Novel methods for improving virion production in baculovirus

提高杆状病毒病毒粒子产量的新方法

• 批准号: 7999941
• 负责人: Angelika Fath-Goodin
• 金额: $ 13.26万
• 依托单位: PARATECHS CORP.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7999941
• 关键词: Accounting; Animal Model; Baculoviruses; Cell Culture Techniques; Cells; Chimeric Proteins; Cytolysis; Data; Development; Drug resistance; Engineering; Environment; Gene Expression; Gene Therapy Agent; Genes; Goals; Health; Horizontal Disease Transmission; Human Resources; Infection; Insecta; Invertebrates; Laboratories; Link; Literature; Mammalian Cell; Marketing; Mediating; Membrane; Membrane Fusion; Methods; Mutation; Nature; Nuclear Inner Membrane; Nucleocapsid; Peptides; Pharmaceutical Preparations; Phase; Phenotype; Production; Proteins; Protocols documentation; Recombinant Proteins; Recombinants; Relative (related person); Research; Sorting - Cell Movement; Structure; System; Technology; Testing; Time; Vertebral column; Viral; Viral Genome; Virion; Virus; cell transformation; experience; expression vector; gene therapy; human disease; improved; novel; novel strategies; promoter; protein aminoacid sequence; public health relevance; recombinant virus; research study; systems research; therapeutic vaccine; tissue culture; tissue/cell culture; vector; virus envelope

DESCRIPTION (provided by applicant): Baculovirus expression vectors have been successfully used to produce thousands of proteins for vaccines, therapeutics, and structure-function studies. Baculoviruses have also been adapted for use as mammalian expression systems and potential gene therapy agents. This proposal explores a novel strategy for purification, quantitation, and storage of high titer baculovirus stocks that are needed for mammalian transduction. Baculovirus infection of tissue culture cells is mediated by a form of virus that buds from cells. These viruses are highly infectious due to the presence of fusion proteins in their envelopes. Most of the replicative potential of the cell, however, is directed toward packaging of occlusion-derived viruses (ODV), which lack fusion peptides and have low infectivity in cell culture. The recent discovery of a peptide sequence that directs proteins to the envelope of ODV suggests that it should be possible to increase infectivity of these viruses. This would offer a number of advantages because ODV are not only more numerous, they are simpler to purify, concentrate and titer. ODV are also highly stable and do not lose titer over time, unlike budded virus. To test this idea, recombinant viruses will be produced with the baculovirus GP64 or VSV-G fusion protein linked to a sorting motif that targets heterologous proteins to ODV membranes. These recombinants will be screened for correct targeting of the fusion proteins and their infectivity relative to non-recombinant virus determined in vertebrate and invertebrate tissue culture systems. Experiments will also be conducted to discern whether Vankyrin-enhanced (VE) technology, currently marketed by ParaTechs as a method to increase baculovirus-mediated gene expression, would further boost yields of ODV and their infectivity. If successful, Phase II experiments would focus on producing an engineered viral backbone containing appropriate mammalian promoters and offering simple strategies for producing viral recombinants. Phase II would also expand the range of mammalian cells and explore animal models for gene therapy applications. This strategy of producing ODV that is highly infectious in tissue culture will add value to baculoviruses as mammalian expression vectors and gene therapy agents. The potential advantages offered with respect to simple concentration of viral stocks, rapid determination of titer, and long term storage without loss of infectivity represents a significant commercial opportunity and fully warrants undertaking this research. PUBLIC HEALTH RELEVANCE: Baculoviruses have been successfully adapted for use as mammalian expression systems; these BacMam vectors have allowed functional analyses of proteins not previously possible using other approaches. Whole viral genomes can also be delivered by baculovirus transduction, thus enabling the development of anti-viral drugs and examination of the mechanisms of drug resistance for significant human disease agents. This proposal seeks to develop new strategies for the production, purification, quantitation and storage of infectious baculovirus, which will add value to these expression systems.

描述（由申请人提供）：杆状病毒表达载体已成功地用于生产数千种用于疫苗，疗法和结构功能研究的蛋白质。杆状病毒也已被调整为用作哺乳动物表达系统和潜在的基因治疗剂。该提案探讨了哺乳动物转导所需的纯化，定量和存储纯化，定量和存储的新策略。 组织培养细胞的杆状病毒感染是由细胞芽的病毒形式介导的。由于其信封中存在融合蛋白，这些病毒具有高度感染性。然而，细胞的大多数复制潜力都针对闭塞病毒（ODV）的包装，这些病毒缺乏融合肽，在细胞培养中具有低感染性。最近发现将蛋白质引导到ODV包膜的肽序列的发现表明，应该有可能增加这些病毒的感染性。这将提供许多优势，因为ODV不仅要花很多时间，而且更简单地净化，集中和滴答效果。 ODV也很稳定，并且随着时间的流逝不会失去滴度，与芽的病毒不同。 为了测试这一想法，将用杆状病毒GP64或VSV-G融合蛋白与与ODV膜有关的分类基序连接的重组病毒。这些重组将被筛选，以正确靶向融合蛋白及其感染性，相对于在脊椎动物和无脊椎动物组织培养系统中确定的非重组病毒。还将进行实验，以识别目前由Paratechs销售的Vankyrin增强（VE）技术是否会增加杆状病毒介导的基因表达，将进一步提高ODV的产量及其感染性。如果成功的话，II期实验将集中于生产装有适当的哺乳动物启动子的工程病毒主链，并提供产生病毒重组者的简单策略。第二阶段还将扩大哺乳动物细胞的范围，并探索用于基因治疗应用的动物模型。 这种在组织培养中产生高度感染力的ODV的策略将为肉毒杆菌病毒作为哺乳动物表达载体和基因治疗剂增加价值。关于简单浓度的病毒库存，滴定滴度的快速确定以及长期存储而没有感染感染性的潜在优势代表了一个很大的商业机会，并且完全保证进行这项研究。 公共卫生相关性：杆状病毒已成功地用于哺乳动物表达系统；这些BACMAM载体已允许使用其他方法对蛋白质进行功能分析。整个病毒基因组也可以通过杆状病毒转导，从而实现抗病毒药物的发展以及对重要人类疾病药物的耐药性机制的检查。该建议旨在为传染病病毒的生产，纯化，定量和存储制定新的策略，这将为这些表达系统增加价值。