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.

Keywords: Gene editing; glyco-engineering; plant-derived pharmaceuticals; transcription activator-like effector nucleases.

Figure 1

Targeting mutations in the FucT1/FucT2 and XylT1/XylT2 genes. (a) The FucT and XylT genes are responsible for the attachment of core α(1,3)‐fucose and β(1,2)‐xylose residues to N‐glycans in plants. (b) Schematics of the FucT1/FucT2 and XylT1/XylT2 genes. The FucT1/FucT2 genes contain 7 exons and 6 introns, and the XylT1/XylT2 genes contain 3 exons and 2 introns, represented by the open boxes and angled lines, respectively. TALEN target sites are indicated by arrowheads in the first exon of each gene. (c) DNA sequences of TALEN target sites in the FucT1/FucT2 and (d) the XylT1/XylT2 genes. Each TALEN target site consists of two TALEN recognition sequences, denoted in upper‐case letters; the spacer sequences are shown in lower case. DNA sequence polymorphisms between FucT1 and FucT2 are indicated.

Figure 2

Genetically transmissible mutations induced by TALENs in N. benthamiana plants. (a) TALEN‐induced mutations in FucT1/FucT2 or XylT1/XylT2 knockouts. The plants NB13‐105a and NB13‐213a contain mutations in both alleles of FucT1 and FucT2; the plants NB15‐11d and NB12‐113c have mutations in both alleles of XylT1 and XylT2. TALEN recognition sequences are indicated in upper case; the spacer sequences are in lower case. Deletions are indicated by dashes, and the size of each deletion in base pairs is shown on the right. (b) TALEN‐induced mutations in the plant NB14‐29a that has mutations in all alleles of FucT1, FucT2, XylT1 and XylT2.

Figure 3

N‐glycosylation profiles of endogenous proteins in the FucT1/FucT2 and XylT1/XyT2 mutant plants. (a) Changes in levels of α(1,3)‐fucose in N‐glycans in the endogenous proteins of the FucT1/FucT2 knockout plants. (b) Changes in levels of β(1,2)‐xylose in the endogenous proteins of the XylT1/XylT2 knockout plants. (c) Levels of α(1,3)‐fucose and β(1,2)‐xylose in N‐glycans in the endogenous proteins of the plant with all FucT and XylT genes knocked out.

Figure 4

Comparison of the N‐glycan profile of the rituximab antibody produced in wild‐type and NB14‐29aT2 plant lines. Numbers represent the average percentage of each glycoform identified from three lots of antibodies produced in each plant line.