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

Award Information
Agency: Department of Health and Human Services
Branch: National Institutes of Health
Contract: 1R43GM136071-01
Agency Tracking Number: R43GM136071
Amount: $224,597.00
Phase: Phase I
Program: SBIR
Solicitation Topic Code: 300
Solicitation Number: PA16-157
Solicitation Year: 2016
Award Year: 2020
Award Start Date (Proposal Award Date): 2020-03-01
Award End Date (Contract End Date): 2021-02-28
Small Business Information
Laramie, WY 82072-3037
United States
DUNS: 968784103
HUBZone Owned: No
Woman Owned: No
Socially and Economically Disadvantaged: No
Principal Investigator
 (970) 491-8243
Business Contact
Phone: (307) 760-8194
Research Institution

Project Summary / AbstractThe 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
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 MGAT1genes in our proprietary Sf-RVN and Tn-NVN cell lines. We will then obtain single cell cloneswithout wild-type MGAT1 gene sequences.
2) Next, in Aim 2, we will confirm these clones produce proteins with the expected glycosylationpattern, and screen for productivity, growth, and viability. We will produce a non-cGMP cell bank forthe 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, thenew 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 geneknockouts in the cell lines most commonly used for the baculovirus / insect cell platform.Project NarrativeInsect cells are the most commonly used eukaryotic platform for the production of proteins for
structural biology, but there are no insect cell lines that are useful for the production of glycoproteins for
this purpose. Here, we proposed to develop and test two new insect cell lines with CRISPR-Cas9-induced
knockouts in the MGAT1 N-glycan processing gene as powerful tools to facility glycoprotein structural
biology through their ability to produce glycoproteins that can be deglycosylated under non-denaturing
conditions. Additionally, as proof of concept we will produce two protein therapeutics that require high
mannose glycans for macrophage or dendritic cell targeting in order to demonstrate the value of the new
cell lines for the production of such therapeutics.

* Information listed above is at the time of submission. *

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