Cited by 151Virus-based gene therapy researchViral Infectious Diseases and Gene Expression in InsectsRNA Interference and Gene Delivery

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Production, Purification and Characterization of Adeno-Associated Vectors

Ayuso, Eduard, Mingozzi, Federico, Bosch, Fatima·2010·Current Gene Therapy

DOI 10.2174/156652310793797685

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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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Background
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Background
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Oligophrenin-1 moderates behavioral responses to stress by regulating parvalbumin interneuron activity in the medial prefrontal cortex
Wang, M., Gallo, N. B., Tai, Y., Li, B., & Aelst, L. V. (2021) · Neuron · doi:10.1016/j.neuron.2021.03.016 · Cited by 21
Improved adeno‐associated virus empty and full capsid separation using weak partitioning multi‐column AEX chromatography
Di, W., Koczera, K., Zhang, P., Chen, D. P., Warren, J. C., & Huang, C. (2023) · Biotechnology Journal · doi:10.1002/biot.202300245 · Cited by 21
“[21,22] After 5 days, a nuclease treatment was performed and then the cells were harvested and clarified through a series of depth filters.”
Method
Insight and Development of Advanced Recombinant Adeno-Associated Virus Analysis Tools Exploiting Single-Particle Quantification by Multidimensional Droplet Digital PCR
Zanker, J., Lázaro-Petri, S., Hüser, D., Heilbronn, R., & Savy, A. (2021) · Human Gene Therapy · doi:10.1089/hum.2021.182 · Cited by 18
Chromatography in Downstream Processing of Recombinant Adeno‐Associated Viruses: A Review of Current and Future Practises
Lorek, J. K., Isaksson, M., & Nilsson, B. (2025) · Biotechnology and Bioengineering · doi:10.1002/bit.28932 · Cited by 21
“…in time ‐ consuming assays for batch release but increasingly short timelines in product development demand for economical analytical methods with higher throughput (Ayuso, Mingozzi, and Bosch 2010; Kotin and Snyder 2017; Snyder, Audit, and Francis 2011; Wright and Zelenaia 2011; Zhao et al. 2020).”
Inhibition of a transcriptional repressor rescues hearing in a splicing factor–deficient mouse
Nakano, Y., Wiechert, S. C., Fritzsch, B., & Bánfi, B. (2020) · Life Science Alliance · doi:10.26508/lsa.202000841 · Cited by 18
“7m8 serotype, 0.5 – 3 × 10 13 viral genome/ml) were produced in HEK293 cells at Vigene Biosciences using the triple transfection method (Ayuso et al, 2010).”
Influential
Postinjury Induction of Activated ErbB2 Selectively Hyperactivates Denervated Schwann Cells and Promotes Robust Dorsal Root Axon Regeneration
Han, S. B., Kim, H., Lee, H., Grove, M., Smith, G. M., & Son, Y. (2017) · Journal of Neuroscience · doi:10.1523/jneurosci.0903-17.2017 · Cited by 18
“Recombinant self complementary adeno-associated virus 2 (scAAV2) carrying GFP was generated by helper virus-free system (Ayuso et al., 2010) as described previously (Liu et al., 2016).”
Method
Recent advances in upstream process development for production of recombinant adeno‐associated virus
Ou, J., Tang, Y., Xu, J., Tucci, J., Borys, M., & Khetan, A. (2023) · Biotechnology and Bioengineering · doi:10.1002/bit.28545 · Cited by 17
Reduction of Recombinant Adeno-Associated Virus Vector Adsorption on Solid Surfaces by Polyionic Hydrophilic Complex Coating
Ramy, S., Ueda, Y., Nakajima, H., Hiroi, M., Hiroi, Y., Torisu, T., & Uchiyama, S. (2021) · Journal of Pharmaceutical Sciences · doi:10.1016/j.xphs.2021.10.022 · Cited by 16
N-glycomic profiling of capsid proteins from Adeno-Associated Virus serotypes
Xie, Y., & Butler, M. (2023) · Glycobiology · doi:10.1093/glycob/cwad074 · Cited by 16
