(49f) Accelerated Virus-Less Generation of Stable Insect Sf9 Cell Lines for High-Yield Production of Influenza Vaccines

Current manufacturing of vaccines in insect Sf9 cell line relies on the Baculovirus Expression Vector System, which is limited by virus-induced cell lysis. Thus, there is an interest in the field towards virus-less gene expression to simplify the costly product purification process. Current virus-less methods are based on random integration of the gene of interest into the genome, which generates a heterogeneous cell population with a wide distribution in both protein expression and growth rate. This population is unstable over time hampering the reproducibility of vaccines production. Therefore, a high-yield stable cell line is typically isolated by limiting dilution, a low-throughput process that can take up to 8 weeks. To accelerate the generation of stable cell lines, a potential solution is to use site-specific genome editing tools such as CRISPR to rapidly create more homogenous, high-yield populations. However, the lack of genomic information of the Sf9 cell line has hindered the design of key CRISPR components and limited its utilization.

Taking advantage of the recently published Sf9 genome, we not only knocked out the fused lobes gene to enable expression of human-like glycoprotein, but also achieved targeted insertion of a short DNA sequence. As in other higher eukaryotes, however, a substantial proportion of CRISPR-mediated double stranded breaks resulted in non-templated repair by nonhomologous end joining (NHEJ). To increase the insertion efficiency, we aimed to knock out this competing repair pathway. We successfully employed bioinformatics tools and identified hypothetical genes responsible for NHEJ. More importantly, we confirmed their biological function, as their knockout via CRISPR resulted in stable Sf9 cell lines with improved efficiency in site-specific gene integration through homologous directed repair (HDR). These engineered Sf9 cell lines have enabled rapid generation of high-yield stable cell lines without the need for limiting dilution. This system facilitates high-throughput production of recombinant HA to develop improved vaccines for each year’s influenza season and offers an attractive platform to advance the development and manufacturing of other protein therapeutics.