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. 2008 Nov;82(22):11009-15.
doi: 10.1128/JVI.01608-08. Epub 2008 Sep 17.

Generation of a conditionally replicating adenovirus based on targeted destruction of E1A mRNA by a cell type-specific MicroRNA

Erkko Ylösmäki  1 , Tanja Hakkarainen, Akseli Hemminki, Tapio Visakorpi, Raul Andino, Kalle Saksela
Affiliations

Generation of a conditionally replicating adenovirus based on targeted destruction of E1A mRNA by a cell type-specific MicroRNA

Erkko Ylösmäki et al. J Virol. 2008 Nov.

Abstract

MicroRNAs have emerged as important players in tissue-specific mammalian gene regulation and have also been exploited in experimental targeting of gene expression. We have constructed a recombinant adenovirus that contains sequences complementary to the liver-specific microRNA 122 (miR122) in the 3' untranslated region of the E1A gene. In Huh7 cells, which resemble normal hepatocytes in expressing high levels of miR122, this feature resulted in strongly reduced levels of E1A mRNA and protein. This property allowed us to generate a novel recombinant adenovirus that was severely attenuated in cells of hepatic origin but replicated normally in other cells. This strategy may be useful in circumventing liver toxicity associated with the systemic delivery of oncolytic adenoviruses. These data provide the first example of exploiting differential microRNA expression patterns to alter the natural tropism of a DNA virus. In addition, these results suggest that other microRNAs expressed in a tissue- or transformation-specific manner may also be used for the targeting of adenoviral replication and that the same principle may be applied to other viruses that have shown promise as oncolytic or gene delivery platforms.

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Figures

FIG. 1.
FIG. 1.
Characterization of miR122 target sequences as cell type-specific suppressors of reporter gene expression. (A) Schematic illustration of the FFluc and Renilla luciferase constructs used and sequence of the miR122 target site inserted in different copy numbers into FFluc constructs. (B) Effects of the indicated combinations of miR122 target sequences in the 5′ and/or 3′ UTR of FFluc constructs on the ratio of FFluc versus Renilla luciferase activity in cotransfected A549 (striped bars) or Huh7 (black bars) cells. The ratio for cells transfected with an unmodified FFluc construct (control) was set to 100%, and the other values for the same cell type are expressed relative to this reference. The numbers of miR122 target sequences and their positioning are indicated under the corresponding bars. (C) Suppression of the negative effect of miR122 target sites by an miR122 inhibitor. Huh7 cells were transfected with a Renilla luciferase construct and an unmodified or miR122 target site-containing FFluc construct (3x3′, corresponding to the 3′ three-target-site construct indicated in panel B), together with (+) or without (−) a synthetic antagomir for miR122. The FFluc/Renilla luciferase activity ratio obtained with unmodified FFluc in the absence of the antagomir was set to 100%, and the other values are expressed relative to this reference.
FIG. 2.
FIG. 2.
Inhibitory effects of engineered miR122 target sites on E1A protein (A) and mRNA (B) expression in infected liver cells. (A) Huh7 and A549 cells were infected with Ad5/3-Δ24 (Ad5/3) or Ad5/3-122 (122) at a MOI of 0.05 or left uninfected (−). Three days later, the cells were harvested and analyzed by Western blotting for E1A protein expression (top panels) and total immunoreactive adenoviral protein expression (bottom panels). α-E1A, anti-E1A antibody; α-Ad, antiadenovirus antibody. (B) Real-time RT-PCR was used to quantify E1A mRNA from total RNA extracted from Huh7 cells infected with Ad5/3-Δ24 or Ad5/3-122 at a MOI of 0.05. The relative expression of E1A mRNA at 1, 2, and 3 days (d) postinfection with these two viruses was determined based on an E1A mRNA dilution standard and the parallel quantification of GAPDH mRNA from the same specimens.
FIG. 3.
FIG. 3.
Abolishment of total adenoviral protein expression in liver cells by miR122 target sites. (A) Placing of an out-of-frame translational initiation site upstream of the E1A open reading frame results in a cell type-independent reduction in E1A translation without reducing overall adenoviral protein expression. Huh7 and A549 cells were infected with Ad5/3-Δ24 (Ad5/3) or Ad5/3K (AdK) at a MOI of 0.05. Cells were analyzed 3 days postinfection for adenoviral protein expression as described in the legend to Fig. 2. α-E1A, anti-E1A antibody; α-Ad, antiadenovirus antibody. (B) Liver-specific loss of detectable adenoviral protein expression can be achieved by the introduction of miR122 target sites into the Ad5/3K virus background. Huh7 and A549 cells were infected with Ad5/3K or its derivative Ad5/3K-122 (K122) at a MOI of 0.05 and analyzed 3 days (3d) later for adenoviral protein expression as described in the legend to Fig. 2.
FIG. 4.
FIG. 4.
Cell type- and miR122-dependent CPE of an adenovirus carrying miR122 target sites. (A) The indicated cell lines were infected with Ad5/3K (AdK) or Ad5/3K-122 (K122) at a MOI of 0.05. Cells were photographed 4 days later (or 6 days later in the case of Huh7 cells) to document the appearance of a CPE. Uninfected control cell cultures are shown for comparison. (B) Huh7 cells infected with Ad5/3K-122 as described in panel A following pretreatment with the miRIDIAN miR122 inhibitor. +, present; −, absent.
FIG. 5.
FIG. 5.
Effect of miR122 target sites on viral replication in liver-derived and control cells. Huh7 and A549 cells were infected with Ad5/3K (AdK) or Ad5/3K-122 (K122) at a MOI of 0.05, and the titers of cell-associated infectious virus at three (A549) or five (Huh7) days postinfection were determined by a standard 50% tissue culture infective dose limiting dilution method. Values are expressed as PFU per milliliter.
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