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）：杆状病毒表达载体系统（BEVS）已成功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的一个限制是杆状病毒倾向于积累转座子插入到fp25k基因中，导致“少多面体（FP）”表型。这种突变改变了病毒生产的平衡，从封闭衍生病毒（在组织培养中不具有传染性）转向芽化病毒（BV），这是用于杆状病毒表达的病毒形式。较高水平的BV对BEVS使用者是有利的，但FP突变体也缺乏驱动靶基因表达的多面蛋白启动子的转录。杆状病毒还会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/或表达所需的病毒基因的缺失。促进DIP形成的一个因素是转位到fp25k。本提案的目标是限制换位到fp25k的有害影响，同时利用