Glycoengineered extracellular vesicles, methods of production and uses thereof

Abstract

The present invention relates to glycoengineered extracellular vesicles, as well as methods of production of such glycoengineered extracellular vesicles, methods of glycoengineering extracellular vesicles, and uses of glycoengineered extracellular vesicles.

Description

[0001] Glycoengineered extracellular vesicles, methods of production and uses thereof

[0002] Technical field

[0003] The present invention relates to glycoengineered extracellular vesicles, as well as methods of production of such glycoengineered extracellular vesicles, methods of glycoengineering extracellular vesicles, and uses of glycoengineered extracellular vesicles.

[0004] Background

[0005] Extracellular vesicles (EVs), which include exosomes, are membrane-bound vesicles secreted by various cell types and play important roles in health and disease by regulating cell-cell communication. The bilayer lipid structure enables encapsulation and safe transportation of biomolecules, including nucleic acid, proteins, chemotherapeutics, toxins, and metabolites. Among these EVs, a subset population with diameters ranging from 30 to 250 nanometers is commonly referred to as small EVs (sEVs) which primarily consist of exosomes. There is a growing interest in the development of sEVs-based therapies and some progresses have been made in regenerative medicine and immunotherapies. In vitro and in vivo studies have suggested that sEVs derived from immune cells, such as natural killer cells, chimeric antigen receptor-T cells, and dendritic cells, can be used as a therapeutic approach for cancer treatments. These sEVs have the ability to target cancer cells or regulate the immune response. EVs in comparison to their parental cells can show similar therapeutic effects in combination with additional benefits such as low toxicity, low immunogenicity and a safer profile due to non-self proliferation.

[0006] Extracellular vesicles (EVs) are particles that are released from the cells, they are delimited by a lipid bilayer, and cannot replicate on their own. This includes both engineered EVs and EVs produced under various cell culture conditions. EVs differ from non-vesicular extracellular particles (NVEPs) that are made from cell derived components, such as proteins nucleic acids and lipids, and are released from the cell but do not have a lipid bilayer. NVEPs are smaller than EVs and may not be detected by some EV characterization methods, thus their quantity in an extracellular particle preparation may remain unknown. They may contain DNA, RNA, proteins and other molecules from the cell which they can deliver to their target cells, also over larger distances within the body. Delivery platforms such as LNPs (Lipid NanoParticles also called liposomes), polymeric nanoparticles and inorganic nanoparticles — collectively known as nanomedicines — carry various payloads such as chemotherapies, RNA and small molecules and deliver the drugs to specific cells or organs. While there are more than 20 nanomedicines FDA-approved for various diseases including cancer, hepatitis C and haemophilia, as well as Covid-vaccines, they have important limitations.

[0007] As LNPs above, exosomes can also be externally loaded with drugs, genetic material and other small molecules. And they are prevalent in many body fluids such as serum and milk. While Exosomes are similar in size to nanoparticles (-30-250 nm) they are thought to have many advantages over the more traditional nanomedicine delivery tools. It is thought that three key characteristics could give exosomes the clear edge over existing platforms: they may be less immunogenic, less toxic and more able to cross biological barriers such as the blood-brain barrier (BBB). Because exosomes are less immunogenic, they might be particularly useful for repeat drug dosing. Repetitive dosing with gene therapies that are delivered by viral vectors such as AAVs can result in an immune response against the viral vector, and preclinical work suggests that there is a finite number of doses that patients can receive. The same is true for nanoparticles, which the body eventually recognizes as foreign. Furthermore, the preferred natural tropism and isogenic behaviour of exosomes gives them an important additional targeting advantage. While exosomes are still in earlier development their therapeutic promises are very high, for drug delivery but also more direct for regenerative medicine and wound healing, as cell therapy surrogates for immunotherapy, such as dendritic cell based exosomes (dexosomes) for targeted immune stimulation, for direct killing of tumor cells in processes similar as “ADCC”, for delivery of cytokines, antagonists and agonsits, and many more. Furthest developments are in early clinical study phases and include different mode-of-actions delivered by exosomes such as delivery of small molecules, siRNA, RNA, DNA, cytokines and enzymes. While the field for exosome therapies and drug delivery is wide, it is still in development and many improvements are desired including their targeting, delivery, efficacy, manufacturing etc.

[0008] Glycans, also known as carbohydrates, are essential components of cell surfaces and most secreted proteins in most organisms. The diversity and abundance of glycans contribute to their critical roles in numerous biological interactions. Glycoconjugates are built up and modified via highly regulated and sequential enzymatic processes, which requires the participation of various glycosyl hydrolases and glycosyltransferases. Approximately 85% of extracellular protein are estimated to be glycosylated. The surface of human cells is heavily covered with various glycoconjugates, the major types include glycoproteins (N- and O-glycans), glycolipids including glycosphingolipids, and proteoglycans with glycosaminoglycans (GAGs). Similar to their parental cells, the surface of EVs have also been found to carry various glycans. However, a rising challenge lies in understanding the regulation of EV glycosylation and its consequent effects. To gain first insights into the role of glycans for EVs, enzymatic removal of surface glycans has been used indicating roles of certain sugar moieties in the cellular uptake and biodistribution of EVs. It has recently been found that displaying a certain O-glycan (sialyl Lewis X and Lewis X) on CD63, a surface marker of sEV, through virus mediated genetic addition of one fucosyltransferase in the cell, yields sEVs with increased levels of the sugar and targeting to cell receptors recognizing SLeX (Zheng et al., 2022).

