Patent Application
20100154081
The current invention relates to the field of molecular farming, i.e. the use of plant cells or plants as bioreactors to produce biopharmaceuticals, particularly polypeptides and proteins with pharmaceutical interest such as therapeutic proteins, which have an altered glycosylation pattern that resembles mammalian glycosylation. The invention may also be applied to alter the glycosylation pattern of proteins in plants for any purpose, including modulating the activity or half-life of endogenous plant proteins or proteins introduced in plant cells.
The plant-specific N-glycosylation pathway appears to be one of the major drawbacks impeding the use of recombinant glycoproteins, particularly human glycoproteins, produced in plant cells or plants for therapeutic purposes.
The most important differences between the N-glycosylation pattern in plant glycoproteins and mammalian glycoproteins are the presence of β(1,2) xylosyl and core-α (1,3) fucosyl residues, and the absence of terminal β(1,4) galactosyl residues and sialic acid. β(1,2) xylosyl and core-α (1,3) fucosyl residues are thus absent in humans, and administration of plant-made therapeutical glycoproteins to humans, particularly over longer period could lead to immunogenic or allergic reactions. Moreover, the absence of terminal β(1,4) galactose in plant-made antibodies appears to result in a less efficient immune response than when using a corresponding antibody produced in mammalian cell cultures.
To mimic the N-glycosylation pattern of mammalian glycoproteins in plant-made glycoproteins, the level of the enzymes responsible for incorporation of β(1,2) xylosyl and core-α (1,3) fucosyl residues (i.e. β(1,2) xylosyltransferase (“XylT”) and α (1,3) fucosyltransferase (“FucT”)) needs to be reduced or eliminated in the host plant cells. In addition, the host plant cells need to be supplemented with β(1,4) galactosyltransferase (“GalT”) to achieve N-terminal incorporation of β(1,4) galactosyl in glycoproteins.
EP 1151109 describes the identification of core α (1,3) fucosyltransferase genes from Arabidopsis.
EP 1263968 describes the identification of β(1,2) xylosyltransferase gene from Arabidopsis.
Patent application PCT/EP2007/002322, published as WO2007/107296 describes the identification of β(1,2) xylosyltransferase genes from Nicotiana species.
Cox et al. (2006) (Nature BioTechnology 24, 1591-1597) report glycan optimization of a human monoclonal antibody in Lemnaceae by co-expressing the heavy and light chains of the mAbs with an RNA interference construct targeting expression of the endogenous α (1,3) fucosyltransferase and β(1,2) xylosyltransferase genes.
Koprivova et al. (2004) (Plant Biotechnology Journal 2, 517-523) describe the generation of targeted knockouts of Physcomitrella patens (a moss) wherein both the α (1,3) fucosyltransferase and β(1,2) xylosyltransferase genes were disrupted. The N-glycans of endogenous glycoproteins of these mutant moss plants lacked α (1,3) fucosyl and β(1,2) xylosyl residues. In addition, no such residues were detected on secreted human glycosylated growth factor expressed in such moss cells.
Strasser et al. (2004) (FEBS Letters 561, 132-136) report on the generation of Arabidopsis thaliana knock-out plants which completely lack both XylT and FucT activity. To this end, a triple knock-out plant was generated carrying knock-out mutations in the β(1,2) xylosyltransferase gene as well as in both genes encoding α (1,3) fucosyltransferase (FucTA and FucTB) present in Arabidopsis thaliana. Analysis of the N-glycans in such plants revealed the complete absence of α (1,3) fucosyl and β(1,2) xylosyl residues. Furthermore, less N-glycan heterogeneity was observed in the triple knock-out plants than in wild-type plants, and a high proportion of complex N-glycans carried terminal β N-acetylglucosamine residues on both the α1,3- and α1,6-linked mannoses.
Expression of β(1,4) galactosyltransferase (GalT) in plants has also been reported.
WO00/34490 provides a method for manufacturing a glycoprotein having a human-type sugar chain comprising a step in which a transformed plant cell is obtained by introducing into a plant cell a gene of a glycosyltransferase such as GalT and the gene of an exogenous glycoprotein, and a step in which the obtained transformed plant cell is cultivated.
