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  • br Correspondence Cristina Fillat Institut d Investigacions

    2021-03-03


    Correspondence: Cristina Fillat, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, Spain. E-mail: [email protected]
    www.moleculartherapy.org
    A
    AdwtE miRLib
    MIA PaCa-2
    L-ITR
    miRLib R-ITR
    E1Ap E1A MLP L1
    EGFP CMVp
    B
    R-ITR
    AdwtE
    hTR R-ITR
    AdwtE hTR
    C
    Mock
    AdwtE
    C
    miRNAexpression 10
    E
    AdwtE
    AdwtE hTR
    AdwtE
    AdwtE hTR 
    D
    Mock
    AdwtE
    C
    expression
    miRNA
    AdwtE hTR
    EGFPintensity(AdwtEmiR/AdwtE)
    MIA PaCa-2
    AdwtE hTR
    * EGFPintensity(AdwtEmiR/AdwtE) 6
    Figure 1. miR-99b and miR-485 Identified through a Bioselection Strategy as Enhancers of the Adenoviral Replication
    (A) Schematic representation of the miRNA adenoviral library bioselection procedure. 243 human miRNAs under the control of a CMV promoter were cloned into the adenoviral genome (between the E4 gene and R-ITR) to generate a library (AdwtE miRLib). The library was bioselected through a replication-based strategy in PANC-1 and MIA PaCa-2 cell lines (see the Materials and Methods for a detailed description). As viruses encoding miR-99b and miR-485 were bioselected in both cell lines, they were
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    Molecular Therapy
    engineered as oncolytic agents for therapeutics against different can-cer types, including PDAC.17,18 Upon adenoviral infection, cellular
    miRNA profiles suffer profound changes that influence viral replica-tion.10,11,19 For this reason, dysregulated miRNA profiles in cancer
    cells may generate an inadequate environment for adenoviral activity. Recently, Hodzic and coworkers11 identified a group of miRNAs that sensitize 3X FLAG Peptide to Ad5-induced cell death. However, several of these miRNAs triggered premature cell death that interfered with the adenoviral cycle, and only miR-26b was found to be an enhancer of Ad5 propagation. Intriguingly, and contrary to what was expected, miR-26b was highly expressed in PC-3 cells, and Ad5 infection only caused a slight reduction of its expression, thus confirming the complexity of miRNA-virus interactions.11
    Here we describe the identification of human miRNAs that favor adenoviral activity in pancreatic cancer, using a replication-based bio-selection strategy. We identified miR-99b and miR-485 as enhancers of the adenovirally mediated oncolysis in PDAC, whereby they facil-itate the formation of mature virions by means of negatively regu-lating the expression of specific transcription factors. Arming the ICOVIR15 oncolytic adenovirus with miR-99b or miR-485 resulted in new viruses that greatly improved the antitumor efficacy. We believe that this approach could be used to bypass the complexity of the host-virus interactions, as miRNA sequences are codified directly within the adenoviral genome, giving rise to a single thera-peutic agent.
    RESULTS
    In Vitro Bioselection of the miRNA Adenoviral Library AdwtE miRLib in PDAC Cell Lines
    To identify miRNAs that enhance adenoviral activity, we generated a miRNA adenoviral library (AdwtE miRLib). The AdwtE miRLib consisted of a pool of adenoviruses coding for up to 243 different hu-man primary miRNA (pri-miRNA) genomic sequences under the control of the cytomegalovirus (CMV) promoter. These sequences were introduced next to the right inverted terminal repeat (R-ITR) of a wild-type adenovirus 5 expressing the EGFP gene under the con-trol of the major later promoter (MLP) (AdwtE) (Figure 1A). The AdwtE miRLib was subjected to 20 rounds of bioselection in PANC-1 and MIA PaCa-2 PDAC cell lines. After this, adenoviral clones were isolated by the plaque assay, and the miRNAs present were identified by Sanger sequencing (Figure 1A). Although several miRNAs were detected to be enriched following PANC-1 and MIA PaCa-2 bioselection, adenoviruses encoding miR-99b and miR-485 were enriched in the two cell lines (Figure 1A; Table S1). In MIA
    Next, we took advantage of the miRNome profile of 11 PDAC and 3 normal pancreatic tissues, performed by next-generation sequencing (NGS) (Illumina Genome Analyzer), in the context of a previous study focused on the identification of miRNA biomarkers for early disease detection.15 Analysis of miR-99b and miR-485 in this NGS dataset revealed that both miRNAs’ 30 arms (3p), miR-99b-3p and miR-485-3p, were significantly downregulated in PDAC tumors (Fig-ure S1A). Using The Cancer Genome Atlas (TCGA) dataset (https:// tcga-data.nci.nih.gov/docs/publications/tcga/?), we classified patients into groups with high or low expression for each miRNA, and we evaluated relative survival. Interestingly, low miRNA expression of both miRNAs correlated with poor clinical outcome (Figures S1B and S1C). These results suggest that miR-99b and miR-485 may play a role in PDAC tumorigenesis.