[0009] Investigations of glycosylation and its role is therefore still very limited and technologies precisely targeting certain glycan chains or sugar moieties and one or more defined enzymes in the glycosylation machinery single are desirable to gain in-depth knowledge regarding EV glycosylation and the resulting effects.

[0010] Summary

[0011] The inventors have found that glycosylation pattern of EVs is a key determinant for their functions, including their ability to interact with target cells, modulate immune responses, influence cell behaviour, and participate in disease processes. Understanding and modifying these glycosylation patterns can have significant implications for the development of EV based therapies and diagnostic tools.

[0012] Herein are provided methods for modulating the glycosylation of extracellular vesicles by modifying the cell producing said extracellular vesicles. Thus are provided herein glycoengineered extracellular vesicles, which glycoengineering can confer or improve properties of interest of the extracellular vesicles.

[0013] Herein is provided a method of obtaining an extracellular vesicle, said method comprising the steps of: a. providing a cell wherein at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or the activity of a product encoded by said at least one nucleic acid has been modified, resulting in a modified glycosylation of extracellular vesicles produced by the cell; and b. isolating the extracellular vesicle from said cell.

[0014] Thus, it will be evident to the skilled person that the extracellular vesicles obtained by the method described herein are glycoengineered extracellular vesicles.

[0015] Thus, herein is provided a method of obtaining an extracellular vesicle, wherein said extracellular vesicle is glycoengineered, said method comprising the steps of: a. providing a cell wherein at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or the activity of a product encoded by said at least one nucleic acid has been modified, resulting in a modified glycosylation of extracellular vesicles produced by the cell; and b. isolating the extracellular vesicle from said cell.

[0016] The method of obtaining these glycoengineered extracellular vesicles can for example be used to study the role of any single enzyme, particularly of the glycosylation machinery, such as any glycosyltransferase, combinations of various glycosyltransferases or other enzymes in the glycosylation machinery, such as glycosidases, enzymes of the precursor pathways and chaperons both in cells and EVs. The method can for example also be used to investigate how selective glycoengineering affects EV generation and EV biology, the molecular composition on EVs, and the differences in glycosylation between the cells and EVs.

[0017] Also provided is an isolated glycoengineered extracellular vesicle with a modified property of interest, preferably wherein the isolated glycoengineered extracellular vesicle has been obtained by the methods described herein. Such glycoengineered extracellular vesicles display a glycosylation profile which is different from the glycosylation profile observed on extracellular vesicles produced by a cell which has not been modified as described herein. By modifying the surface glycosylation pattern of the EVs through selective glycoengineering it is possible to change the properties of the isolated EV. Such properties can, for example, be related to the EVs ability to: facilitate binding and internalization of the target cell; stimulate or suppress immune responses; load and release of cargo molecules; facilitate tissue repair and wound healing and affect disease progression such as for example cancer metastasis. Since the role of carbohydrates for EVs is largely unexplored, understanding the effect of the altered glycosylation patterns and selective glycoengineering opens up for developing improved applications of EVs.

[0018] Also provided are isolated glycoengineered extracellular vesicles, having a modified glycosylation compared to another extracellular vesicle obtained from a reference cell, wherein the modified glycosylation and the reference cell are described herein.

[0019] Also provided is a composition comprising an isolated glycoengineered extracellular vesicle obtained via the methods described herein, or the isolated extracellular vesicle as described herein.

[0020] Also provided is a kit comprising an isolated glycoengineered extracellular vesicle as described herein, preferably wherein said extracellular vesicle is obtained via the methods described herein, and optionally further comprising instructions for use.

[0021] Also provided is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated extracellular vesicle, wherein the isolated extracellular vesicle has been obtained by the methods described herein, for use in medicine.

[0022] Also provided is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated extracellular vesicle, wherein the isolated extracellular vesicle has been obtained by the methods described herein, for use in a method of treating and / or preventing a disease and / or a medical condition in a subject in need thereof.

[0023] Also provided is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to deliver a molecule of interest to a target cell, wherein the molecule of interest is preferably selected from the group consisting of: a nucleic acid sequence, such as a DNA molecule, such as an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule, a gRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide including short peptides, oligopeptides or proteins; and a dye. The polypeptide may be an enzyme, an antibody, a cytokine or fragments thereof.

[0024] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to modulate the expression of a target nucleic acid, such as to increase the expression of a target nucleic acid, such as to decrease the expression of a target nucleic acid, and / or to modulate the activity of a product encoded by a target nucleic acid, such as to increase the activity of a polypeptide encoded by a target nucleic acid, or such as to decrease the activity of a product encoded by a target nucleic acid.