“…nine AAV serotypes Nine AAV serotypes were produced by using helper-free transient triple plasmid transfection of human embryo kidney (HEK) Expi293F™ cells in suspension culture as described previously with minor revision (Ayuso et al. 2010; Chahal et al. 2014; Grieger et al. 2016; Su et al. 2020).”
Method
Multi‐omics kinetic analysis of recombinant adeno‐associated virus production by plasmid transfection of HEK293 cells
Lu, M., Lee, Z., & Hu, W. (2024) · Biotechnology Progress · doi:10.1002/btpr.3428 · Cited by 17
“15 rAAV produced in insect cells also pose additional concerns about different posttranslational modifications as compared with human cell produced product.”
BackgroundMethod
A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency
Marwidi, Y., Nguyen, H.-O. B., Santos, D., Wangzor, T., Bhardwaj, S., Ernie, G., Prawdzik, G., Lew, G., Shivak, D. A., Trias, M., et al. (2024) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2024.101228 · Cited by 15
Stronger together: Analytical techniques for recombinant adeno associated virus
Hutanu, A., Boelsterli, D., Schmidli, C., Montealegre, C., Thai, M. H. N. D., Bobály, B., Koch, M., & Schwarz, M. A. (2021) · Electrophoresis · doi:10.1002/elps.202100302 · Cited by 14
Gene therapy for haemophilia A and B, from basic principles to clinical implementation: An illustrated review
Ay, C., Frenzel, L., Pinachyan, K., & Quellec, S. L. (2023) · Haemophilia · doi:10.1111/hae.14907 · Cited by 14
Co-targeting myelin inhibitors and CSPGs markedly enhances regeneration of GDNF-stimulated, but not conditioning-lesioned, sensory axons into the spinal cord
Zhai, J., Kim, H., Han, S. B., Manire, M., Yoo, R., Pang, S., Smith, G. M., & Son, Y. (2021) · eLife · doi:10.7554/elife.63050 · Cited by 14
“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).”
Method
Angiotensin 1-7 Overexpression Mediated by a Capsid-optimized AAV8 Vector Leads to Significant Growth Inhibition of Hepatocellular Carcinoma In vivo
Mao, Y., Pei, N., Chen, X., Chen, H., Yan, R., Bai, N., Li, A. I., Li, J., Zhang, Y., Du, H., et al. (2018) · International Journal of Biological Sciences · doi:10.7150/ijbs.22235 · Cited by 14
Drug product Formulation and Fill/Finish Manufacturing Process Considerations for AAV-Based Genomic Medicines
Som, M., Gikanga, B., Kanapuram, V., & Yadav, S. (2024) · Journal of Pharmaceutical Sciences · doi:10.1016/j.xphs.2024.03.024 · Cited by 13
Unlocking DOE potential by selecting the most appropriate design for rAAV optimization
Tzimou, K., Catalán-Tatjer, D., Nielsen, L. K., & Lavado‐García, J. (2024) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2024.101329 · Cited by 13
Separation of full, empty, and partial adeno-associated virus capsids via anion-exchange chromatography with continuous recycling and accumulation
Lee, Y. S., Lee, J., Fang, K., Gee, G. V., Rogers, B. J., McNally, D., & Yoon, S. (2024) · Journal of Chromatography B · doi:10.1016/j.jchromb.2024.124206 · Cited by 12
Oligodendrocyte precursor cells stop sensory axons regenerating into the spinal cord
Kim, H., Skuba, A., Xia, J., Han, S. B., Zhai, J., Hu, H., Kang, S. H., & Son, Y. (2023) · Cell Reports · doi:10.1016/j.celrep.2023.113068 · Cited by 11
Tuning capsid formation dynamics in recombinant adeno‐associated virus producing synthetic cell lines to enhance full particle productivity
Lu, M., Lin, Y., Kuo, H., Cai, W., Ye, Q., Zhao, L., & Hu, W. (2024) · Biotechnology Journal · doi:10.1002/biot.202400051 · Cited by 11
Rescue of lysosomal acid lipase deficiency in mice by rAAV8 liver gene transfer
Laurent, M., Harb, R., Jenny, C., Oustelandt, J., Jimenez, S., Cosette, J., Landini, F., Ferrante, A., Corre, G., Vujić, N., et al. (2025) · Communications Medicine · doi:10.1038/s43856-025-00816-8 · Cited by 13
“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 .”
Influential
Overview of analytics needed to support a robust gene therapy manufacturing process
