Quantitation of Trace Levels of DNA Released from Disrupted Adeno-Associated Virus Gene Therapy Vectors
| Autor: | Yu Zoe Zhang, Jared S. Bee, Megan Kuhn Phillippi, Kristin B. O'berry, Roberto A DePaz, Akanksha Kaushal, Tristan Marshall |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Treatment response
viruses Genetic enhancement Genetic Vectors Pharmaceutical Science 02 engineering and technology Vectors in gene therapy medicine.disease_cause 030226 pharmacology & pharmacy Virus 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Freezing medicine Distribution (pharmacology) Adeno-associated virus Chemistry DNA Genetic Therapy Dependovirus 021001 nanoscience & nanotechnology Molecular biology Capsid 0210 nano-technology |
| Zdroj: | Journal of pharmaceutical sciences. 110(9) |
| ISSN: | 1520-6017 |
| Popis: | Adeno-associated virus (AAV) vectors for gene therapy have potential to provide a durable treatment response for a number of diseases with unmet need. DNA is released from AAV capsids at high temperatures. Less is known about DNA release that may occur under conditions relevant to clinical and commercial manufacturing, storage, and distribution. In this work we developed and applied a sensitive fluorescent dye-based method to quantitate trace levels of DNA released from AAV capsids. The method was used to characterize the impact of manufacturing process steps on the increase (up to 1.5%) and removal (down to 0.2%) of free DNA. Free DNA increased by 0.3% per day at 37 °C and by 0.4% per freeze/thaw cycle in a phosphate-buffered saline formulation. When stored for 2 years at different temperatures, free DNA remained low (0.6%) at both ≤ -60 °C and at 2-8 °C but was higher (2.6%) when the same sample was stored at -20 °C. The dye-based method may be used to further characterize release of free DNA for different processes, formulations, and stress conditions. Overall, release of free DNA was a relatively minor degradation pathway under the conditions studied in this work. |
| Databáze: | OpenAIRE |
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