Enhanced baculovirus vectors with higher titers and increased genome stability

具有更高滴度和更高基因组稳定性的增强型杆状病毒载体

• 批准号: 8976604
• 负责人: Angelika Fath-Goodin
• 金额: $ 46.17万
• 依托单位: PARATECHS CORP.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8976604
• 关键词: Achievement; Address; Adoption; Ankyrins; Baculoviruses; Biocontrols; Cell Line; Cells; Cessation of life; Cloning; Coupled; Cytolysis; Defective Viruses; Engineering; Equilibrium; Funding; Gene Expression; Gene Targeting; Genes; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Goals; Health; Heavy Metals; Infection; Insect Viruses; Insecta; Link; Maintenance; Mammalian Cell; Marketing; Methods; Modification; Mutation; Phase; Phenotype; Process; Production; Proteins; Recombinant Proteins; Regulation; Research Personnel; Staging; Structure; System; Technology; Testing; Time; Toxic effect; Transcriptional Regulation; Transgenes; United States National Institutes of Health; Vertebral column; Viral; Viral Genes; Viral Proteins; Viral Vector; Virus; base; commercialization; cost; design; ecdysone receptor; expression vector; improved; in vivo; innovative technologies; mutant; prevent; promoter; protein expression; protein function; protein structure function; therapeutic vaccine; tissue culture; user-friendly; vaccine trial; vector

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.

描述（由申请人提供）：已成功利用杆状病毒表达载体系统（BEV）生产数千种蛋白质作为疫苗和疗法，以及蛋白质结构和功能的研究。 BEV的一个局限性是杆状病毒将转座子插入到FP25K基因中的倾向，导致“少数Polyhedra（FP）”表型。该突变将病毒产生的平衡从组织培养中没有传染性的闭塞病毒转移到芽病毒（BV），这些病毒是杆状病毒表达中使用的病毒的形式。较高水平的BV对于BEVS使用者将是有利的，但FP突变体也缺乏从驱动靶基因表达的多艾同学启动子的转录。杆状病毒还迅速积累有缺陷的干扰颗粒（DIP），这通常与靶基因表达的急剧下降有关，这是由于靶基因和/或其表达所需的病毒基因的删除所致。促进倾角形成的一个因素是将其转换为FP25K。该提案的目的是将换位的有害影响限制为FP25K，同时利用 消除FP25K活性的事实显着增加了BV的产生。在第一阶段，探索了两种策略 - 用于控制FP25K的诱导构建体和一个与组成性表达FP25K的细胞系相结合的FP25K缺失突变体 - 用于调节FP25K表达。这两种策略都试图在杆状病毒感染的扩增阶段产生高滴度病毒，并在重组蛋白表达阶段从多沙同行蛋白启动子转移到高水平的转录。结果记录了从病毒中删除FP25K的I期目标的实现，并从工程细胞系中从病毒中删除了互补的表达，从而可以预测对好友病毒和重组蛋白产生的预测控制。但是，由于重金属诱导剂对昆虫细胞的毒性，诱导构建体没有提供足够的FP25K控制。因此，在第二阶段，我们将（1）开发 使用基于Ecdysone受体的诱导启动子，改进了具有更严格的转录调控的诱导构建体； （2）使用表达细胞内和分泌的黄色荧光蛋白（YFP）对BEVS和BACMAM（哺乳动物表达）技术表达细胞内和分泌的黄色荧光蛋白（YFP）的病毒验证FP25K调节的有益作用； （3）为简化的BEV和Bacmam克隆开发用户友好的FP25K突变型骨架，并通过几个医学上相关的转基因演示其实用性； （4）在Vankyrin增强BEVS（VE-BEVSTM）技术的背景下测试FP25K表达系统。 Paratechs的VE-BEVS产物延迟了杆状病毒感染细胞的死亡和裂解，从而将靶蛋白表达提高到20倍。许多 BEVS用户希望同时合并Vankyrin和FP25K技术；因此，确定它们是否兼容很重要。综上所述，II期目标的完成将使BEV的用户能够控制和优化BV和重组蛋白表达的产生。