Means for producing glycoproteins with paucimannosidic n-glycans

Abstract

The present invention relates to the field of expression of recombinant glycoproteins in plants. In particular, the present invention relates to a method of producing a genetically modified plant capable of producing glycoproteins harbouring paucimannosidic N-glycans, to a method of producing a glycoprotein of interest harbouring paucimannosidic N-glycans and to genetically modified plants, plant cells, protoplasts, protonemas, gametophytes, sporophytes or spores produced with or used in such methods.

Description

Means for producing glycoproteins with paucimannosidic N-glycans

[0001] The present invention relates to the field of expression of recombinant glycoproteins in plants. In particular, the present invention relates to a method of producing a genetically modified plant capable of producing glycoproteins harbouring paucimannosidic N- glycans, to a method of producing a glycoprotein of interest harbouring paucimannosidic N- glycans and to genetically modified plants, plant cells, protoplasts, protonemas, gametophytes, sporophytes or spores produced with or used in such methods.

[0002] Lysosomal storage diseases (LSD) are a group of approximately 50 rare inherited metabolic disorders that result from defects in lysosomal function. Lysosomes are responsible for digesting various molecules involving several critical enzymes. If one of these enzymes is defective, because of a mutation, the large molecules accumulate within the cell, eventually killing it. Lysosomal storage disorders are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of lipids, glycoproteins or mucopolysaccharides. Treatment of lysosomal storage diseases is mostly symptomatic, with enzyme replacement therapy (ERT) being the most common. ERT requires administration of active lysosomal proteins into the cells via an uptake route. Inter alia, plantbased expression systems have been utilized to produce lysosomal enzymes for therapeutic use (WO 2016 / 146760). Relative to mammalian cell-based systems, plant-based systems have several advantages including lower production costs, eliminated risk of contamination by mammalian pathogens and, in the case of moss, a relatively easier manipulation of the N- glycosylation pathway.

[0003] The inventors of WO 2016 / 146760 reported that the production of a transgenic lysosomal protein, a-galactosidase, in bryophyte cells led to the formation of glycoproteins with a high degree of paucimannosidic glycosylation, i.e. a glycosylation terminating with few, e.g. 2 mannose residues, in a branched -Man<(Man)2 structure. These structures proved to be highly effective for the uptake, especially in cells affected by lysosomal storage disease. While this altered glycosylation is unnatural for the lysosomal proteins, the altered proteins were still very effective therapeutic proteins.

[0004] However, the N-glycosylation pathway of plants such as the moss P. patens does not provide consistently high degrees of paucimannosidic glycans on glycoproteins. While a- galactosidase expressed in P. patens yielded up to 80% of the desirable paucimannosidic glycosylation, alpha-glucosidase mainly exhibited the GnGn glycoform and the inventors of WO 2016 / 146760 had to treat the alpha-glucosidase in vitro with P-N-acetylglucosaminidase to compensate for this deficiency in paucimannosidic glycans. Hence, while plants and in particular moss can on its own trim complex N-glycans by removing terminal N- acetylglucos amine (GlcNAc), thereby giving rise to N-glycans with terminal mannose, said approach fails in some cases for unknown reasons.

[0005] Therefore, there is a clear need in the art for new means to produce glycoproteins such as lysosomal proteins with paucimannosidic glycans in plants.

[0006] This problem is solved by the subject-matter as set forth in the appended claims and in the description below.

[0007] As will be shown in the following, the inventors of the present invention have surprisingly found that co-expressing an invertebrate P-N-acetylglucosaminidase in the plant producing the glycoprotein of interest accomplishes what the endogenous plant P-N- acetylglucosaminidases could not achieve. The inventors have demonstrated this by expressing the P-N-acetylglucosaminidase of the fall armyworm (Spodoptera frugiperda) in the moss P. patens. On this basis, plants can now be used to consistently produce glycoproteins such as lysosomal proteins with paucimannosidic glycosylation irrespective of the actual type of glycoprotein product.

[0008] Therefore, the present invention relates in a first aspect to a method of producing a genetically modified plant capable of producing glycoproteins harbouring paucimannosidic N-glycans, wherein the method comprises the step of introducing a nucleic acid encoding a P- N-acetyl-hexosaminidase into said plant, wherein the P-N-acetyl-hexosaminidase is an invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with an invertebrate P-N-acetyl-hexosaminidase.

