Journal: Biomedicines
Article Title: Engineering and Preclinical Evaluation of Western Reserve Oncolytic Vaccinia Virus Expressing A167Y Mutant Herpes Simplex Virus Thymidine Kinase
doi: 10.3390/biomedicines8100426
Figure Lengend Snippet: Rationale for engineering modified HSV- tk armed OVV, WOTS-418 and characterization. ( A ) Wild type HSV-tk transgene instability in VV backbone. Wild type HSV-tk or modified HSV-tk 418m containing shuttle plasmid (pOTS or pOTS-418) was used for HR with VV. Firefly luciferase positive HR lysate were further screened with or without BrdU TK negative selection pressure. Isolated firefly luciferase positive singles plaques were further checked for HSV-tk expression by western blotting (detail screening and selection procedure described in the methods section). ( B ) WOTS-418 engineering map. Transgene insertion location in WR VV backbone is shown (top) and the diagram is showing the partial and representative location of the wild type HSV-tk DNA sequence (bottom). The middle panel is showing the representative modified HSV- tk 418m DNA sequence, indicating the location of altered nucleotides and DNA sequence confirmation. The promoters used for HSV-tk 418m and firefly luciferase were pSE/L and p 7.5, respectively. ( C ) Monitoring long-term HSV-tk 418m gene stability. To evaluate the long-term transgene stability, WOTS-418 virus samples from two different time points (first amplification and tenth amplification) were used. A549 cells were infected with WOTS-418 (0.1 PFU/cell), WR VV and HSV-1. 24 h post infection ( p.i.) , cell pellets were used to measure HSV- tk expression by western blotting. WR VV: Wild type western reserve vaccinia virus (Negative control); HSV-1: Wild type HSV-1 virus (Positive control); Anti-HSV-1 thymidine Kinase; sc-28037, vN-20: SCBT, Dallas, TX, USA; Anti-GAPDH; MB001: Bioworld, Louis Park, MN, USA; Secondary antibody anti-goat; A50-101P: Bethyl, Montgomery, TX, USA; Secondary antibody anti-mouse; ADI-SAB-100-J: Enzo, Executive Blvd Farmingdale, NY, USA. Protein Marker: ExcelBand 3-Color PreStained Protein Markers, #2700 (Green BioResearch LLC, Baton Rouge, LA, USA), a mixture of blue, red, and green stained recombinant proteins (5 to 245 kDa), was used as size standards in western blotting. ( D ) WOTS-418 DNA stability: Samples from C was also used for long-term DNA stability confirmation, which was evaluated by HindIII restriction digestion assay (detail procedure described in the methods section). Blue arrows indicate the alteration pattern of DNA band size after recombination. ( E ) GCV antiviral potency comparison between OTS-412 and WOTS-418: Quantitative analysis of viral DNA copies, measured in fold inhibition, was done following virus + GCV treatment and virus without GCV treatment. NCI-H460 and A549 cells were infected with either OTS-412 or WOTS-418 (0.1 PFU/cell) and co-treated with GCV (100 μM). 72 h p.i. , samples were harvested, and viral copy numbers were measured by quantitative polymerase chain reaction (qPCR). Data presented as mean ± SEM ( n = 2) and compared using two-tailed Student’s t -test ( p -value). ( F ) WR, WR-GFP, and WOTS-418 cytotoxicity comparison (left panel). Three human cancer cells (HeLa, NCI-H460, and HCT 116) and three murine cancer cells (Renca, CT-26.WT, and 4T1) were infected with WR, WR-GFP, WOTS-418 at a dose 0.1 PFU/cell for human cancer and 1 PFU/cell for murine cancer cells. 72 h p.i. samples were evaluated for cytotoxicity by CCK-8 assay. Data are presented as mean± SEM ( n = 2). WR VV, WR-GFP, and WOTS-418 virus yield in various cancer cell lines (right panel): After cytotoxicity experiment, samples were harvested and subjected to three freeze and thaw cycles. Next, samples were sonicated with a cup sonicator (100% power for 30 s, 3 times with 1-min interval) and reinfected (same volume, 30 μL) in pre-seeded HeLa cells (10,000 cells/well) in 96-well plates and incubated at 37 °C with 5% CO 2 . 72 h p.i., CCK-8 assay was performed to evaluate that viral cytotoxicity of the three viruses. Data are presented as mean ± SEM ( n = 3). ( G ) WR vs. WOTS-418 safety assessment: Non-tumor bearing BALB/c syngeneic mice were treated intranasally with WR VV (1 × 10 5 PFU and 1 × 10 7 PFU) or WOTS-418 (1 × 10 7 PFU and 5 × 10 8 PFU). After WR VV or WOTS-418 treatment, survival and body weight were monitored. Survival curve demonstrating overall survival and p < 0.0001 was determined by log-rank (Mantel-Cox) test ( n ≥ 3).
Article Snippet: Human osteosarcoma (143B and U-2 OS), human cervical adenocarcinoma (HeLa and HeLa S3), human epithelial lung carcinoma (A549), murine colorectal cancer cell (CT-26.WT), and murine breast carcinoma (4T1) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA).
Techniques: Modification, Plasmid Preparation, Luciferase, Selection, Isolation, Expressing, Western Blot, Sequencing, Virus, Amplification, Infection, Negative Control, Positive Control, Marker, Staining, Recombinant, Comparison, Inhibition, Real-time Polymerase Chain Reaction, Two Tailed Test, CCK-8 Assay, Sonication, Incubation