WO02/057468 describes a method for the secretory production of a glycoprotein having a human-type sugar chain, comprising a step of introducing a gene of an enzyme capable of performing a transfer reaction of a galactose residue to a non-reducing terminal acetyl-glucosamine residue, and a gene of a heterologous glycoprotein, to obtain a transformed plant cells, a step of culturing the plant cell and a step of recovering the culture medium of the plant cell.
WO01/31405 describes a plant comprising a functional mammalian enzyme providing N-glycan biosynthesis that is normally not present in plants (such as a galactosyltransferase) said plant comprising additionally at least a second mammalian protein or functional fragment thereof that is normally not present in plants.
WO01/29242 describes a process for the production of proteins or polypeptides using genetically manipulated plants or plant cells, as well as genetically manipulated plants and plant cells per se. The described plants include transgenic plants comprising Mouse GalT, Bovine GalT or Human GalT.
WO03/078637 describes methods for optimizing glycan processing in organisms (and in particular plants) so that a glycoprotein having complex bi-antennary glycans and thus containing galacose residues on both arms and which are devoid of (or reduced in) xylose and fucose can be obtained.
Palacpac et al. (1999) (Proc. Natl. Acad. Sci. USA 96, 4692-4697) describes stable expression of human β(1,4) galactosyltransferase in plant cells, and the ensuing modified N-linked glycosylation patterns.
Bakker et al. (2001) (Proc. Natl. Acad. Sci. USA 96, 4692-4697 also describes stable expression of human β(1,4) galactosyltransferase in tobacco cells and partially galactosylated N-glycans (30%) on a monoclonal antibody expressed in such cells.
Bakker et al. (2006) described a tobacco plant expressing a hybrid β(1,4) galactosyltransferase and demonstrated that a mAB purified from leaves of plants expressing the hybrid enzyme displayed a N-glycan profile that featured high levels of galactose, undetectable xylose and a trace of fucose.
WO2004/057002 describes bryophyte plants and bryophyte plant cells comprising dysfunctional fucT and XylT genes and an introduced glycosyltransferase gene, such as a mammalian galactosyltransferase.
Huether et al. (2005) (Plant Biology, 7, 292-299) describes glycoengeering of moss lacking plant-specific sugar residues. Described are transgenic strains of the moss Physcomitrella patens in which the α (1,3) fucosyltransferase and β(1,2) xylosyltransferase genes were knocked out by targeted insertion of the human β(1,4) galactosyltransferase coding sequence in both of the plant genes.
The prior art however did not describe higher plant cells or plants which have no functional α (1,3) fucosyltransferase and β(1,2) xylosyltransferase activity while at the same time comprising a functional β(1,4) galactosyltransferase expressed under control of a plant-expressible promoter. To the contrary, the closest prior art document WO2004/057002 indicated that in higher plants it was not thought possible to suppress the activities of β(1,2) xylosyltransferase and of α (1,3) fucosyltransferase and moreover stresses the important differences between mosses and higher plants on the biochemical level.
Finally, none of the prior art documents demonstrated that plant glycoproteins obtained from plant cells or plants wherein β(1,2) xylosyltransferase and α (1,3) fucosyltransferase was eliminated and wherein further a β(1,4) galactosyltransferase was provided would exhibit a N-glycan profile with a higher level of galactosylation of the glycoproteins than in glycoproteins obtained from plants which have a normal β(1,2) xylosyltransferase and of α (1,3) fucosyltransferase activity level. Such a result is unexpected and inherently unpredictable, as the glycosylation pathway in eukaryotic organisms, but specifically in plants appears to be highly regulated and wherein modulation of the level of one glycotransferase appears to interact with the activity of other glycotransferase. See e.g. Bakker et al. 2006 (supra) where expression of a hybrid galactosyltransferase not only resulted in a N-glycan profile featuring high levels of galactose but also reduced levels of xylose and fucose.
The current invention provides method and means to alter the N-glycosylation pattern of glycoproteins in higher plant cells and plants as will become apparent from the following description, examples, drawings and claims provided herein.