[0009] In a second aspect, the present invention relates to a method of producing a genetically modified plant capable of producing glycoproteins harbouring paucimannosidic N- glycans, wherein the method comprises the step of introducing a nucleic acid encoding a glycoprotein of interest into said plant, wherein said plant comprises a nucleic acid encodingan invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with an invertebrate P-N-acetyl-hexosaminidase.

[0010] In a third aspect, the present invention relates to a method of producing a glycoprotein of interest harbouring paucimannosidic N-glycans, wherein the method comprises the following steps: a) expressing said glycoprotein of interest in a genetically modified plant, wherein the genetic modification of the plant comprises at least the presence of a nucleic acid sequence encoding a P-N-acetyl-hexosaminidase, wherein the P-N-acetyl-hexosaminidase is an invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with such invertebrate P-N-acetyl-hexosaminidase, and wherein said P-N-acetyl- hexosaminidase or variant thereof is expressed in said genetically modified plant in parallel with expression of the glycoprotein of interest, and b) optionally isolating the glycoprotein of interest from said genetically modified plant.

[0011] In a fourth aspect, the present invention relates to genetically modified plant, plant cell, protoplast, protonema, gametophyte, sporophyte or spore, wherein the genetically modified plant cell, protonema, gametophyte, sporophyte or spore is a plant cell, protoplast, protonema, gametophyte, sporophyte or spore comprising a nucleic acid encoding a P-N-acetyl- hexosaminidase, wherein the P-N-acetyl-hexosaminidase is an invertebrate P-N-acetyl- hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with an invertebrate P -N- acetyl-hexo saminidase .

[0012] In a fifth aspect, the present invention relates to a glycoprotein harbouring paucimannosidic N-glycans, wherein the glycoprotein is obtainable or obtained with the method according to the third aspect of the invention.

[0013] As mentioned above, the present invention relates in a first aspect to a method of producing a genetically modified plant capable of producing glycoproteins harbouring paucimannosidic N-glycans, wherein the method comprises the step of introducing a nucleic acid encoding a P-N-acetyl-hexosaminidase into said plant, wherein the P-N-acetyl- hexosaminidase is an invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with an invertebrate P-N-acetyl-hexosaminidase. The plant for use in the method of the first aspect of the invention may be any plant capable of producing proteins harbouring complex N-glycans. The plant may for example be Arabidopsis, such as Arabidopsis thaliana, rice, in particular Oryza sativa, tobacco, such as Nicotiana benthamianaor Nicotiana labacum, or a bryophyte such as Leptobryum pyriforme, Sphagnum magellanicum or Physcomitrium patens (P. patens). Preferably, the plant is P. patens. As will be understood by those skilled in the art, “introducing a nucleic acid” and the like means in the context of generating a genetically modified plant that the nucleic acid is introduced by means of genetic engineering. It is not meant to cover conventional breeding techniques. Introducing the nucleic acid encoding the invertebrate P-N-acetyl-hexosaminidase or variant thereof into said plant renders said plant a transgenic, genetically modified plant. However, this does not preclude that the plant may have already been a genetically modified plant from the start, i.e. before said nucleic acid encoding the invertebrate P-N-acetyl-hexosaminidase or variant thereof is introduced into said plant. In fact, it is even preferred if the plant has already been genetically modified. For example, the plant may be a bryophyte, in particular P. patens, where alphal,3- fucosyltransferase and / or betal,2-xylosyltransferase activity is suppressed or eliminated. In particular, the plant may be a P. patens plant where the endogenous alpha- 1,3- fucosyltransferase and beta-l,2-xylosyltransferase genes have been inactivated by knock-out. Such genetic modifications are particularly preferred because it prevents formation of planttype glycosylations that may be immunogenic in a mammal such as a human. The plant may also be already genetically modified to express a glycoprotein of interest, for example mammalian glycoproteins, in particular human glycoproteins such as human lysosomal a-glucosidase. The transformation of the plant with the nucleic acid encoding the exogenous invertebrate P-N-acetyl-hexosaminidase may be accomplished by standard means in the art, for instance by way of homologous recombination or non-homologous recombination. Homologous recombination and non-homologous recombination are particularly suitable means for transforming P. patens.