[0025] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to modulate the expression of a target nucleic acid, such as to increase the expression of a target nucleic acid, or such as to decrease the expression of a target nucleic acid.

[0026] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to modulate the activity of a product encoded by a target nucleic acid, such as to increase the activity of a product encoded by a target nucleic acid, such as to decrease the activity of a product encoded by a target nucleic acid.

[0027] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to edit a target nucleic acid in a target cell.

[0028] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to deliver a compound, such as a small molecule, such as a toxin, to a target cell, such as an animal cell, a plant cell or a yeast cell. Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to deliver a compound, to improve transfer of a nucleic acid of interest to a target cell compared to the transfer of said nucleic acid of interest to said target cell by another extracellular vesicle, wherein the other extracellular vesicle is an extracellular vesicle from a reference cell in which expression of the at least one nucleic acid and / or the activity of the product encoded by the at least one nucleic acid has not been modified, preferably wherein the improved transfer leads to increased expression of the nucleic acid of interest compared to the expression obtained when using the other extracellular vesicle.

[0029] Also provided herein is a method of modulating the tropism and / or target specificity of an extracellular vesicle, wherein the method comprises or consists of the method of obtaining an isolated glycoengineered extracellular vesicle as described herein, wherein said glycoengineered extracellular vesicle and has a modified glycosylation on its surface.

[0030] Also provided herein is a method of modifying, such as increasing or decreasing, the total uptake of a target molecule in a target cell, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as described herein.

[0031] Also provided herein is a method of modifying, such as increasing or decreasing, the uptake of a target molecule per second in a target cell, wherein the method comprises or consists of the method of obtaining a glycoengineered extracellular vesicle as described herein.

[0032] Also provided herein is a method of modifying, such as increasing or decreasing, the delivery efficiency of a target molecule to a target cell, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as described herein, and wherein the method further comprises loading said target molecule in the glycoengineered extracellular vesicle and contacting said extracellular vesicle with the target cell. Also provided herein is a method of modifying, such as increasing or decreasing, the loading of a target molecule to a glycoengineered extracellular vesicle, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as defined herein, and further comprises loading said target molecule in the extracellular vesicle.

[0033] Also provided herein is a method of increasing the transfer of a target nucleic acid to a target cell or a method of increasing the expression of a product encoded by a target nucleic acid in a target cell, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as defined herein, and further comprises: a. loading said target nucleic acid in the extracellular vesicle; and b. contacting the extracellular vesicle with the target cell, whereby the target nucleic acid is transferred to the target cell and / or whereby the product encoded by the target nucleic acid is expressed.

[0034] Also provided herein is a method to identify a glycoengineered extracellular vesicle with a glycosylation pattern of interest comprising the steps of: a. providing a glycoengineered extracellular vesicle, wherein the glycoengineered extracellular vesicle has been obtained by a cell, wherein expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as described herein; b. optionally, determining whether the glycoengineered extracellular vesicle has the glycosylation pattern of interest, such as by contacting the extracellular vesicle with a polypeptide, such as a lectin, such as an antibody, specific for the glycosylation pattern of interest, and / or such as by performing glycoanalytics of the extracellular vesicle, and / or by testing the extracellular vesicle in an assay.

[0035] Also provided is a method to identify a nucleic acid of interest, wherein modification of the expression of the nucleic acid of interest in a cell and / or modification of the activity of a product encoded by the nucleic acid of interest in said cell results in a modified glycosylation on the surface of an extracellular vesicle produced by the cell, said method comprising the steps of: a. obtaining a glycoengineered extracellular vesicle from a cell, wherein expression of a nucleic acid sequence in the cell and / or the activity of a product encoded by the nucleic acid has been modified as described herein; b. optionally, determining whether the glycoengineered extracellular vesicle has the glycosylation pattern of interest, such as by contacting the extracellular vesicle with a polypeptide, such as a lectin, such as an antibody, specific for the glycosylation pattern of interest; and / or such as by performing glycoanalytics of the extracellular vesicle; and / or by testing the glycoengineered extracellular vesicle in an assay; and c. concluding that the nucleic acid sequence is a nucleic acid sequence of interest.

[0036] Also provided herein is a method of obtaining a glycoengineered extracellular vesicle, preferably a glycoengineered small extracellular vesicle, with a modified property of interest, said method comprising the steps of: a. providing a cell wherein i. at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or wherein the activity of a product encoded by said at least one nucleic acid has been modified; and / or ii. wherein at least one nucleic acid has been introduced, resulting in a modified glycosylation of extracellular vesicles produced by the cell, thereby obtaining a glycoengineered extracellular vesicle with a modified property of interest; and b. isolating the extracellular vesicle from said cell, wherein the property of interest is: i. modified nucleic acid transfer, such as gene, RNA or DNA transfer; ii. modified expression of a transferred nucleic acid; iii. increased editing of a target nucleic acid such as a gene; iv. modified target specificity or tropism; v. total uptake of a molecule by the glycoengineered extracellular vesicle; or vi. increased delivery of a molecule to a target, and wherein the property of interest is modified compared to an extracellular vesicle obtained from a reference cell in which said at least one nucleic acid, said expression of said at least one nucleic acid sequence and / or said activity of the product encoded by said at least one nucleic acid sequence has not been modified.