It is one object of the invention to provide a method to produce glycoproteins with altered glycosylation profile in higher plant cells, said method comprising the steps of providing a plant cell wherein said plant cell has a reduced level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity, preferably no detectable β(1,2) xylosyltransferase and no detectable α (1,3) fucosyltransferase activity and a functional β(1,4) galactosyltransferase activity, such as a mammalian or human β(1,4) galactosyltransferase and cultivating said cell and isolating glycoproteins from said cell. The reduced level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity may be the result of a null mutation in the endogenous β(1,2) xylosyltransferase and a (1,3) fucosyltransferase encoding genes or may be achieved by transcriptional or post-transcriptional silencing of the expression of endogenous β(1,2) xylosyltransferase and a (1,3) fucosyltransferase encoding genes. The β(1,4) galactosyltransferase is preferably expressed from a chimeric gene comprising a plant-expressible promoter operably linked to a DNA region encoding said β(1,4) galactosyltransferase and a DNA region involved in transcription termination and polyadenylation. The DNA region encoding the β(1,4) galactosyltransferase may be a nucleotide sequence encoding the amino acid sequence of SEQ ID No 11 such as the nucleotide sequence of SEQ ID No 10 from nucleotide position 523 to nucleotide position 1719. The glycoprotein may be a glycoprotein foreign to the higher plant cell and may be expressed from a chimeric gene comprising a plant expressible promoter operably linked to a coding region encoding the glycoprotein. The glycoprotein may also be expressed using a viral RNA vector. Preferred glycoproteins are therapeutic proteins such as monoclonal antibodies.
It is another object of the invention to provide a glycoprotein obtained by the methods described herein. The glycoproteins provided are derived from a higher plant cell having a complex N-glycan profile devoid of β(1,2) xylosyl and α (1,3) fucosyl and further comprising terminally linked β(1,4) galactosyl residues. In the glycoproteins according to the invention, a β(1,4) galactosyl residue may have been transferred to at least 30%, preferably at least 40% of the terminally linked N-acetylglucosamine residues, particularly when assessing the total glycoproteins, and not only secreted proteins.
In yet another embodiment, the invention provides cells of a higher plant comprising a reduced level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity or no β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity and a functional β(1,4) galactosyltransferase activity. The cells of the higher plant may comprise a chimeric gene including a plant-expressible promoter operably linked to a DNA region encoding a β(1,4) galactosyltransferase such as a mammalian or human or a hybrid β(1,4) galactosyltransferase and a DNA region involved in transcription termination and polyadenylation. The reduced level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity in the plant cell may be the result of a null mutation in the endogenous β(1,2) xylosyltransferase and α (1,3) fucosyltransferase encoding gene or may be achieved by transcriptional or post-transcriptional silencing of the expression of endogenous β(1,2) xylosyltransferase and α (1,3) fucosyltransferase encoding gene.
In yet another embodiment a higher plant consisting essentially of the plant cells described is provided.
The invention also provides a method to modify the N-glycosylation pattern of glycoproteins in higher plant cells, comprising the step of generating a plant cell wherein the plant cell has a reduced level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity and a functional β(1,4) galactosyltransferase.
The current invention is based on the observation that N-glycans from glycoproteins derived from higher plant cells containing a chimeric plant-expressible huGalT which contained no functional β(1,2) xylosyltransferase or core-α (1,3) fucosyltransferase comprised more β(1,4) galactosyl residues than N-glycans from glycoproteins derived from transgenic higher plant cells which have a functional β(1,2) xylosyltransferase and core-α (1,3) fucosyltransferase and further contain a chimeric plant-expressible huGalT.
In a first embodiment, the invention thus provides a method to produce glycoproteins with altered glycosylation profile in higher plant cells comprising the steps of providing a plant cell wherein said plant cell has a reduced level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity and a functional β(1,4) galactosyltransferase activity; followed by cultivating the obtained cell and isolating glycoproteins from said cell.
As used herein “a higher plant cell” is a cell of plant belonging to the Angiospermae or the Gymospermae, but excluding Algae and Bryophyta. Preferably, the higher plant cell is a cell of a plant belonging to the Brassicaceae or the Solanaceae, including Arabidopsis or Nicotiana spp.
The level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity can conveniently be reduced or eliminated by identifying plant cells having a null mutation in all of the genes encoding β(1,2) xylosyltransferase and in all of the genes encoding a (1,3) fucosyltransferase.