[0014] The method according to the first aspect of the invention involves introducing into the plant a nucleic acid encoding an invertebrate P-N-acetyl-hexosaminidase. The invertebrate P-N-acetyl-hexosaminidases catalyses the enzymatic trimming of complex N-glycans by removing terminal N- acetylgluco s amine (GlcNAc, G) moieties, leading to paucimannosidic N- glycans with terminal mannose moieties instead. Such paucimannosidic N-glycans comprise the structure of formula 1 :(formula 1) wherein a square represents N- acetylgluco s amine (GlcNAc), a circle represents mannose (Man), and a circle with a T represents a terminal mannose. This formula 1, also referred herein as “MM” glycan, represents a core structure that may be further modified - in paucimannosidic N-glycans this further modification is also possible as long as the T- mannoses remain terminal, i.e. are at the non-reducing ends of the sugar chains. The terminal mannoses may be methylated, especially O-methylated. Common modifications are where one or more of the GlcNAc or Man subunits may be al,3-fucosylated, al,6-fucosylated and / or pi,2-xylosylated. al, 3- fucosylations and al,6-fucosylatations are found commonly on the reducing GlcNAc. A pi,2- xylosylation is usually found at the non-terminal mannose (circle without T in formula 1. According to the invention, preferably a al,3-fucosylation and / or pi,2-xylosylation is prevented or reduced, e.g. by way of using a respective knock out plant (e.g. as shown in WO 2004 / 057002 or Cox et al., Nature Biotechnology 24(12), 2006: 1591-7). A paucimannosidic N-glycan structure can also be represented by formula (2):(formula 2)Formula 2 further shows the type of carbohydrate subunit connectivity. The GlcNAc to the right is bound to the amino acid sequence of the glycoprotein. The reducing and non-reducing ends of an oligosaccharide are conventionally drawn with the reducing-end monosaccharide residue furthest to the right and the non-reducing end furthest to the left (as e.g. in formula 2). Note, the reducing GlcNAc is shown left in the short formulas given herein, such as -GlcNAc2-Man3. In an N-glycan, the reducing -GlcNAc is bound to an asparagine in the amino acid chain of the lysosomal protein. In paucimannosidic glycoforms, two non-reducing mannose termini exist (left in formula 2, top in formula 1). Formula (2) is the common core of most N-glycans, including high-mannose and complex N-glycans. In case of paucimannosidic structures, bothupper and lower Man to the left as shown in formula (2) are terminal, whereas in high mannose and complex N-glycans at least one Man contains a further bond to another Man or GlcNAc. Glycoproteins harbouring MGn glycans (one additional terminal N-acetylglucosamine at one terminal Man as shown in formula 2) or GG glycans (one additional terminal N- acetylglucos amine at each terminal Man as shown in formula 2), these MGn and GG glycans are converted to the structures of formula 1 or 2 in presence of the invertebrate N- P-N-acetyl- hexosaminidases. Possible further modifications of glycoproteins, in particular in plants such as P. patens, is the presence of methylated hexoses (Hex), preferably methylated mannose (Man). Usually, the methylation is a methylation of an oxygen of mannose, in particular a 2-0- methylation.

[0015] The P-N-acetyl-hexosaminidase encoded by the nucleic acid to be introduced into the plant (e.g. P. patens) may be a P-N-acetyl-hexosaminidase naturally occurring in invertebrates or a variant of an invertebrate P-N-acetyl-hexosaminidase exhibiting at least 80% sequence identity with an invertebrate P-N-acetyl-hexosaminidase known to the skilled person. The level of sequence identity may be for example be preferably at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99%. Preferred variant sequences are fragments of the parent molecule, retaining the general activity of the parent P-N-acetyl-hexosaminidase molecule. However, the level of said activity can be the same, higher or lower as the one of the respective parent molecule. Other preferred variant sequences are those resulting from conservative amino acid substitutions within the parent sequence. Possible sites for variation in the sequence can also be gleaned from comparisons of P-N-acetyl-hexosaminidase sequences of species within the same genus (e.g. Spodoptera), where variations in sequence are typically indicative of possible substitutions sites.

[0016] As used herein, the term "% sequence identity", has to be understood as follows: Two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may then be determined over the whole length of each of the sequences being compared. In the above context, an amino acid sequence having a "sequence identity" of at least, for example, 95% to a query amino acid sequence, is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence except that the subject amino acid sequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtain an amino acid sequence having a sequence of at least 95% identity to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted or substituted with another amino acid or deleted. Methods for comparing the identity of two or more sequences are well known in the art. In addition, if reference is made herein to a sequence sharing "at least" at certain percentage of sequence identity, then 100% sequence identity are preferably not encompassed.

