Production, purification and characterization of adeno-associated vectors
Abstract
The use of recombinant adeno-associated viral vectors (rAAVs) as gene transfer tools has increased dramatically during the past several years, establishing AAV as the vector of choice for many therapeutic applications. With the steady advance of the field toward clinical studies, and the isolation and engineering of several novel AAV serotypes, efficient, scalable, and versatile production and purification methods are continuously under development. Here, we review the current state of the art in the various production and purification methods for rAAVs. Classical parameters and methodologies to characterize rAAV stocks will be also discussed. Keywords: Gene therapy, helper viruses, empty capsids, GMP, gradient vs. column purification, Parvoviridae, AAV serotype 2, chromosome 19, ITR, p5, p19, Rep 78, Rep 68, p5 promoter, p19 promoter, cap gene, VP1, VP2, VP3, adenoviruses (Ad), herpes simplex virus (HSV), vaccinia virus, and cytomegalovirus (CMV), AAV7, AAV8, AAV9, baculovirus vectors, virus-associated (VA) RNA, E2A, E4, Hela cells, HEK 293 cells, E1B genes, HeLa, A549, HEK293 cell lines, RPE65 deficiency, polyethylene glycol (PEG), Cesium chloride (CsCl) isopycnic gradient centrifugation, Column chromatography, AVB Sepharose High performance medium, Nhydroxysuccinimide (NHS)-activated Sepharose High Performance, human factor IX transgene, CD8 T cell, benzonase, anion-exchange chromatography, Vector Titration, ELISA, Transduction Titer, polyacrylamide gel electrophoresis (PAGE)
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Quality attributes of rAAV are routinely analyzed using time-intensive quality control assays for batch release testing of the final product to ensure they fulfill regulatory specifications for safe use.
“Quality attributes of rAAV are routinely analyzed using time-intensive quality control assays for batch release testing of the final product to ensure they fulfill regulatory specifications for safe use.16, 17, 18, 19”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [16]
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“15 rAAV produced in insect cells also pose additional concerns about different posttranslational modifications as compared with human cell produced product.”
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Stronger together: Analytical techniques for recombinant adeno associated virus
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“Recombinant self-650 complementary adeno-associated virus 2 (scAAV2) carrying eGFP was generated by helper 651 virus-free system (Ayuso et al., 2010) as described previously (Liu et al., 2014).”
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Unlocking DOE potential by selecting the most appropriate design for rAAV optimization
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Oligodendrocyte precursor cells stop sensory axons regenerating into the spinal cord
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Rescue of lysosomal acid lipase deficiency in mice by rAAV8 liver gene transfer
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“The fi nal product was formulated in sterile phosphate buffered saline containing 0.001% of Pluronic (F68; Sigma Aldrich, Saint Louis, MO), and stored at − 80 °C 16,17 .”
InfluentialOverview of analytics needed to support a robust gene therapy manufacturing process
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Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production
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Residual DNA impurities in AAV vectors—nature and transcription
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Strategies and methods for the detection and identification of viral vectors
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AAV Vectors for Efficient Gene Delivery to Rodent Hearts
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Gene therapy using Aβ variants for amyloid reduction.
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Synergism of dual AAV gene therapy and rapamycin rescues GSDIII phenotype in muscle and liver
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“The most common platform used to manufacture rAAV vectors is transient transfection of human embryonic kidney 293 (HEK293) cells (Ayuso et al. 2010; Fu et al. 2023b; Ou et al. 2024).”
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Live Imaging of Mitochondrial ROS Production and Dynamic Redox Balance in Neurons
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- Authors
- Ayuso, E., Mingozzi, F., & Bosch, F.
- Affiliations
- Universitat Autònoma de Barcelona (ES)
- Venue
- Current Gene Therapy
- Publisher
- Bentham Science Publishers Ltd.
- Published
- 2010-12-01
- Volume
- 10
- Issue
- 6
- Pages
- 423-436
- ISSN
- 1566-5232 (ISSN-L)
- OpenAlex
- W2128086887
- Cited by
- 151
- References
- 0
- Citations by year
- 2026: 7 · 2025: 10 · 2024: 15 · 2023: 15 · 2022: 7 · 2021: 23 · 2020: 12 · 2019: 10 · 2018: 11 · 2017: 19 · 2016: 5 · 2015: 6 · 2014: 2 · 2013: 6 · 2012: 1
- FWCI
- 1.62
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Current Gene Therapy (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Ministerio de Ciencia e Innovación
- Instituto de Salud Carlos III
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- adeno-associated virus vectors, AAV production, AAV purification, GMP manufacturing, AAV serotypes, AAV8, AAV9, helper virus, empty capsids, virus-associated RNA, AAV gene therapy, human factor IX, PEGylation, column chromatography, AAV serotype 2
- MeSH terms
- Adenoviridae, Animals, Biotechnology, Cell Line, Clinical Trials as Topic, Genetic Vectors, Humans, Serotyping
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