Human mrc5 fibroblasts

BQ788, ACT-132577, macitentan, and bosentan were from MedChemExpress LCC (MedChemExpress LCC, Princeton, NJ, USA). Ambrisentan was from Ark Pharm Inc. (Ark Pharm Inc., Microsoft Libertyville, IL, USA). Ganciclovir was from Hoffmann La Roche (Stockholm, Sweden). Human primary RPE-1 retinal pigment epithelial cells (a kind gift from Dr. Richard J. Stanton and Dr. Derrick Dargan, Medical Research Council Centre for Virology, UK [32 (link),33 (link)]) were cultured in RPMI with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL). Human MRC5 fibroblasts (ATCC, US) were cultured in minimum essential medium with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL). Primary human umbilical vein endothelial cells (HUVECs) were harvested with collagenase as described [34 (link)] or purchased (Lonza, Basel, Switzerland) and cultured in endothelial cell growth medium (Lonza). All cells were cultured at 37 °C in 5% CO₂/95% air.

Selective replication of TILT-123 was studied in A549 lung adenocarcinoma cells (positive control, purchased from ATCC, Manassas, VA, USA), human primary hepatocytes (Lonza Verviers SPRL, Bruxelles, Belgium), human MRC-5 fibroblasts (ATCC), and human vascular endothelial HUVEC cells (Millipore). The cells were plated on 24-well plates and infected with TILT-123, wild type Adenovirus 5 (VR-1516, ATCC), Ad5/3 replicative control virus (a similar selection device as TILT-123 but no cytokine payload), or Ad5/3-Luc1 non-replicative control virus [10 (link),19 (link)]. Infected cells and cell culture supernatants were harvested 72 h post-infection by centrifugation.
The virus was released from the cells by repeating freeze–thaw cycles four times in total. DNA was extracted from the samples with the phenol/chloroform/isoamyl alcohol (25:24:1) method and the virus copy numbers determined by detecting the adenovirus E4 copy number in samples by qPCR [20 (link)]. Infectious virus particles were determined with TCID50 assay, where a sample dilution series was plated on A549 cells and cytopathic effect formation was followed for ten days. TILT-123 transgene production was measured from the cell culture supernatants with ELISA, as described above.

Human SUM159 cells were a gift from Dr. Carol Otey (UNC-Chapel Hill, Chapel Hill, NC) and were cultured in Ham’s F12 media (Corning) supplemented with 10% fetal bovine serum (FBS, Rocky Mountain Biologicals), 0.5 μg/ml hydrocortisone (Sigma), and 2.5 μg/ml insulin (Life Technologies). Human MRC5 fibroblasts were purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) supplemented with 10% FBS. All cells were grown at 37 °C and 5% CO₂. All experiments were conducted with early passage cells that were passaged no more than 15 times. Mycoplasma was tested regularly by staining with Hoechst 33342 (Anaspec).