[0017] The invertebrate P-N-acetyl-hexosaminidase or variant thereof encoded by the nucleic acid introduced into the plant (e.g. P. patens) may preferably be an insectan P-N-acetyl- hexosaminidase, or a variant thereof exhibiting at least 80% sequence identity with an insectan P-N-acetyl-hexosaminidase. For example, the level of sequence identity may be preferably at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99%. Even more preferably, the invertebrate P-N-acetyl-hexosaminidase or variant thereof is an insectan P-N-acetyl- hexosaminidase derived from the family of Noctuidae, such as Mythimna, Trichoplusia, Chrysodeixis, Helicoverpa, and Spodoptera, or a variant of an insectan P-N-acetyl- hexosaminidase of the family of Noctuidae, such as Mythimna, Trichoplusia, Chrysodeixis, Helicoverpa, and Spodoptera, exhibiting at least 80% sequence identity with an insectan P-N- acetyl-hexosaminidase of the family of Noctuidae. The level of sequence identity may for example be at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99%. Even more preferably, the invertebrate P-N-acetyl-hexosaminidase or variant thereof is a P-N-acetyl- hexosaminidase of an insect of the genus Spodoptera, such as Spodoptera litura, Spodoptera littoralis, Spodoptera exigua or Spodoptera frugiperda. These Spodoptera P-N-acetyl- hexosaminidases share high levels of sequence identity with the P-N-acetyl-hexosaminidase used in the examples of the present invention. The invertebrate P-N-acetyl-hexosaminidase may also be a variant a P-N-acetyl-hexosaminidase of an insect of the genus Spodoptera, such as Spodoptera litura, Spodoptera littoralis, Spodoptera exigua or Spodoptera frugiperda exhibiting at least 80% sequence identity with at least one of these Spodoptera P-N-acetyl- hexosaminidases. The level of sequence identity may for example be at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99%. Even more preferably, the P-N-acetyl- hexosaminidase is a P-N-acetyl-hexosaminidase of Spodoptera frugiperda or a variant thereofexhibiting at least 80% sequence identity with at least one of these Spodoptera P-N-acetyl- hexosaminidases. The level of sequence identity may for example be preferably at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99%. A particularly preferred example of the P-N-acetyl-hexosaminidase for use in the present invention is the P-N-acetyl- hexosaminidase according to SEQ ID NO:1 or a variant thereof exhibiting at least 80% sequence identity with SEQ ID NO:1. The level of sequence identity may for example be preferably at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99% with SEQ ID NO:1. Said sequence may also be found under GenBank accession ABB76926.1 or DQ249309.1, with the signal peptide sequence (aa 1-18) removed. Preferably, the P-N-acetyl- hexosaminidase protein is a soluble P-N-acetyl-hexosaminidase protein, i.e. does not comprise a transmembrane. It is understood that the invertebrate P-N-acetyl-hexosaminidase or variant thereof does not occur naturally in plants or vertebrate animals such as mammals. Otherwise, it would not be an invertebrate P-N-acetyl-hexosaminidase or variant thereof.

[0018] The nucleic acid encoding the invertebrate P-N-acetyl-hexosaminidase may provide genetic elements allowing the expression of the P-N-acetyl-hexosaminidase in the plant (e.g. P. patens). These genetic elements may comprise for example a suitable promoter, 5’ and 3’ UTRs and a terminator. The promotor is preferably a strong promoter such as endogenous actin promoter of the plant (e.g. of P. patens).

[0019] As disclosed above, the present invention relates in a second aspect to a method of producing a genetically modified plant capable of producing glycoproteins harbouring paucimannosidic N-glycans, wherein the method comprises the step of introducing a nucleic acid encoding a glycoprotein of interest into said plant, wherein said plant comprises a nucleic acid encoding an invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with an invertebrate P-N-acetyl-hexosaminidase. Essentially, this second aspect of the invention relates to the situation where a given plant has already been genetically modified in advance to express an invertebrate P-N-acetyl-hexosaminidase, and where the genetic information encoding a given glycoprotein of interest is introduced at a later stage. It is immediately apparent, that whatever has been set forth above in the context of the first aspect of the invention is equally applicable to the second aspect of the invention. In particular, the plant can be as defined above and is most preferably P. patens. The plant mayalso be a plant obtained with the method according to the first aspect of the invention. The invertebrate P-N-acetyl-hexosaminidase can also take the form as outlined in the embodiments above and is most preferably the P-N-acetyl-hexosaminidase according to SEQ ID NO: 1 or a variant thereof exhibiting at least 80%, more preferably at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99% sequence identity with SEQ ID NO: 1.

