Establishment of an Sf9 VSV-G cell-line for pseudotyping baculoviral vectors to enhanced transduction efficiency in mammals

PLASTINE MARÍA DEL PILAR; AMALFI SABRINA; TABOGA OSCAR; ALFONSO VICTORIA

Simposio; Simposio Fronteras en Biociencia 4; 2023

Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)

Baculoviruses show potential as gene delivery vectors in mammals; however, their transduction efficiency varies depending on the cell line. A widely employed strategy to improve cell tropism and increase transduction efficiency is the pseudotyping of viral vectors. In this study, we aimed to develop a stable Sf9 insect cell line that inducibly expresses the G-protein of the vesicular stomatitis virus (VSV-G) to pseudotype budded baculoviruses. Initially, VSV-G gene was cloned into a plasmid under the control of the very late, strong, and infection-inducible pXXL promoter, which was previously constructed in our laboratory. The Sf9-VSVG stable cell line was obtained by blasticidin resistance selection and was subsequently diluted to establish oligoclonal cell lines. Since VSV-G is a fusogenic protein, we selected specific clones based on the size and number of syncytia observed after baculovirus infection. In addition, the sub-clones were evaluated by the VSV-G expression levels in the cell extracts after infection and for the final selection of a single cellular clone, the incorporation of VSV-G into purified viral particles was assessed. Next, to enhance the cell line performance, the infection conditions under which functional pseudotyped baculoviruses are obtained were optimized. Finally, different baculoviruses expressing eGFP under the control of mammalian promoters were pseudotyped and the expression of the reporter gene was quantified in mammalian cells of diverse origins using flow cytometry. The transduction efficiency of pseudotyped baculovirus consistently increased in all tested mammalian cell lines compared with control viruses. These findings demonstrate the feasibility and advantages of improving gene delivery performance without the need to insert the pseudotyping gene into the baculoviral genomes.