Oncolytic adenovirus is one of the most promising treatments against cancer and is widely evaluated clinically. During high titer production, “Wild-type-” like replication-competent adenovirus (RCA) contaminants can be generated through recombination events due to the DNA sequence similarity between oncolytic virus and host cells. These RCA contaminants raise various safety concerns in clinics. Cell culture-based methods have been developed to detect RCA contaminants in replication-deficient adenovirus vectors. These methods were based on that only RCA contaminants, but not the vectors, are able to grow in and lyse the test cell line. However, these methods are not suitable for distinguishing RCA contaminants from the oncolytic adenovirus products because both can replicate in test cell lines. Herein, we reported a qPCR-based method to quantify RCA contaminants quickly and reliably in E1B-deleted oncolytic adenovirus products. This method is based on specific detection of the E1B gene, which can be acquired during production via recombination events between viral and host cell DNA. The assay is sensitive with the limit of detection at 10 VP of the RCA contaminants and the limit of quantification at 75 VP of the RCA contaminants in each 40 µL qPCR reaction. We have also validated the method on virus batches produced in the non-GMP and GMP conditions. Our results showed that this qPCR-based method was reliable and robust for detecting and quantifying RCA contaminants in oncolytic adenovirus products. The method may also be adapted for other oncolytic adenoviruses products by switching primer sets.

Background: Oncolytic viruses are promising tools for immune stimulatory gene therapy of cancer, but their clinical effect on solid tumors have so far been limited. Transduction of the target tumor cells is limited by both extracellular matrix that blocks viral spread within the solid tumor tissue and electrostatic forces that inhibit virus from binding its entry receptor on the cell surface. The enzymes hyaluronidase and collagenase and the polycations diethylaminoethyl (DEAE)-dextran, branched Polyethylenimine (PEI) and protamine sulfate have previously shown potential to improve gene transfer in different forms of viral gene therapy, since they may help the virus to overcome these barriers. In this study, we compared the transduction-enhancing potential of these substances when used as vehicles for adenoviral transduction in solid tumor tissue.

Methods: Subcutaneous tumors of pancreatic ductal adenocarcinoma were established in mice and treated with a mix of adenoviral vector Adf35(GFP-Luc) and either one of the selected vehicles. Transduction efficacy was determined by quantification of the viral transgene expression level using live imaging.

Results: Addition of hyaluronidase tripled the transgene expression of Adf35(GFP-Luc) when compared to virus alone. No such positive effect was seen for the other tested vehicles.

Conclusions: Out of the tested candidates, hyaluronidase showed the best potential to facilitate viral spread in tumor tissue and transduction of tumor cells. Therefore, hyaluronidase may be used as vehicle to improve clinical efficacy of oncolytic virotherapies.