Improvement of the Nicotiana tabacum BY-2 suspension cell platform for the production of therapeutic glycoproteins

Abstract

Plant suspension cells, especially Nicotiana tabacum Bright Yellow 2 (BY- 2), are promising hosts for the production of pharmacological glycoproteins such as antibodies or vaccines. However, improvement has still to be made regarding the product quality and the production yield. The recombinant proteins produced in plants carry N-glycans with nonhuman residues (β(1,2)-xylose and core α(1,3)-fucose), which can greatly impact the immunogenicity, allergenicity, or activity of the pharmacological proteins. We therefore sought to inactivate by CRISPR/Cas9 the genes coding for the glycosyltransferases that are responsible for adding these plant-like residues. We first demonstrated that this genome editing technique was efficient in BY-2 cells by inactivating a gene coding for a fluorescent protein (mCherry). We then targeted two genes coding for β(1,2)-xylosyltransferase (XylT) and four genes coding for α(1,3)-fucosyltransferase (FucT) in BY-2 cells. Knockout lines completed devoid of β(1,2)-xylose and α(1,3)-fucose were obtained as demonstrated by Western blotting and mass spectrometry. PCR and DNA sequencing confirmed that XylT and FucT genes had been hit. We eventually expressed a human IgG2 in a knockout line and demonstrated the absence of β(1,2)-xylose or α(1,3)-fucose residues on its glycosylation moiety. In a second part of this project, two different approaches were investigated to increase the transcription rate of a transgene and thus the production yield of the protein of interest. The first one consisted of finding strong constitutive transcription promoters by assuming that the most abundant transcripts in BY-2 cells possess very active promoters. A list of highly abundant transcripts in BY-2 and Arabidopsis cells was obtained thanks to RNA sequencing. The second approach consisted of using a deconstructed geminivirus-based vector coupled with an ethanolinducible system in order to induce the formation of a high copy number of replicons containing a gene of interest. However, the system turned out to be leaky, resulting in low expression levels of the gene of interest. Nevertheless, replicon formation was observed and we therefore came to the conclusion that the system could become efficient by replacing the ethanol inducible promoter by a tightly controlled one.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 201