Effects of microRNA binding sites on expression of CMV promoter driven luciferase plasmids <i>in vitro</i>.

<p>Cells were seeded in triplicate in 12 well plates. After 24 h 0.5 µg of plasmid DNA (containing 0 (black), 4 (light grey) or 8 (white) sense mir-122 binding sites, or 4 antisense binding sites (dark grey)) was mixed with 2.5 ul DOTAP (Roche) reagent. 24 h following transfection cells were lysed and relative luminescence was measured using 25 µl cell lysate. N = 3, Error bars +/−standard deviation and data is shown as RLU/µg cell protein, determined by BCA assay. (** P<0.005).</p

Regulation of E1A expression.

<p>(A) Structures of viruses engineered and used in this study. (i) Ad5-WT (ii) Four tandem repeats of binding sites for mir-122 were inserted into the 3′ UTR of E1A. (iii) Luciferase coding sequence was inserted into Ad5 to generate a fusion with the E1A coding sequence. (iv) Four tandem repeats of binding sites for mir-122 were inserted into 3′ UTR of luciferase in the virus shown in iii. (B) A549 cells were seeded at 5×10⁴ cells per well and transfected with pre-mir122 (Ambion) or pre-mir negative control (Ambion). Immediately following transfection Ad-E1A-Luc-mir122 was added at 10 vp/cell in 450 µl DMEM media (10% FCS). 18 h later, 30 pmol/well of pre-cursor mir122 and negative control precursor microRNAs were added to each well in addition to the 500 µl described above. Luciferase readings were performed at 24 h (p = <0.0005). (C–E) Time course of luciferase expression of Ad5-E1A-Luc (solid squares) and Ad5-E1A-Luc-mir (open squares) in mir-122-negative OVCAR3 (C) and A549 (D) cells, and in mir-122-positive Huh7 cells (E) in vitro. (*** P<0.0005).</p

Effects of including binding sites for microRNA122a on expression of plasmids in vivo following hydrodynamic delivery to mice.

<p>(A) Imaging luminescence (8 h from mice administered pCMV-Luc not containing (left panel) and containing (right panel) four binding sites for mir-122 (plasmids pCMV-Luc and pCMV-Luc-mir in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#ppat-1000440-g001" target="_blank">Figure 1</a>).The animal on the right side of all images is a control treated with PBS, but mock injected with luciferin, used to provide a background reading. (B) Imaging luminescence (8 h from mice administered pE1A-Luc fusion constructs not containing (left panel) and containing (right panel) four binding sites for microRNA122a (plasmids pE1A-Luc and pE1A-luc-mir in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#ppat-1000440-g001" target="_blank">Figure 1</a>). The animal on the right is an untreated control. The two images in A are directly comparable with each other, as also the two images in B; however scaling is different between (A) and (B) in order to accommodate substantially different signal intensities from these plasmids. (C) Time course of luciferase expression from CMV promoter-driven and E1A promoter driven constructs shown in A and B. Black = 8 h, Grey = 24 h, White = 48 h. n = 4 throughout (except pCMV-Luc-mir122 where n = 3), error bars show standard deviation.</p

Plasmid construction.

<p>pCIK-Lux (referred to as pCMV-Luc) was cleaved with Not1 and concatamers of mir-122 binding sites (4 or 8 sense, or 4 antisense; the sequence of the 4 sense insert is shown at the top of the figure) inserted into the luciferase 3′UTR. Both pCMV-Luc and the version containing 4 microRNA sites (pCMV-Luc-mir) were modified with the C terminal half of E1A expression cassette, isolated from pAd5WT (Ad5 wild-type) by PCR. Both resulting constructs were then cloned into pAd5Kpn1, which contains the E1A promoter and coding sequence, to produce E1A promoter regulated E1A-luciferase fusion constructs termed pE1A-Luc and pE1A-Luc-mir.</p

Assessment of hepatotoxicity of wild-type Ad5 modified with microRNA binding sites.

<p>(A) Measurement of serum ALT (black bars) and AST (grey bars) 72 h following intravenous administration of 5×10¹⁰ viral particles of wild-type Ad5 and Ad5-mir122. Analysis was performed as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#s4" target="_blank">Methods</a> section. (B) Adenovirus genomes in murine liver were measured by real time PCR 72 h following intravenous administration of 5×10¹⁰ viral particles of wild-type Ad5 WT and Ad5-mir122, as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000440#s4" target="_blank">Methods</a> section. (C) Assessment of liver histology. The left liver lobe from each mouse was immersed in 10% buffered formalin overnight at room temperature, embedded in wax and sectioned using a vibratome. Sections were stained with haematoxylin and eosin and analysed by light microscopy at ×40 magnification. Mice were treated with PBS, non-replicating E1, E3-deleted Ad5 expressing GFP (Ad5-GFP), wild-type Ad5, or wild-type Ad5 modified to contain 4 mir-122 binding sites, as indicated. (*** P<0.0005).</p