Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors
Robust and accurate quantification of recombinant adeno-associated virus (rAAV) vectors’ infectivity is essential for pre-clinical and clinical development of AAV gene therapy programs. The industry standard method for rAAV titration is the 50% tissue culture infectious dose (TCID50) assay using HeL...
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Elsevier
2025-06-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050125000877 |
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| author | Chloé Fustinoni Xiang Liu Zhi Chen Ryan Cawood Weimin Valenti Maria I. Patrício Weiheng Su |
| author_facet | Chloé Fustinoni Xiang Liu Zhi Chen Ryan Cawood Weimin Valenti Maria I. Patrício Weiheng Su |
| author_sort | Chloé Fustinoni |
| collection | DOAJ |
| description | Robust and accurate quantification of recombinant adeno-associated virus (rAAV) vectors’ infectivity is essential for pre-clinical and clinical development of AAV gene therapy programs. The industry standard method for rAAV titration is the 50% tissue culture infectious dose (TCID50) assay using HeLa-based cell lines that stably encode the rep and cap genes from AAV serotype 2. Co-infection with wild-type (WT) adenoviruses provides the helper functions for expression of these genes, and the use of quantitative PCR (qPCR)/droplet digital PCR (ddPCR) serves as the endpoint method for the detection of infectious events. However, TCID50 assays using these HeLa-based rep cap trans-complementing cell lines have traditionally been regarded as challenging due to high variability, stability of the integrated genes, and safety concerns associated with the use of WT helper viruses. Here we developed a novel method for infectious titration of rAAV using our vector “tetracycline-enabled self-silencing adenovirus” (TESSA); we engineered it to deliver and express the AAV2 rep genes and adenoviral helper functions for rAAV genome replication, independent of the cell type. This approach allows the infectious titration of rAAV serotypes in cell lines permissive to adenovirus but without the production of adenoviral particles for improved safety, therefore benefiting GMP analytical requirements for rAAV gene therapies. |
| format | Article |
| id | doaj-art-e6ca7724d6474ae185bd3f70f11ec6a9 |
| institution | OA Journals |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-e6ca7724d6474ae185bd3f70f11ec6a92025-08-20T02:02:44ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-06-0133210149210.1016/j.omtm.2025.101492Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectorsChloé Fustinoni0Xiang Liu1Zhi Chen2Ryan Cawood3Weimin Valenti4Maria I. Patrício5Weiheng Su6OXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford OX4 4HG, Oxfordshire, UKWuxi ATU Co., No. 860, Xinyang Road, Lingang, Shanghai, ChinaWuxi ATU Co., No. 860, Xinyang Road, Lingang, Shanghai, ChinaOXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford OX4 4HG, Oxfordshire, UKWuXi Advanced Therapies, 4701 League Island Blvd, Philadelphia, PA 19112, USAOXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford OX4 4HG, Oxfordshire, UKOXGENE, A WuXi Advanced Therapies Company, Medawar Centre, Robert Robinson Avenue, Oxford OX4 4HG, Oxfordshire, UK; Corresponding author: Weiheng Su, Medawar Centre, Robert Robinson Avenue, Oxford, OX4 4HG, UK.Robust and accurate quantification of recombinant adeno-associated virus (rAAV) vectors’ infectivity is essential for pre-clinical and clinical development of AAV gene therapy programs. The industry standard method for rAAV titration is the 50% tissue culture infectious dose (TCID50) assay using HeLa-based cell lines that stably encode the rep and cap genes from AAV serotype 2. Co-infection with wild-type (WT) adenoviruses provides the helper functions for expression of these genes, and the use of quantitative PCR (qPCR)/droplet digital PCR (ddPCR) serves as the endpoint method for the detection of infectious events. However, TCID50 assays using these HeLa-based rep cap trans-complementing cell lines have traditionally been regarded as challenging due to high variability, stability of the integrated genes, and safety concerns associated with the use of WT helper viruses. Here we developed a novel method for infectious titration of rAAV using our vector “tetracycline-enabled self-silencing adenovirus” (TESSA); we engineered it to deliver and express the AAV2 rep genes and adenoviral helper functions for rAAV genome replication, independent of the cell type. This approach allows the infectious titration of rAAV serotypes in cell lines permissive to adenovirus but without the production of adenoviral particles for improved safety, therefore benefiting GMP analytical requirements for rAAV gene therapies.http://www.sciencedirect.com/science/article/pii/S2329050125000877rAAVinfectious titerTCID50TESSATREAT assay |
| spellingShingle | Chloé Fustinoni Xiang Liu Zhi Chen Ryan Cawood Weimin Valenti Maria I. Patrício Weiheng Su Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors Molecular Therapy: Methods & Clinical Development rAAV infectious titer TCID50 TESSA TREAT assay |
| title | Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors |
| title_full | Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors |
| title_fullStr | Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors |
| title_full_unstemmed | Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors |
| title_short | Self-silencing adenovirus enables precise infectious titration of recombinant adeno-associated viral vectors |
| title_sort | self silencing adenovirus enables precise infectious titration of recombinant adeno associated viral vectors |
| topic | rAAV infectious titer TCID50 TESSA TREAT assay |
| url | http://www.sciencedirect.com/science/article/pii/S2329050125000877 |
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