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

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

Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 cells by a multiplex CRISPR/Cas9 strategy results in glycoproteins without plant-specific glycans

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with planttypical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)- fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells. © 2017 Mercx, Smargiasso, Chaumont, De Pauw, Boutry and Navarre

Evolutionary and Predictive Functional Insights into the Aquaporin Gene Family in the Allotetraploid Plant Nicotiana tabacum

Aquaporins (AQPs) are a class of integral membrane proteins that facilitate the membrane diffusion of water and other small solutes. Nicotiana tabacum is an important model plant, and its allotetraploid genome has recently been released, providing us with the opportunity to analyze the AQP gene family and its evolution. A total of 88 full-length AQP genes were identified in the N. tabacum genome, and the encoding proteins were assigned into five subfamilies: 34 plasma membrane intrinsic proteins (PIPs); 27 tonoplast intrinsic proteins (TIPs); 20 nodulin26-like intrinsic proteins (NIPs); 3 small basic intrinsic proteins (SIPs); 4 uncharacterized X intrinsic proteins (XIPs), including two splice variants. We also analyzed the genomes of two N. tabacum ancestors, Nicotiana tomentosiformis and Nicotiana sylvestris, and identified 49 AQP genes in each species. Functional prediction, based on the substrate specificity-determining positions (SDPs), revealed significant differences in substrate specificity among the AQP subfamilies. Analysis of the organ-specific AQP expression levels in the N. tabacum plant and RNA-seq data of N. tabacum bright yellow-2 suspension cells indicated that many AQPs are simultaneously expressed, but differentially, according to the organs or the cells. Altogether, these data constitute an important resource for future investigations of the molecular, evolutionary, and physiological functions of AQPs in N. tabacum

Gene Inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 Suspension Cells

Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices (GMPs). Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry) with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar) gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells

Data_Sheet_1_Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9.docx

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the N-glycosylation pathway including the presence of β(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for Arabidopsis thaliana and Nicotiana benthamiana. Such engineering of N-glycosylation would also be desirable for Nicotiana tabacum, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated N. tabacum cv. SR-1 β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four FucT and two XylT genes. These two enzymes are responsible for generating non-human N-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing β(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing Nicotiana tabacum as a biologics platform for Global Health.</p