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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-05-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2022.883249/full |
| _version_ | 1818552008832974848 |
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| author | Menghan Gao Erik Yngve Di Yu Chuan Jin |
| author_facet | Menghan Gao Erik Yngve Di Yu Chuan Jin |
| author_sort | Menghan Gao |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-12T09:07:29Z |
| format | Article |
| id | doaj.art-b7a6aad03e0e4f74a46e114f70b2394a |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-12T09:07:29Z |
| publishDate | 2022-05-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-b7a6aad03e0e4f74a46e114f70b2394a2022-12-22T00:29:38ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2022-05-01910.3389/fmolb.2022.883249883249A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus ProductsMenghan GaoErik YngveDi YuChuan JinOncolytic 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.https://www.frontiersin.org/articles/10.3389/fmolb.2022.883249/fullreplication-competent adenovirusconditionally replicating adenovirusquantificationclinical productionqPCRRCA contaminants |
| spellingShingle | Menghan Gao Erik Yngve Di Yu Chuan Jin A qPCR-Based Method for Quantification of RCA Contaminants in Oncolytic Adenovirus Products Frontiers in Molecular Biosciences replication-competent adenovirus conditionally replicating adenovirus quantification clinical production qPCR RCA contaminants |
| 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 | replication-competent adenovirus conditionally replicating adenovirus quantification clinical production qPCR RCA contaminants |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2022.883249/full |
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