Genes encoding α (1,3) fucosyltransferase in plants are well known and include the following database entries identifying experimentally demonstrated and putative FucT cDNA and gene sequences, parts thereof or homologous sequences: NM 112815 (Arabidopsis thaliana), NM103858 (Arabidopsis thaliana), AJ 618932 (Physcomitrella patens) At1g49710 (Arabidopsis thaliana) and At3g19280 (Arabidopsis thaliana). DQ789145 (Lemna minor), AY557602 (Medicago truncatula) Y18529 (Vigna radiata) AP004457 (Oryza sativa), AJ891040 encoding protein CAI70373 (Populus alba×Populus tremula) AY082445 encoding protein AAL99371 (Medicago sativa) AJ582182 encoding protein CAE46649 (Triticum aestivum) AJ582181 encoding protein CAE46648 (Hordeum vulgare) (all sequences herein incorporated by reference).
Genes encoding β(1,2) xylosyltransferase in plants are well known and include the following database entries identifying experimentally demonstrated and putative XylT cDNA and gene sequences, parts thereof or homologous sequences: AJ627182, AJ627183 (Nicotiana tabacum cv. Xanthi), AM179855 (Solanum tuberosum), AM179856 (Vitis vinifera), AJ891042 (Populus alba×Populus tremula), AY302251 (Medicago sativa), AJ864704 (Saccharum officinarum), AM179857 (Zea mays), AM179853 (Hordeum vulgare), AM179854 (Sorghum bicolor), BD434535, AJ277603, AJ272121, AF272852, AX236965 (Arabidopsis thaliana), AJ621918 (Oryza sativa), AR359783, AR359782, AR123000, AR123001 (Soybean), AJ618933 (Physcomitrella patens) and At5g55500 (Arabidopsis thaliana) as well as the nucleotide sequences from Nicotiana species described in application PCT/EP2007/002322 (all sequences herein incorporated by reference).
Based on the available sequences, the skilled person can isolate genes encoding a (1,3) fucosyltransferase or genes encoding β(1,2) xylosyltransferase from plants other than the plants mentioned above. Homologous nucleotide sequence may be identified and isolated by hybridization under stringent conditions using as probes identified nucleotide sequences.
“Stringent hybridization conditions” as used herein means that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at approximately 65° C., preferably twice for about 10 minutes. Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), particularly chapter 11.
Nucleotide sequences obtained in this way should be verified for encoding a polypeptide having an amino acid sequence which is at least 80% to 95% identical to a known α (1,3) fucosyltransferase or β(1,2) xylosyltransferase from plants.
For the purpose of this invention, the “sequence identity” of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (×100) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970) The computer-assisted sequence alignment above, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wis., USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3. Sequences are indicated as “essentially similar” when such sequence have a sequence identity of at least about 75%, particularly at least about 80%, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical. It is clear than when RNA sequences are the to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
Other sequences encoding α (1,3) fucosyltransferase or β(1,2) xylosyltransferase may also be obtained by DNA amplification using oligonucleotides specific for genes encoding α (1,3) fucosyltransferase or β(1,2) xylosyltransferase as primers, such as but not limited to oligonucleotides comprising about 20 to about 50 consecutive nucleotides from the known nucleotide sequences or their complement.
The art also provides for numerous methods to isolate and identify plant cells having a mutation in a particular gene.
Mutants having a deletion or other lesion in the α (1,3) fucosyltransferase or β(1,2) xylosyltransferase encoding genes can conveniently be recognized using e.g. a method named “Targeting induced local lesions in genomes (TILLING)”. Plant Physiol. 2000 June; 123(2):439-42. Plant cells having a mutation in the desired gene may also be identified in other ways, e.g. through amplification and nucleotide sequence determination of the gene of interest. Null mutations may include e.g. genes with insertions in the coding region or gene with premature stop codons or mutations which interfere with the correct splicing. Mutants may be induced by treatment with ionizing radiation or by treatment with chemical mutagens such as EMS.
