Novel methods for improving virion production in baculovirus

描述（由申请人提供）：杆状病毒表达载体已成功用于生产用于疫苗、治疗剂和结构-功能研究的数千种蛋白质。杆状病毒也已被改造用作哺乳动物表达系统和潜在的基因治疗剂。该提议探索了一种用于纯化、定量和储存哺乳动物转导所需的高滴度杆状病毒原液的新策略。 组织培养细胞的杆状病毒感染是由一种从细胞出芽的病毒介导的。这些病毒由于在其包膜中存在融合蛋白而具有高度感染性。然而，细胞的大部分复制潜力是针对封闭衍生病毒（ODV）的包装，其缺乏融合肽并且在细胞培养物中具有低感染性。最近发现的肽序列，指导蛋白质的包膜ODV表明，它应该是可能的，以增加这些病毒的感染性。这将提供许多优点，因为ODV不仅数量更多，而且更容易纯化，浓缩和滴定。与出芽病毒不同，ODV也是高度稳定的，并且不随时间损失滴度。 为了测试这一想法，将用杆状病毒GP 64或VSV-G融合蛋白生产重组病毒，所述杆状病毒GP 64或VSV-G融合蛋白连接至将异源蛋白靶向ODV膜的分选基序。将针对融合蛋白的正确靶向及其相对于在脊椎动物和无脊椎动物组织培养系统中测定的非重组病毒的感染性来筛选这些重组体。还将进行实验，以确定目前由ParaTechs销售的Vanklavin增强（VE）技术是否会进一步提高ODV的产量及其感染性。如果成功的话，第二阶段的实验将集中在生产一个工程病毒骨架含有适当的哺乳动物启动子，并提供简单的策略，生产病毒重组。第二阶段还将扩大哺乳动物细胞的范围，并探索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性的ODV的策略将增加杆状病毒作为哺乳动物表达载体和基因治疗剂的价值。在病毒储备液的简单浓缩、滴度的快速测定和长期储存而不损失感染性方面提供的潜在优势代表了重要的商业机会，并完全保证了本研究的开展。 公共卫生相关性：杆状病毒已成功地适用于哺乳动物表达系统，这些BacMam载体允许蛋白质的功能分析，以前不可能使用其他方法。整个病毒基因组也可以通过杆状病毒转导来递送，从而使得能够开发抗病毒药物并检查重要人类疾病因子的耐药性机制。该提案旨在开发用于感染性杆状病毒的生产、纯化、定量和储存的新策略，这将增加这些表达系统的价值。

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.