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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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Items 101–151 of 151
Gene therapy in neuromuscular disorders
Mendonça, R. D. H., & Zanoteli, E. (2022) · Arquivos de Neuro-Psiquiatria · doi:10.1590/0004-282x-anp-2022-s135 · Cited by 9
“6 (8-34) days of life at dosing and mean weight of 3.”
BackgroundAAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines
Luo, Y., Frederick, A., Martin, J. M., Scaria, A., Cheng, S. H., Armentano, D., Wadsworth, S. C., & Vincent, K. A. (2017) · Human Gene Therapy Methods · doi:10.1089/hgtb.2016.158 · Cited by 7
“6,7 The most generally adopted vector production method, plasmid DNA transfection in HEK293 cells, was used to generate the vector used in the clinical trials for Leber congenital amaurosis Type 2 8 and hemophilia B. 9 Recombinant viral vector–based protocols such as the baculovirus-and HSV-based…”
Cross-species genetic screens to identify kinase targets for APP reduction in Alzheimer's disease
Huichalaf, C. H., Al‐Ramahi, I., Park, K., Grunke, S. D., Lü, N., de Haro, M., Merz, K. E., Gallego‐Flores, T., Perez, A. M., Jung, S. Y., et al. (2019) · Human Molecular Genetics · doi:10.1093/hmg/ddz034 · Cited by 7
Rigorous anterograde trans-monosynaptic tracing of genetic defined neurons with retargeted HSV1 H129Preprint
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Cagin, U. (2021) · Advances in experimental medicine and biology · doi:10.1007/978-3-030-55035-6_15 · Cited by 5
Manufacturing Considerations and Challenges for AAV Ocular Gene Therapy
Rodrigues, G. A., Shalaev, E., Karami, T. K., Cunningham, J., Rivers, H. M., Feng, S., Shah, D., & Slater, N. K. H. (2021) · AAPS advances in the pharmaceutical sciences series · doi:10.1007/978-3-030-76367-1_22 · Cited by 5
Tejero, M., Duzenli, O. F., Caine, C., Kuoch, H., & Aslanidi, G. (2023) · Genes · doi:10.3390/genes14101866 · Cited by 5
AAV Vector-Based Gene Therapy, Progress and Current Challenges
Kuranda, K., & Mingozzi, F. (2017) · Springer eBooks · doi:10.1007/978-3-319-53457-2_5 · Cited by 5
Self-citationKrüger‐Haag, A., Lehmann, C., Schmidt, E., Sonntag, F., Hörer, M., & Kochanek, S. (2019) · The Journal of Gene Medicine · doi:10.1002/jgm.3094 · Cited by 5
“In this respect, various manufacturing platforms for AAV vectors have been established.(6-9) On the one hand, one major method currently employed for large scale AAV vector production is an insect cell line‐ based platform using baculovirus infection.”
MethodA synthetic platform for developing recombinant adeno‐associated virus type 8 producer cell lines
Lin, Y., Kuo, H., Lu, M., Mahl, T., Aslanidi, G., & Hu, W. (2025) · Biotechnology Progress · doi:10.1002/btpr.70009 · Cited by 4
“15 These viral vector systems use a packaging vector of the virus and two recombinant viral vectors, one carrying the rAAV genome and the other encoding the Rep and Cap proteins.”
Aponte, J. J., Barajas, D., Peltier, J., Bardliving, C. L., Shamlou, P. A., & Gold, D. (2018) · Biotechnology Progress · doi:10.1002/btpr.2725 · Cited by 4
“Adenovirus, Herpesvirus, and Baculovirus complementation systems constitute robust platforms for vector production at preclinical and clinical scale.(6,7) Several process optimization studies achieved a considerable improvement in vector particle formation by focusing on many bioengineering aspects such as cell growth…”
BackgroundViral Vectors in Neurobiology: Therapeutic and Research Applications
Coura, R. (2012) · InTech eBooks · doi:10.5772/32492 · Cited by 4
“Their primary applications are in gene therapy, especially oncolytic gene therapy, and vaccination (Ayuso et al., 2010). www.intechopen.com Molecular Virology 80”
BackgroundEmmerling, V. V., Holzmann, K., Lanz, K., Kochanek, S., & Hörer, M. (2013) · BMC Proceedings · doi:10.1186/1753-6561-7-s6-p12 · Cited by 4
“Furthermore, rAAV vectors have favourable properties such as non-pathogenicity of AAV, low B-/T-cell immunogenicity against transgenes delivered and long-term transgene expression from a non-integrating vector [5,9].”