[0037] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in medicine, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified glycosylation; and contacting said glycoengineered extracellular vesicle with a therapeutic molecule or active ingredient, thereby obtaining an isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0038] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in the treatment of a disease and / or a medical condition, in wound healing, in regenerative medicine or in gene therapy in a subject in need thereof, comprising the steps of: providing an isolated glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a therapeutic molecule or active ingredient, optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; and administering said isolated glycoengineered extracellular vesicle or composition to a subject in need thereof; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0039] Also provided herein is the use of an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for the delivery of a molecule to a cell, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified extracellular glycosylation; and contacting said glycoengineered extracellular vesicle with a molecule or active ingredient, thereby obtaining an isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0040] Also provided herein is a method to identify an isolated glycoengineered extracellular vesicle, as defined herein, with a glycosylation pattern of interest comprising the steps of: a. providing an isolated glycoengineered extracellular vesicle, wherein the extracellular vesicle has been obtained from a cell, wherein the expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as defined herein; b. optionally, determining the extracellular surface glycosylation pattern, c. loading the isolated extracellular vesicle with a molecule of interest d. providing a target cell e. contacting said isolated extracellular vesicles with said target cell f. analyzing target cellular properties of interest, such as target specificity or tropism, total uptake of a molecule, uptake of a molecule per second, delivery efficiency or expression of a nucleic acid or polypeptide.

[0041] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in medicine, the use comprising the steps of:

[0042] - providing a glycoengineered extracellular vesicle having a modified glycosylation and comprising a therapeutic molecule or active ingredient;

[0043] - optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0044] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in the treatment of a disease and / or a medical condition, in wound healing, in regenerative medicine or in gene therapy in a subject in need thereof, comprising the steps of: providing an isolated glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a therapeutic molecule or active ingredient;

[0045] - optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; and preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0046] Also provided herein is the use of an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for the delivery of a molecule to a cell, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a molecule, such as a therapeutic molecule, or active ingredient;

[0047] - optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0048] Description of Drawings

[0049] Figure 1

[0050] Graphic illustration of the strategy for cell-based glycoengineering of extracellular vesicles. The glycosylation machinery of parental cells is modified to produce specific glycan structures. As a result of the modification, the secreted extracellular vesicles are also affected, resulting in glycoengineered extracellular vesicles with a modified surface glycosylation pattern.

[0051] Figure 2

[0052] Glycan binding assay of glycoengineered cells using lectins or antibodies with Flow Cytometry Analysis. The assay was conducted using two doses, 0.3 ng or 1.0 ng, of each lectin PHA-L (fig 2A), VVA (fig 2B), PNA (fig 2C), CTB (fig 2D), antibody (CS-56) (fig 2E) and SNA (fig 2F). The mean fluorescence intensity (MFI) was measured for each binding, and the results of three technical replicates are presented as the mean ± standard deviation (SD). Three independent experiments were conducted, a representative experiment is presented using three different clones for each glycoengineering.

[0053] Figure 3

[0054] Nanoparticle tracking analysis (NTA) of the extracellular vesicles produced from glycoengineered cell lines. The NTA assay was performed to determine the size distribution (fig 3A) and concentration (fig 3B) of extracellular vesicles. The extracellular vesicles used for the assay were isolated from three different clones for each glycoengineering method and the results of those three replicates are presented as the mean ± standard deviation (SD); Western blot analysis was performed to check the exosome biomarker, syntenin-1 , expression in extracellular vesicles and cell lysates (fig 3C).

[0055] Figure 4

[0056] Latex beads capture of genetically modified extracellular vesicles and staining using lectins or antibodies with Flow Cytometry Analysis. The assay was conducted using two concentrations, 0.3 ng or 1.0 ng, of each lectin PHA-L (fig 4A), VVA (fig 4B), PNA (fig 4C), CTB(fig 4D), antibody (CS-56) (fig 4E) and SNA (fig 4F) . The mean fluorescence intensity (MFI) was measured for each binding, and the results of three technical replicates are presented as the mean ± standard deviation (SD). Three independent experiments were conducted, and a representative experiment is presented using three different clones for each glycoengineering.

[0057] Figure 5

[0058] Viable cell density, cells / ml (fig 5A) and cell viability % (fig 5B).

[0059] HEK293F cells were knocked out in MGAT1, GNE, C1GALT1C1, B4GALT7, and B4GALT5 / 6. The extracellular vesicles used for the assay were isolated from three different clones for each glycoengineering method and the results of those three replicates are presented as the mean ± standard deviation (SD). DO: day 0, when the cells are seeded; D2: day 2; D4: day 4; D6: day 6.

[0060] Figure 6

[0061] Clonal variations in the yield of extracellular vesicles among different clones including clones from the same knockout. HEK293F cells were knocked out in MGAT1, GNE, C1GALT1C1 , B4GALT7, and B4GALT5 / 6. Extracellular vesicles were isolated from three different clones for each knock-out and the extractible amount of extracellular vesicles is shown as ng / cell (Fig 6A) and as ug / ml (Fig 6B).

