Filling Adeno-Associated Virus Capsids: Estimating Success by Cryo-Electron Microscopy.
Hum Gene Ther
; 30(12): 1449-1460, 2019 12.
Article
em En
| MEDLINE
| ID: mdl-31530236
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Capsídeo
/
Dependovirus
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Microscopia Crioeletrônica
/
Proteínas do Capsídeo
Tipo de estudo:
Risk_factors_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article