Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy

Suriyasri Subramanian, Anna C. Maurer, Carol M. Bator, Alexander M. Makhov, James F. Conway, Kevin B. Turner, James H. Marden, Luk H. Vandenberghe, Susan L. Hafenstein

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1449-1460
Number of pages12
JournalHuman Gene Therapy
Volume30
Issue number12
DOIs
StatePublished - Dec 2019

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All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

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. (2019). Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy. Human Gene Therapy, 30(12), 1449-1460. https://doi.org/10.1089/hum.2019.041