[0062] Figure 7

[0063] Modulation and improvement of cellular uptake of extracellular vesicles through cellular glycoengineering. The extracellular vesicles were labelled with a fluorescence dye using the standard technology for exosome uptake and targeting assays. The labelled extracellular vesicles were tested for various time intervals, after 7 h and after 20h, for the uptake into target cells. The assay is normalized to the same amount of extracellular vesicles put into the labelling (fig 7A), or normalized to the same total fluorescence of the extracellular vesicles after the labelling and purification of the exosomes (fig 7B).

[0064] Figure 8

[0065] The speed of EV uptake can be improved and modulated. The uptake of fluorescently labelled extracellular vesicles isolated from parental HEK293F cell knocked out in MGATI(ANG), C1GALT1C1(AOG), B4GALT5 / 6(AGSL), B4GALT7(AGAG) or GNE(ASA). Mean Fluorescent Intensity (MFI) after 1 , 2.5, 5 and 24 h (Fig 8a) percentage of positive cells after 1 , 2.5, 5 and 24 h (fig 8b).

[0066] MFI in HEK 293F, KHYG-1 and NK92 cells knocked out in MGATI(ANG), C1GALT1C1(AOG), B4GALT5 / 6(AGSL), B4GALT7(AGAG) or GNE(ASA) after 7h (fig 80, fig 8e) and 20 h (fig 8d, fig 8f).

[0067] Figure 9

[0068] Improved DNA oligonucleotides-based genetic transfer to target cells. DNA oligonucleotides conjugated with a fluorescence label were loaded onto extracellular vesicles (EV) isolated from MGATI(ANG), C1GALT1C1(AOG), B4GALT5 / 6(AGSL), B4GALT7(AGAG) and GNE(ASA) knock-outs. Loaded extracellular vesicles were incubated with target cells for a 16 h and the fluorescent signal in the target cells was determined through FACS fluorescence intensity analysis. Controls are shown as no extracellular vesicles (No EV) and extracellular vesicles isolated from WT cells (WT EV).

[0069] Figure 10 Improved plasmid DNA-based genetic transfer and improved expression of DNA- encoded proteins to target cells. A DNA plasmid encoding GFP (green fluorescence protein) was loaded onto extracellular vesicles isolated from MGATI(ANG), C1GALT1C1(AOG), B4GALT5 / 6(AGSL), B4GALT7(AGAG) and GNE(ASA) knockouts. Loaded GE-exosomes were incubated with target cells and the expression of GFP in each cell through FACS fluorescence intensity analysis was determined after 24 hours. Controls are shown as no extracellular vesicles (No EV) and extracellular vesicles isolated from WT cells (WT EV) (fig 10a). Fold change in GFP positive cells loaded with extracellular vesicles isolated from MGATI(ANG), C1GALT1C1(AOG), B4GALT5 / 6(AGSL), B4GALT7(AGAG) and GNE(ASA) knock-out cells (fig 10b).

[0070] Figure 11

[0071] Improved RNA-based genetic transfer. Fold change in Texas Red positive cells, as a result from the delivery of fluorescently labelled siRNA by extracellular vesicles isolated from MGATI(ANG), C1GALT1C1(AOG), B4GALT5 / 6(AGSL), B4GALT7(AGAG) and GNE(ASA) knock-out cells. Data is normalized to WT fluorescent levels.

[0072] Figure 12

[0073] The figure illustrates the comparison of InDeis percentages in KHYG-1 cells caused by Cas9 and gRNA plasmids delivered through HEK WT EV, HEK ASA EV and HEK AGAG EV.

[0074] Figure 13

[0075] Improved plasmid DNA-based gene transfer and enhanced expression of DNA- encoded proteins in BCi NS1.1 cells. A GFP-encoding plasmid was loaded onto EVs isolated from wild-type (WT EV) and B4GALT7 knockout (AGAG EV) HEK293F cells, with Liposome used as a control. BCi NS1.1 cells were incubated with two different doses of loaded WT and AGAG EVs, as well as the Liposome control. GFP expression was assessed after 24 hours using fluorescence microscopy. Representative images are shown for 15 pg WT EV (13A), 60 pg WT EV (13B), 15 pg AGAG EV (13C), 60 pg AGAG EV (13D), and 15 pg Liposome (13E). B4GALT7 knockout HEK293F cells with stable GFP expression were used to isolate GFP-encapsulated sEVs, which were then applied to the cells and GFP abundance was assessed after 16 hours using fluorescence microscopy (13F). The GFP fluorescence intensity was quantified using Imaged software. The images were split into blue, red, and green channels. A threshold of 120 was applied to distinguish GFP-positive cells from the background. The “Analyze Particles” function was used to measure the integrated density of different areas, and the total integrated density for each image is presented in 13 G.

[0076] Figure 14

[0077] TEER measurements of electrical resistance from the apical to the basolateral side suggest that all sEVs exhibit low cytotoxicity.

