br Quantification of Viral Genomes and
Quantification of Viral Genomes and Infective Viral Particles
Cellular DNA and viral DNA were obtained from cellular extracts us-ing UltraClean BloodSpin DNA Isolation Kit. Adenoviral genomes were analyzed by qPCR on a ViiA7 System (Applied Biosystems), us-ing SYBER Green I Master plus mix (Roche) and the primer set 4 (Table S4). The adenoviral copy number was quantified with a stan-dard curve of adenovirus DNA dilutions (using 102–107 copies) in a background of genomic DNA. Viral DNA was expressed relative to the cellular DNA content, determined by qPCR using albumin 12 intron primers (primer set 5; Table S4) and a standard curve of genomic DNA and corrected by the number of Methylpiperidino pyrazole per well. Infective viral particles were determined by viral DNA quantification in A549 at 4 hr PI.
Total RNA was obtained from cell cultures using miRNeasy Mini RNA Extraction Kit (QIAGEN). About 10 ng total RNA was reverse transcribed using Applied Biosystems TaqMan MicroRNA Reverse Transcription Kit and stem-loop primers (Thermo Fisher Scientific), according to the manufacturers’ instructions. Applied Biosystems TaqMan Universal PCR Master Mix No AmpErase UNG, qPCR probes (Thermo Fisher Scientific), and 1.5 mL RT reaction were used for the qPCR amplification reaction, performed as indicated by the manufacturer. Expression data were normalized to small nucleolar RNA U6 expression (RNU6B). The following stem-loop primers and qPCR probes were purchased from Applied Biosystems TaqMan MicroRNA Assay (Thermo Fisher Scientific): RNU6B (001093), hsa-miR-99b* (002196), hsa-miR-99b (000436), hsa-miR-485-3p (001277), and hsa-miR-485-5p (001036). Reactions were per-formed on a ViiA7 System.
In Vivo Competition Assay
Subcutaneous tumors were generated in 8-week-old male athymic nude Foxn 1nu nu/nu mice (ENVIGO) by injecting 2 106 PANC-1 or MIA PaCa-2 cells embedded in a ratio 1:1 of Matrigel (BD Biosciences) into each flank. Tumors were measured twice per week, and their volumes were calculated using the formula V = D d2 pO6. Mice were randomly assigned to different groups of treatment. Once tumor volume reached a median of 200 mm3, mice were intravenously administered a 1:1 mixture of AdwtE:AdwtE miR-99b or AdwtE:AdwtE miR-485 (2 1010 vp/ animal). Animals were sacrificed 12 days after virus administration and tumors were collected. To determine the number of infective par-ticles of each virus, tumors were mechanically homogenized in PBS 1 , followed by three freeze-thaw cycles. Cell debris was eliminated by centrifugation, and a fraction of the supernatant was titrated (as above) using specific primer sets for each virus (Table S2). The results were corrected by the sample protein content, as determined by the BCA Protein Assay Kit (Thermo Fisher Scientific). Animal proced-ures met the guidelines of European Community Directive 86/609/ EEC and were approved by the Local Ethical Committee.
Antitumoral Efficacy Study
PANC-1 subcutaneous tumors were established as described above. Mice were randomly assigned to different groups of treatment, and they were intravenously administered with a single dose of saline so-lution or virus (2 1010 or 4 1010 vp/animal) when tumor volumes reached a median of 100 mm3. Tumors were measured twice per week. Animals were euthanized 40 days after virus administration and tumors were collected.
Experimental data are represented by the mean ± SEM of at least three independent experiments. Statistical analysis was performed on GraphPad Prism version (v.)6.01. Statistical differences were eval-uated using a 2-tailed non-parametric Mann-Whitney test or a one-sample t test; p < 0.05 was taken as the level of significance. The in vivo tumor growth statistical analysis was evaluated using R v.2.14.1 soft-
ware with a linear mixed-effect model using the Ime4 package. Statis-tical differences were evaluated using a multiple comparison of means by Tukey contrasts; p < 0.05 was taken as the level of significance.
Supplemental Information includes eight figures, five tables, and Sup-plemental Materials and Methods and can be found with this article online at https://doi.org/10.1016/j.ymthe.2018.09.016.
M.R.-R. designed and performed the experiments and contributed to manuscript writing. G.R. contributed to the CRISPR-Cas9 experi-ments and cytotoxicity studies. M.A.M. contributed to target valida-tion and nanoparticle experiments. M.G. provided miRNA NGS data of PDAC and healthy tissue. R.A. provided some reagents and exper-tise and contributed to manuscript writing. C.F. coordinated the study and wrote the manuscript.