Accurate Quantification and Characterization of Adeno-Associated Viral Vectors

Frontiers in Microbiology
David DobnikMaja Ravnikar

Abstract

One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors' characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by ...Continue Reading

References

Sep 1, 2004·BioTechniques·Peter M Vallone, John M Butler
Jun 25, 2008·Molecular Therapy : the Journal of the American Society of Gene Therapy·Philippe Moullier, Richard O Snyder
Jul 6, 2013·Medical Science Monitor Basic Research·Feng WangRuian Xu
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Aug 17, 2014·Current Opinion in Virology·Eric Zinn, Luk H Vandenberghe
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May 3, 2018·Methods in Molecular Biology·David DobnikJana Žel

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Citations

Mar 30, 2020·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·Fengting YuFujie Zhang
May 6, 2020·Human Gene Therapy·Jian XuYongling Zhu
Jun 21, 2020·Clinical Infectious Diseases : an Official Publication of the Infectious Diseases Society of America·Guillermo Aquino-Jarquin
Feb 5, 2021·Journal of Medical Virology·Amir Asri KojabadMajid Mirzaei Nodooshan
Mar 20, 2021·Molecular Therapy. Methods & Clinical Development·Andreas L GimpelRichard D Braatz
Jan 6, 2021·Journal of Microbiology and Biotechnology·Changwoo ParkSeil Kim
May 1, 2021·Genes·Cristina Martinez-Fernandez de la CamaraRobert E MacLaren
Jul 24, 2021·International Journal of Pharmaceutics·Yu Zoe ZhangTristan Marshall
Jul 27, 2021·Biotechnology and Bioengineering·Andrew D Tustian, Hanne Bak
Jul 31, 2021·Human Gene Therapy·David Nathan Quan, Joseph Shiloach

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Methods Mentioned

BETA
dot-blot
ELISA
PCR
enzyme linked immunosorbent
transmission electron microscopy
transfection

Software Mentioned

R studio
AutoDimer
Mfold
QuantaSoft
Primer Express
Excel

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