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- W4386996941 abstract "The widespread successful use of recombinant Adeno-associated virus (rAAV) in gene therapy has driven the demand for scale-up manufacturing methods of vectors with optimized yield and transduction efficiency. The Baculovirus/Sf9 system is a promising platform for high yield production; however, a major drawback to using an invertebrate cell line compared to a mammalian system is a generally altered AAV capsid stoichiometry resulting in lower biological potency. Here, we introduce a term of the structural and biological “fitness” of an AAV capsid as a function of two interdependent parameters: (1) packaging efficiency (yield), and (2) transduction efficiency (infectivity). Both parameters are critically dependent on AAV capsid structural proteins VP1/2/3 stoichiometry. To identify an optimal AAV capsid composition, we developed a novel Directed Evolution (DE) protocol for assessing the structural and biological fitness of Sf9-manufactured rAAV for any given serotype. The approach involves the packaging of a combinatorial capsid library in insect Sf9 cells, followed by a library screening for high infectivity in human Cre–recombinase-expressing C12 cells. One single DE selection round, complemented by Next-Generation Sequencing (NGS) and guided by in silico analysis, identifies a small subset of VP1 translation initiation sites (known as Kozak sequence) encoding “fit” AAV capsids characterized by a high production yield and superior transduction efficiencies." @default.
- W4386996941 created "2023-09-25" @default.
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- W4386996941 date "2023-09-23" @default.
- W4386996941 modified "2023-09-30" @default.
- W4386996941 title "Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System" @default.
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- W4386996941 doi "https://doi.org/10.3390/v15101983" @default.
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