High-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling Article Swipe
Hao Liu
,
Nan Liu
,
Chen Zhou
,
Ailing Du
,
Mayank Kapadia
,
Phillip W.L. Tai
,
Erik S. Barton
,
Xiupeng Chen
,
Dan Wang
·
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1038/s44321-025-00248-w
YOU?
·
· 2025
· Open Access
·
· DOI: https://doi.org/10.1038/s44321-025-00248-w
Triple transfection of HEK293 cells is the most widely used method for producing recombinant adeno-associated virus (rAAV), a leading gene delivery vector for human gene therapy. Despite its tremendous success, this approach generates several vector-related impurities that could potentially compromise the safety and potency of rAAV. In this study, we introduce a method for high-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling (AAVPure Mfg ). Compared with traditional triple transfection, AAVPure Mfg substantially improves vector purity by reducing prokaryotic DNA contaminants by 10- to 50-fold and increasing the full capsid ratio up to threefold. Mechanistically, Bxb1-mediated excision of the transgene cassette generates a minicircle cis construct devoid of bacterial sequences and ensures synchronized colocalization of trans and cis constructs in productive cells. Furthermore, we developed iterations that enhance vector genome homogeneity and streamline the production of rAAV with various transgenes, serotypes, and ITR configurations. Overall, our findings demonstrate that AAVPure Mfg overcomes the inherent limitations associated with triple transfection, offering a broadly applicable and easy-to-implement method for producing high-purity rAAV with reduced plasmid costs.
Related Topics
Concepts
Minicircle
Recombination
Coupling (piping)
Vector (molecular biology)
Biology
Production (economics)
Physics
Recombinant DNA
Genetics
Materials science
Gene
Metallurgy
Economics
Macroeconomics
Metadata
- Type
- article
- Language
- en
- Landing Page
- https://doi.org/10.1038/s44321-025-00248-w
- OA Status
- gold
- Cited By
- 1
- References
- 49
- Related Works
- 10
- OpenAlex ID
- https://openalex.org/W4410441954
All OpenAlex metadata
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- OpenAlex ID
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https://openalex.org/W4410441954Canonical identifier for this work in OpenAlex
- DOI
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https://doi.org/10.1038/s44321-025-00248-wDigital Object Identifier
- Title
-
High-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic couplingWork title
- Type
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articleOpenAlex work type
- Language
-
enPrimary language
- Publication year
-
2025Year of publication
- Publication date
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2025-05-16Full publication date if available
- Authors
-
Hao Liu, Nan Liu, Chen Zhou, Ailing Du, Mayank Kapadia, Phillip W.L. Tai, Erik S. Barton, Xiupeng Chen, Dan WangList of authors in order
- Landing page
-
https://doi.org/10.1038/s44321-025-00248-wPublisher landing page
- Open access
-
YesWhether a free full text is available
- OA status
-
goldOpen access status per OpenAlex
- OA URL
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https://doi.org/10.1038/s44321-025-00248-wDirect OA link when available
- Concepts
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Minicircle, Recombination, Coupling (piping), Vector (molecular biology), Biology, Production (economics), Physics, Recombinant DNA, Genetics, Materials science, Gene, Metallurgy, Economics, MacroeconomicsTop concepts (fields/topics) attached by OpenAlex
- Cited by
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1Total citation count in OpenAlex
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2025: 1Per-year citation counts (last 5 years)
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49Number of works referenced by this work
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10Other works algorithmically related by OpenAlex
