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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：
CAROL D BLAIR
金额：
$ 22.46万
依托单位：
GLYCOBAC, LLC
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：
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

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.

项目摘要/摘要