean values ± SD (n = 6). (e) Ad-specific CTL-mediated lysis of the hepatocytes transduced with Ad vectors. C57BL/6 mice were intravenously administered Ad- at a dose of 1 × 1010 IFU/mouse. Ten days after the injection, the splenocytes were harvested and incubated with Ad- at an MOI of 10 for 4 days. Primary mouse hepatocytes were transduced with Ad-L2 or Ad-E4-122aT-L2 at an MOI of 10 for 24 hours and were incubated with the splenocytes at 37 °C. LDH levels in the medium were measured 4 hours after incubation. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-L2.
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fig4: Ad vector-induced immune responses following intravenous administration of Ad vectors into mice. (a) Hexon-specific IFN-γ+ CD8+ T cells in the splenocytes. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Fifteen days after the injection, the splenocytes were harvested. The splenocytes were incubated with hexon peptide for 6 hours. (b) Infiltration of lymphocytes into the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, liver mononuclear cells were isolated and analyzed for cells expressing CD3, CD4, and CD8 by fluorocytometry. The data are expressed as the mean values ± SD (n = 6). N. S., not significant. (c) IFN-γ and chemokine mRNA levels in the liver after administration of Ad-L2 and Ad-E4-122aT-L2. C57BL/6 mice were treated with Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, IFN-γ and chemokine mRNA levels in the liver were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (n = 3–6). (d) Anti-Ad antibody levels in the serum following intravenous administration of Ad vectors. C57BL/6 mice were intravenously administered as described above. Anti-Ad antibody levels in the serum were determined by ELISA 14 days after administration. The data are expressed as the mean values ± SD (n = 6). (e) Ad-specific CTL-mediated lysis of the hepatocytes transduced with Ad vectors. C57BL/6 mice were intravenously administered Ad- at a dose of 1 × 1010 IFU/mouse. Ten days after the injection, the splenocytes were harvested and incubated with Ad- at an MOI of 10 for 4 days. Primary mouse hepatocytes were transduced with Ad-L2 or Ad-E4-122aT-L2 at an MOI of 10 for 24 hours and were incubated with the splenocytes at 37 °C. LDH levels in the medium were measured 4 hours after incubation. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-L2.

Mentions: In order to determine whether suppression of the E4 gene expression by incorporation of the miR-122a–targeted sequences in the 3′-UTR of the E4 gene would lead to a low level of cellular immune responses against Ad protein, the numbers of CTLs against the hexon, which is one of the major capsid proteins and the dominant epitope of Ad vector,29 in the splenocytes were determined 15 days following administration by an intracellular cytokine staining assay. All the Ad vectors induced elevation in the numbers of hexon-specific CD8+ T cells producing IFN-γ, but these cell numbers were not significantly different between the Ad vectors examined (Figure 4a).


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-induced immune responses following intravenous administration of Ad vectors into mice. (a) Hexon-specific IFN-γ+ CD8+ T cells in the splenocytes. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Fifteen days after the injection, the splenocytes were harvested. The splenocytes were incubated with hexon peptide for 6 hours. (b) Infiltration of lymphocytes into the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, liver mononuclear cells were isolated and analyzed for cells expressing CD3, CD4, and CD8 by fluorocytometry. The data are expressed as the mean values ± SD (n = 6). N. S., not significant. (c) IFN-γ and chemokine mRNA levels in the liver after administration of Ad-L2 and Ad-E4-122aT-L2. C57BL/6 mice were treated with Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, IFN-γ and chemokine mRNA levels in the liver were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (n = 3–6). (d) Anti-Ad antibody levels in the serum following intravenous administration of Ad vectors. C57BL/6 mice were intravenously administered as described above. Anti-Ad antibody levels in the serum were determined by ELISA 14 days after administration. The data are expressed as the mean values ± SD (n = 6). (e) Ad-specific CTL-mediated lysis of the hepatocytes transduced with Ad vectors. C57BL/6 mice were intravenously administered Ad- at a dose of 1 × 1010 IFU/mouse. Ten days after the injection, the splenocytes were harvested and incubated with Ad- at an MOI of 10 for 4 days. Primary mouse hepatocytes were transduced with Ad-L2 or Ad-E4-122aT-L2 at an MOI of 10 for 24 hours and were incubated with the splenocytes at 37 °C. LDH levels in the medium were measured 4 hours after incubation. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-L2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362365&req=5

fig4: Ad vector-induced immune responses following intravenous administration of Ad vectors into mice. (a) Hexon-specific IFN-γ+ CD8+ T cells in the splenocytes. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Fifteen days after the injection, the splenocytes were harvested. The splenocytes were incubated with hexon peptide for 6 hours. (b) Infiltration of lymphocytes into the liver following Ad vector administration. C57BL/6 mice were intravenously administered Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, liver mononuclear cells were isolated and analyzed for cells expressing CD3, CD4, and CD8 by fluorocytometry. The data are expressed as the mean values ± SD (n = 6). N. S., not significant. (c) IFN-γ and chemokine mRNA levels in the liver after administration of Ad-L2 and Ad-E4-122aT-L2. C57BL/6 mice were treated with Ad vectors at 1 × 1010 IFU/mouse. Ten days after administration, IFN-γ and chemokine mRNA levels in the liver were determined by real-time RT-PCR. The data are expressed as the mean values ± SD (n = 3–6). (d) Anti-Ad antibody levels in the serum following intravenous administration of Ad vectors. C57BL/6 mice were intravenously administered as described above. Anti-Ad antibody levels in the serum were determined by ELISA 14 days after administration. The data are expressed as the mean values ± SD (n = 6). (e) Ad-specific CTL-mediated lysis of the hepatocytes transduced with Ad vectors. C57BL/6 mice were intravenously administered Ad- at a dose of 1 × 1010 IFU/mouse. Ten days after the injection, the splenocytes were harvested and incubated with Ad- at an MOI of 10 for 4 days. Primary mouse hepatocytes were transduced with Ad-L2 or Ad-E4-122aT-L2 at an MOI of 10 for 24 hours and were incubated with the splenocytes at 37 °C. LDH levels in the medium were measured 4 hours after incubation. The data are expressed as the mean values ± SD (n = 4). *P < 0.05 in comparison with Ad-L2.
Mentions: In order to determine whether suppression of the E4 gene expression by incorporation of the miR-122a–targeted sequences in the 3′-UTR of the E4 gene would lead to a low level of cellular immune responses against Ad protein, the numbers of CTLs against the hexon, which is one of the major capsid proteins and the dominant epitope of Ad vector,29 in the splenocytes were determined 15 days following administration by an intracellular cytokine staining assay. All the Ad vectors induced elevation in the numbers of hexon-specific CD8+ T cells producing IFN-γ, but these cell numbers were not significantly different between the Ad vectors examined (Figure 4a).

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