Enhanced preparation of adeno-associated viral vectors by using high hydrostatic pressure to selectively inactivate helper adenovirus
| Autor: | Joshua N. Leonard, Peter Ferstl, David V. Schaffer, Antonio Delgado |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Virus Cultivation
viruses Hydrostatic pressure Genetic Vectors Bioengineering Gene delivery Biology medicine.disease_cause Transfection Applied Microbiology and Biotechnology Virology Virus Viral vector Adenoviridae Plasmid Helper virus medicine Pressure Vector (molecular biology) Helper Viruses Biotechnology |
| Zdroj: | Biotechnology and bioengineering. 97(5) |
| ISSN: | 0006-3592 |
| Popis: | Gene delivery vectors based on adeno-associated virus (AAV) have significant therapeutic potential, but much room for improvement remains in the areas of vector engineering and production. AAV production requires complementation with either helper virus, such as adenovirus, or plasmids containing helper genes, and helper virus-based approaches have distinct advantages in the use of bioreactors to produce large quantities of AAV vectors for clinical applications. However, helper viruses must eventually be inactivated and removed from AAV preparations to ensure safety. The current practice of thermally inactivating adenovirus is problematic as it can also inactivate AAV. Here, we report a novel method using high hydrostatic pressure (HHP) to selectively and completely inactivate helper adenovirus without any detectable loss of functional AAV vectors. The pressure inactivation kinetics of human adenovirus serotype 5 and the high-pressure stabilities of AAV serotypes 2 and 5 (AAV2, AAV5), which were previously unknown, were characterized. Adenovirus was inactivated beyond detection at 260 MPa or higher, whereas AAV2 was stable up to approximately 450 MPa, and surprisingly, AAV5 was stable up to at least 700 MPa. The viral genomic DNA of pressure-inactivated AAV2 was made sensitive to DNAse I digestion, suggesting that gross changes in particle structure had occurred, and this hypothesis was further supported by transmission electron microscopy. This approach should be useful in the laboratory- and clinical-scale production of AAV gene delivery vectors. Moreover, HHP provides a tool for probing the biophysical properties of AAV, which may facilitate understanding and improving the functions of this important virus. |
| Databáze: | OpenAIRE |
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