[0078] Detailed description

[0079] Definitions

[0080] Glycoengineering: the term herein refers to a method of changing the glycosylation, glycans or sugar moieties of molecules, cells or EV such as of glycoproteins, glycolipids, proteoglycans / glycosaminoglycans and include in an extension a method of changing the properties of a surface comprising proteins, lipids and / or proteoglycans / GAGs such as the surface of a cell, i.e. the cell membrane, or the membrane of an extracellular vesicle. A modified glycosylation can be any change in at least one glycan structure in quality or quantity. This can be a large change (e.g. removal of complex N-glycans by change to mannose structures on N-glycans, removal of a group of a certain sugar moieties (e.g. all sialylation), to removal of a certain sugar moiety in a certain linkage at a certain position (e.g. removal of core- fucose on N-glycans), exchange of a sugar linkage to another (e.g. alpha 2-6 to alpha 2-3 sialic acids) or any combinations thereof including secondary effects (e.g. removal of a certain GalNAc transferase for O-glycosylation may result in the lack of glycosylation normally caused by another GalNAc transferase at another site because the present of the first GalNAc is a prerequisite of the initiation of the 2nd site). It also includes further glycan modifications such as sulfation or phosphorylation.

[0081] Glycoengineering is typically achieved by in vivo technologies, such as by gene editing, or by enzymatic methods, for example removal of glycosylation by enzymes. In the context of the present disclosure, glycoengineering refers only to genetic methods, unless otherwise indicated. Such genetic methods include Genome Editing, but also RNA Interference, RNA Editing , Chemical Inhibition / Activation, Epigenetic Regulations. The term “specific glycoengineering” refers to a targeted glycoengineering, i.e. the modification of a specific type of glycosylation, which in some cases may impact other types of glycosylation. Modified glycosylation: the term herein is to be construed as a change in glycosylation properties on a surface comprising proteins, such as the surface (internal or external) of an extracellular vesicle. A glycosylation pattern can be modified quantitatively, i.e. the extent of glycosylation on the protein is different after glycoengineering compared to the extent of glycosylation in a reference cell, qualitatively, i.e. the type of glycosylation is different in the glycoengineered extracellular vesicle, or both, i.e. both the amount of glycosylation and the type of glycosylation are different in the glycoengineered extracellular vesicle.

[0082] Herein are provided methods for modulating the glycosylation of extracellular vesicles by genetically modifying the cell producing said extracellular vesicles. Thus are provided herein glycoengineered extracellular vesicles. Glycoengineering of the extracellular vesicles can confer or improve properties of interest of the extracellular vesicles.

[0083] The terms identity and homology, with respect to a polynucleotide (or polypeptide), are defined herein as the percentage of nucleic acids (or amino acids) in the candidate sequence that are identical or homologous, respectively, to the residues of a corresponding native nucleic acids (or amino acids), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity I similarity I homology, and considering any conservative substitutions according to the NCIIIB rules (hftp: / / www.chem. qmul.ac.uk / iubmb / misc / naseq.html; NC-llIB, Eur J Biochem (1985)) as part of the sequence identity. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art. Generally, a given identity between two sequences implies that the homology between these sequences is at least equal to the identity; for example, if two sequences are 70% identical to one another, they cannot be less than 70% homologous to one another - but could be sharing 80% homology. Throughout the present disclosure, the term “at least 70%” when referring to a percentage of sequence homology or of sequence identity shall refer to a sequence homology or identity of at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%.

[0084] Glycosylation

[0085] Herein are provided methods to modify the glycosylation pattern or glycosylation profile of extracellular vesicles produced by a cell. By genetically modifying a cell producing extracellular vesicles, extracellular vesicles with a modified glycosylation are obtained.

[0086] Thus, it will be evident to the skilled person that the isolated extracellular vesicles obtained by the method described herein are glycoengineered extracellular vesicles.

[0087] The inventors have surprisingly found that modifying a cell by modifying one or more nucleic acids involved in glycosylation, and / or by introducing a nucleic acid involved in glycosylation, such as a nucleic acid encoding an enzyme involved in glycosylation, the glycosylation pattern displayed by extracellular vesicles produced by the cell can differ from the glycosylation pattern displayed by extracellular vesicles produced by the unmodified cell. The glycosylation pattern can be tailored as described herein to obtain glycoengineered extracellular vesicles, and can then be tested for properties of interest.

[0088] It will be understood by the skilled person that the term “a nucleic acid involved in glycosylation” encompasses any nucleic acid which participates directly or indirectly in the biochemical process of glycosylation. Examples of said nucleic acid involved in glycosylation comprise any nucleic acid encoding for a glycosylation enzyme, or any nucleic acid which is capable of modulating transcription of a glycosylation enzyme, or the activity of a glycosylation enzyme.

[0089] Thus, in some embodiments, the isolated glycoengineered extracellular vesicle and the cell from which the glycoengineered extracellular vesicle is isolated have a different glycosylation pattern. Glycosylation involves several pathways, in particular:

[0090] N-glycosylation,

[0091] O-linked glycosylation,

[0092] Glycolipids,

[0093] Glycosaminoglycans, Proteoglycans, Sialylation, Fucosylation.

