Use of a viral mucin-like protein to convert adherent cells to suspension culture

使用病毒粘蛋白样蛋白将贴壁细胞转化为悬浮培养物

• 批准号: 8121973
• 负责人: Angelika Fath-Goodin
• 金额: $ 20.1万
• 依托单位: PARATECHS CORP.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8121973
• 关键词: Achievement; Adherent Culture; Adhesions; Adopted; Area; Baculoviruses; Biological Products; Cell Adhesion; Cell Line; Cells; Characteristics; Data; Development; Future; Genes; Goals; Growth; Health; Hemocytes; Human; Human Resources; Individual; Insect Viruses; Insecta; Kinetics; Literature; Mammalian Cell; Marketing; Mediating; Medicine; Mesenchymal Stem Cells; Methods; Monitor; Mucins; Pharmaceutical Preparations; Phase; Platelet Factor 4; Production; Property; Proteins; Protocols documentation; Recombinant Proteins; Relative (related person); Research Personnel; Sf9 cell line; Stem cells; Subunit Vaccines; Surface; Suspension Culture; Suspension substance; Suspensions; System; Technology; Testing; Therapeutic; Time; Tissue Engineering; Transgenic Organisms; Undifferentiated; Vaccine Production; Vaccines; Viral; Viral Proteins; base; cell growth; cell transformation; cell type; continuous cell line; cost; embryonic stem cell; experience; expression vector; gene delivery system; high risk; high standard; improved; novel; protein expression; scale up; success; tissue culture; tissue/cell culture; vector

DESCRIPTION (provided by applicant): Most cell culture lines are anchorage-dependent and require surface attachment for proliferation. For industrial production, increased surface area can be provided by microcarrier beads, but the associated increases in cost and the resulting complexity of manipulation may preclude their use. Due to these limitations, anchorage-independent cells are preferred for the production of biopharmaceuticals, but appropriate anchorage-independent cells are not available for all applications; e.g., production of vaccines and cell-type specific proteins. This proposal explores the use of a novel insect virus protein to transform adherent cells to cells that can thrive in suspension culture. ParaTechs has identified a cell line from Agrotis ipsilon (black cutworm), which provides levels of recombinant protein expression that are 3-to-10 times higher than the standard Sf9 cell line. Unfortunately, the cells are strongly adherent, which makes them unsuitable for large-scale protein production. We intend to stably transform the A. ipsilon cells with an insect virus gene that causes a loss of adhesion in hemocytes. We predict that expression of this protein in transformed cells will enable them to grow in suspension culture. In combination with our Vankyrin-Enhanced Baculovirus Expression Vector System (VE-BEVS), which has the ability to increase protein production per cell by a factor of 4 to 22, we anticipate achieving a level of protein expression per cell that is at least 12-to-220 times higher than currently possible. This dramatic increase in yield will be significant for all BEVS users, from individual researchers to large biopharmaceutical companies. We also will apply this technology to adherent mammalian tissue cultures. A simple method for converting mammalian cells from anchorage-independent to suspension culture would make a significant contribution to the production of vaccines and bio-therapeutics, and would result in improved human health. ParaTechs personnel are experienced baculovirologists and cell biologists. The protocols use standard technologies that are routinely adopted in our company. We do not anticipate difficulty with the experimentation. Current literature strongly supports our hypothesis and we are confident of our success. PUBLIC HEALTH RELEVANCE: The future of human medicine will involve a dramatic increase in development of protein-based drugs and subunit vaccines, whose production will require large-scale propagation of tissue culture cells in suspension culture. Most continuous cell lines, however, are anchorage-dependent, and no method exists to routinely and easily transform adherent cells to suspension culture. ParaTechs will test an insect virus gene that causes hemocytes to lose adhesion for the ability to adapt anchorage- dependent insect and mammalian cells to suspension culture. Achievement of this goal would make a significant contribution to biopharmacology and human health.

描述（由申请方提供）：大多数细胞培养系具有锚定依赖性，需要表面附着才能增殖。对于工业生产，微载体珠粒可以提供增加的表面积，但是相关的成本增加和由此产生的操作复杂性可能妨碍它们的使用。由于这些限制，优选锚定非依赖性细胞用于生物药物的生产，但是合适的锚定非依赖性细胞并非可用于所有应用;例如，生产疫苗和细胞类型特异性蛋白质。该提案探索了使用一种新的昆虫病毒蛋白将贴壁细胞转化为可以在悬浮培养中茁壮成长的细胞。 ParaTechs已经鉴定了来自小地老虎（小地老虎）的细胞系，其提供的重组蛋白表达水平比标准Sf 9细胞系高3至10倍。不幸的是，细胞粘附性很强，这使得它们不适合大规模生产蛋白质。我们打算稳定地改造A。带有昆虫病毒基因的细胞，导致血细胞粘附力的丧失。我们预测这种蛋白质在转化细胞中的表达将使它们能够在悬浮培养中生长。结合我们的Vankelovirus增强型杆状病毒表达载体系统（VE-BEVS），该系统能够将每个细胞的蛋白质产量提高4至22倍，我们预计每个细胞的蛋白质表达水平至少比目前可能的水平高12至220倍。这种产量的大幅增加对所有BEVS用户来说都是重要的，从个人研究人员到大型生物制药公司。我们也将把这项技术应用于贴壁哺乳动物组织培养。一种将哺乳动物细胞从非贴壁依赖性转化为悬浮培养的简单方法将对疫苗和生物治疗剂的生产做出重大贡献，并将改善人类健康。 ParaTech的工作人员是经验丰富的杆状病毒学家和细胞生物学家。这些协议使用我们公司常规采用的标准技术。我们预计试验不会有困难。目前的文献强烈支持我们的假设，我们有信心我们的成功。 公共卫生相关性：人类医学的未来将涉及基于蛋白质的药物和亚单位疫苗的开发的急剧增加，其生产将需要在悬浮培养中大规模繁殖组织培养细胞。然而，大多数连续细胞系是贴壁依赖性的，并且没有方法常规且容易地将贴壁细胞转化为悬浮培养物。ParaTechs将测试一种昆虫病毒基因，该基因使血细胞失去粘附能力，以使依赖锚定的昆虫和哺乳动物细胞适应悬浮培养。这一目标的实现将对生物药理学和人类健康做出重大贡献。