Enhancing mammalian glycoprotein production in the baculovirus expression vector

描述（由申请人提供）：杆状病毒表达载体系统（BEVS）是一种经过验证的、强大的、通用的真核蛋白表达方法。它被用于为众多研究项目生产疫苗、诊断和生物活性蛋白。然而，像所有的表达系统一样，BEVS也有它的缺点。一个是由于病毒诱导的细胞死亡和裂解，表达是短暂的。paratech公司已经将一款解决这一问题的产品商业化。表达病毒锚蛋白基因的细胞系显示杆状病毒感染细胞的延迟死亡和裂解，从而显著提高重组蛋白的生产。这种活性被称为vankyrin-enhanced BEVS (VE-BEVSTM)，可将靶蛋白的表达提高22倍。杆状病毒表达的第二个限制是昆虫细胞缺乏产生最终唾液化的复杂n -聚糖的能力，这限制了BEVS用于表达人类治疗性蛋白的有效性。glybac LLC开发了一种转基因昆虫细胞系（SfSWT4），该细胞系表达六种哺乳动物糖基化酶，允许合成终端唾液蛋白。该II期方案将ParaTechs的VE技术与glybac的细胞系相结合，以优化“人源化”n -聚糖的表达。在I期，在不同启动子的控制下，用几种vankyrin基因转染转基因细胞系SfSWT4。筛选多克隆细胞以增强糖蛋白表达。数据显示，受感染的细胞寿命更长，产生的唾液化蛋白更多，证实了我们的假设。II期将通过以下方式扩展这些研究：(1)根据FDA用于生产疫苗和生物制剂的细胞指南克隆和表征VE-SWTTM细胞；(2)研究VE病毒载体和VE- swt细胞系之间的协同效应，以提供更高水平的增强；(3) VE-SWTTM细胞中医学相关糖蛋白的表达增强。这些II期研究将显著扩展ParaTechs的VE-BEVS技术和GlycoBac的糖工程细胞系的应用。两家公司的员工都有使用这些技术的经验。初步研究表明，这项技术有很大的机会实现预期的效果。此外，两家公司之前发布的转化细胞系的营销经验表明，对扩展技术的需求很大。基于ParaTechs， Inc.和GlycoBac， LLC的声誉，以及对表达重组人源糖蛋白的改良细胞系不断增长的需求，这项新技术应该相对容易商业化。

DESCRIPTION (provided by applicant): The baculovirus expression vector system (BEVS) is a proven, powerful and versatile method of eukaryotic protein expression. It is used to produce vaccines, diagnostics, and biologically active proteins for a multitude of research projects. Like all expression systems, however, BEVS has its disadvantages. One is the fact that expression is short-lived due to virus-induced cell death and lysis. ParaTechs has already commercialized a product that addresses this shortcoming. Cell lines that express a viral ankyrin gene show delayed death and lysis of baculovirus-infected cells, thereby significantly enhancing recombinant protein production. This activity, referred to as vankyrin-enhanced BEVS (VE-BEVSTM), boosts target protein expression up to 22-fold. A second limitation of baculovirus expression is that insect cells lack the ability to produce terminally sialylated, complex N-glycans, which limits the usefulness of BEVS for the expression of human therapeutic proteins. GlycoBac LLC has developed a transgenic insect cell line (SfSWT4) that expresses six mammalian glycosylation enzymes, allowing synthesis of terminally sialyated proteins. This Phase II proposal combines ParaTechs' VE technology with GlycoBac's cell line to optimize expression of "humanized" N-glycans. In Phase I, the transgenic cell line SfSWT4 was transformed with several vankyrin genes under the control of different promoters. Polyclonal cells were screened for enhanced glycoprotein expression. Data demonstrating that infected cells lived longer and produced more authentically sialylated protein confirmed our hypothesis. Phase II will extend these studies by (1) cloning and characterization of VE-SWTTM cells according to FDA guidelines for cells used to produce vaccines and biological; (2) examining synergistic effects between VE virus vectors and VE-SWT cell lines to provide greater levels of enhancement; and (3) demonstrating enhanced expression of medically relevant glycoproteins in VE-SWTTM cells. These Phase II studies will significantly expand the applications of ParaTechs' VE-BEVS technology and GlycoBac's glycoengineered cell lines. Personnel at both companies have experience with the techniques to be used. Preliminary studies indicate that this technology has a significant chance of performing as envisioned. Furthermore, prior marketing experience with transformed cell lines previously released from the two companies suggests a significant demand for the expanded technology. This new technology should be relatively easy to commercialize based on the established reputations of ParaTechs, Inc. and GlycoBac, LLC and the growing demand for improved cell lines to express recombinant humanized glycoproteins. PUBLIC HEALTH RELEVANCE: The inability of insect cells to produce terminally sialylated, complex N-glycans limits the usefulness of the baculovirus expression system for the production of human therapeutic proteins. This proposal addresses that deficiency and aims to develop new cell lines that produce authentic "humanized" N-glycans in the context of ParaTechs' vankyrin-enhanced baculovirus technology. Successful completion of these objectives will produce cells that provide the highest levels of accurately processed secreted and transmembrane proteins and will be marketed to individual researchers and pharmaceutical companies engaged in structure-function studies of the human secretome and development of protein therapeutics.