br AdwtE miR mRNAexpression AdwtEmiR

AdwtE miR-485 mRNAexpression(AdwtEmiR/AdwtE) 3
Hexon
Fiber
Hexon
Fiber
MIA PaCa-2
Figure 4. The Increase in AdwtE miR-99b and AdwtE miR-485 Infective Particles Is Facilitated by Higher Expression Levels of Viral Genes
C
expressionmiR/AdwtE) 6
Mock Ad wtE
miR
iR
AdwtE
m
AdwtE
HEXON
PENTON
FIBER
GAPDH  (D) protein levels in PANC-1 and MIA PaCa-2 491-72-5 infected (at 1 and 5 IFU/cell, respectively) with AdwtE, AdwtE miR-99b, or AdwtE miR-485. Representative western blot im-ages are shown. Quantification of signal was normalized to cellular GAPDH in each replicate. The dashed line repre-sents AdwtE values. Data are shown as mean ± SEM for at least four independent biological replicates. Significance was assessed by comparison to AdwtE-infected cells using a one-sample t test. *p < 0.05, **p < 0.01, ***p < 0.001.
Protein(AdwtE 2
Hexon Penton Fiber Hexon
Penton Fiber
MIA PaCa-2
MIA PaCa-2
Mock Ad wtE
iR iR
AdwtE m AdwtE m
HEXON
PENTON
FIBER
GAPDH 
ICOVIR15 miR-99b and ICOVIR15 miR-485 displayed a significant increase in infective par-ticles released from PANC-1 cells, and they had a 2-fold increase in cytotoxicity as compared to the parental virus ICOVIR15 (Figures 6A and 6B). Similar data were obtained from MIA PaCa-2 cells (Figure S7).
D
miR / AdwtE) expression
(AdwtE Protein 
MIA PaCa-2
iR iR
m AdwtE m
A
dwtE
GAPDH 
To evaluate the antitumor effects of ICOVIR15
-99b -485 miR viruses, mice with subcutaneous PANC-1
tumors were treated with a systemic dose of sa-
wtE miR wtE miR
Ad Ad
(vp)/animal. Treatment with ICOVIR15 miR vi-
GAPDH ruses strongly inhibited tumor growth that was
consistently higher than inhibition observed us-
ing the parental ICOVIR15 (Figure 6C). Interest-
ingly, ICOVIR15 miR viruses administered
miR-99b and miR-485 Confer a Superior In Vitro and In Vivo Activity to the Oncoselective Adenovirus ICOVIR15
We next evaluated whether the improved adenoviral fitness triggered by miR-99b and miR-485 translates into enhanced antitumor activity. To this end, we armed ICOVIR15 with the candidate miRNAs. ICOVIR15 is an Ad5 derivative oncolytic adenovirus that keeps intact all the virus functions, with modifications in the E1A and fiber genes. The E1A region contains a 24-bp deletion and incorporates E2F-responsive elements to redirect E1A transcription toward deregula-tion of the Rb/p16 pathway. The fiber contains an RGD motif inserted in the HI-loop region.22  at 2 1010 vp/animal displayed the same antitumoral activity as ICOVIR15 administered at the double dose (4 1010 vp/animal) (Fig-ure 6D). The presence of replicating active adenovirus in tumors was determined by analyzing E1A expression by qRT-PCR and by immu-nostaining at the end of the experiment. Tumors treated with ICOVIR15 miR viruses, and especially with ICOVIR15 miR-485, dis-played increased E1A mRNA levels (Figure 6E). Larger areas of adeno-virus replication were also observed in the tumors that were treated with ICOVIR15 miR viruses (Figure 6F). These data strongly suggest that miR-99b and miR-485 confer enhanced potency to the ICOVIR15 oncolytic adenovirus through increased adenoviral release.
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
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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