[0020] The nucleic acid encoding the glycoprotein of interest according to the second aspect of the invention may encode a glycoprotein of any source, preferably a mammalian, especially human glycoprotein or a glycoprotein of a non-human animal, such as a rodent, a dog, cat, horse, cow, camel, or pig. The glycoprotein is preferably a lysosomal protein, preferably a mammalian lysosomal protein, even more preferably a human lysosomal protein. The (preferably human) lysosomal protein may be selected from the group consisting of a glucosidase (GAA), a-galactosidase, preferably a- galactosidase A (GLA); B-glucoceramidase, P-glucosidase (glucocerebrosidase); a-mannosidase; aspartylglucosaminidase; P-mannosidase; acid xeremidase; a-ducosidase; P-galactosidase, P -hexosaminidase activator protein; galactocerebrosidase, galactoceramidase; lysosomal acid lipase (LAL); a-iduronidase; iduronate-2-sulfatase; glucosamine-N-sulfatase, heparansulfatsulfamidase (SGSH); a-N- acetyl-glucosaminidase (NAGLU); a-glucosaminide-N-acetyltransferase; N- acetygalactosamine-6-sulfatase; P-galactosidase; N-acetygalactosamine-4-sulfatase; P- glucoronidase; neuraminidase; sphingomyelinase, sphingomyelin phosphodiesterase; mammalian, in particular human P-hexosaminidase, or its a subunit; alpha-N- acetylgalactosaminidase (NAGA), a-galactosaminidase; P-Hexosaminidase A, in particular mammalian P-Hexosaminidase A; galactose-6-sulfate sulfatase and hyaluronidase. A particularly preferred example for the glycoprotein of interest is a glucosidase (GAA) according to SEQ ID NO:2. Typically, the nucleic acid encoding the glycoprotein of interest will also provide for its expression in the plant (e.g. P. patens), e.g. by providing suitable promoter, UTR, terminator, or localisation sequences.

[0021] According to the third aspect, the present invention relates to a method of producing a glycoprotein of interest harbouring paucimannosidic N-glycans, wherein the method comprises the following steps: a) expressing said glycoprotein of interest in a genetically modified plant, wherein the genetic modification of the plant comprises at least the presence of a nucleic acid sequence encoding a P-N-acetyl-hexosaminidase, wherein the P-N-acetyl-hexosaminidase is an invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with such invertebrate P-N-acetyl-hexosaminidase, and wherein said P-N-acetyl-hexosaminidase or variant thereof is expressed in said genetically modified plant in parallel with expression of the glycoprotein of interest, and b) optionally isolating the glycoprotein of interest from said genetically modified plant. It is evident that the third aspect of the invention reflects the actual production of a glycoprotein in a plant expressing in parallel the invertebrate P-N-acetyl-hexosaminidase as discussed for the preceding aspects. As a consequence, the plant can take for example the form of any of the embodiments discussed above for the plant of the first aspect of the invention. In particular, the plant can be P. patens. The plant may also be a plant obtained with the method according to the first aspect of the invention. Likewise, the invertebrate P-N-acetyl-hexosaminidase may be as discussed in detail above for the first aspect of the invention and is preferably the P-N-acetyl-hexosaminidase according to SEQ ID NO: 1 or a variant thereof exhibiting at least 80%, more preferably at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99% sequence identity with SEQ ID NO:1. The glycoprotein of interest may be a glycoprotein as discussed above in the context of the second aspect of the invention. A particularly preferred glycoprotein to be produced with the method according to the third aspect of the invention is a glucosidase (GAA) according to SEQ ID NO:2. In some embodiment the glycoprotein of interest is isolated from said genetically modified plant.

