Engineering pseudotyped baculoviruses for gene editing in mammalian cells

Abstract

Baculoviruses (BVs) are a promising gene delivery tool, capable of accommodating large DNA cargoes and providing an all-in-one solution for precise gene editing when coupled with CRISPR technology. For effective transduction into mammalian cells, BVs require pseudotyping, with commonly used proteins being GP64 (the native baculovirus envelope protein) and Vesicular Stomatitis Virus G glycoprotein (VSV-G). While both offer broad tropism, VSV-G plays a critical role in facilitating endosomal escape. However, its expression is linked to cytotoxicity in mammalian cells.
Current recombinant baculoviruses use a polyhedrin (polH) promoter to express VSV-G, one of the strongest baculoviral promoters. This one-year MScR project aimed to assess whether regulating VSV-G expression through alternative promoters could reduce cytotoxicity without compromising transduction efficiency. Five promoters (gp64, vp39, orf-81, orf-13, and lef8) were chosen based on mRNA expression levels, and plasmids were designed to replace the polH promoter.
Recombinant viruses were produced in Sf21 cells, and western blot analysis revealed that all alternative promoters significantly reduced VSV-G expression, with orf-81 and orf-13 showing expression levels below 5%. Despite this decrease, transduction efficiency achieved in HEK293T cells remained comparable to polH VSV-G BVs with significantly improved cell viability observed across most cell lines tested, including A549 and HeLa. In primary HUVEC, Orf-81 and Orf-13 VSV-G pseudotyped viruses even exhibited higher transduction efficiency and less cytotoxicity compared to polH VSV-G viruses.
Concentration studies showed that non-pseudotyped, orf-81, and orf-13 VSV-G pseudotyped viruses retained stability over time, while polH VSV-G viruses exhibited up to a 50% loss in titre. Cryo-EM imaging revealed that VSV-G hyperexpression disrupted viral morphology, resulting in membrane envelope enlargement and viral aggregation. This could explain the faster decline in viral titres following concentration.
Future work could involve expanding gene-editing experiments to additional cell lines and quantifying the gene-editing events via DNA sequencing. This would be followed by the development of a new bacmid containing an orf-13 VSV-G expression cassette for improved gene delivery applications.

Date of Award	17 Jun 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Imre Berger (Supervisor) & Mark D Szczelkun (Supervisor)