Stability of the adeno-associated virus 8 reference standard material
Abstract
Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 10 12 vector genome (vg)/ml, 9.38 ± 11.4 × 10 8 infectious units (IU)/ml and 5.76 ± 2.39 × 10 11 total particles (p)/ml as determined by qPCR, TCID 50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions ( <-70° C). The use of RSM is strongly recommended and endorsed by regulatory agencies to normalize laboratory internal controls and to provide accurate titration of AAV vectors lots.
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Sandwich ELISA was used for characterization of AAV2 and AAV8 reference standard material, demonstrating acceptable precision in the multi-laboratory study, with repeatability of 10% to 15% CV and reproducibility of around 40% CV.
“Sandwich ELISA was used for the characterization of AAV2 and AAV8 RSM, demonstrating acceptable precision in the multi-laboratory study, with repeatability of 10%–15% CV and reproducibility of around 40% CV.37, 38, 39”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [39]
qPCR is the standard procedure for genome titration of rAAV reference standard material, but large multi-laboratory studies highlighted its low precision, with repeatability as poor as greater than 30% CV and reproducibility at 70% to 100% CV.
“qPCR is the standard procedure for genome titration of rAAV reference standard material (RSM), but large multi-laboratory studies highlighted its low precision, with repeatability, indicated by the coefficient of variation (CV), as poor as >30% CV and reproducibility at 70%–100% CV.37, 38, 39”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [39]
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- Authors
- Penaud-Budloo, M., Broucque, F., Harrouet, K., Bouzelha, M., Saleun, S., Douthe, S., D’Costa, S., Ogram, S., Adjali, O., Blouin, V., Lock, M., Snyder, R. O., & Ayuso, E.
- Affiliations
- Inserm (FR)
- University of Pennsylvania (US)
- Corresponding author
- Ayuso, Eduard
- Venue
- Gene Therapy
- Published
- 2019-03-29
- Volume
- 26
- Issue
- 5
- Pages
- 211-215
- ISSN
- 0969-7128 (ISSN-L)
- 1476-5462
- OpenAlex
- W2933022733
- Cited by
- 14
- References
- 12
- Citations by year
- 2026: 1 · 2024: 3 · 2023: 2 · 2022: 3 · 2021: 3 · 2020: 2
- FWCI
- 1.21
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Gene Therapy (publishedVersion)
- PubMed (publishedVersion)
- HAL (submittedVersion)
- Funders
- Agence Nationale de la Recherche (ANR-10-DPBS-0001)
- Institut National de la Santé et de la Recherche Médicale
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- adeno-associated virus 8, gene therapy vectors, storage conditions, AAV serotype 2, infectious units
- MeSH terms
- Dependovirus, Genetic Vectors, Humans, Reference Standards, Virus Replication, Cryopreservation, Practice Guidelines as Topic, Capsid Proteins, Protein Stability, HEK293 Cells
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