[0022] The fourth aspect of the invention relates to genetically modified plant, plant cell, protoplast, protonema, gametophyte, sporophyte or spore, wherein the genetically modified plant cell, protonema, gametophyte, sporophyte or spore is a plant cell, protoplast, protonema, gametophyte, sporophyte or spore comprising a nucleic acid encoding a P-N-acetyl- hexosaminidase, wherein the P-N-acetyl-hexosaminidase is an invertebrate P-N-acetyl- hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with an invertebrate P-N-acetyl-hexosaminidase. The plant may be a plant obtainable by or obtained by the method according to the first or second aspect of the invention. In particular, the plant can be P. patens. The plant cell, protoplast, protonema, gametophyte, sporophyte or spore may be a plant cell, protoplast, protonema, gametophyte, sporophyte or spore obtained from a plant obtainable by or obtained by a method according to the first or second aspect of the invention. The plant cell, protoplast, protonema, gametophyte, sporophyte or spore may be a plant cell, protoplast, protonema, gametophyte, sporophyte or spore of P. patens. The invertebrate P-N-acetyl-hexosaminidase encoded by the nucleic acid comprised by the genetically modified plant, plant cell, protoplast, protonema, gametophyte, sporophyte or spore may be as discussed in detail above for the first aspect of the invention and is preferably the P-N-acetyl- hexosaminidase according to SEQ ID NO: 1 or a variant thereof exhibiting at least 80%, more preferably at least 85%, more preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, and most preferably at least 98% or at least 99% sequence identity with SEQ ID NO:1. The genetically modified plant, plant cell, protoplast, protonema, gametophyte, sporophyte or spore may optionally additionally comprise a nucleic acid encoding a glycoprotein of interest. The glycoprotein of interest may be a glycoprotein as discussed above in the context of the second and third aspect of the invention, respectively, in particular a human lysosomal protein. A particularly preferred glycoprotein in such scenario is a glucosidase (GAA) according to SEQ ID NO:2.

[0023] The glycoprotein harbouring paucimannosidic N-glycans according to the fifth aspect of the invention may be as discussed above in the context of the second and third aspect of the invention, respectively, and may in particular be a human lysosomal protein. A particularly preferred glycoprotein in such scenario is a glucosidase (GAA) according to SEQ ID NO:2. The inventive glycoprotein according to the fifth aspect of the invention is characterized by the presence of paucimannosidic N-glycans, some of which may potentially be additionally methylated. Preferably, the inventive glycoprotein according to the fifth aspect of the invention comprises a proportion of at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50% and even more preferably about 60% paucimannosidic N-glycans as compared to the total of N-glycans present on the glycoprotein of interest. In a particularly preferred embodiment, the inventive glycoprotein is a glucosidase (GAA) and comprises non-methylated paucimannosidic N- glycans in the range of 15% to 45%.

[0024] For sake of completeness it is noted once more that whatever has been set forth herein for a specific aspect of the invention will likewise be applicable to other aspects of the invention. Therefore, unless technically indicated otherwise, any embodiment disclosed herein for one specific aspect of the invention will likewise be applicable to any of the other aspects of the invention.

[0025] The term "comprising", as used herein, shall not be construed as being limited to the meaning "consisting of" (i.e. excluding the presence of additional other matter). Rather,"comprising" implies that optionally additional matter may be present. The term "comprising" encompasses as particularly envisioned embodiments falling within its scope "consisting of" (i.e. excluding the presence of additional other matter) and "comprising but not consisting of" (i.e. requiring the presence of additional other matter), with the former being more preferred.Figures

[0026] In the following a brief description of the appended figures will be given. The figures are intended to illustrate aspects of the present invention in more detail. However, they are not intended to limit the overall scope of the invention.

[0027] Fig. 1 illustrates the enzymatic activity of the P-N-acetyl-hexosaminidase of .S'. frugiperda (.S' / Hcxo), which is the cleavage of GlcNAc residues (squares) from GnGn glycans.

[0028] Fig. 2 A) Proportions of different glycans on GAA for parental strain and two clones of P. patens expressing .S' / Hcxo (SEQ ID NO:1); GnAF: glycans terminated with GlcNAc and the LewisA-Structure (Fucocl -4(Gal|31 -3)-GlcNAc-) ; GnGn: glycans terminated with two GlcNAc; GnM: glycans terminated with GlcNAc and mannose; Man4-6: glycans terminated with 4 to 6 mannoses; MM: paucimannosidic glycans; B) HILIC analysis of clone T7#006:

[0029] Fig. 3 illustrates a more detailed glycan profiling on basis of LC-ESI-MS for the parental P. patens clone and clone T7#006. GnAF: glycans terminated with GlcNAc and the LewisA-Structure (Fucocl-4(Gal|31-3)-GlcNAc-); MAF: glycans terminated with Mannose and the LewisA-Structure (Fucocl-4(Gal|31-3)-GlcNAc-); GnGn: glycans terminated with two GlcNAc; GnM: glycans terminated with GlcNAc and mannose; GnM*2Me: double methylated glycans terminated with GlcNAc and mannose; GnM*lMe: single methylated glycans terminated with GlcNAc and mannose;Man6: glycans terminated with 6 mannoses; MM: non-methylated paucimannosidic glycans; MM*2Me: double methylated paucimannosidic glycans; MM*lMe: single methylated paucimannosidic glycansExamples

