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TitleFilling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy.
Journal, issue, pagesHum Gene Ther, Vol. 30, Issue 12, Page 1449-1460, Year 2019
Publish dateNov 14, 2019
AuthorsSuriyasri 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 /
PubMed AbstractAdeno-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, ...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.
External linksHum Gene Ther / PubMed:31530236 / PubMed Central
MethodsEM (single particle)
Resolution3.26 - 3.42 Å
Structure data

EMDB-20624:
Empty AAV3B
Method: EM (single particle) / Resolution: 3.26 Å

EMDB-20625:
AAV3B genome containing
Method: EM (single particle) / Resolution: 3.42 Å

Source
  • Homo sapiens (human)

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