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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.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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Adeno-Associated Virus (AAV) as a Vector for Gene Therapy
Naso, M. F., Tomkowicz, B., Perry, W. L., & Strohl, W. R. (2017) · BioDrugs · doi:10.1007/s40259-017-0234-5 · Cited by 1,362
Emerging issues in AAV-mediated in vivo gene therapy
Colella, P., Ronzitti, G., & Mingozzi, F. (2018) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2017.11.007 · Cited by 881
“AAV vectors can be produced at high yields by either transient triple transfection of mammalian cells,(16) infection of packaging mammalian and insect cells,(17) or other methods (reviewed by Ayuso et al.(18)).”
BackgroundMethodSelf-citationAAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer
Costa Verdera, H., Kuranda, K., & Mingozzi, F. (2020) · Molecular Therapy · doi:10.1016/j.ymthe.2019.12.010 · Cited by 641
Self-citationHuman Immune Responses to Adeno-Associated Virus (AAV) Vectors
Ronzitti, G., Gross, D.-A., & Mingozzi, F. (2020) · Frontiers in Immunology · doi:10.3389/fimmu.2020.00670 · Cited by 357
“An important focus in the field is the continuous improvement of the rAAV manufacturing processes to increase vector yields and purity while reducing costs (17, 18, 21, 22).”
BackgroundSelf-citationManufacturing challenges and rational formulation development for AAV viral vectors
Srivastava, A., Mallela, K. M. G., Deorkar, N., & Brophy, G. (2021) · Journal of Pharmaceutical Sciences · doi:10.1016/j.xphs.2021.03.024 · Cited by 269
Metadata
- 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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