Insect cell lines for glycoprotein structural biology and mannose-dependent protein drugs

用于糖蛋白结构生物学和甘露糖依赖性蛋白药物的昆虫细胞系

• 批准号: 9908833
• 负责人: CAROL D BLAIR
• 金额: $ 22.46万
• 依托单位: GLYCOBAC, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9908833
• 关键词: Antibiotics; Baculoviruses; Biological; Biotechnology; CRISPR/Cas technology; Cell Line; Cells; Chimeric Proteins; Clone Cells; Complex; Crystallization; Custom; Dendritic Cells; Deposition; Disease; Engineering; Enzymes; Freezing; Genes; Genome; Glycoproteins; Growth; Human; Immunologic Adjuvants; Insecta; Knock-out; Licensing; Malignant Neoplasms; Mammalian Cell; Mannose; Membrane Glycoproteins; Modeling; Pattern; Pharmaceutical Preparations; Phase; Polysaccharides; Population; Production; Productivity; Property; Proteins; Provenge; Research; Research Personnel; Small Business Innovation Research Grant; Structural Protein; Structure; Surface; Suspension Culture; System; Testing; Therapeutic; Universities; Virus; Wyoming; X-Ray Crystallography; cell bank; clinical efficacy; experience; flexibility; glucosylceramidase; glycoprotein structure; glycosylation; improved; innovation; interest; knockout gene; macrophage; protein structure; structural biology; structured data; sugar; therapeutic protein; therapeutic target; three dimensional structure; tool; transcriptome; vector

Project Summary / Abstract The objective of the proposed research is to create and validate new insect cell lines that can be used to produce glycoproteins (proteins with attached sugars to their surface) specifically for structural biology research. We will engineer insect cell lines so they produce sugar structures that can be removed under non-denaturing conditions, thereby retaining the three-dimensional structure of the proteins. As a result, better quality structural data can be obtained for these proteins. This includes for example membrane glycoproteins, glycoproteins involved in diseases such as cancer, and glycoprotein complexes. We will achieve our objective through three Specific Aims: 1) In Aim 1, we will generate custom CRISPR-Cas9 vectors and use them to disrupt the MGAT1 genes in our proprietary Sf-RVN and Tn-NVN cell lines. We will then obtain single cell clones without wild-type MGAT1 gene sequences. 2) Next, in Aim 2, we will confirm these clones produce proteins with the expected glycosylation pattern, and screen for productivity, growth, and viability. We will produce a non-cGMP cell bank for the best performers for distribution and deposit cells at the ATCC. 3) Finally, in Aim 3, we will use the new cell lines to produce two commercial protein drugs that require mannose-terminated glycans for efficacy. We will confirm the new cell lines can provide the increased mannose content that is associated with efficacy. The significance of the proposed research is that although insect cells are the most commonly used eukaryotic system used to produce proteins for structural research, they are not suitable for the production of glycoproteins for this purpose – even though about one half of all proteins are glycoproteins! Apart from the utility of the new cell lines for structural biology research, we believe they will have value for the production of glycoprotein therapeutics (“biologics”) that rely on mannose-rich glycans for efficacy. Such biologics include enzyme replacement therapeutics (targeting macrophages), as well as immunostimulants (targeting dendritic cells). The innovative aspects of the proposed research are: 1) We will use our proprietary insect cell lines that are free of persistent adventitious viruses. Thus, the new cell lines will have a superior biosafety profile for the production of proteins therapeutics. 2) We will use our newly developed CRISPR-Cas9 tools that are the first to enable targeted gene knockouts in the cell lines most commonly used for the baculovirus / insect cell platform.

项目总结/摘要 这项研究的目的是建立和验证新的昆虫细胞系， 用于生产糖蛋白（表面附着糖的蛋白质），专门用于结构 生物学研究。我们将改造昆虫细胞系，使它们产生糖结构， 在非变性条件下除去，从而保留了所述化合物的三维结构。 proteins.因此，可以获得这些蛋白质的更好质量的结构数据。这包括例如 例如膜糖蛋白、与疾病如癌症有关的糖蛋白和糖蛋白 配合物 我们将通过三个具体目标实现我们的目标： 1)在目标1中，我们将生成定制的CRISPR-Cas9载体，并使用它们来破坏MGAT 1。 我们专有的Sf-RVN和Tn-NVN细胞系中的基因。然后我们将获得单细胞克隆 没有野生型MGAT 1基因序列。 2)接下来，在目标2中，我们将确认这些克隆产生具有预期糖基化的蛋白质 模式，并筛选生产力，生长和生存能力。我们将生产非cGMP细胞库， 在ATCC分配和存款细胞的最佳执行者。 3)最后，在目标3中，我们将使用新的细胞系生产两种商业蛋白质药物， 需要甘露糖封端的聚糖来发挥功效。我们将确认新的细胞系可以提供 与功效相关的甘露糖含量增加。 这项研究的意义在于，尽管昆虫细胞是最常见的细胞， 用真核系统来生产蛋白质用于结构研究，它们不适合于 糖蛋白的生产-即使大约一半的蛋白质是 糖蛋白！除了新细胞系在结构生物学研究中的用途外，我们相信它们 将具有生产依赖于富含甘露糖的糖蛋白治疗剂（“生物制剂”）的价值 聚糖的功效。这样的生物制剂包括酶替代治疗剂（靶向巨噬细胞）， 以及免疫刺激剂（靶向树突细胞）。 拟议研究的创新方面是： 1)我们将使用我们专有的不含持久性外源病毒的昆虫细胞系。因此 新的细胞系将具有用于生产蛋白质治疗剂的上级生物安全特性。 2)我们将使用我们新开发的CRISPR-Cas9工具，这是第一个能够实现靶向基因表达的工具。 最常用于杆状病毒/昆虫细胞平台的细胞系中的敲除。