Ramsey, J. P., Khatwani, S. L., Lin, M. J., Boregowda, R., Surosky, R. T., & Ramelmeier, R. A. (2021) · Current Opinion in Biomedical Engineering · doi:10.1016/j.cobme.2021.100339 · Cited by 12
Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production
Cervera, L., & Kamen, A. (2017) · Methods in molecular biology · doi:10.1007/978-1-4939-7312-5_10 · Cited by 11
Residual DNA impurities in AAV vectors—nature and transcription
Jen, H., Wilkinson, P. F., Lu, X., & Zhang, W. (2025) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2025.101503 · Cited by 10
Strategies and methods for the detection and identification of viral vectors
Stellberger, T., Koehler, N., Dinkelmeier, A., Draxler, J., Haase, M., Hellinckx, J., Pavlovic, M., Busch, U., & Baiker, A. E. (2017) · Virus Genes · doi:10.1007/s11262-017-1492-5 · Cited by 10
AAV Vectors for Efficient Gene Delivery to Rodent Hearts
López-Gordo, E., Kohlbrenner, E., Katz, M. G., & Weber, T. (2019) · Methods in molecular biology · doi:10.1007/978-1-4939-9139-6_19 · Cited by 10
Popularizing Recombinant Baculovirus-derived OneBac System for Laboratory Production of all Recombinant Adeno-associated Virus Vector Serotypes
Wu, Y., Han, Z., Duan, M., Jiang, L., Tian, T., Jin, D., Wang, Q., & Xu, F. (2021) · Current Gene Therapy · doi:10.2174/1566523221666210118111657 · Cited by 10
Gene therapy using Aβ variants for amyloid reduction
Park, K., Wood, C. A., Li, J., Taylor, B. C., Oh, S.-W., Young, N. L., & Jankowsky, J. L. (2021) · Molecular Therapy · doi:10.1016/j.ymthe.2021.02.026 · Cited by 11
Knockout of Pro‐Apoptotic BAX and BAK1 Genes in HEK293T Cells Enhances Adeno‐Associated Virus (AAV) Production: AAV2 and AAV9
Park, S.-J., Shin, S., Han, G., & Lee, G. M. (2025) · Biotechnology Journal · doi:10.1002/biot.202400529 · Cited by 9
Ultrafiltered recombinant AAV8 vector can be safely administered in vivo and efficiently transduces liver
Kleven, M. D., Gomes, M. M., Wortham, A. M., Enns, C. A., & Kahl, C. A. (2018) · PLoS ONE · doi:10.1371/journal.pone.0194728 · Cited by 9
Synergism of dual AAV gene therapy and rapamycin rescues GSDIII phenotype in muscle and liver
Jauze, L., Vie, M., Miagoux, Q., Rossiaud, L., Vidal, P., Montalvo-Romeral, V., Saliba, H., Jarrige, M., Polvèche, H., Nozi, J., et al. (2024) · JCI Insight · doi:10.1172/jci.insight.172614 · Cited by 10
Development of an HEK293 Suspension Cell Culture Medium, Transient Transfection Optimization Workflow, and Analytics for Batch rAAV Manufacturing
Green, E. A., Fu, Q., Ndahiro, N., Leibiger, T. M., Wang, Y., Lee, Y.-S., Lee, K. H., Betenbaugh, M. J., Yoon, S., & McNally, D. J. (2025) · Biotechnology and Bioengineering · doi:10.1002/bit.28980 · Cited by 9
“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).”
A novel protocol for in‐depth analysis of recombinant adeno‐associated virus capsid proteins using UHPLC–MS/MS
Zarei, M., Wang, P., Jonveaux, J., Haller, F. M., Gu, B., Koulov, A. V., & Jahn, M. (2021) · Rapid Communications in Mass Spectrometry · doi:10.1002/rcm.9247 · Cited by 8
AAV2/9-mediated gene transfer into murine lacrimal gland leads to a long-term targeted tear film modification
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Live Imaging of Mitochondrial ROS Production and Dynamic Redox Balance in Neurons
Can, K., Kügler, S., & Müller, M. (2017) · Neuromethods · doi:10.1007/978-1-4939-6890-9_9 · Cited by 8

Authors: Ayuso, E., Mingozzi, F., & Bosch, F.
Affiliations: Universitat Autònoma de Barcelona (ES)
ORCID: Ayuso, Eduard
Mingozzi, Federico
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)
DOI: 10.2174/156652310793797685
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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Production, Purification and Characterization of Adeno-Associated Vectors

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