Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls
Open accessAbstract
Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro , based on detection of vector genome replication in trans -complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells. In the present study, we have compared these methods for the titration of infectious rAAV8 vector particles, and, to assess their ability to discriminate infectious and non-infectious rAAV serotype 8 particles, we have generated a VP1-defective AAV8-GFP vector. Since VP1 is required to enter the cell nucleus, the lack of VP1 should drastically reduce the infectivity of rAAV particles. The AAV8 reference standard material was used as a positive control. Our results demonstrated that methods based on measurement of rAAV biological activity (i.e., vector genome replication or transgene expression) were able to accurately discriminate infectious versus non-infectious particles, whereas methods simply measuring intracellular vector genomes were not. Several cell fractionation protocols were tested in an attempt to specifically measure vector genomes that had reached the nucleus, but genomes from wild-type and VP1-defective AAV8 particles were equally detected in the nuclear fraction by qPCR. These data highlight the importance of using suitable controls, including a negative control, for the development of biological assays such as infectious unit titration.
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Infectious titer, a measure of biological activity in vitro, takes 1 to 3 days to quantify and exhibits variability of multiple orders of magnitude, making it impractical for timely characterization of product potency.
“In contrast, the infectious titer—a measure of biological activity in vitro—takes 1–3 days to quantify and exhibits variability of multiple orders of magnitude;28,34 thus, it is impractical for timely characterization of product potency.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [34]
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- Authors
- François, A., Bouzelha, M., Lecomte, E., Broucque, F., Penaud-Budloo, M., Adjali, O., Moullier, P., Blouin, V., & Ayuso, E.
- Affiliations
- Inserm (FR)
- Corresponding author
- François, Achille
- Ayuso, Eduard
- Venue
- Molecular Therapy - Methods & Clinical Development
- Publisher
- Elsevier BV
- Published
- 2018-09
- Volume
- 10
- Pages
- 223-236
- ISSN
- 2329-0501 (ISSN-L)
- PMC
- PMC6090651
- OpenAlex
- W2883800592
- Cited by
- 50
- References
- 48
- Citations by year
- 2026: 2 · 2025: 9 · 2024: 7 · 2023: 12 · 2022: 5 · 2021: 8 · 2020: 4 · 2019: 3
- FWCI
- 2.29
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Molecular Therapy — Methods & Clinical Development (publishedVersion, cc-by-nc-nd)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- HAL (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Commissariat Général à l'Investissement
- Association Française contre les Myopathies
- Centre hospitalier universitaire Sainte-Justine
- Agence Nationale de la Recherche
- 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
- recombinant adeno-associated virus, AAV8, trans-complementation, transgene expression, nuclear fractionation, negative control
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