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. 2015 Mar 20;6(8):6179-90.
doi: 10.18632/oncotarget.3350.

Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells

Xavier Bofill-De Ros  1   2 , Eneko Villanueva  1 , Cristina Fillat  1   2
Affiliations

Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells

Xavier Bofill-De Ros et al. Oncotarget. .

Abstract

Tissue-specific detargeting by miRNAs has been demonstrated to be a potent strategy to restrict adenoviral replication to cancer cells. These studies have generated adenoviruses with miRNA target sites placed in the 3'UTR of early gene products. In this work, we have studied the feasibility of providing tissue-specific selectivity to replication-competent adenoviruses through the regulation of the late structural protein fiber (L5 gene). We have engineered a 3'UTR containing eight miR-148a binding sites downstream the L5 coding sequence (Ad-L5-8miR148aT). We present in vitro and in vivo evidences of Ad-L5-8miR148aT miRNA-dependent regulation. In vitro data show that at 72 hours post-infection miR-148a-regulation impaired fiber expression leading to a 70% reduction of viral release. The application of seven consecutive rounds of infection in miR-148a cells resulted in 10.000-fold reduction of viral genomes released. In vivo, liver production of infective viral particles was highly impaired, similarly to that triggered by an adenovirus with miRNA target sites regulating the early E1A gene. Noticeably, mice treated with Ad-L5-8miR148aT showed an attenuation of adenoviral-induced hepatotoxicity but retained full lytic activity in cancer cells and exhibited robust antitumoral responses in patient-derived xenografts. Thus, miRNA-control of late proteins constitutes a novel strategy to provide selectivity to adenoviruses.

Keywords: L5 gene; fiber protein; miRNAs; oncolytic adenovirus; pancreatic cancer.

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Figures

Figure 1
Figure 1. miR-148a regulates fiber expression from cells infected with Ad-L5-8miR148aT
(A). Scheme of miRNA target sites engineered in the L5 viral gene. B, C. (Upper panels) Representative Western blots of E1A and Fiber from (B) MIA PaCa-2 miR-148a and MIA PaCa-2 miR-SC or (C) PANC-1 and RWP-1 cells infected with 50 vp/cell of Ad-wt and Ad-L5-8miR148aT for 24 and 72h. (Lower panels) Histograms representing the densitometric analysis of Fiber and E1A signals, normalized against GAPDH and expressed as relative to Fiber and E1A of Ad-wt infected cultures. Data is shown as the mean ± SEM of four independent experiments.
Figure 2
Figure 2. miR-148a regulates viral release from cells infected with Ad-L5-8miR148aT
A, B. Quantification of viral production from supernatants obtained from cells infected with 50 vp/cell of Ad-wt and Ad-L5-8miR148aT at 72h post-infection by (A) hexon immunostaining, (B) qPCR. Data is shown as the mean ± SEM of five (A) and four (B) independent experiments. ** p<0.01 and *** p<0.001. (C). Quantification of viral production in supernatant (extracellular) and pellets (intracellular) of cells infected with 50 vp/cell of Ad-wt and Ad-L5-8miR148aT at 48h post-infection by qPCR. Data is shown as the mean ± SEM of three independent experiments. * p<0.05. (D). Quantification of viral production in supernatant of cells infected with Ad-wt, Ad-E1A-8miR148aT and Ad-L5-8miR148aT upon several passages by qPCR. Data is shown in arbitrary units (AU) as the mean ± SEM of four independent experiments, calculated as the 2ΔCt between the miRNA-targeted viruses and Ad-wt. Differences between slopes were analyzed using F-test for nonlinear models *** p<0.001.
Figure 3
Figure 3. Fiber expression is directly or indirectly regulated, in miR-148a positive tissues, following Ad-L5-8miR148aT or Ad-E1A-8miR148aT systemic delivery
(A). Representative Western blots of Fiber in liver extracts from wild type mice intravenously injected with 2×1010 vp/mice of Ad-wt, Ad-E1A-8miR148aT and Ad-L5-8miR148aT. Quantification of Fiber signal normalized to GAPDH expression (n=10). Values are expressed relative to Fiber content from Ad-wt treated mice. * and ** denote p<0.05 and p<0.01, respectively. (B). Relative expression of E1A, Hexon and Fiber compared to Ad-wt assessed by RT-qPCR in liver, pancreas and kidneys (n=10/treatment). * and ** denote p<0.05 and p<0.01, respectively.
Figure 4
Figure 4. Ad-L5-8miR148aT replication is attenuated in mice liver and displays reduced hepatotoxicity following systemic delivery
A viral dose of 2×1010vp of Ad-wt, Ad-E1A-8miR148aT or Ad-L5-8miR148aT was intravenously delivered to wild-type mice C57BL/6 mice (n=10). Four days later liver and blood samples were collected. (A). Relative viral replication compared to Ad-wt assessed by genomic qPCR of the L3 gene in liver, pancreas and kidneys (n=10/treatment). * p<0.05. (B). Viral production from livers of mice treated with Ad-wt, Ad-E1A-8miR148aT and Ad-L5-8miR148aT assessed by hexon immunostaining (n=10/treatment). ** p<0.01. (C). Assessment of hepatotoxicity by the determination of AST, ALT and total bilirubin in the serum. Dashed lines correspond to the reference values for C57BL/6 mice. *, ** and *** denote p<0.05, p<0.01 and p<0.001, respectively.
Figure 5
Figure 5. Ad-L5-8miR148aT shows oncolytic potency in vitro and strong antitumoral activity in RWP-1 xenografts and PDX models
(A). Cytotoxicity assays in the indicated cell lines. Half growth inhibitory concentration (IC50) was calculated for each cell line from dose-response curves. Data is shown as the mean ± SEM of four independent experiments. (B). Cytotoxic effects of Ad-wt, Ad-E1A-8miR148aT and Ad-L5-8miR148aT supernatants obtained after 7 consecutives passages of amplification in MIA PaCa-2 miR-148a and MIA PaCa-2 miR-SC cells. Representative image obtained by methylene blue staining of the culture. (C). RT-qPCR expression of miR-148a from non-tumor human pancreas (n=11) and from RWP-1 xenograft (n=4) and CP13 (n=4), CP15 (n=4) PDX. (D). Follow-up of tumor volumes. RWP-1 xenografts were treated intratumorally with a single injection of 5×1010vp/tumor Ad-L5-8miR148aT (n=8), Ad-E1A-8miR148aT (n=8) and Ad-wt (n=8) or with saline (n=8). ** p<0.01. E. Follow-up of tumor volumes. CP13 PDX were treated intratumorally with a single injection of 5×1010vp/tumor Ad-L5-8miR148aT (n=10) or with saline (n=10). *** p<0.001. F. Follow-up of tumor volumes. CP15 PDX were treated intratumorally (n=12) or intravenously (n=10) with a single injection of 5×1010vp/tumor Ad-L5-8miR148aT or with saline (n=10). *** p<0.001.
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