The level of β(1,2) xylosyltransferase and α (1,3) fucosyltransferase activity can also be conveniently be reduced or eliminated by transcriptional or post-transcriptional silencing of the expression of endogenous β(1,2) xylosyltransferase and α (1,3) fucosyltransferase encoding genes. To this end, a silencing RNA molecule is introduced in the plant cells targeting the endogenous β(1,2) xylosyltransferase and α (1,3) fucosyltransferase encoding genes. As used herein, “silencing RNA” or “silencing RNA molecule” refers to any RNA molecule, which upon introduction into a plant cell, reduces the expression of a target gene. Such silencing RNA may e.g. be so-called “antisense RNA”, whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene. However, antisense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals. Silencing RNA further includes so-called “sense RNA” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid. Other silencing RNA may be “unpolyadenylated RNA” comprising at least 20 consecutive nucleotides having 95% sequence identity to the complement of the sequence of the target nucleic acid, such as described in WO01/12824 or U.S. Pat. No. 6,423,885 (both documents herein incorporated by reference). Yet another type of silencing RNA is an RNA molecule as described in WO03/076619 (herein incorporated by reference) comprising at least 20 consecutive nucleotides having 95% sequence identity to the sequence of the target nucleic acid or the complement thereof, and further comprising a largely-double stranded region as described in WO03/076619 (including largely double stranded regions comprising a nuclear localization signal from a viroid of the Potato spindle tuber viroid-type or comprising CUG trinucleotide repeats). Silencing RNA may also be double stranded RNA comprising a sense and antisense strand as herein defined, wherein the sense and antisense strand are capable of base-pairing with each other to form a double stranded RNA region (preferably the said at least 20 consecutive nucleotides of the sense and antisense RNA are complementary to each other). The sense and antisense region may also be present within one RNA molecule such that a hairpin RNA (hpRNA) can be formed when the sense and antisense region form a double stranded RNA region. hpRNA is well-known within the art (see e.g. WO99/53050; herein incorporated by reference). The hpRNA may be classified as long hpRNA, having long, sense and antisense regions which can be largely complementary, but need not be entirely complementary (typically larger than about 200 bp, ranging between 200-1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 by (see WO04/073390, herein incorporated by reference). Silencing RNA may also be artificial micro-RNA molecules as described e.g. in WO2005/052170, WO2005/047505 or US 2005/0144667 (all documents incorporated herein by reference)
In another embodiment, the silencing RNA molecules are provided to the plant cell or plant by producing a transgenic plant cell or plant comprising a chimeric gene capable of producing a silencing RNA molecule, particularly a double stranded RNA (“dsRNA”) molecule, wherein the complementary RNA strands of such a dsRNA molecule comprises a part of a nucleotide sequence encoding a XylT or FucT protein.
The plant cells according to the invention also need to comprise a β(1,4) galactosyltransferase activity. Conveniently, such activity may be introduced into plant cells by providing them with a chimeric gene comprising a plant-expressible promoter operably linked to a DNA region encoding a β(1,4) galactosyltransferase and optionally a 3′ end region involving in transcription termination and polyadenylation functional in plant cells.
As used herein, the term “plant-expressible promoter” means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Hapster et al., 1988), the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue-specific or organ-specific promoters including but not limited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et al., 1996), stem-specific promoters (Keller et al., 1988), leaf specific promoters (Hudspeth et al., 1989), mesophyl-specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al., 1989), tuber-specific promoters (Keil et al., 1989), vascular tissue specific promoters (Peleman et al., 1989), stamen-selective promoters (WO 89/10396, WO 92/13956), dehiscence zone specific promoters (WO 97/13865) and the like.
Regions encoding a β(1,4) galactosyltransferase are preferably obtained from mammalian organisms, including humans, but may be obtained from other organisms as well. NM022305 (Mus musculus) NM146045 (Mus musculus) NM 004776 (Homo sapiens) NM 001497 (Homo sapiens) are a few database entries for genes encoding a β(1,4) galactosyltransferase. Others database entries for β(1,4) galactosyltransferases include AAB05218 (Gallus gallus), XP693272 (Danio rerio), CAF95423 (Tetraodon nigroviridis) or NP001016664 (Xenopus tropicalis) (all sequences herein incorporated by reference). The β(1,4) galactosyltransferase may be a hybrid β(1,4) galactosyltransferase i.e. a galactosyltransferase comprising a transmembrane region from another glycosyltransferase as described by e.g. by WO03/078637.
