SynBac: Synthetic Baculovirus Genome for Next-generation Drug Discovery
Lead Research Organisation:
University of Bristol
Department Name: Biochemistry
Abstract
Baculovirus is a highly efficient delivery system for recombinant genes into eukaryotic cells, with great impact on the production of eukaryotic proteins, including high-value drug targets. Vaccines against cervicular cancer and others are produced by this method. More recently, baculovirus has emerged as a versatile tool for gene therapy. We contributed to the field the award-winning MultiBac technology for multiprotein complex research.
These applications, at the forefront of modern biology, rely on a large baculovirus genome (130 kb) derived from AcMNPV. This genome has been intensively researched, mainly by entymologists. Genes essential for propagation in nature and in cell culture were delineated and DNA elements which impede applications in the laboratory. Genetic alterations of the wild-type viral genome have been performed, by classical knock-out technologies, to improve gene insertion, delivery and protein production properties. Such alterations require excessive effort by specialists. Therefore, it is currently not possible to fully exploit the vast potential of the baculovirus system.
In the present project, we boldly propose to fully reverse the current approach. We will design in silico and construct in vitro new, fully synthetic customized baculovirus genomes which will be, for the first time, in a streamlined, highly versatile format for multigene transfer and the production of high-value, next generation recombinant protein targets for drug discovery. We will apply state-of-the-art genome editing tools, notably CRISPR-Cas9, to inform our approach by systematically disrupting genes and non-coding regions including gene regulatory elements. We further aim to address the "scale-up problem" which currently impedes pharma-scale biologics production by baculoviral systems. As proof-of-concept, we already created a partly synthetic hybrid genome by replacing a large part (20 kb) of wild-type with designer DNA. Rigorous testing of this prototype compellingly validated our approach.
The CASE fellow will:
(1) utilize computational biology, comparative bioinformatics and data mining to create blueprints for optimized minimal baculovirus genomes.
(2) Synthesize designer genomes in large fragments, and use advanced recombination technologies to assemble these into functional genomes (AstraZeneca platform).
(3) Exploit cutting-edge CRISPR-Cas9 tool to edit genes and regulatory DNA elements in the baculoviral genome in a parallelized fashion (AstraZeneca platform) and implement this information in the synthetic design.
(4) Address the "scale-up problem" by reconfiguring the very late phase of baculoviral life cycle ("hyperburst management").
(5) Rigorously validate novel designer genomes experimentally.
These will be the first fully synthetic baculoviral genomes, with the potential to transform academic and industrial R&D applications.
These applications, at the forefront of modern biology, rely on a large baculovirus genome (130 kb) derived from AcMNPV. This genome has been intensively researched, mainly by entymologists. Genes essential for propagation in nature and in cell culture were delineated and DNA elements which impede applications in the laboratory. Genetic alterations of the wild-type viral genome have been performed, by classical knock-out technologies, to improve gene insertion, delivery and protein production properties. Such alterations require excessive effort by specialists. Therefore, it is currently not possible to fully exploit the vast potential of the baculovirus system.
In the present project, we boldly propose to fully reverse the current approach. We will design in silico and construct in vitro new, fully synthetic customized baculovirus genomes which will be, for the first time, in a streamlined, highly versatile format for multigene transfer and the production of high-value, next generation recombinant protein targets for drug discovery. We will apply state-of-the-art genome editing tools, notably CRISPR-Cas9, to inform our approach by systematically disrupting genes and non-coding regions including gene regulatory elements. We further aim to address the "scale-up problem" which currently impedes pharma-scale biologics production by baculoviral systems. As proof-of-concept, we already created a partly synthetic hybrid genome by replacing a large part (20 kb) of wild-type with designer DNA. Rigorous testing of this prototype compellingly validated our approach.
The CASE fellow will:
(1) utilize computational biology, comparative bioinformatics and data mining to create blueprints for optimized minimal baculovirus genomes.
(2) Synthesize designer genomes in large fragments, and use advanced recombination technologies to assemble these into functional genomes (AstraZeneca platform).
(3) Exploit cutting-edge CRISPR-Cas9 tool to edit genes and regulatory DNA elements in the baculoviral genome in a parallelized fashion (AstraZeneca platform) and implement this information in the synthetic design.
(4) Address the "scale-up problem" by reconfiguring the very late phase of baculoviral life cycle ("hyperburst management").
(5) Rigorously validate novel designer genomes experimentally.
These will be the first fully synthetic baculoviral genomes, with the potential to transform academic and industrial R&D applications.
People |
ORCID iD |
| Imre Berger (Primary Supervisor) | |
| Barbara Gorda (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M009122/1 | 01/10/2015 | 31/03/2024 | |||
| 1864756 | Studentship | BB/M009122/1 | 01/10/2016 | 31/03/2021 | Barbara Gorda |
| Description | 67 single gene deletions in the genome of the AcMNPV baculovirus have been generated to enable to classify if these genes are essential or non essential for the propagation of the virus and protein expression in cell culture. The single gene deletion viruses were tested and genes grouped depending on the performance of the virus. Currently all the single gene deletions which resulted in a functional virus are being re tested to confirm results. 2020 update- functional single gene deletions were used as potential sites for the relocation of the Tn7 site. 5 sites were tested in total, with 4 of the sites undergoing futher validation currently in terms of virus stability and protein expression yields. 2021 update- the Tn7 site was successfully relocated to 4 locations in the baculovirus genome, additionally the site-specific recognition sites RoxP and VloxP were inserted to provide alternative ways to insert auxiliary DNA sequences. The new baculovirus SynBac1 variants with alternative Tn7 sites were tested and validated in terms of protein expression, where equal amounts of proteins are observed as when compared to the control genome, EMBacY. The RoxP and VloxP sites were validated by inserting donor plasmids with fluorescent proteins and observing fluorescence. The stability of the new SynBac1 variants was tested and preliminary data point that SynBac1 variants have higher stability than EMBacY, however virus titer information need to be determined beforehand. |
| Exploitation Route | The outcome of the single gene deletions will enable us to decide which genes can be deleted in the baculoviral genome to reach our objective of generating a minimal functional baculoviral genome. The data generated might be of interests to entomologists studying the function of genes in the AcMNPV baculovirus which infects insects. Utilize SynBac1 baculovirus genome as the next generation baculovirus vector expression system |
| Sectors | Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |