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（由申请人提供）：大多数细胞培养线依赖于锚固，需要表面附着以进行增殖。对于工业生产，微载体珠可以提供表面积增加，但是相关的成本增加和由此产生的操纵复杂性可能会排除其使用。由于这些局限性，与生物药物的生产相比，独立于锚固的细胞是优选的，但是所有应用都没有适当的锚定细胞。例如，生产疫苗和细胞型特异性蛋白。该建议探讨了新型昆虫病毒蛋白的使用将粘附细胞转化为可以在悬浮培养中繁殖的细胞。 Paratechs已从Agrotis Ipsilon（黑色cut虫）鉴定出细胞系，该细胞系提供的重组蛋白表达水平比标准SF9细胞系高3至10倍。不幸的是，这些细胞非常粘附，这使得它们不适合大规模蛋白质产生。我们打算用昆虫病毒基因稳定地转化A. ipsilon细胞，从而导致血细胞粘附损失。我们预测该蛋白在转化的细胞中的表达将使它们能够在悬浮培养物中生长。结合我们的Vankyrin增强的杆状病毒表达载体系统（VE-BEV），它具有将每个细胞蛋白质产生的能力提高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高12至220倍。从个人研究人员到大型生物制药公司，所有BEVS使用者的产量急剧提高将很大。我们还将将该技术应用于依从性哺乳动物组织培养物。一种将哺乳动物细胞从独立于锚定为悬浮培养的简单方法将对疫苗和生物治疗药的产生做出重大贡献，并会改善人类健康。 伞兵人员是经验丰富的杆状化病毒学家和细胞生物学家。协议使用我们公司常规采用的标准技术。我们预计实验不会遇到困难。当前的文献强烈支持我们的假设，我们对我们的成功充满信心。 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基疫苗的发展，其生产将需要大规模传播悬浮培养中的组织培养细胞。然而，大多数连续的细胞系都是锚定依赖性的，并且没有任何方法可以常规，容易地将粘附的细胞转化为悬浮培养物。 paratechs将测试一种昆虫病毒基因，该基因会导致血细胞失去粘附，以使依赖锚定的昆虫和哺乳动物细胞适应悬浮培养。实现这一目标将为生物药理学和人类健康做出重大贡献。