Small-Scale Recombinant Adeno-Associated Virus Purification

Recombinant adeno-associated virus (rAAV) vectors have become increasingly popular in research and clinical trials due to their efficient gene transfer and long-term expression in tissues including brain. In addition, rAAV has demonstrated an impressive safety profile in gene therapy trials. The eme...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1382; p. 95
Main Authors Burger, Corinna, Nash, Kevin R
Format Journal Article
LanguageEnglish
Published United States 2016
Subjects
Dependovirus - genetics
Dependovirus - isolation & purification
Genetic Vectors
HEK293 Cells
Humans
Triiodobenzoic Acids - chemistry
Viral Load
Tissue culture
Adeno-associated virus
Iodixanol gradient purification
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Summary:Recombinant adeno-associated virus (rAAV) vectors have become increasingly popular in research and clinical trials due to their efficient gene transfer and long-term expression in tissues including brain. In addition, rAAV has demonstrated an impressive safety profile in gene therapy trials. The emergence of rAAV serotypes with different cell tropisms and distribution properties has allowed scientists to tailor serotypes to specific experimental needs. AAV does not have a cytopathic effect; therefore, purification methods require extraction of the viral vector from the cell. This involves gradient ultracentrifugation of the cellular extract sometimes followed by chromatography. This chapter describes a small-scale production method for rAAV purification from ten to twenty 15 cm plates of human embryonic kidney-derived 293B cells (HEK 293) cells that can yield approximately 300 μl of a 5 × 10(12) to 1 × 10(13) genome copies/ml viral preparation final concentration.
ISSN:1940-6029
DOI:10.1007/978-1-4939-3271-9_7