Aim
Generate a CHO MGAT1- cell line via CRISPR gene knockout that maintains growth properties suitable for use in protein manufacturing. In addition, transiently produce rgp120 using the engineered CHO MGAT1- cell line and characterize protein glycosylation patterns and bN-mAb binding.
CHO Electroporation
• CHO-S or CHO MGAT1- cells were resuspended in MaxCyte electroporation buffer at 2 x 108 cells/mL.
• For CHO MGAT1- construction, CRISPR/Cas9 exonuclease with guide sequence plasmid was added to resuspended CHO-S at a final concentration of 300 μg/mL.
• For transient expression of HIV gp120, an expression plasmid encoding the gp120 from the A244 HIV strain were added to CHO MGAT1- cells.
• Post electroporation, cells were seeded at 4 x 106 cells/mL in OPTI-CHO media and cultured in 125-mL Erlenmeyer shake flasks.
• For gp120 production, cultures were supplemented with 1 mM sodium butyrate 24 hours post electroporation and the temperature lowered to 32°C.
Full methods for CHO MGAT1- screening and selection, as well as glycosylation analysis and binding assays are detailed in PLoS Biol, 16(8): e2005817, 2018.
Results
CRISPR Delivery & Creation of CHO MGAT1- Cell Line
CHO-S cells were electroporated with a plasmid encoding CRISPR/cas9, tracrRNA, and a complete guide RNA targeting the MGAT1 enzyme. Clones were initially screened for staining with fluorescein-labeled Galanthus nivalis lectin (GNA), a lectin that recognizes glycans with terminal mannose but not complex, sialic acid-containing glycans. As anticipated, the parental CHO-S cell line did not bind GNA. The four GNA-binding clones with the fastest growth rates were transiently transfected with a plasmid encoding A244-rgp120. Secreted rgp120 was purified and analyzed for overall titer and binding to the glycan-dependent bN-mAb PG9.
The selected CHO MGAT1- cell line had rgp120 yields comparable to the parental CHO-S line, as well as similar doubling times and the ability to grow at high density. These characteristics were far superior to the yield, growth time, and cell density restrictions of the GnTI- 293 HEK cell line used in previous studies.3 Sequence confirmation of the selected clone was performed to ensure disruption of the MGAT1 gene.

Figure 1: Identification of MGAT1- CHO Cells via GNA Binding of Oligomannose
CHO-S parental cells or those electroporated with MGAT-targeting CRISPR machinery were incubated with fluorescein-labeled GNA to identify cells with surface proteins containing terminal mannose residues.

Transient Expression of rgp120
A224-rgp120 transiently produced using the selected MGAT1- CHO cell line or parental CHO-S cells was characterized by MALDI-TOF mass spectroscopy to detail glycan composition. MGAT1-deficiency resulted in rgp120 containing >99% oligomannose glycans compared to only 25% when produced in CHO-S cells.
The homogeneity of glycosylation resulting from MGAT1-deficiency also positively impacted purification. The rgp120 in the RV144 HIV clinical trial showed extensive net charge heterogeneity that required immuno-affinity chromatography for purification greatly reducing yield and increasing
manufacturing complexity. rgp120 produced by MGAT1-deficient cells was secreted at high titers with homogeneous glycosylation allowing for purification using more conventional chromatography.
Figure 2: MGAT1-deficient CHO Cells Produce rgp120 Containing >99% Oligomannose Glycans
Purified rgp120 from the selected CHO MGAT1- cell line or the parental CHO-S cell line were analyzed via MALDI-TOF mass spectroscopy.

MGAT1-deficiency Produces rgp120 With Improved Binding to Multiple bN-mAbs
Binding of rgp120 produced in CHO-S or MGAT1- CHO cells to a panel of bN-mAbs was assessed via fluorescence immunoassay.3 All but a single antibody had modest to significant improvements in binding to rgp120 produced in the MGAT-deficient cells. These results suggest that altering glycosylation while leaving the amino acid sequence unchanged can positively impact bN-mAb binding. Examination of the effects of oligomannose engineering on in vivo immunogenicity and vaccine efficacy are critical next steps.
Conclusion
MaxCyte’s high performance cell engineering technology delivered the CRISPR/cas9 machinery that resulted in levels of efficiency and CHO-S cell viability that enabled the rapid generation of an MGAT1-deficient CHO cell line via precision CRISPR gene disruption. The established MGAT1- CHO cell line maintained robust growth in serum-free medium, was able to grow at high cell densities in suspension and produced rgp120 at high titers upon transient transfection. rgp120 produced using the newly engineered MGAT1- CHO cells gave rise to the desired early oligomannose glycans which in turn enhanced the binding of key bN-mAbs. This cell line promises to be a critical piece in the clinical translation of more efficacious HIV vaccines.
Figure 3: Improved Binding of V1/V2 and V3 Domain bN-mAbs to rgp120 Produced in MGAT1-deficient CHO Cells
A224-rgp120 purified from CHO-S or MGAT1- CHO cells were coated onto microtiter plates and binding of a panel antibodies with broadly neutralizing capacity examined via FIA.

References:
1. High-mannose Glycan-dependent Epitopes Are Fequently Targeted in Boad Neutralizing Antibody Responses during HIV-1 Infection. (2012) J Virol,
86(4):2153-2164.
2. Glycan Modification to the gp120 Immunogens Used in the RV144 Vaccine Trial Improve Binding to Broadly Neutralizing Antibodies. (2018) PLoS ONE,
13(4):e0196370.
3. CRISPR/Cas9 Gene Editing for the Creation of an MGAT1-deficienct CHO Cell Line to Control HIV-1 Vaccine Glycosylation. (2018) PLoS Biol, 16(8): e2005817.