Productivity and genetic stability of a novel baculovirus vector for multigene expression from independent transgene loci

The baculovirus expression vector system is an established platform for large-scale production of (glyco)proteins, subunit vaccines, virus-like particles, and recombinant adeno-associated virus (rAAV) vectors. We engineered a novel bacmid vector (BAC6) to improve genetic stability by deletion of the non-homologous repeat (hr) origin of DNA replication (ori) and to preserve product integrity and recovery through deletion of chitinase and cathepsin. Tn7-based transposition in E. coli and homologous recombination in insect cells were combined in BAC6 to drive expression from the odv-e56 and polyhedrin loci, respectively. Virus growth kinetics of BAC6 were similar to the parental bacmid, and genetic stability was investigated for at least eight serial passages at high multiplicity of infection. Next-generation sequencing was used to identify mutations, deletions, and defective interfering particle (DIP) formation, which became apparent only in later passages. With BAC6, the enrichment of DIPs originating from the non-hr ori was prevented. BAC6 versatility was demonstrated by high-yield (12 mg/L) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike production in suspension Sf9 insect cells. Finally, rAAV production with BAC6, simultaneously employing both transgene insertion sites, resulted in yields of 5.8e10 AAV capsids/mL and 2.3e10 genome copies/mL. BAC6 provides insertion of multiple transgenes at two different loci and is non-inferior to commercial baculovirus expression vectors with regard to genetic stability and productivity.