title: Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.
abstract: Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Stoddard
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Zachary L
authors.initials: ZL
authors.lastname: Demorest
authors.affiliation: Medicago Inc., Québec, QC, Canada.
authors.forename: Pierre-Olivier
authors.initials: PO
authors.lastname: Lavoie
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Song
authors.initials: S
authors.lastname: Luo
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Benjamin M
authors.initials: BM
authors.lastname: Clasen
authors.affiliation: Cellectis SA, Paris, France.
authors.forename: Frederic
authors.initials: F
authors.lastname: Cedrone
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Erin E
authors.initials: EE
authors.lastname: Ray
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Coffman
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Aurelie
authors.initials: A
authors.lastname: Daulhac
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Ann
authors.initials: A
authors.lastname: Yabandith
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Retterath
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Luc
authors.initials: L
authors.lastname: Mathis
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.affiliation: Medicago Inc., Québec, QC, Canada.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: D'Aoust
authors.affiliation: Cellectis Plant Sciences Inc., New Brighton, MN, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D001483:Base Sequence; D005643:Fucose / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D006031:Glycosylation; D009154:Mutation / Q000235:genetics; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D011523:Protoplasts / Q000378:metabolism; D000069283:Rituximab / Q000096:biosynthesis; D014026:Nicotiana / Q000235:genetics*; D000069896:Transcription Activator-Like Effector Nucleases / Q000378:metabolism; D014170:Transformation, Genetic; D014994:Xylose / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D010940:Plant Proteins; D011134:Polysaccharides; D005643:Fucose; D000069283:Rituximab; D014994:Xylose; D000069896:Transcription Activator-Like Effector Nucleases
keywords: Gene editing; glyco-engineering; plant-derived pharmaceuticals; transcription activator-like effector nucleases