BackgroundLin, Y., Kuo, H., Lu, M., Rungkittikhun, C., & Hu, W. (2024) · Biotechnology and Bioengineering · doi:10.1002/bit.28885 · Cited by 3
“This method is simple to implement and can produce high levels of rAAV (Ayuso, Mingozzi, and Bosch 2010; Merten 2016), but the process requires the production of multiple plasmids and transient transfection in large ‐ scale reactors is cumbersome (Shupe et al. 2022).”
Virus-Based Neural Circuit Tracing
Xu, F., & Liu, Q. L. (2024) · Advances in neurobiology · doi:10.1007/978-3-031-69188-1_5 · Cited by 3
Recent developments in gene therapy research in India
Selot, R., & Ghosh, A. (2024) · Journal of Biosciences · doi:10.1007/s12038-024-00423-0 · Cited by 3
“…require highly stringent quality checks, irrespective of the method of production (Ghosh et al. predominantly cellular virus with ultra-structural and size similarity to cellular organelles and this requires multi-step purification without breakage of capsid or transgene (Ayuso et al. 2010).”
High-efficiency HDR in immortalized cell lines by crude rAAV mediated donor template deliveryPreprint
Mehta, S., Buyanbat, A., Zheng, G., Liu, N., & Orkin, S. H. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.05.02.490359 · Cited by 3
“As such, save a few reports in permissive cell lines, rAAV has been underutilized for KI of large DNA fragments into cell lines [20-24].”
BackgroundOptimization and scale up strategies for reproducible AAV enrichment step on CIMmultus® QA HR line
Žigon, R., Prebil, S. D., Švigelj, T., Kozjak, K., Žvanut, T., Leskovec, M., & Štrancar, A. (2026) · Gene Therapy · doi:10.1038/s41434-026-00594-5 · Cited by 2
“First is the method itself, where the traditional puri fi cation methods, such as caesium chloride gradient ultracentrifugation are labour-intensive process that requires manual handling and multiple steps, making it less suitable for large-scale production [9].”
Purifying Viral Vectors: A Review of Chromatography Solutions
Crowley, L., Labisch, J. J., Leskovec, M., Sbaizero, M. T., McLaughlin, K., Nestola, P., & Boulais, A. (2023) · Springer eBooks · doi:10.1007/978-3-031-28489-2_8 · Cited by 2
Ye, Q., Li, R., Xiao, Z., Na, D., Cui, N., Zhao, L., & Tan, W. (2025) · Biochemical Engineering Journal · doi:10.1016/j.bej.2025.109652 · Cited by 1
Wu, S., Xue, L., Li, X., Wang, Y., Zhu, Y., Luo, Y., Sun, J., Jin, T., Shu, W., & Wang, Z. (2025) · Immunologic Research · doi:10.1007/s12026-025-09609-6 · Cited by 1
“These vectors can be prepared through various methods, with the most common involving the utilization of three distinct DNA plasmids encoding the vector genome, rep and cap genes sourced from specific AAV serotypes, and helper plasmids for transfection into HEK293 cell lines [10, 11].”
