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数千种用于疫苗、治疗和结构功能研究的蛋白质。杆状病毒也被用于哺乳动物表达系统和潜在的基因治疗剂。本提案探讨了一种新的策略，用于纯化、定量和储存高滴度的杆状病毒，这是哺乳动物转导所需要的。杆状病毒感染组织培养细胞是由一种从细胞中冒出的病毒介导的。由于包膜中存在融合蛋白，这些病毒具有高度传染性。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，这种病毒缺乏融合肽，在细胞培养中具有低传染性。最近发现的一个肽序列可以将蛋白质引导到ODV的包膜上，这表明它应该有可能增加这些病毒的传染性。这将带来许多好处，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也高度稳定，不会随着时间的推移而失去滴度。为了验证这一想法，重组病毒将与杆状病毒GP64或VSV-G融合蛋白连接到一个针对ODV膜的异源蛋白的分类基序。这些重组体将在脊椎动物和无脊椎动物组织培养系统中进行筛选，以确定融合蛋白的正确靶向性及其相对于非重组病毒的感染性。此外，还将进行实验，以确定Vankyrin-enhanced （VE）技术是否会进一步提高ODV的产量及其传染性。该技术目前由ParaTechs公司作为一种增加杆状病毒介导基因表达的方法进行销售。如果成功，第二阶段的实验将集中于生产含有适当哺乳动物启动子的工程病毒骨架，并为生产病毒重组提供简单的策略。第二阶段还将扩大哺乳动物细胞的范围，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传染性的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。病毒库的简单浓缩、滴度的快速测定和不丧失传染性的长期储存方面的潜在优势代表了一个重要的商业机会，完全值得进行这项研究。

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.