Galli A, Della Latta V, Bologna C, Pucciarelli D, Cipriani F, Backovic A, Cervelli T
Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, Pisa, Italy.
J Appl Microbiol. 2017 Aug;123(2):414-428. doi: 10.1111/jam.13511. Epub 2017 Jul 13.
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 (ssDNA) formation in Saccharomyces cerevisiae.
Yeast expression plasmids have been developed in which the transcription of AAV capsid proteins (VP1,2,3) is driven by the constitutive ADH1 promoter or galactose-inducible promoters. Optimal VP1,2,3 expression was obtained from GAL1/10 bidirectional promoter. Moreover, we demonstrated that AAP is expressed in yeast and virus-like particles (VLPs) assembled inside the cell. Finally, the expression of two Ad proteins, E4orf6 and E1b55k, had no effect on AAV ssDNA formation.
This study confirms that yeast is able to form AAV VLPs; however, capsid assembly and ssDNA formation are less efficient in yeast than in human cells. Moreover, the expression of Ad proteins did not affect AAV ssDNA formation.
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.
2型腺相关病毒（AAV）是一种无致病性的细小病毒，是基因治疗的一种有前景的工具。我们旨在构建用于衣壳蛋白最佳表达和组装的质粒，并评估腺病毒（Ad）蛋白对酿酒酵母中AAV单链DNA（ssDNA）形成的影响。
已开发出酵母表达质粒，其中AAV衣壳蛋白（VP1、2、3）的转录由组成型ADH1启动子或半乳糖诱导型启动子驱动。从GAL1/10双向启动子获得了最佳的VP1、2、3表达。此外，我们证明了AAP在酵母中表达，并且细胞内组装了病毒样颗粒（VLP）。最后，两种Ad蛋白E4orf6和E1b55k的表达对AAV ssDNA的形成没有影响。
本研究证实酵母能够形成AAV VLP；然而，衣壳组装和ssDNA形成在酵母中比在人类细胞中效率更低。此外，Ad蛋白的表达不影响AAV ssDNA的形成。
需要新的基于AAV的基因治疗载体（rAAV）制造策略来降低成本和生产时间。我们的研究探索了酵母作为rAAV生产替代系统的可行性。