Wang, D., Ta, V., Wang, H., Ju, J., Wang, C., Chehadeh, C., Torreblanca-Zanca, A., Magaña, Y., Castle, M. J., Wang, S., et al. (2026) · Alzheimer s & Dementia · doi:10.1002/alz.71450 · Cited by 1
Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment
Gstöttner, C., Hutanu, A., Boon, S., Raducanu, A., Richter, K., Haindl, M., Ruppert, R., & Domínguez‐Vega, E. (2023) · Analytical Chemistry · doi:10.1021/acs.analchem.3c00222 · Cited by 1
Phase-Appropriate Potency Assay Development for AAV-Based Gene Therapy Products
Peters, E. E., Pach, R., & Datta, S. (2023) · Springer eBooks · doi:10.1007/978-3-031-28489-2_10 · Cited by 1
Culturing of Animal Cells, Stem Cells, Tissues, and Organs for Industrial Products: An Introduction
Akram, Z. (2024) · Springer eBooks · doi:10.1007/978-3-031-50989-6_15 · Cited by 1
Optimization of Production of Recombinant Adeno-Associated Virus Serotype 9
Aliev, T. I., & Рамисович, И. И. (2025) · Molecular Biology · doi:10.1134/s0026893325700244 · Cited by 1
Gautier, B., Hajjar, H., Soares, S., Berthelot, J., Deck, M., Abbou, S., Campbell, G. R., Fovet, C.-M., Schütza, V., Jouvenel, A., et al. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.01.29.924605 · Cited by 1
“Vector production was performed following the CPV facility protocol as previously described [2].”
MethodImmunogenicity of AAV Gene Therapy Products
Jawa, V., & Wu, B. (2024) · Wiley eBooks · doi:10.1002/9781119852810.ch5 · Cited by 1
Strategien zum Nachweis und der Identifizierung viraler Vektoren
Stellberger, T., Köhler, N., Dinkelmeier, A., Haase, M., Hellinckx, J., Pavlovic, M., Busch, U., & Baiker, A. E. (2017) · Journal of Consumer Protection and Food Safety · doi:10.1007/s00003-016-1064-y
“Das Vektorplasmid trägt dabei das von inverted terminal repeats (ITRs) flankierte Transgen, das Verpackungsplasmid kodiert für die AAV-Leserahmen rep und cap und das Helferplasmid stellt die Gene bereit, die für die adenovirale Helferfunktionen erforderlich sind (Ayuso et al. 2010).”
BackgroundSchleef, M. (2019) · WORLD SCIENTIFIC (EUROPE) eBooks · doi:10.1142/9781786346889_0013
DETERMINANTS OF AAV TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER
Albright, B. H. (2018) · Carolina Digital Repository (University of North Carolina at Chapel Hill) · doi:10.17615/vnpr-wx33
Zhai, J., Kim, H., Han, S. B., Manire, M., Yoo, R., Pang, S., Smith, G. M., & Son, Y. (2020) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2020.09.11.293894
“Production and microinjection of AAV2-GFP, LV-chABC and LV-GDNF Recombinant self-complementary adeno-associated virus 2 (scAAV2) carrying eGFP was generated by helper virus-free system (Ayuso et al., 2010) as described previously (Liu et al., 2014).”
Characterization of Adeno-Associated Virus (AAV) Gene Therapy Products
Rathore, A. S., Auclair, J. R., & Kaur, A. (2023) · LCGC North America · doi:10.56530/lcgc.na.st3379l8
Hope Unlocked: Gene Therapy with Viral Vectors and Gene Editing
Thiaville, J. J., Dias, M. S., Kato, S. E. M., Basak, S., Rengarajan, S., Silva, H. P., Rivas, N., & D’Costa, S. M. (2025) · Springer eBooks · doi:10.1007/978-3-031-81787-8_5
The Deubiquitinating Enzyme Otub2 Modulates Pancreatic Beta-Cells Function and Survival
Oshry, M., Isaac, R., Boura‐Halfon, S., Casana, E., Sampson, S. R., Lev, S., Bosch, F., Zick, Y., & Vinik, Y. (2026) · Frontiers in Bioscience-Landmark · doi:10.31083/fbl44406
Self-citationCharacterisation of AAV capsid-specific CD8+ T cell responses in human subjects
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Les agrégats de PABPN1 dans la dystrophie musculaire oculopharyngée
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Développement d'un traitement de thérapie génique pour la glycogénose de type III
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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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