[0030] In the following, specific examples illustrating embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific examples described herein. Indeed, various modifications of the invention in additionto those described herein will become readily apparent to those skilled in the art from the foregoing description and the examples below. All such modifications fall within the scope of the appended claims.Example 1: Cell line engineering

[0031] The transgene encoding the mature P-N-acetyl-hexosaminidase of .S'. frugiperda minus the first 18 amino acids encoding the signal peptide (.S' / Hcxo; see also GenBank accession number DQ249309.1 and SEQ ID NO:1) was synthesized by Invitrogen GeneArt Gene Synthesis Services (Thermo Fisher Scientific; Regensburg, Germany), and was transferred by restriction and ligation into a standard expression vector containing genetic elements necessary for strong expression as described by Hintze et al. (Int. J. Mol. Sci., 2020, 21, 2642). Expression elements derived by PCR or extracted from cloned DNA vectors were used for the PEG- mediated transformation of P. patens protoplasts (see Koprivova et al.; Plant Biotechnol. J. 2, 517-523). The parental cell line used for transformation expresses GAA (SEQ ID NO:2) and has been modified to eliminate the xylT and fucT genes (Koprivova et al., 2004) but still expresses A-acetylglucosaminyltransferase I (GnT-I) and therefore synthesizes mainly GnGn glycans. After transformation, the resulting P. patens clones were genotyped by PCR. Transgene expression was verified by real-time PCR at Microsynth (Balgach, Switzerland).Example 2: Production and glycan analysis

[0032] Moss culture, SDS-PAGE, column purification, and N-glycan analysis by HILIC- HPLC were performed as previously described (Hintze et al., Int. J. Mol. Sci., 2020, 21, 2642). Moss strains were screened in 180-mL shake-flask cultures and the inventors used 1-L cultures in a stirred-tank bioreactor to represent production conditions.

[0033] Moss strains were screened for the presence of paucimannosidic N-glycans by LC-ESI-MS using in-gel digested GAA samples. Secreted protein was separated by SDS- PAGE, and GAA bands were excised for further processing. Protein was S-alkylated with iodoacetamide and digested in-gel with trypsin (Promega) and / or GluC and / or chymotrypsin (both Roche). The digested samples were loaded onto a BioBasic-18 C18 column (150 x 0.32 mm, 5 pm, Thermo Fisher Scientific) using 80 mM ammonium formiate buffer as the aqueous solvent A and 100% acetonitrile as solvent B. The inventors established a gradient from 5% to32% B in 35 min, and 32% to 75% B in 15 min to elute large peptides at a flow rate of 6 pL / min. The peptides were detected using a QTOF-MS (Bruker maXis 4G) equipped with the standard ESI source in positive ion / DDA mode (= switching to MS / MS mode for eluting peaks). MS scans were recorded in the range 150-2200 Da, and the four highest peaks were selected for fragmentation. Alternatively, the samples were separated by nano-flow HPLC (300 nL / min) on a Thermo Acclaim PepMap column (25 cm x 0.075 mm ID, 2 pm particle size) followed by detection using the Bruker maXis 4G equipped with a captive spray ESI source in positive ion / DDA mode as above. MS scans were recorded in the range 150-2200 Da, and the six highest peaks were selected for fragmentation. The inventors used the ESI calibration mixture (Agilent) for instrument calibration. Manual glycopeptide searches were carried out using DataAnalysis 4.0 (Bruker) followed by conversion to mgf files, which are suitable for MS / MS ion searches with GPM. To quantify the different glycoforms, the peak areas of extracted ion chromatograms representing the first four isotopic peaks were summed using the quantification software Quant Analysis (Bruker). The inventors detected and quantified the GAA glycopeptides LENLSSSEMGYTATLTR (SEQ ID NOG), QVVENMTR (SEQ ID NO:4), GVFITNETGQPLIGK (SEQ ID NOG), NNTIVNELVR (SEQ ID NOG) and VTVLGVATAPQQVLSNGVPVSNFTYSPDTK (SEQ ID NO:7).

[0034] Growth and productivity of the SfHexo transformed moss cultures remained in the same range as the parental P. patens culture without the SfHexo transgene, i.e. expression of the SfHexo transgene did neither negatively impact growth nor negatively impact productivity.

