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Strategies to optimize capsid protein expression and single‐stranded DNA formation of adeno‐associated virus in Saccharomyces cerevisiae
Author(s) -
Galli A.,
Della Latta V.,
Bologna C.,
Pucciarelli D.,
Cipriani F.,
Backovic A.,
Cervelli T.
Publication year - 2017
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13511
Subject(s) - capsid , plasmid , saccharomyces cerevisiae , adeno associated virus , biology , yeast , recombinant dna , dna , gene , transcription (linguistics) , virus like particle , promoter , microbiology and biotechnology , gene expression , expression vector , virus , virology , vector (molecular biology) , genetics , linguistics , philosophy
Aims Adeno‐associated virus type 2 ( AAV ) is a nonpathogenic parvovirus that is a promising tool for gene therapy. We aimed to construct plasmids for optimal expression and assembly of capsid proteins and evaluate adenovirus (Ad) protein effect on AAV single‐stranded DNA (ss DNA ) formation in Saccharomyces cerevisiae . Methods and Results Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins ( VP 1,2,3) is driven by the constitutive ADH 1 promoter or galactose‐inducible promoters. Optimal VP 1,2,3 expression was obtained from GAL 1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus‐like particles ( VLP s) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ss DNA formation. Conclusions This study confirms that yeast is able to form AAV VLP s; however, capsid assembly and ss DNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ss DNA formation. Significance and Impact of the Study New manufacturing strategies for AAV ‐based gene therapy vectors ( rAAV ) are needed to reduce costs and time of production. Our study explores the feasibility of yeast as alternative system for rAAV production.