Recombinant AAV genome size effect on viral vector production, purification, and thermostability
Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfectio...
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| Format: | Article |
| Language: | English |
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Elsevier
2024-03-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050124000044 |
| _version_ | 1797328737370374144 |
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| author | Nermin Ibreljic Benjamin E. Draper Carl W. Lawton |
| author_facet | Nermin Ibreljic Benjamin E. Draper Carl W. Lawton |
| author_sort | Nermin Ibreljic |
| collection | DOAJ |
| description | Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfection using AAV plasmids containing the same GFP transgene with DNA stuffers for variable-size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography, and the results suggest that the triple transfection condition significantly affects the AEX retention time and resolution between the full and empty capsid peaks. Charge detection-mass spectrometry was performed on all AEX full-capsid peak samples showing a wide distribution of empty, partial, full length, and copackaged DNA in the capsids. The AEX-purified samples were then analyzed by differential scanning fluorimetry, and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content. |
| first_indexed | 2024-03-08T06:56:00Z |
| format | Article |
| id | doaj.art-79bcb01b76c746f388173992dbe362e4 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-03-08T06:56:00Z |
| publishDate | 2024-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-79bcb01b76c746f388173992dbe362e42024-02-03T06:35:45ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012024-03-01321101188Recombinant AAV genome size effect on viral vector production, purification, and thermostabilityNermin Ibreljic0Benjamin E. Draper1Carl W. Lawton2Department of Chemical Engineering, University of Massachusetts Lowell, One University Avenue, Lowell, MA 01854, USA; Sarepta Therapeutics, 55 Blue Sky Drive, Burlington, MA 01803, USA; Corresponding author: Nermin Ibreljic, Department of Chemical Engineering, University of Massachusetts Lowell, One University Avenue, Lowell, MA 01854, USA.Megadalton Solutions, 3750 E. Bluebird Lane, Bloomington, IN 47401, USADepartment of Chemical Engineering, University of Massachusetts Lowell, One University Avenue, Lowell, MA 01854, USAAdeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. The present work studied the effect of genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension-adapted HEK293 cells via triple transfection using AAV plasmids containing the same GFP transgene with DNA stuffers for variable-size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography, and the results suggest that the triple transfection condition significantly affects the AEX retention time and resolution between the full and empty capsid peaks. Charge detection-mass spectrometry was performed on all AEX full-capsid peak samples showing a wide distribution of empty, partial, full length, and copackaged DNA in the capsids. The AEX-purified samples were then analyzed by differential scanning fluorimetry, and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.http://www.sciencedirect.com/science/article/pii/S2329050124000044gene therapyadeno-associated virusviral genome titercapsid titertriple transfectionanion exchange chromatography |
| spellingShingle | Nermin Ibreljic Benjamin E. Draper Carl W. Lawton Recombinant AAV genome size effect on viral vector production, purification, and thermostability Molecular Therapy: Methods & Clinical Development gene therapy adeno-associated virus viral genome titer capsid titer triple transfection anion exchange chromatography |
| title | Recombinant AAV genome size effect on viral vector production, purification, and thermostability |
| title_full | Recombinant AAV genome size effect on viral vector production, purification, and thermostability |
| title_fullStr | Recombinant AAV genome size effect on viral vector production, purification, and thermostability |
| title_full_unstemmed | Recombinant AAV genome size effect on viral vector production, purification, and thermostability |
| title_short | Recombinant AAV genome size effect on viral vector production, purification, and thermostability |
| title_sort | recombinant aav genome size effect on viral vector production purification and thermostability |
| topic | gene therapy adeno-associated virus viral genome titer capsid titer triple transfection anion exchange chromatography |
| url | http://www.sciencedirect.com/science/article/pii/S2329050124000044 |
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