SummaryBiopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core a(1,3)-fucose and b(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 a(1,3)-fucosyltransferase (FucT) and the two b(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 b(1,2)-xylose and had a significant reduction in core a(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 a(1,3)-fucose and b(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.

Summary Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N‐glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N‐glycosylation in alfalfa. The first was a knock‐down of two plant‐specific N‐glycan maturation enzymes, β1,2‐xylosyltransferase and α1,3‐fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human β1,4‐galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N‐acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock‐down and knock‐in strategies strongly, but not completely, inhibited the biosynthesis of α1,3‐fucose‐ and β1,2‐xylose‐containing glycoepitopes in transgenic alfalfa. However, recombinant human β1,4‐galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N‐glycans.