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
基本信息
- 批准号:9908833
- 负责人:
- 金额:$ 22.46万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AntibioticsBaculovirusesBiologicalBiotechnologyCRISPR/Cas technologyCell LineCellsChimeric ProteinsClone CellsComplexCrystallizationCustomDendritic CellsDepositionDiseaseEngineeringEnzymesFreezingGenesGenomeGlycoproteinsGrowthHumanImmunologic AdjuvantsInsectaKnock-outLicensingMalignant NeoplasmsMammalian CellMannoseMembrane GlycoproteinsModelingPatternPharmaceutical PreparationsPhasePolysaccharidesPopulationProductionProductivityPropertyProteinsProvengeResearchResearch PersonnelSmall Business Innovation Research GrantStructural ProteinStructureSurfaceSuspension CultureSystemTestingTherapeuticUniversitiesVirusWyomingX-Ray Crystallographycell bankclinical efficacyexperienceflexibilityglucosylceramidaseglycoprotein structureglycosylationimprovedinnovationinterestknockout genemacrophageprotein structurestructural biologystructured datasugartherapeutic proteintherapeutic targetthree dimensional structuretooltranscriptomevector
项目摘要
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.
项目概要/摘要
拟议研究的目的是创建并验证新的昆虫细胞系
用于生产糖蛋白(表面附着有糖的蛋白质),专门用于结构
生物学研究。我们将改造昆虫细胞系,使它们产生可用于
在非变性条件下去除,从而保留了三维结构
蛋白质。因此,可以获得这些蛋白质的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以及糖蛋白
复合物。
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 CRISPR-Cas9 载体并使用它们来破坏 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具有预期糖基化的蛋白质
生产力、增长和生存能力的模式和筛选。我们将生产一个非 cGMP 细胞库
ATCC 分销和存款单元表现最佳的机构。
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才能发挥功效。我们将确认新的细胞系可以提供
增加与功效相关的甘露糖含量。
这项研究的意义在于,尽管昆虫细胞是最常见的
使用真核系统来生产用于结构研究的蛋白质,它们不适合
为此目的生产糖蛋白——尽管所有蛋白质中约有一半是
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
对于依赖富含甘露糖的糖蛋白疗法(“生物制剂”)的生产具有价值
聚糖的功效。此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)。
拟议研究的创新点是:
1) 我们将使用不含持久性外来病毒的专有昆虫细胞系。因此,
新的细胞系将为蛋白质治疗药物的生产提供卓越的生物安全性。
2) 我们将使用我们新开发的CRISPR-Cas9工具,该工具是第一个启用目标基因的工具
杆状病毒/昆虫细胞平台最常用的细胞系中的基因敲除。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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CAROL D BLAIR的其他文献
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{{ truncateString('CAROL D BLAIR', 18)}}的其他基金
Glycoengineered insect cells for commercial biologics manufacturing
用于商业生物制品制造的糖基工程昆虫细胞
- 批准号:
9331691 - 财政年份:2013
- 资助金额:
$ 22.46万 - 项目类别:
Human monoclonal antibodies (huMAbs) for medically important arboviruses
针对医学上重要的虫媒病毒的人单克隆抗体 (huMAb)
- 批准号:
8261430 - 财政年份:2011
- 资助金额:
$ 22.46万 - 项目类别:
Human monoclonal antibodies (huMAbs) for medically important arboviruses
针对医学上重要的虫媒病毒的人单克隆抗体 (huMAb)
- 批准号:
7675651 - 财政年份:2009
- 资助金额:
$ 22.46万 - 项目类别:
Specialized Biodefense Training in Basic Research
基础研究专业生物防御培训
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7126298 - 财政年份:2005
- 资助金额:
$ 22.46万 - 项目类别:
INTERFERENCE TO DENGUE VIRUS REPLICATION IN MOSQUITOES
干扰蚊子中的登革热病毒复制
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2069088 - 财政年份:1993
- 资助金额:
$ 22.46万 - 项目类别:
Interference to Dengue Virus Replication in Mosquitoes
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- 批准号:
6477987 - 财政年份:1993
- 资助金额:
$ 22.46万 - 项目类别:
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