Ad5mir122 shows reduced E1A activity in primary hepatocytes.

<p>Primary human hepatocytes were infected with either Ad5mir122luc or Ad5WTluc at 1 vp/cell. Luciferase levels are shown 3 days post infection (n = 3) as relative light units per mg total protein. Error bars represent standard deviation.</p

MicroRNA mediated knockdown of both E1A mRNA and protein measured by RT-QPCR and western blot after intravenous injection of 2×10¹⁰ vp of Ad5mir122.

<p>A) RT QPCR for the 13S E1A mRNA transcript in the livers of mice 48 hrs after intravenous injection with 2×10¹⁰ vp of Ad5mir122, Ad5WT, Ad5Luc or PBS. Ad5mir122 shows significantly reduced E1A mRNA during liver infection when compared to Ad5WT (N = 3). Statistical analysis was performed using a two tailed student T-Test (*P = <0.05). B) Western blot to confirm that all E1A proteins variants are knocked down. Each lane represents protein extracted from an individual mouse. Control lanes containing liver from mice treated with either an E1A deleted Ad5Luc vector or PBS show no E1A signal. Ad5WT treatment shows 3 clearly defined bands corresponding to proteins produced from the 13S (36 kDa), 12S (26 kDa) and a smaller fainter band which may represent the 11S or the 10S E1A transcript product. Treatment with Admir122 shows significant knockdown in E1A protein levels for all splice variants. The blot was exposed for 1, 5 and 10 minutes with the 5 minute exposure presented here. Molecular weights were calculated against a dual colour molecular weight ladder (Bio-Rad).</p

Pharmacodynamics led dose escalation study to determine the optimal treatment dose with Ad5mir122 and Ad5WT in tumour bearing mice.

<p>A) Serum Alanine Transaminase (ALT) levels from mice receiving Ad5mir122, Ad5WT or PBS. ALT values for each group are shown at days 2, 5 and 8 (n = 3) after the first injection. Data is presented as ALT units per litre using the equation in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#s2" target="_blank">materials and methods</a>. B) Luciferase imaging 2 days after the first injection of Ad5mir122 (left panel) or Ad5WT (right panel). C) Luciferase imaging eight days after the first injection. Mice which received a single injection of Ad5WT are shown in the right panel and mice receiving three injections of Ad5mir122 are shown in the left panel. Images are all presented on the same scale. D) Dosage and treatment schedule for Ad5mir122 and Ad5WT. Viral doses are indicated above each line and include 10% of a Luciferase reporter virus (Ad5mir122luc). All mice were treated with bisphosphonate liposomes at day -1.</p

The level and activity of mature mir122 <i>in vivo</i> is unaffected by Ad5mir122.

<p>Mice were injected with 2×10¹⁰ vp of either Ad5mir122, Ad5WT, an E1A deleted Ad5luc vector or PBS. Quantification of mir122 mature RNA levels was performed using a Taqman microRNA assay specific for mir122. CT values were corrected against the levels of the microRNA, let7a, as a reference gene using the method published by Pfaffl M <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016152#pone.0016152-Pfaffl1" target="_blank">[25]</a>. A) RT-QPCR for mir122 showing nine superimposed amplification curves (from three mice) in each treatment group, before correction against let7a. Samples were reverse transcribed using equal amounts of total RNA (5 ng) and RT-PCR was performed using equal amounts of cDNA. CT values shown here represent the average of the reactions from three mice plus or minus standard deviation. B) The total number of mRNA changes recorded by genome wide mRNA profiling of extracted murine hepatic RNA. Positive signals are those in which the median mRNA level changed by ≧2-fold from all mice in each group in comparison to median mRNA level in mice treated with PBS (n = 3 for all groups). The total number of genes altered is calculated using the average of the three independent replicates and therefore no error bars are shown. C) Western blot analysis of the mir122 regulated protein Aldolase A in mice treated as above. Liver protein extracts were subjected to a BCA protein assay and equal loading was confirmed by Ponceau stain (data not shown).</p

Ad5mir122 kills mir122 negative cells with Ad5WT potency.

<p>Comparison of Ad5mir122 and Ad5WT in cancer cell lines incubated at a multiplicity of infection of 100 viral particles per cell. The percentage cell survival is shown 6 days post infection (N = 5) using an MTS cell survival assay. Statistical analysis was performed using one way ANOVA (* = p<0.05, NS  =  not significant).</p