Enhancing mammalian glycoprotein production in the baculovirus expression vector

增强杆状病毒表达载体中哺乳动物糖蛋白的产量

• 批准号: 7909460
• 负责人: Angelika Fath-Goodin
• 金额: $ 14.8万
• 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: 2010-05-01 至 2011-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7909460
• 关键词: Address; Ankyrins; Baculovirus Expression System; Baculoviruses; Cell Death; Cell Line; Cells; Cessation of life; Collaborations; Complex; Cytolysis; Data; Development; Diagnostic; Disadvantaged; Engineering; Enzymes; Funding; Gene Expression; Genes; Glycoproteins; Goals; Human; Human Resources; Individual; Infection; Insecta; Insecticides; Integral Membrane Protein; Life; Marketing; Methods; Pharmacologic Substance; Phase; Polysaccharides; Population; Positioning Attribute; Process; Production; Proteins; Recombinant Proteins; Research Personnel; Research Project Grants; Staging; Structure; System; Techniques; Technology; Testing; Therapeutic Human Experimentation; Transgenic Organisms; Vaccines; Viral; Virus; Virus Diseases; cost; experience; expression vector; glycosylation; glycosyltransferase; phase 1 study; phase 2 study; promoter; protein expression; public health relevance; response; therapeutic protein

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 20-fold, depending on the protein and method of vankyrin delivery. 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. This proposal addresses that issue and aims to develop new cell lines that produce authentic "humanized" N-glycans in the context of vankyrin-enhanced baculovirus technology. To obtain this goal, three different insect cell lines that have been previously engineered to produce mammalian glycosylation enzymes will be co-infected with baculoviruses that encode a vankyrin gene and different test glycoproteins. The transgenic cell line with the highest vankyrin response will be transformed with two different vankyrin genes under the control of several promoters. Polyclonal cells will be screened for enhanced glycoprotein expression, and the most promising cell population will be cloned. Different monoclonal lines will be tested for production of complex terminally sialylated glycoproteins, as compared to the non-transformed parental line. These cells will provide the highest yields 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. These Phase I studies will significantly expand the applications of ParaTechs' vankyrin- enhanced BEVS technology. ParaTechs personnel and the Jarvis group have experience with all of the techniques to be used in these studies and do not anticipate difficulty achieving the objectives. The data derived from funding of this Phase I proposal will position ParaTechs to initiate Phase II studies to demonstrate the utility of these cell lines for production of transmembrane and secreted proteins in collaboration with potential end users. PUBLIC HEALTH RELEVANCE: The inability of insect cells to produce terminally sialylated, complex N-glycans limits the usefulness of 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.

描述（由申请人提供）：杆状病毒表达载体系统（BEVS）是一种经证实的、功能强大且通用的真核蛋白表达方法。它被用于生产疫苗，诊断和生物活性蛋白质，用于许多研究项目。然而，与所有表达系统一样，BEVS也有其缺点。一个是由于病毒诱导的细胞死亡和裂解，表达是短暂的。ParaTechs已经将一种解决这一缺陷的产品商业化。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，从而显著增强重组蛋白的产生。这种活性被称为vankyrin增强的BEVS（VE-BEVSTM），根据vankyrin递送的蛋白质和方法，将靶蛋白表达提高高达20倍。杆状病毒表达的第二个限制是昆虫细胞缺乏产生末端唾液酸化的复合N-聚糖的能力，这限制了BEVS用于表达人治疗性蛋白质的有用性。该提案解决了这一问题，旨在开发新的细胞系，在vanklavin增强的杆状病毒技术的背景下产生真正的“人源化”N-聚糖。为了实现这一目标，将用编码vankyrin基因和不同测试糖蛋白的杆状病毒共感染先前已被工程化以产生哺乳动物糖基化酶的三种不同昆虫细胞系。具有最高vankyrin应答的转基因细胞系将在几种启动子的控制下用两种不同的vankyrin基因转化。将筛选多克隆细胞以增强糖蛋白表达，并克隆最有希望的细胞群。与未转化的亲本系相比，将检测不同单克隆系的复杂末端唾液酸化糖蛋白的产生。这些细胞将提供最高产量的精确加工的分泌和跨膜蛋白，并将销售给从事人类分泌蛋白结构-功能研究和蛋白质治疗开发的个人研究人员和制药公司。这些第一阶段的研究将大大扩展ParaTechs的vankyrin增强BEVS技术的应用。ParaTechs人员和Jarvis小组对这些研究中使用的所有技术都有经验，预计实现目标不会有困难。从第一阶段提案的资助中获得的数据将使ParaTechs能够启动第二阶段研究，以证明这些细胞系与潜在最终用户合作生产跨膜和分泌蛋白的实用性。 公共卫生相关性：昆虫细胞不能产生末端唾液酸化的复合N-聚糖，限制了杆状病毒表达系统用于生产人治疗性蛋白质的有用性。该提案解决了这一缺陷，并旨在开发新的细胞系，在ParaTechs vankrobin增强杆状病毒技术的背景下产生真正的“人源化”N-聚糖。这些目标的成功完成将产生提供最高水平的精确加工的分泌和跨膜蛋白的细胞，并将销售给从事人类分泌组的结构-功能研究和蛋白质疗法开发的个体研究人员和制药公司。