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| abstract_inverted_index.triple | 72, 161 |
| abstract_inverted_index.vector | 22, 57, 78, 132 |
| abstract_inverted_index.widely | 9 |
| abstract_inverted_index.(rAAV), | 17 |
| abstract_inverted_index.50-fold | 88 |
| abstract_inverted_index.AAVPure | 74, 153 |
| abstract_inverted_index.Despite | 27 |
| abstract_inverted_index.broadly | 165 |
| abstract_inverted_index.enhance | 131 |
| abstract_inverted_index.ensures | 115 |
| abstract_inverted_index.genetic | 64 |
| abstract_inverted_index.leading | 19 |
| abstract_inverted_index.plasmid | 176 |
| abstract_inverted_index.potency | 44 |
| abstract_inverted_index.reduced | 175 |
| abstract_inverted_index.several | 34 |
| abstract_inverted_index.various | 142 |
| abstract_inverted_index.(AAVPure | 66 |
| abstract_inverted_index.Abstract | 0 |
| abstract_inverted_index.Compared | 69 |
| abstract_inverted_index.Overall, | 148 |
| abstract_inverted_index.approach | 32 |
| abstract_inverted_index.cassette | 104 |
| abstract_inverted_index.coupling | 65 |
| abstract_inverted_index.delivery | 21 |
| abstract_inverted_index.excision | 100 |
| abstract_inverted_index.findings | 150 |
| abstract_inverted_index.improves | 77 |
| abstract_inverted_index.inherent | 157 |
| abstract_inverted_index.offering | 163 |
| abstract_inverted_index.reducing | 81 |
| abstract_inverted_index.success, | 30 |
| abstract_inverted_index.therapy. | 26 |
| abstract_inverted_index.bacterial | 112 |
| abstract_inverted_index.construct | 109 |
| abstract_inverted_index.developed | 128 |
| abstract_inverted_index.formation | 62 |
| abstract_inverted_index.generates | 33, 105 |
| abstract_inverted_index.introduce | 51 |
| abstract_inverted_index.overcomes | 155 |
| abstract_inverted_index.producing | 13, 171 |
| abstract_inverted_index.sequences | 113 |
| abstract_inverted_index.transgene | 103 |
| abstract_inverted_index.utilizing | 59 |
| abstract_inverted_index.applicable | 166 |
| abstract_inverted_index.associated | 159 |
| abstract_inverted_index.compromise | 40 |
| abstract_inverted_index.constructs | 122 |
| abstract_inverted_index.impurities | 36 |
| abstract_inverted_index.increasing | 90 |
| abstract_inverted_index.iterations | 129 |
| abstract_inverted_index.minicircle | 61, 107 |
| abstract_inverted_index.production | 58, 138 |
| abstract_inverted_index.productive | 124 |
| abstract_inverted_index.serotypes, | 144 |
| abstract_inverted_index.streamline | 136 |
| abstract_inverted_index.threefold. | 97 |
| abstract_inverted_index.tremendous | 29 |
| abstract_inverted_index.demonstrate | 151 |
| abstract_inverted_index.high-purity | 55, 172 |
| abstract_inverted_index.homogeneity | 134 |
| abstract_inverted_index.limitations | 158 |
| abstract_inverted_index.potentially | 39 |
| abstract_inverted_index.prokaryotic | 82 |
| abstract_inverted_index.recombinant | 14 |
| abstract_inverted_index.traditional | 71 |
| abstract_inverted_index.transgenes, | 143 |
| abstract_inverted_index.Furthermore, | 126 |
| abstract_inverted_index.contaminants | 84 |
| abstract_inverted_index.synchronized | 116 |
| abstract_inverted_index.transfection | 2 |
| abstract_inverted_index.Bxb1-mediated | 99 |
| abstract_inverted_index.substantially | 76 |
| abstract_inverted_index.transfection, | 73, 162 |
| abstract_inverted_index.colocalization | 117 |
| abstract_inverted_index.vector-related | 35 |
| abstract_inverted_index.configurations. | 147 |
| abstract_inverted_index.Mechanistically, | 98 |
| abstract_inverted_index.adeno-associated | 15 |
| abstract_inverted_index.easy-to-implement | 168 |
| abstract_inverted_index.recombination-dependent | 60 |
| cited_by_percentile_year.max | 95 |
| cited_by_percentile_year.min | 91 |
| countries_distinct_count | 1 |
| institutions_distinct_count | 9 |
| citation_normalized_percentile.value | 0.89265983 |
| citation_normalized_percentile.is_in_top_1_percent | False |
| citation_normalized_percentile.is_in_top_10_percent | True |