Rapid mass production of human influenza vaccine in insects using genome editing

摘要 正如最近的COVID-19大流行所表明的那样，疫苗的快速大规模生产 对于新出现的传染病来说是至关重要的。使用杆状病毒/昆虫 用于体外蛋白表达的细胞表达系统已经成为疫苗的游戏规则改变者 但它也带来了挑战：转染可能很困难，需要重复 最终产品需要多个纯化步骤来去除残留物 杆状病毒我们提出了一种替代方法，可以绕过相关的缺点 用杆状病毒/昆虫细胞表达系统：在可扩展的昆虫中的体内疫苗生产 非哺乳动物遗传模型系统。我们的目标是通过使用CRISPR-Cas9 将人类和禽流感的病毒抗原基因插入昆虫体内的系统， 大规模生产用于饲料和/或废物管理。黄粉虫（Tenebrio molitor） 和丽蝇（Phormia regina）是操纵蛋白质表达的有前途的生物反应器 由于其高代谢和繁殖力，易于饲养，高弹性和多功能性， 在大规模生产系统中（这两种昆虫的大规模生产设施已经存在）。不像 现有的转基因昆虫宿主，如卷心菜尺蠖幼虫、粉虫和绿头苍蝇 是群居的，并且可以以每天几吨的规模成本有效地大量饲养。通过 利用CRISPR-Cas9技术进行重组蛋白表达， 在这些昆虫的抗原，我们将工程师的抗原的生命阶段依赖激活 生产利用现有的技术，以促进快速抗原蛋白纯化，我们将 确认抗原生产并对其进行表征。最后，我们将量化影响（如果有） 修饰的基因组没有与大规模生产和验证成本相关的表型 生产方面的考虑。这项工作将为稳定供应低成本和高质量的食品奠定基础。 可定制的抗原，使用昆虫生物质作为负担得起的和可扩展的生物反应器。

ABSTRACT As the recent COVID-19 pandemic has made clear, the rapid mass production of vaccines for emerging infectious diseases is of paramount importance. The use of the baculovirus/insect cell expression system for in vitro protein expression has been a game changer for vaccine production, but it also introduces challenges: transfection can be difficult, needs to be repeated with every batch, and the final product requires multiple purification steps to remove the residual baculovirus. We propose an alternative approach that could side-step the drawbacks associated with baculovirus/insect cell expression system: in vivo vaccine production in a scalable insect non-mammalian genetic model system. We aim to achieve this by using the CRISPR-Cas9 system to insert viral antigen genes for human and avian influenza into insects that are already mass produced for feed and/or waste management. Both the yellow mealworm (Tenebrio molitor) and the blow fly (Phormia regina) are promising bioreactors for manipulating protein expression in vivo due to their high metabolism and fecundity, ease of rearing, high resilience, and versatility in mass production systems (large-scale production facilities already exist for both insects). Unlike existing transgenic insect hosts such as cabbage looper larvae, both mealworms and blowflies are gregarious and can be cost effectively mass-reared at a scale of several tons per day. By using the CRISPR-Cas9 technique for recombinant protein expression to express influenza antigens in these insects, we will engineer life-stage dependent activation of the antigen production. Using existing technologies to facilitate rapid antigen protein purification, we will confirm the antigen production and characterize it. Finally, we will quantify what impact (if any) the modified genome has on phenotypes relevant to mass production and validate cost considerations for production. This work will set the stage for a steady supply of low-cost and customizable antigens, using insect biomass as an affordable and scalable bioreactor.