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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...

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Autores principales: Gao, Menghan, Yngve, Erik, Yu, Di, Jin, Chuan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9169249/
https://www.ncbi.nlm.nih.gov/pubmed/35677885
http://dx.doi.org/10.3389/fmolb.2022.883249
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author Gao, Menghan
Yngve, Erik
Yu, Di
Jin, Chuan
author_facet Gao, Menghan
Yngve, Erik
Yu, Di
Jin, Chuan
author_sort Gao, Menghan
collection PubMed
description 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.
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spelling pubmed-91692492022-06-07 A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products Gao, Menghan Yngve, Erik Yu, Di Jin, Chuan Front Mol Biosci Molecular Biosciences 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. Frontiers Media S.A. 2022-05-23 /pmc/articles/PMC9169249/ /pubmed/35677885 http://dx.doi.org/10.3389/fmolb.2022.883249 Text en Copyright © 2022 Gao, Yngve, Yu and Jin. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Gao, Menghan
Yngve, Erik
Yu, Di
Jin, Chuan
A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products
title A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products
title_full A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products
title_fullStr A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products
title_full_unstemmed A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products
title_short A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products
title_sort qpcr-based method for quantification of rca contaminants in oncolytic adenovirus products
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9169249/
https://www.ncbi.nlm.nih.gov/pubmed/35677885
http://dx.doi.org/10.3389/fmolb.2022.883249
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