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蛋白质，用作疫苗和治疗剂以及用于蛋白质结构和功能的研究。BEVS的一个限制是杆状病毒倾向于将转座子插入fp 25 k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在组织培养中不具有感染性的闭塞衍生病毒转移到出芽病毒（BV），出芽病毒是用于杆状病毒表达的病毒形式。较高水平的BV对于BEVS使用者是有利的，但FP突变体也缺乏来自驱动靶基因表达的多角体蛋白启动子的转录。杆状病毒还快速积累缺陷型干扰颗粒（DIP），其通常与靶基因表达的急剧降低有关，这是由于靶基因和/或其表达所需的病毒基因的缺失。促进DIP形成的一个因素是转座到fp 25 k中。该提案的目标是限制转置到fp 25 k的有害影响，同时利用 事实上，FP 25 K活性的消除显著增加了BV的产生。在第一阶段，两种策略-一种诱导型结构控制fp 25 k和fp 25 k缺失突变体加上组成型表达FP 25 K的细胞系-进行了探索，用于调节FP 25 K的表达。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角体蛋白启动子切换到高水平转录。结果证明了I期目标的实现，其中从病毒中缺失fp 25 k并从工程化细胞系中互补表达，从而能够预测出芽病毒和重组蛋白生产的控制。然而，由于重金属诱导剂对昆虫细胞的毒性，诱导型构建体不能提供足够的FP 25 K控制。因此，在第二阶段，我们将（1）开发一个 使用基于蜕皮激素受体的诱导型启动子，具有更紧密转录调控的改进的诱导型构建体;（2）使用表达BEVS和BacMam的细胞内和分泌的黄色荧光蛋白（YFP）的病毒，验证FP 25 K调控的有益效果（哺乳动物表达）技术;（3）开发用于简化BEVS和BacMam克隆的用户友好的fp 25 k突变体骨架，并证明它们与几种医学相关转基因的效用;以及（4）在vankle-enhanced BEVS（VE-BEVSTM）技术的背景下测试fp 25 k表达系统。ParaTechs的VE-BEVS产品延迟杆状病毒感染细胞的死亡和裂解，从而将靶蛋白表达提高20倍。许多 BEVS用户希望同时采用vankyrin和FP 25 K技术;因此，确定它们是否兼容非常重要。总的来说，II期目标的完成将使BEVS的用户能够控制和优化BV的生产和重组蛋白的表达。