According to the invention, the N-glycan profile of glycoproteins may be altered or modified. The glycoproteins may be glycoproteins endogeneous to the cell of the higher plant, and may result in altered functionality, folding or half-life of these proteins. Glycoproteins also include proteins which are foreign to the cell of the higher plant, i.e. which are not normally expressed in such plant cells in nature. These may include mammalian or human proteins, which can be used as therapeutics such as e.g. monoclonal antibodies. Conveniently, the foreign glycoproteins may be expressed from chimeric genes comprising a plant-expressible promoter and the coding region of the glycoprotein of interest, whereby the chimeric gene is stably integrated in the genome of the plant cell. Methods to express foreign proteins in plant cells are well known in the art. Alternatively, the foreign glycoproteins may also be expressed in a transient manner, e.g. using the viral vectors and methods described in WO02/088369, WO2006/079546 and WO2006/012906 or using the viral vectors described in WO89/08145, WO93/03161 and WO96/40867 or WO96/12028. The methods of the invention lead to the presence of a higher proportion of glycoproteins with a human glycosylation profile, such as complex biantennary glycosylated proteins having galactosyl residues with a β(1,4) linkage to both (pre)terminal GlcNac residues, and without fucosyl residue linked α1,3 to the core structure or β1,2 linked xylosyl residues (AA in abbreviated glycan structure nomenclature—see Table 2).
The methods and means described herein are believed to be suitable for all plant cells and plants, gymnosperms and angiosperms, both dicotyledonous and monocotyledonous plant cells and plants including but not limited to Arabidopsis, alfalfa, barley, bean, corn or maize, cotton, flax, oat, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco and other Nicotiana species, including Nicotiana benthamiana, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut and watermelon Brassica vegetables, sugarcane, vegetables (including chicory, lettuce, tomato) and sugarbeet.
Methods for the introduction of chimeric genes into plants are well known in the art and include Agrobacterium-mediated transformation, particle gun delivery, microinjection, electroporation of intact cells, polyethyleneglycol-mediated protoplast transformation, electroporation of protoplasts, liposome-mediated transformation, silicon-whiskers mediated transformation etc. The transformed cells obtained in this way may then be regenerated into mature fertile plants.
Gametes, seeds, embryos, progeny, hybrids of plants, or plant tissues including stems, leaves, stamen, ovaria, roots, meristems, flowers, seeds, fruits, fibers comprising the chimeric genes of the present invention, which are produced by traditional breeding methods are also included within the scope of the present invention.
As used herein “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.
The following non-limiting Examples describe the identification of XylT−, FucT−, GalT+ plant cells and analysis of the glycoproteins contained therein.
Unless stated otherwise in the Examples, all recombinant techniques are carried out according to standard protocols as described in “Sambrook J and Russell D W (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, New York” and in “Ausubel F A, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A and Struhl K (eds.) (2006) Current Protocols in Molecular Biology. John Wiley & Sons, New York”. Standard materials and references are described in “Croy R D D (ed.) (1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd., Oxford and Blackwell Scientific Publications, Oxford” and in “Brown T A, (1998) Molecular Biology LabFax, 2nd Edition, Academic Press, San Diego”. Standard materials and methods for polymerase chain reactions (PCR) can be found in “McPherson M J and Møller S G (2000) PCR (The Basics), BIOS Scientific Publishers Ltd., Oxford” and in “PCR Applications Manual, 3rd Edition (2006), Roche Diagnostics GmbH, Mannheim or www.roche-applied-science.com”
Throughout the description and Examples, reference is made to the following sequences:
A. thaliana lines containing a T-DNA insertion in either the XylT gene (At5g55500), the FucTA gene (At3g19280) and the FucTB gene (At1g49710) are available in the public A. thaliana T-DNA insertion database SIgnAL (http://signal.salk.edu) as Salk—42226, Salk—87481 and Salk—63355 respectively.
