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  • br miR b and miR human genomic sequences were

    2022-07-26


    miR-99b and miR-485 human genomic sequences were incorporated into an ICOVIR15 genome under a CMV promoter next to the R-ITR. First, we tested the in vitro activity of these miR candidate-en-coding viruses. In line with previous results with AdwtE miR viruses,  DISCUSSION
    Oncolytic adenoviruses are therapeutics under clinical development. Nonetheless, although they display good safety profiles, their onco-lytic activity does not fully eliminate tumors,17 highlighting the
    www.moleculartherapy.org
    A
    expressionmiR/AdwtE) 2.0
    ELF4
    B
    expressionmockinfected) 2.0
    Protein(infected/ 1.0
    ELF4
    AdwtE
    AdwtE hTR
    MDM2
    KLF8
    AdwtE AdwtE miR miR AdwtE
    Mock AdwtE
    ELF4
    GAPDH
    MDM2
    GAPDH
    KLF8
    -TUBULIN 
    Figure 5. miR-99b and miR-485 Downregulate Genes Involved in Transcriptional Regulation
    (A) qPCR analysis of ELF4, MDM2, and KLF8 mRNA levels
    values are expressed relative to cellular GAPDH in each
    replicate. The dashed line represents mock-infected
    values. (B) Quantification of ELF4, MDM2, and KLF8
    AdwtE hTR. Representative western blot images are
    shown. Quantification of signal was normalized to cellular
    GAPDH for each replicate. The dashed line represents
    mock-infected values. (C) Validation of the miR-99b target
    at KLF8 30 UTR. HEK293T Midostaurin were co-transfected with a psiCHECK-2 reporter plasmid, containing wild-type
    expression plasmid (miRVec control, miRVec-99b, or
    miRVec-485). Renilla and luciferase activities were eval-
    uated 72 hr post-transfection. (D and E) Relative infective
    viral particle release in control PANC-1 cells (Lenticrv2)
    and three CRISPR/Cas9-modified PANC-1 clones for
    C
    D
    E
    activity control)
    ELF4cr
    KLF8cr
    Renilla/Fireflyluciferase (+miRVecmiR/+miRVec
    IFUrelease(Clone/Lenticrv2) 8
    IFUrelease
    KLF KLF8
    ELF KLF8
    ELF4 (D) and KLF8 (E). Cells were seeded in triplicate and
    tants were collected and titrated by viral infectious units.
    The dashed line represents PANC-1 Lenticrv2 cells
    at least three independent biological replicates. Signifi-
    cance was assessed by comparison to mock-infected
    cells using a one-sample t test and to infected cells using a
    two-tailed Mann-Whitney test. For (C), data are shown as
    mean ± SEM for at least three independent biological
    replicates. Significance was assessed by comparison to
    miRVec control-transfected cells using a one-sample t test. For (D), data are shown as mean ± SEM for at least five independent biological replicates. Significance was assessed by comparison to PANC-1 Lenticrv2 cells using a one-sample t test. *p < 0.05, **p < 0.01, ***p < 0.001.
    need for novel adenoviral designs with increased potency. miRNAs have been shown to play a role in the progression of the adenoviral cycle.9–11 However, it remains unclear how adenoviral replication is affected by the extensively deregulated miRNA profile of tumor cells. We hypothesized that, by expressing specific miRNAs, it would be feasible to re-establish the levels of key cellular genes relevant for pro-ductive viral infection, leading to improved adenoviral oncolysis.
    Adenoviruses have the potential of being armed with sequences of in-terest that provide additional functions. We performed an approach based on the generation of an adenoviral miRNA library encoding 243 human miRNAs, followed by a replication-based bioselection strategy that identified miR-99b and miR-485 as enhancers of the adenoviral activity in pancreatic cancer cells. A similar strategy has been previously used in the context of an in vivo alpha virus infection to identify interactions between the virus and host factors impacting viral replication.23 However, the designs of these approaches were