[0094] Several classes of glycans are thus produced: N-linked glycans through N- glycosylation; O-linked glycans through O-linked glycosylation; phosphoglycans; C- linked glycans; glypiation; in other words, carbohydrates covalently linked to a nonsugar moiety (lipids or proteins). The major glycoconjugates are glycoproteins, glycopeptides, peptidoglycans, glycolipids, and lipopolysaccharides.

[0095] Glycosylation is a complex post-translational modification. A given protein (whether comprised in an extracellular vesicle or not) can display many forms of glycosylation.

[0096] Thus, in the context of the present disclosure, a modified glycosylation pattern or a modified glycosylation refers to any modification of glycosylation, either direct (for example by manipulating genes encoding proteins involved directly in a specific type of glycosylation, such as genes encoding enzymes such as glycosyltransferases) or indirect (for example by manipulating genes encoding proteins involved in the regulation of glycosylation) i.e. a modification of least one type of glycosylation. A modified glycosylation can be a modification of the type of glycosylation; by way of example, O-linked glycosylation can be hampered in the modified extracellular vesicle, e.g. there is reduced O-glycosylation in the cell (and hence on the extracellular vesicle) in general, but individual subbranches of that type of glycosylation can be modified in another way, e.g. there is reduction of O-GalNAc but increase of O-Man; or the glycosylation happens to a different extent or on different residues.

[0097] In some embodiments, the extracellular vesicle is glycoengineered by modifying the cell from which the extracellular vesicle is obtained, in particular the modified glycosylation can be a modification of one or more of: a. N-glycosylation, such as modified high-mannose N-glycans, modified hybrid N- glycans and / or modified complex N-glycans; b. O-linked glycosylation, such as modified O-GalNAc, modified O-GIcNAc, modified O-Man, modified O-Gal, modified O-Fuc, and / or modified O-GIc; c. glycolipids such as glycosphingolipids; d. glycosaminoglycans; e. proteoglycans; and f. sialylation.

[0098] Thus, in some embodiments, the modified glycosylation of extracellular vesicles produced by the cell can be a modification of one or more of: a. N-glycosylation, such as modified high-mannose N-glycans, modified hybrid N- glycans and / or modified complex N-glycans; b. O-linked glycosylation, such as modified O-GalNAc, modified O-GIcNAc, modified O-Man, modified O-Gal, modified O-Fuc, and / or modified O-GIc; c. glycolipids such as glycosphingolipids; d. glycosaminoglycans; e. proteoglycans; and f. sialylation.

[0099] It will be evident that the cell may be modified so that several types of glycosylation are impacted simultaneously. For example, the cell may be modified so that the extracellular vesicle (i.e., the glycoengineered extracellular vesicle) displays two modifications of glycosylation, for example: a. N-glycosylation and O-linked glycosylation; b. N-glycosylation and glycosphingolipids; c. N-glycosylation and glycosaminoglycans; d. N-glycosylation and proteoglycans; e. N-glycosylation and modified glycan chains resulting from modified sialylation f. O-linked glycosylation and glycolipids; g. O-linked glycosylation and glycosaminoglycans; h. O-linked glycosylation and proteoglycans; i. O-linked glycosylation and modified glycan chains resulting from modified sialylation; j. glycolipids and glycosaminoglycans; k. glycolipids and proteoglycans; l. glycolipids and modified glycan chains resulting from modified sialylation; and m. glycosaminoglycans and modified glycan chains resulting from modified sialylation; n. glycosaminoglycans and proteoglycans; o. glycosaminoglycans and sialylation.

[0100] In some embodiments, the modified glycosylation is a modification of three types of glycosylation, and the extracellular vesicle displays modified: a. N-glycosylation, O-linked glycosylation and glycolipids; b. N-glycosylation, O-linked glycosylation and glycosaminoglycans; c. N-glycosylation, O-linked glycosylation and proteoglycans; d. N-glycosylation, O-linked glycosylation and modified glycan chains resulting from modified sialylation; e. N-glycosylation, glycolipids and glycosaminoglycans; f. N-glycosylation, glycolipids and proteoglycans; g. N-glycosylation, glycolipids and modified glycan chains resulting from modified sialylation; h. N-glycosylation, glycosaminoglycans and proteoglycans; i. N-glycosylation, glycosaminoglycans and modified glycan chains resulting from modified sialylation; j. N-glycosylation, proteoglycans and modified glycan chains resulting from modified sialylation; k. O-linked glycosylation, glycolipids and glycosaminoglycans; l. O-linked glycosylation, glycolipids and proteoglycans; m. O-linked glycosylation, glycolipids and modified glycan chains resulting from modified sialylation; n. O-linked glycosylation, glycosaminoglycans and proteoglycans; o. O-linked glycosylation, glycosaminoglycans and modified glycan chains resulting from modified sialylation; p. Glycolipids, glycosaminoglycans and proteoglycans; q. Glycolipids, glycosaminoglycans and modified glycan chains resulting from modified sialylation; or r. Glycosaminoglycans, proteoglycans and modified glycan chains resulting from modified sialylation. In some embodiments, the modified glycosylation is a modification of four or five types of glycosylation selected from modified N-linked glycosylation, modified O-linked glycosylation, modified glycolipids, modified glycosaminoglycans, modified proteoglycans, and modified glycan chains resulting from modified sialylation.

