Enhanced baculovirus vectors with higher titers and increased genome stability

DESCRIPTION provided by applicant The baculovirus expression vector system BEVS has been successfully utilized to produce thousands of proteins for use as vaccines and therapeutics as well as for studies of protein structure and function One limitation of BEVS is the propensity of baculoviruses to accumulate transposon insertions into the fp k gene leading to the andquot few polyhedra FP andquot phenotype This mutation shifts the balance of virus production from occlusion derived viruses which are not infectious in tissue culture to budded viruses BV which are the form of virus that is used in baculovirus expression Higher levels of BV would be advantageous for BEVS users but FP mutants are also deficient in transcription from the polyhedrin promoter that drives expression of target genes Baculoviruses also rapidly accumulate defective interfering particles DIP which are often linked to a sharp decrease in target gene expression due to deletion of the target gene and or the viral genes needed for its expression One factor promoting DIP formation is transposition into fp k The goal of this proposal is to limit deleterious effects of transposition into fp k while taking advantage of the fact that elimination of the FP K activity significantly increases BV production During Phase I two strategies an inducible construct for controlling fp k and an fp k deletion mutant coupled with a cell line constitutively expressing FP K were explored for regulating FP K expression Both strategies sought to produce high titer virus during the amplification stage of baculovirus infection and enable a switch to high level transcription from the polyhedrin promoter during the recombinant protein expression phase Results document achievement of Phase I objectives with deletion of fp k from the virus and complementary expression from an engineered cell line enabling the predicted control of budded virus and recombinant protein production However the inducible construct did not provide sufficient FP K control due to the toxicity of the heavy metal inducer to insect cells Therefore in Phase II we will develop an improved inducible construct with tighter transcriptional regulation using an ecdysone receptor based inducible promoter validate the beneficial effects of FP K regulation using viruses that express intracellular and secreted yellow fluorescent protein YFP for BEVS and BacMam mammalian expression technology develop user friendly fp k mutant backbones for simplified BEVS and BacMam cloning and demonstrate their utility with several medically relevant transgenes and test the fp k expression system in the context of vankyrin enhanced BEVS VE BEVSTM technology ParaTechsandapos VE BEVS products delay death and lysis of baculovirus infected cells thereby boosting target protein expression up to fold Many BEVS users would want to incorporate both vankyrin and FP K technologies therefore it is important to determine whether they are compatible Taken together completion of Phase II objectives will enable users of the BEVS to control and optimize production of BV and recombinant protein expression PUBLIC HEALTH RELEVANCE The baculovirus expression vector system BEVS is commonly used for production of proteins for structure function studies vaccines and therapeutics Unfortunately many researchers are not aware that certain mutations rapidly accumulate leading to lower levels of target gene expression ParaTechs has developed a strategy to prevent these mutations while simultaneously providing higher levels of virus production stable genome maintenance and target gene expression