Brought to you by Applied Stemcell, Inc.
Introduction
Applied StemCell’s (ASC) proprietary TARGATT™ gene editing technology allows for an irreversible, site-specific gene insertion at a safe-harbor locus. Because the insertion occurs at a defined safe-harbor locus, the cell lines are stable and there is consistent expression across constructs. The system permits large fragment knockin up to 20 kb, and the technology functions in non-dividing cells. TARGATT™ enables a single copy insertion, and we have observed high integration efficiencies and medium to high levels of protein expression.
ASC has used TARGATT™ to engineer CHO and HEK293 master cell lines for bioproduction, to establish a mammalian cell-based library for antibody screening, and to construct a naïve IgG library system.
Figure 1: TARGATT™ gene insertion schematic. The technology allows site-specific, irreversible gene insertion at a safe-harbor locus.
ASC engineered a TARGATT™ Master CHO Cell Line with a landing pad at a safe-harbor locus that permits a single copy insertion and expression of any gene of interest. The stable cell line grants high efficiency for gene insertion. We have measured the integration efficiency of the TARGATT™ Master CHO Cell Line and compared it to a control, random integration, using GFP as a reporter. Before drug selection, we observed a gene insertion efficiency ranging from 13-18%. A gene insertion efficiency of over 97% was reported post-selection.
Figure 2: GFP, western blot & flow cytometry analysis on polyclonal pools.
Figure 3: Schematic of the single-copy insertion experimental design along with florescence imaging and flow cytometry confirming single-copy insertion.
To confirm single-copy insertion in the TARGATT™ CHO Master Cell Line, we designed an experiment with two different donor plasmids, one with a green florescence protein and the other with a red florescence marker, mCherry. A 1:1 mix of the donor plasmids was transfected into the CHO Master Cell Line, and distinct green and red cells were visualized without any overlap. This suggests that integration does in fact occur as a single copy.
Figure 4: The H11 locus and the animals that have an orthologous sequence of the H11 safe-harbor locus.
H11 Safe-Harbor Locus
The single-copy, irreversible insertion results in the H11 safe-harbor locus that is identified in an intergenic region between two highly expressed genes. ASC has identified orthologous sequences of H11 in various cell lines (e.g., human cells, HEK293 cells, iPS cells, and CHO cells) and animals (e.g., mice, pigs, and rats). Insertion at the H11 site leads to little or no phenotypic change. Also, integration does not affect any endogenous gene expression or function, and medium to high levels of transgene expression have been observed.
Safe-Harbor Locus Case Study
In this case study, a collaboration partner collected expression data for the production of 2 antibodies using our TARGATT™ CHO Master Cell Line. A single copy of the transgene for antibody 1 and 2 was inserted into the H11 locus in the master cell line, and the cells were grown in a 17-day fed-batch shake-flask. We detected consistent production of greater than 0.5 g/l in bulk enriched pools with no optimization. These cells have the potential to generate stable CHO libraries for research-scale production.
Figure 5: Case study results depicting the increase of antibody (1 and 2) expression over a 17-day period.
What do current library screening systems lack?
Traditionally, library screening is completed using bacteria or yeast phage display. Bacteria allow for the creation of large libraries in a short period of time while yeast provides a eukaryotic environment. These systems may be cost effective, but they lack post-translational modifications that mammalian cells permit.
Mammalian cells also offer a human like environment, but the current available systems are slow and laborious to work with at a high cost. The available mammalian library systems for screening may provide an environment closer to the human system, but the coverage they allow is very low compared to bacteria and yeast.
Applied StemCell’s Solution
Our goal was to engineer the TARGATT™ system into HEK293 & CHO cells in order to:
Figure 6: Comparison of the currently available library screening systems.
Table 1: A comparison of the TARGATT™ mammalian display with available alternatives display systems.
To address the current library screening and size issues, Applied StemCell is using its TARGATT™ gene editing technology to develop a mammalian display system that can consistently hit within an order of magnitude typical for bacteria and yeast. When comparing the TARGATT™ system for mammalian display to other available systems, it is clear that the TARGATT™ system offers unique features including site-specific and single-copy gene insertion.
The TARGATT™ Screen Master Cell Lines were engineered using a split-cassette selection/screen system. This allows us to obtain clean results with little background. We separated the promoter and the transgene. The promoter was inserted in the chromosome at a safe-harbor locus, and the transgene is carried by the donor plasmid. This system only allows expression of the insert if there is a site-specific gene insertion at the safe-harbor locus that contains the promoter. If random integration were to occur, the gene would not have a promoter and therefore would not be expressed.
Applied StemCell integrated this system into HEK293 and CHO cells. Both cell lines have reported high integration efficiencies and medium to high levels of protein expression.
Figure 7: Schematic of the split-cassette selection/screen system.
TARGATT™ CHO Library Screen Master Cell Line:
Figure 8: The TARGATT™ HEK293 Library Screen Master Cell Line presented >40% integration efficiency without drug selection and >90% with drug selection.
TARGATT™ HEK293 Library Screen Master Cell Line:
Figure 9: The TARGATT™ CHO Library Screen Master Cell Line presented an integration efficiency of ~18% without drug selection and >90% with drug selection.
Compared to the available screening systems, the TARGATT™ screening system has the potential to reach library sizes used in E.coli and surpass moderate yeast libraries with a much higher efficiency.
Figure 10: A size comparison between available screening systems and the TARGATT™ screening system.
- TARGATT™ efficiency: 1,600 – 4000x improvement over FlpIn (40% vs. 0.01% - 0.025%)
- If we match cell counts used in FlpIn projects, TARGATT™ HEK293 can reach library sizes used in E. coli
- Can surpass moderate yeast libraries
Figure 11: Applied StemCell’s platform for antibody discovery and screening.
Figure 12: Sequencing data that displays no repeat sequences for light (top row) and heavy (bottom row) chains.
Applied StemCell is taking the TARGATT™ system to construct a naïve IgG library system for antibody discovery and screening. ASC built the donor library by extracting RNA from a non-immunized human donor (spleen, lymph-node, or bone marrow), generating cDNA, and amplifying the heavy and light chains via PCR. The library was incorporated into a donor vector and Sanger sequencing and NGS was conducted for library QC.
ASC transfected the TARGATT™ HEK293 Library Screen Master Cell Line with the library donor DNA and the TARGATT™ integrase vector. Following a 40-hour period, drug selection was carried out followed by QC for library quality by Sanger sequencing and NGS. Our current data confirms we have high library diversity coverage and no repeat sequences.
Our TARGATT™ system has been integrated into both HEK293 and CHO cells for library screening
We use a validated safe-harbor locus, H11
ASC uses the recombinase serine integrase
High gene integration efficiency
We have a split-cassette system that allows us to achieve clean results with little background
The TARGATT™ system for library screening is advantageous because our coverage is estimated to reach what bacteria and yeast can offer
Table 2: A summary of the TARGATT™ system advantages compared to currently available systems.
Applied StemCell’s TARGATT™ technology allows faster and efficient site-specific integration of large DNA fragments. We incorporated our technology into CHO cells for bioproduction and HEK293/CHO cells for antibody screening and library construction.
While ASC offers TARGATT™ CHO and HEK293 stable cell lines, Applied StemCell can engineer the TARGATT™ system into your favorite cell line so you can use the technology for your specific research needs!
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ASC Licenses Its TARGATT™ CHO Cell Technology for Biotherapeutics Development – Milpitas, California, January 19th, 2021
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