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  • br Together the data suggest that


    Together, the data suggest that miR-99b and miR-485 may be poten-tial regulators of the host response to adenoviral activity. Thus, we considered AdwtE miR-99b and AdwtE miR-485 to be good candi-dates for further characterization.
    Viruses Encoding miR-99b or miR-485 Have Increased Activity in PDAC
    Adenoviral clones encoding either miR-99b or miR-485 were selected, expanded, and purified to generate AdwtE miR-99b and AdwtE miR-485. Two control viruses were also generated: the parental virus (AdwtE) and a similar-sized virus of AdwtE miR en-coding the human telomerase RNA (hTR) (AdwtE hTR) (Figure 1B).
    To verify that candidate miRNAs were expressed from AdwtE miR-99b and AdwtE miR-485 viruses, PANC-1 KN 93 were infected with the corresponding viruses; after 24 hr, specific expression of the cor-responding miRNAs was analyzed. We determined that both the 30 arm (3p) and the 50 arm (5p) of the miRNA chains were overex-pressed, indicating that candidate miRNAs were correctly processed from the viral genome (Figures 1C and 1D).
    Next, we examined AdwtE miR-99b and AdwtE miR-485 viral activ-ities in PANC-1 and MIA PaCa-2 cells in comparison to the control viruses (AdwtE and AdwtE hTR). For this, cells were exposed to three
    chosen for further validation. (B) Schematic representation of the candidate viruses, AdwtE miR-99b and AdwtE miR-485, and the control viruses, AdwtE and AdwtE hTR. (C and D) Expression of miR-99b (5p and 3p) (C) and miR-485 (5p and 3p) (D) in PANC-1 cells at 24 hr PI with mock or 10 IFU/cell AdwtE, AdwtE miR-99b, or AdwtE miR-485. (E and F) AdwtE miR-99b and AdwtE miR-485 activity. PANC-1 (E) and MIA PaCa-2 (F) cells were infected (at 1 IFU/cell or 5 IFU/cell, respectively) with AdwtE, AdwtE miR-99b, AdwtE miR-485, or AdwtE hTR. At 48 hr PI, virus-containing supernatants were collected and used to infect new cells (10% for PANC-1 and 50% for MIA PaCa-2). The procedure was repeated for three sequential steps. EGFP expression was analyzed and quantified at the end of each infection. The dashed line represents AdwtE values. Representative images are shown. Original magnification 4 ; scale bar, 200 mm. Data are shown as mean ± SEM for at least five independent biological replicates. Sig-nificance was assessed using a two-tailed Mann-Whitney test (C and D) and by comparison to AdwtE-infected cells using a one-sample t test and to AdwtE hTR infected cells using a two-tailed Mann-Whitney test (E and F). *p < 0.05, **p < 0.01.
    Intravenous Sample
    subcutaneous injection co-administration collection
    consecutive rounds of infection, and EGFP fluorescence levels were evaluated at the end of each infection. Cells infected with the two AdwtE miR viruses displayed higher EGFP levels after the first round, which increased throughout the infection rounds, reaching levels 10-fold higher than AdwtE in PANC-1 cells and 4-fold higher than AdwtE in MIA PaCa-2 cells (Figures 1E and 1F). Similar results were obtained in CP15-Luc, NP-18, Capan-2, HPAF-II, and Hs766T PDAC cells (Figure S2A).
    IFU/ngprotein (AdwtEmiR/AdwtE) 3 ** IFU/ngprotein(AdwtEmiR/AdwtE) 20
    dwtE R
    AdwtE mi
    MIA PaCa-2
    ** IFU/ngprotein(AdwtEmiR/AdwtE)
    IFU/ngprotein (AdwtEmiR/AdwtE)
    Ad iR
    AdwtE m
    Figure 2. AdwtE miR-99b and AdwtE miR-485 Viruses Have Viral Activity Superior to the Parental Virus AdwtE in an In Vivo Context
    (A) Scheme of the in vivo competition assay. Mice bearing subcutaneous PANC-1
    and MIA PaCa-2 tumors were intravenously administered a 1:1 mixture of
    AdwtE:AdwtE miR-99b or AdwtE:AdwtE miR-485 (2 1010 vp/animal). Tumors were recovered at 12 days PI and the infective viral particles of each virus were
    To assess if the enhanced in vitro activity of AdwtE miR viruses could also be recapitulated in vivo, we performed an in vivo compe-tition assay. Animals with subcutaneous PANC-1 or MIA PaCa-2 tumors in the lateral flanks received a mixture of 1:1 AdwtE:AdwtE miR-99b or AdwtE:AdwtE miR-485 through the lateral tail vein. Af-ter 12 days, the amount of infective particles of each virus present in the tumors was quantified (Figure 2A). Adenoviruses encoding either of the miRNA candidates were more abundant in both PANC-1 and MIA PaCa-2 tumors (Figures 2B and 2C). KN 93 In PANC-1 tumors, AdwtE miR-485 was up to 10-fold superior to AdwtE, while AdwtE miR-99b replicated only 2-fold more than AdwtE (Figure 2B). These results coincide with the bioselection approach in which AdwtE miR-485 was the most represented clone in PANC-1 cells (Table S1). In MIA PaCa-2 tumors, miR-99b and miR-485 similarly improved AdwtE fitness by 4-fold (Figure 2C). These results suggest that miR-99b- and miR-485-encoding viruses may trigger a more potent antitumoral activity than the parental un-modified virus.