Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy
Open accessAbstract
Adeno-associated viruses (AAVs) have been employed successfully as gene therapy vectors in treating various genetic diseases for almost two decades. However, transgene packaging is usually imperfect, and developing a rapid and accurate method for measuring the proportion of DNA encapsidation is an important step for improving the downstream process of large scale vector production. In this study, we used two-dimensional class averages and three-dimensional classes, intermediate outputs in the single particle cryo-electron microscopy (cryo-EM) image reconstruction pipeline, to determine the proportion of DNA-packaged and empty capsid populations. Two different preparations of AAV3 were analyzed to estimate the minimum number of particles required to be sampled by cryo-EM in order for robust calculation of the proportion of the full versus empty capsids in any given sample. Cost analysis applied to the minimum amount of data required for a valid ratio suggests that cryo-EM is an effective approach to analyze vector preparations.
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cryo-EM is used for quantifying content ratio and validated as a release testing assay.
“In addition to TEM, cryogenic electron microscopy (cryo-EM) is used for quantifying the content ratio and validated as a release testing assay.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [88]
TEM low accuracy is often caused by dimly stained, partially filled particles and uneven staining, leading to low contrast and ambiguity in capsid identification.
“Low accuracy is often caused by dimly stained, partially filled particles and uneven staining, leading to low contrast and ambiguity in identification of capsids.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [88]
cryo-EM is more robust than TEM due to interference of cell debris and uneven staining of capsids in TEM.
“Although one study found no significant difference in the measured content ratio between TEM and cryo-EM,89 the latter has been reported to be more robust than the former, owing to the commonly observed interference of cell debris and uneven staining of capsids in TEM.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [88]
Although TEM can be used on non-purified samples, it is commonly used only with purified samples, as proteins and cell debris present in the cell lysate can interfere with accurate identification of rAAV capsids in TEM images.
“Although it has been reported that TEM can be used on non-purified samples, it is commonly used only with purified samples, as proteins and cell debris present in the cell lysate can interfere with the accurate identification of rAAV capsids in TEM images.77,88”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [88]
Multiple thousands of capsids must be imaged and analyzed for statistical significance in TEM.
“Multiple thousands of capsids must be imaged and analyzed for statistical significance, with the capsid content of individual images varying by around 2%.”
From Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies (2021) · cites this paper as [88]
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Gimpel, A. L., Katsikis, G., Sha, S., Maloney, A. J., Hong, M. S., Nguyen, T. N. T., Wolfrum, J., Springs, S. L., Sinskey, A. J., Manalis, S. R., et al. (2021) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2021.02.010 · Cited by 185 · see citing works
- Authors
- Subramanian, S., Maurer, A. C., Bator, C. M., Makhov, A. M., Conway, J. F., Turner, K. B., Marden, J. H., Vandenberghe, L. H., & Hafenstein, S. L.
- Affiliations
- Pennsylvania State University (US)
- Broad Institute (US)
- University of Pittsburgh (US)
- University of Pennsylvania (US)
- Corresponding author
- Hafenstein, Susan L.
- ORCID
- Venue
- Human Gene Therapy
- Publisher
- SAGE Publications
- Published
- 2019-12
- Volume
- 30
- Issue
- 12
- Pages
- 1449-1460
- ISSN
- 1043-0342 (ISSN-L)
- 1557-7422
- PMC
- PMC6921155
- OpenAlex
- W2987565474
- Cited by
- 40
- References
- 70
- Citations by year
- 2026: 3 · 2025: 4 · 2024: 8 · 2023: 7 · 2022: 7 · 2021: 10 · 2020: 1
- FWCI
- 2.71
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Human Gene Therapy (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Pennsylvania Department of Health
- National Institutes of Health (1s10od019995-01)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Structural Biology › Advanced Electron Microscopy Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- adeno-associated virus, DNA encapsidation, empty capsids, gene therapy vectors, cryo-electron microscopy, cost analysis
- MeSH terms
- Dependovirus, Capsid, Genetic Vectors, Humans, Virion, Cryoelectron Microscopy, Capsid Proteins
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