Plant lines carrying a homozygous T-DNA insertion, plant lines carrying a heterozygous T-DNA insertion and plant lines carrying no T-DNA insertion at the desired locus can be discriminated using two PCR reactions for each insertion. The first PCR reaction is determinative for the presence of a T-DNA insertion in the desired locus whereby respectively a primer recognizing a target in the inserted T-DNA (LB; SEQ ID No 7) and a primer recognizing the target flanking the T-DNA specific for the locus (such as FucTA RP of SEQ ID No 2; FucTB RP of SEQ ID No 4 or XylT RP of SEQ ID No 6) are used and PCR fragment of about 400 by is amplified. The second PCR reaction is determinative for the absence of a T-DNA insertion in the desired locus whereby respectively a primer recognizing a target left of the T-DNA insertion (such as FucTA LP of SEQ ID No 1; FucTB LP of SEQ ID No 3 or XylT RP of SEQ ID No 5) and a primer recognizing the target flanking the T-DNA specific for the locus at the other side (such as FucTA RP of SEQ ID No 2; FucTB RP of SEQ ID No 4 or XylT RP of SEQ ID No 6) are used and a PCR fragment of about 900 by is amplified.
Plant lines lacking a T-DNA insertion will react positively in the PCR reaction using the specific LP and RP primers and a fragment of about 900 by will be amplified. Such plants will also react negatively in the PCR reaction using the LB and specific RP primers.
Plant lines homozygous for the T-DNA insertion will react positively in the PCR reaction using the LB and specific RP primers and a fragment of about 400 by will be amplified. Such plants will react negatively in the PCR reaction using the specific LP/RP primer combination.
Plant lines heterozygous for the T-DNA insertion will react positively in the PCR reaction using the LB and specific RP primers and a fragment of about 400 by will be amplified. Such plants will also react positively in the PCR reaction using the specific LP/RP primer combination and a fragment of about 900 by will be amplified.
With a first cross between homozygous FucTA knock-out plants (fafa/FBFB) and FucTB knock-out plants (FAFA/fbfb) a heterozygous FucTA/FucTB knock-out line (FAfa/FBfb) could be identified using the PCR reactions described above.
These plants (XX/FAfa/FBfb) were crossed with a homozygous XylT knock-out line (xx/FAFA/FBFB) and progeny of the plants were screened using the above mentioned PCR reactions for the heterozygosity in all three loci (xX/faFA/fbFB). Such triple knock-out plants were selfed and progeny was screened for homozygosity for the T-DNA insertion in all three loci using the above described PCR reactions.
2 Lines out of 300 were identified as positive in the LB/specific RP primer PCR reaction and negative in the specific LP/specific RP primer PCR reaction. The lines are thus potential candidate triple knock-out homozygous plants.
Proteins were extracted from the triple knock-out homozygous A. thaliana plants described in Example 1, separated on polyacrylamide gel and blotted to a PVDF membrane. The resulting blots were treated in a Western Blot using anti-horse radish peroxidase polyclonal antibodies which had been separated in a fraction recognizing β(1,2) xylosyl residues and a fraction recognizing core-α (1,3) fucosyl residues through affinity chromatography using insect phospholipase bound sepharose.
The result is displayed in
Proteins were extracted from the plant line reacting negatively both for the presence of xylose and fucose residues, treated with pepsine, and peptide-N-glycosidase A to obtain N-glycans which were subjected to MALDI-TOF analysis. The result of this analysis is displayed in
Table 1 represents the relative proportion of the different N-glycans present in the endogenous glycoproteins calculated on the basis of the surface of the different peaks in the MALDI-TOF mass spectra. In wild type plants 32% of the N-glycans present on the endogenous proteins contained one or two terminal GlcNac residues, whereas in the triple knock plants this percentage was 44%.
Isolation of huGalT coding sequence was done by PCR reaction using oligonucleotide primers having the nucleotide sequence of SEQ ID No 8 and SEQ ID No 9. The nucleotide sequence was determined (SEQ ID No 10 from nucleotide position 523 to nucleotide position 1719) and the encoded amino acid sequence was compared to that of the huGalT used by Bakker et al. 2001 (supra) (
Using standard recombinant techniques a chimeric gene was constructed containing the following operably linked DNA regions:
The chimeric DNA encoding huGalT was introduced into a T-DNA vector further comprising a glyphosate-resistance gene and introduced into Agrobacterium tumefaciens comprising a helper Ti-plasmide.