Claims

Claims1. Method of producing a genetically modified plant capable of producing glycoproteins harbouring paucimannosidic N-glycans, wherein the method comprises the step of introducing a nucleic acid encoding a P-N-acetyl-hexosaminidase into said plant, wherein the P-N-acetyl-hexosaminidase is an invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with an invertebrate P-N-acetyl- hexosaminidase.

2. Method of producing a glycoprotein of interest harbouring paucimannosidic N-glycans, wherein the method comprises the following steps: a) expressing said glycoprotein of interest in a genetically modified plant, wherein the genetic modification of the plant comprises at least the presence of a nucleic acid sequence encoding a P-N-acetyl-hexosaminidase, wherein the P-N-acetyl- hexosaminidase is an invertebrate P-N-acetyl-hexosaminidase or a variant thereof exhibiting at least 80% sequence identity with such invertebrate P-N-acetyl- hexosaminidase, and wherein said P-N-acetyl-hexosaminidase or variant thereof is expressed in said genetically modified plant in parallel with expression of the glycoprotein of interest, and b) optionally isolating the glycoprotein of interest from said genetically modified plant.

3. The method according to claim 2, wherein the glycoprotein of interest is a lysosomal protein, in particular wherein the lysosomal protein is selected from the group consisting of a glucosidase (GAA), a-galactosidase, preferably a-galactosidase A (GLA); B- glucoceramidase, P-glucosidase (glucocerebrosidase); a-mannosidase; aspartylglucosaminidase; P-mannosidase; acid xeremidase; a-ducosidase; P-galactosidase, P-hexosaminidase activator protein; galactocerebrosidase, galactoceramidase; lysosomal acid lipase (LAL); a-iduronidase; iduronate-2-sulfatase; glucosamine-N-sulfatase, heparansulfatsulfamidase (SGSH); a-N-acetyl-glucosaminidase (NAGLU); a- glucosaminide-N-acetyltransferase; N-acetygalactosamine-6-sulfatase; P-galactosidase; N-acetygalactosamine-4-sulfatase; P-glucoronidase; neuraminidase; sphingomyelinase, sphingomyelin phosphodiesterase; mammalian, in particular human P-hexosaminidase, or its a subunit; alpha-N-acetylgalactosaminidase (NAGA), a-galactosaminidase; P-Hexosaminidase A, in particular mammalian P-Hexosaminidase A; galactose-6-sulfate sulfatase and hyaluronidase.

4. The method according to claim 1 or claim 3, wherein the P-N-acetyl-hexosaminidase is an insectan P-N-acetyl-hexosaminidase or variant thereof exhibiting at least 80% sequence identity with an insectan P-N-acetyl-hexosaminidase.

5. The method according to claim 4, wherein the P-N-acetyl-hexosaminidase is an P-N-acetyl- hexosaminidase of Spodoptera frugiperda (S. frugiperda).

6. The method according to any one of claims 1 to 3, wherein the invertebrate P-N-acetyl- hexosaminidase comprises the sequence of SEQ ID NO:1 or is a variant thereof exhibiting at least 80% sequence identity with SEQ ID NO:1, preferably exhibiting at least 90% sequence identity with SEQ ID NO:1.

7. The method according to any one of claims 1 to 6, wherein the P-N-acetyl-hexosaminidase is a soluble P-N-acetyl-hexosaminidase.

8. The method according to any one of claims 1 to 7, wherein the P-N-acetyl-hexosaminidase is expressed under the control of the plant’s endogenous act promotor.

9. The method according to any one of claims 1 to 8, wherein the plant is a bryophyte.

10. The method according to any one of claims 1 to 9, wherein the plant is Physcomitrium patens (P. patens).

11. The method according to claim 10, wherein the P-N-acetyl-hexosaminidase is an P-N- acetyl-hexosaminidase of S. frugiperda.

12. The method according to claim 11, wherein the P-N-acetyl-hexosaminidase comprises the sequence of SEQ ID NO:1.

13. Genetically modified plant, plant cell, protoplast, protonema, gametophyte, sporophyte or spore, wherein the genetically modified plant cell, protonema, gametophyte, sporophyte or spore is a plant cell, protoplast, protonema, gametophyte, sporophyte or spore of a plant produced with or used in a method as defined in any one of claims 1 to 12.

14. The genetically modified plant of claim 13, wherein the plant is P. patens and or comprises a nucleic acid encoding a P-N-acetyl -hexosaminidase of S.frugiperda.

15. The genetically modified plant of claim 14, wherein the plant expresses human a glucosidase (GAA), and wherein said human a glucosidase (GAA) harbours paucimannosidic N-glycans.

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