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Hum Gene Ther. 2019 Sep 18. doi: 10.1089/hum.2019.041. [Epub ahead of print]

Filling AAV capsids: estimating success by cryo-EM.

Author information

1
Penn State College of Medicine Department of Microbiology and Immunology, 219261, Hershey, Pennsylvania, United States; sxs1161@psu.edu.
2
Harvard University, 1812, Cambridge, Massachusetts, United States; amaurer@fas.harvard.edu.
3
Penn State, 311285, University Park, Pennsylvania, United States; czb221@psu.edu.
4
University of Pittsburgh, 6614, Pittsburgh, Pennsylvania, United States; amm241@pitt.edu.
5
University of Pittsburgh, 6614, Pittsburgh, Pennsylvania, United States; jxc100@pitt.edu.
6
University of Pennsylvania, 6572, Philadelphia, Pennsylvania, United States; keturner@upenn.edu.
7
Penn State, 311285, University Park, Pennsylvania, United States; jhm10@psu.edu.
8
Mass Eye and Ear / Harvard Medical School, Ophthalmology, 20 Staniford Street, Massachusetts Eye and Ear Infirmary, Schepens Eye Research Institute, Boston, Massachusetts, United States, 02114.
9
Pennsylvania, United States; luk_vandenberghe@meei.harvard.edu.
10
Penn State, 311285, W231 MSC building, University Park, Pennsylvania, United States, 16802-1800.
11
Penn State College of Medicine Department of Microbiology and Immunology, 219261, Hershey, Pennsylvania, United States, 17033-0850; shafenstein@psu.edu.

Abstract

Adeno-associated viruses (AAV) 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 2D class averages and 3D 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 analysed 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 vs 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 analyse vector preparations.

PMID:
31530236
DOI:
10.1089/hum.2019.041

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