SynBac: Synthetic Baculovirus Genome for Next-generation Drug Discovery

杆状病毒是重组基因进入真核细胞的高效传递系统，对真核蛋白（包括高价值药物靶点）的生产具有重要影响。宫颈癌和其他疾病的疫苗就是用这种方法生产的。最近，杆状病毒已成为基因治疗的多功能工具。我们为该领域的多蛋白复合物研究贡献了屡获殊荣的MultiBac技术。这些处于现代生物学前沿的应用依赖于AcMNPV衍生的大型杆状病毒基因组（130 kb）。这个基因组主要由词源学家进行了深入的研究。描述了在自然界和细胞培养中繁殖所必需的基因和妨碍在实验室应用的DNA元素。通过经典的基因敲除技术，已经对野生型病毒基因组进行了遗传改变，以改善基因插入、传递和蛋白质生产特性。这种改变需要专家付出极大的努力。因此，目前还不可能充分利用杆状病毒系统的巨大潜力。在目前的项目中，我们大胆地建议完全扭转目前的做法。我们将在计算机上设计和构建新的、完全合成的定制杆状病毒基因组，这将是第一次以流线型、高度通用的形式进行多基因转移和生产高价值的、用于药物发现的下一代重组蛋白靶点。我们将应用最先进的基因组编辑工具，特别是CRISPR-Cas9，通过系统地破坏基因和非编码区域，包括基因调控元件，来为我们的方法提供信息。我们进一步致力于解决目前阻碍杆状病毒系统生产制药规模生物制品的“规模问题”。作为概念的证明，我们已经用设计的DNA取代了大部分（20 kb）的野生型DNA，创造了一个部分合成的杂交基因组。对这个原型的严格测试有力地验证了我们的方法。CASE研究员将：(1)利用计算生物学、比较生物信息学和数据挖掘来创建优化最小杆状病毒基因组的蓝图。(2)大片段合成设计基因组，并利用先进的重组技术将其组装成功能基因组（阿斯利康平台）。(3)利用尖端的CRISPR-Cas9工具，并行编辑杆状病毒基因组中的基因和调控DNA元件（阿斯利康平台），并在合成设计中实现这些信息。(4)通过重新配置杆状病毒生命周期的最后阶段（“超爆发管理”）来解决“放大问题”。(5)通过实验严格验证新的设计基因组。这将是第一批完全合成的杆状病毒基因组，具有改变学术和工业研发应用的潜力。

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.