Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression
Open accessAbstract
Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion-exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N]2SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1-SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.
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AEC separates empty and full rAAV capsids of serotypes 1, 2, 5, 6, 8, 9 based on isoelectric point differences caused by the encapsulated negatively charged genome.
“AEC has been used to separate empty and full rAAV capsids of several serotypes (1, 2, 5, 6, 8, 9) on a positively charged stationary phase based on the difference in their isoelectric points caused by the encapsulated, negatively charged genome.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [94]
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“It has been reported that the contamination of empty virions inhibits the transduction from AAV full particles after muscular injection (10).”
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- Authors
- Urabe, M., Xin, K.-Q., Obara, Y., Nakakura, T., Mizukami, H., Kume, A., Okuda, K., & Ozawa, K.
- Affiliations
- Jichi Medical University (JP)
- Yokohama City University (JP)
- Corresponding author
- Urabe, Masashi
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2006-04
- Volume
- 13
- Issue
- 4
- Pages
- 823-828
- ISSN
- 1525-0016 (ISSN-L)
- OpenAlex
- W2088763985
- Cited by
- 94
- References
- 14
- Citations by year
- 2026: 3 · 2025: 7 · 2024: 7 · 2023: 17 · 2022: 9 · 2021: 9 · 2020: 5 · 2019: 5 · 2018: 3 · 2017: 3 · 2016: 4 · 2015: 4 · 2014: 1 · 2013: 4 · 2012: 2
- FWCI
- 1.33
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Molecular Therapy (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Funders
- Ministry of Health and Welfare
- 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 › Protein purification and stability
- Keywords
- empty capsids, virus-like particles, anion exchange chromatography, isoelectric point, vector purification, transgene expression, secreted alkaline phosphatase, GFP expression
- MeSH terms
- Dependovirus, Capsid, Chromatography, Ion Exchange, Genetic Vectors, Transduction, Genetic, Gene Expression, Transgenes, Green Fluorescent Proteins
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