Enhanced baculovirus vectors with higher titers and increased genome stability

描述(申请人提供)：杆状病毒表达载体系统(BEVS)已成功地用于生产数千种用于疫苗和治疗以及蛋白质结构和功能研究的蛋白质。BEVS的一个局限性是杆状病毒倾向于将转座子插入到fp25k基因中，导致“少多角体(FP)”表型。这种突变将病毒生产的平衡从组织培养中不具传染性的闭塞衍生病毒转移到芽生病毒(BV)，BV是杆状病毒表达中使用的病毒形式。较高水平的BV对BEVS使用者是有利的，但FP突变体也缺乏来自多角体启动子的转录，而多角体启动子驱动目标基因的表达。杆状病毒还会迅速积累缺陷干扰颗粒(DIP)，这通常与靶基因表达的急剧下降有关，这是由于目标基因和/或其表达所需的病毒基因的缺失。促进倾角形成的一个因素是转位到fp25k。这项提议的目标是限制转座到fp25k的有害影响，同时利用 FP25K活性的消除显著增加了BV的产量。在第一阶段，探索了两种策略--控制fp25k的可诱导构建和fp25k缺失突变体与组成性表达fp25K的细胞系相结合--来调节fp25K的表达。这两种策略都寻求在杆状病毒感染的放大阶段产生高滴度病毒，并在重组蛋白表达阶段从多角体启动子切换到高水平转录。结果证明，通过从病毒中删除fp25k和从工程细胞系中进行互补表达，实现了第一阶段目标，从而能够预测控制萌芽病毒和重组蛋白的生产。然而，由于重金属诱导剂对昆虫细胞的毒性，可诱导构建的载体不能提供足够的FP25K控制。因此，在第二阶段，我们将(1)制定一个 通过使用基于蜕皮激素受体的诱导性启动子，改进了可诱导结构，具有更严格的转录调控；(2)使用表达细胞内和分泌黄色荧光蛋白(YFP)的病毒来验证FP25K调控的有益效果，以用于BEVS和BacMam(哺乳动物表达)技术；(3)开发用户友好的fp25k突变骨架，用于简化BEVS和BacMam的克隆，并展示其与几个医学相关转基因的实用性；以及(4)在vankyrin增强型BEVS(VE-BEVSTM)技术的背景下测试fp25k表达系统。ParaTechs的VE-BEVS产品可以延缓杆状病毒感染细胞的死亡和裂解，从而将目标蛋白的表达提高20倍。许多 BEV用户可能希望同时采用vankyrin和FP25K技术；因此，确定它们是否兼容非常重要。总而言之，完成第二阶段目标将使BEVS的用户能够控制和优化BV和重组蛋白表达的生产。

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 fp25k gene, leading to the "few polyhedra (FP)" 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 fp25k. The goal of this proposal is to limit deleterious effects of transposition into fp25k, while taking advantage of the fact that elimination of the FP25K activity significantly increases BV production. During Phase I, two strategies - an inducible construct for controlling fp25k and an fp25k deletion mutant coupled with a cell line constitutively expressing FP25K - were explored for regulating FP25K 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 fp25k 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 FP25K control due to the toxicity of the heavy metal inducer to insect cells. Therefore, in Phase II, we will (1) develop an improved inducible construct with tighter transcriptional regulation, using an ecdysone receptor-based inducible promoter; (2) validate the beneficial effects of FP25K regulation using viruses that express intracellular and secreted yellow fluorescent protein (YFP) for BEVS and BacMam (mammalian expression) technology; (3) develop user friendly fp25k mutant backbones for simplified BEVS and BacMam cloning and demonstrate their utility with several medically relevant transgenes; and (4) test the fp25k expression system in the context of vankyrin-enhanced BEVS (VE-BEVSTM) technology. ParaTechs' VE-BEVS products delay death and lysis of baculovirus-infected cells, thereby boosting target protein expression up to 20 fold. Many BEVS users would want to incorporate both vankyrin and FP25K 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.