Viruses, Vol. 15, Pages 1983: Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System

Viruses, Vol. 15, Pages 1983: Exploring the Comprehensive Kozak Sequence Landscape for AAV Production in Sf9 System Viruses doi: 10.3390/v15101983 Authors: Oleksandr Kondratov Sergei Zolotukhin 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) an...
Source: Viruses - Category: Virology Authors: Tags: Article Source Type: research