A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products.

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.

A qPCR-Based Method for Quantification of Replication Competent Adenovirus (RCA) in Conditionally Replicating Oncolytic Adenoviruses.

Production of conditionally replicating adenoviruses may unfortunately generate undesired replication-competent adenovirus (RCA) which raises safety concerns in clinical usage. Cell-based assays can detect RCA in batches of nonreplicating adenoviral vectors but cannot distinguish RCA from conditionally replicating oncolytic adenoviruses. Considering the great potential in using oncolytic viruses for cancer treatment, there is a need for comprehensive RCA-detection and -quantification methods. Here, we present a quantitative polymerase chain reaction (qPCR)-based assay that can be used to detect RCA particles in batches of conditionally replicating oncolytic adenoviruses. The assay is quantifying RCA by detection of the specific DNA sequence generated after a recombination event. Results showed that the method can successfully detect low levels of RCA, with a low limit of detection of ten viral particles.

Protein Kinase R Is Constitutively Expressed in the Human Pancreas.

Viral infection of the insulin-producing cells in the pancreas has been proposed in the etiology of type 1 diabetes. Protein kinase R (PKR) is a cytoplasmic protein activated through phosphorylation in response to cellular stress and particularly viral infection. As PKR expression in pancreatic beta-cells has been interpreted as a viral footprint, this cross-sectional study aimed at characterizing the PKR expression in non-diabetic human pancreases. PKR expression was evaluated in pancreas tissue from 16 non-diabetic organ donors, using immunohistochemistry, qPCR, and western blot. Immunohistochemistry and western blot showed readily detectable PKR expression in the pancreatic parenchyma. The qPCR detected PKR mRNA in both endocrine and exocrine samples, with a slightly higher expression in the islets. In conclusion, PKR is constitutively expressed in both endocrine and exocrine parts of the pancreas and its expression should not be interpreted as a viral footprint in pancreatic beta cells.