(127b) 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 successfully employed bioinformatics tools and identified hypothetical genes responsible for nonhomologous end joining (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. Specifically, two Sf9 stable cells lines each producing high-yield influenza hemagglutinin subtype 1 (H1) and subtype 3 (H3) were generated within just one week. In addition to improving the efficiency of HDR for gene knockin, NHEJ-gene KO also improved the efficiency of using CRISPR to knockout other genes that are important for expressing human-like glycosylated H1/H3 proteins for vaccination. This system enables high-throughput production of recombinant HA to develop improved vaccines for each year’s influenza season. Future work will focus on the production of more complex protein therapeutics such as multivalent subunit vaccines or complex virus-like particles. The accelerated generation of insect cell lines offers an attractive platform to advance the development and manufacture of any protein therapeutic.