Using the floral dip method, transgenic A. thaliana plants comprising a plant-expressible huGalT chimeric gene were isolated by spraying the population of potential transgenic plants first with glyphosate, followed by a further identification using PCR reaction with primers specific for the huGalT chimeric gene.
The chimeric gene was introduced both into WT A. thaliana plants as well as into the triple knock-out A. thaliana lines described in Example 1.
Proteins were isolated from leaf material of plants comprising the huGalT chimeric gene as described in Example 4 and analysed by Western blotting using HRP-conjugated RCA120. RCA120 is a lectin from Ricinus communis which binds both β(1,4) and β(1,3) bound galactosyl residues. Since plants also contain β(1,3) bound galactosyl, protein samples treated with a β(1,4) galactosidase were also included in the Western Blot analysis.
The wt and triple knock-out transgenic plants generated in Example 4 originate from independent transformation events, with potentially a different expression pattern of the HuGalT transgene, thereby complicating the comparison of the N-glycan analysis of endogenous glycoproteins isolated from both types of plants.
To generate isogenic transgenic HuGalT lines which differ only in the zygosity status for the T-DNA insertions in xylosyltransferase and fucosyltransferase genes, a β(1,2) xylosyltransferase and α (1,3) fucosyltransferase deficient A. thaliana line as identified in Example 1 (homozygous triple knock out mutant) was transformed with a chimeric HuGalT gene as described in Example 3 using the floral dip method.
Glyphosate tolerant progeny plants were verified for the intact presence of the HuGalT chimeric gene using PCR amplification specific for HuGalT and CaMV35S promoter. The candidate transformants were also verified for homozygous triple knock-out status as described in Example 1. By Southern blot analysis, candidate transformed plants which had a single insertion of the HuGalT comprising T-DNA were selected. By Western blotting using HRP-conjugated RCA120 as described in Example 5, candidate transformed plants with a good expression level of HuGalT, as judged from the difference in RCA120 binding before and after treatment with β(1,4) galactosidase, were selected.
Transformed A. thaliana plants, homozygous for the presence of T-DNA insertions in the xylosyl and fucosyltransferase genes, comprising a single chimeric HuGalT gene with good expression in hemizygous state (xx/fafa/fbfb/HuGalT−) were crossed with isogenic wild type A. thaliana plants (XX/FaFa/FbFfb/—). Progeny plants may have the following genotypes:
The zygosity status for either the chimeric gene or the T-DNA insertions in the xylosyl and fucosyltransferase genes was checked as described above. The HuGalT chimeric gene in the triple knock-out parent line and the transgenic hemizygous progeny plants (genotype a above) are integrated at the same locus in the genome and thus allow a comparison of the N-glycan profiles of the glycoproteins of plants differing in xylosyl/fucosyl transferase activity without potentially distortion caused by the “position effect” of the HuGalT gene. Analysis of the N-glycan profile of the plant with a genotype as described in b above, allows to verify that plants with a hemizygous knock-out in xylosyl/fucosyltransferase genes behave as a wt plant with regard to the N-glycan profile of endoglycoproteins.
Endoglycoproteins were prepared as described in Example 1 from
The following conclusions can be drawn from the analysis:
A foreign glycoprotein encoding chimeric gene is generated using standard recombinant DNA techniques. The chimeric gene is introduced into a T-DNA vector further comprising a chimeric gene encoding a selectable marker protein, such a chimeric phosphinotricin resistance marker gene and transgenic A. thaliana plants comprising such T-DNAs are generated.
The transgenic plant expressing the foreign glycoprotein are crossed with a XylT−, FucTA−, FucTB− plant expressing huGalT as described in Example 4 or 5 and progeny plants are selected by spraying with glufosinate and glyphosate. The surviving plants are screened by PCR for the presence of both transgenes. Glycoproteins isolated from the identified progeny plants have no β(1,2) xylosyl- or α (1,3) fucosyl-residues and exhibit a high amount of β(1,4) galactosyl residues in their N-linked glycan structures. The glycan structures exhibit the desired AA-type glycans.
| Number | Date | Country | Kind |
|---|---|---|---|
| 07010060.7 | May 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP08/04049 | 5/20/2008 | WO | 00 | 11/20/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60939596 | May 2007 | US |
