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

项目摘要/摘要 这项拟议的研究的目标是创造和验证新的昆虫细胞系，这些细胞系可以 用来生产糖蛋白(表面有糖基的蛋白质)，专门用于结构 生物学研究。我们将改造昆虫细胞系，使它们产生能够 在非变性条件下去除，从而保留了 蛋白质。因此，可以获得这些蛋白质更高质量的结构数据。这包括用于 例如膜糖蛋白、与癌症等疾病有关的糖蛋白和糖蛋白 复合体。 我们将通过三个具体目标来实现我们的目标： 1)在目标1中，我们将生成定制的CRISPR-Cas9载体，并使用它们来扰乱MGAT1 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MGAT1基因序列。 2)接下来，在目标2中，我们将确认这些克隆产生预期糖基化的蛋白质 模式，并筛选生产力、增长和生存能力。我们将生产一种非cGMP细胞库 在ATCC分配和存放细胞方面表现最好的人。 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 需要甘露糖封端的葡聚糖才能有效。我们将确认新的细胞系可以提供 甘露糖含量增加，这与疗效有关。 这项拟议研究的意义在于，尽管昆虫细胞是最常见的 用于生产用于结构研究的蛋白质的真核系统，它们不适合 为此目的生产糖蛋白-即使大约一半的蛋白质是 糖蛋白！除了新的细胞系对结构生物学研究的效用外，我们相信它们 将对依赖于富含甘露糖的糖蛋白疗法(“生物制品”)的生产具有价值 多聚糖的功效。这类生物制剂包括酶替代疗法(针对巨噬细胞)， 以及免疫刺激剂(针对树突状细胞)。 拟议研究的创新方面包括： 1)我们将使用我们的专有昆虫细胞系，这些细胞系没有持续的不定病毒。因此， 新的细胞系将具有更高的生物安全性，可用于生产蛋白质疗法。 2)我们将使用我们新开发的CRISPR-CAS9工具，这些工具是第一个启用靶向基因的工具 最常用于杆状病毒/昆虫细胞平台的细胞系中的敲除。

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.