Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high-resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations but also identify partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Sub-picomole quantities of sample are sufficient for MP analysis, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor the physical attributes of rAAV vectors.
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This work was supported by the intramural program of the NHLBI, NIH. We thank Suja Hiriyanna (Ocular Gene Therapy Core, NEI, NIH) for generating the virus constructs.
This work was supported by the Intramural Research Program of the NHLBI, NIH (HL000338).
The authors declare no competing interests.
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Wu, D., Hwang, P., Li, T. et al. Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther 29, 691–697 (2022). https://doi.org/10.1038/s41434-021-00311-4