[0101] Assays and markers for determining glycosylation profile

[0102] In order to test whether an extracellular vesicle has been successfully glycoengineered, methods are available to the skilled person.

[0103] Markers specific for extracellular vesicles can be employed to confirm that the glycoengineered extracellular vesicle obtained by the present method also is recognised by such markers. Thus, the glycoengineered extracellular vesicle preferably binds to syntenin-1, ALIX and / or CD63; most preferably it binds at least to syntenin-1. This can be determined by e.g. Western blot or other methods known in the art.

[0104] Markers for extracellular vesicles can be internal or external.

[0105] Thus, in some embodiments, the isolated extracellular vesicle binds to at least one marker specific for extracellular vesicles, such as syntenin-1 , ALIX and / or CD63.

[0106] It will be evident that where the marker cannot be detected by binding of a marker molecule that the marker can also be determined by proteomics technologies such as mass spectrometry. Thus, in some embodiments, the glycosylation pattern on a glycoengineered extracellular vesicle can be determined via proteomics, such as mass spectrometry.

[0107] In addition, markers or assays specific types of glycosylation can be employed to determine the glycosylation profile of the glycoengineered extracellular vesicle.

[0108] For determining the glycosylation profile, a carbohydrate binding molecule known to those skilled in the art such as lectins (there many different known and described) or carbohydrate-binding antibodies able to distinguish between different glycoforms can be used. For determining whether N-glycosylation of the glycoengineered extracellular vesicle is modified compared to a reference extracellular vesicle, i.e. an extracellular vesicle which has been obtained from a reference cell as described herein, a marker such as PHA-L can be used. In some embodiments, the modified glycosylation is modified N-glycosylation, and a marker specific for N-glycosylation, such as PHA-L, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, i.e. compared to a reference extracellular vesicle. In some embodiments, the modified glycosylation is modified N-glycosylation, and a marker specific for N-glycosylation, such as a carbohydrate-binding molecule, e.g. an antibody, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell.

[0109] For determining whether O-linked glycosylation of the glycoengineered extracellular vesicle is modified compared to a reference extracellular vesicle, i.e. an extracellular vesicle which has been obtained from a reference cell as described herein above, a marker such as lectin VVA, core-1 structure antibodies such as A78-G / A7, an anti-Tn antibody or PNA can be used. In some embodiments, the modified glycosylation is modified O-linked glycosylation, and a marker specific for O-linked glycosylation, such as lectin VVA, core-1 structure antibodies such as A78-G / A7, an anti-Tn antibody or PNA, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, i.e. compared to a reference extracellular vesicle. In some embodiments, the modified glycosylation is modified O-linked glycosylation, and a marker specific for O- linked glycosylation, such as a carbohydrate-binding molecule, e.g. an antibody, shows modified binding to the glycoengineered extracellular vesicle, compared to the binding of same marker to another extracellular vesicle obtained from a reference cell.

[0110] For determining whether glycolipids, such as glycosphingolipids, of the glycoengineered extracellular vesicle are modified compared to a reference extracellular vesicle, i.e. an extracellular vesicle which has been obtained from a reference cell as described herein above, a marker such as GM1 can be used. In some embodiments, the modified glycosylation is modified glycolipids, such as glycosphingolipids, and a marker specific for glycolipids, such as glycosphingolipids, such as GM1, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, i.e. compared to a reference extracellular vesicle. In some embodiments, the modified glycosylation is modified glycolipids, such as glycosphingolipids, and a marker specific for glycolipids, such as glycosphingolipids, such as a carbohydrate-binding molecule, e.g. an antibody, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell.

[0111] For determining whether glycosaminoglycans of the glycoengineered extracellular vesicle are modified compared to a reference extracellular vesicle, i.e. an extracellular vesicle which has been obtained from a reference cell as described herein above, a marker such as a marker for chondroitin sulfate, for example an anti-chondroitin sulfate antibody, can be used. In some embodiments, the modified glycosylation is modified glycosaminoglycans, and a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, for example an anti-chondroitin sulfate antibody, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, i.e. compared to a reference extracellular vesicle. In some embodiments, the modified glycosylation is modified glycosaminoglycans, and a marker specific for glycosaminoglycans, such as a carbohydrate-binding molecule, e.g. an antibody, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell.

[0112] For determining whether sialylation or fucosylation of the glycoengineered extracellular vesicle is modified compared to a reference extracellular vesicle, i.e. an extracellular vesicle which has been obtained from a reference cell as described herein above, which results in a modification in the length of glycan chains, a marker such as lectin SNA or LCA, can be used. In some embodiments, the modified glycosylation is modified sialylation or fucosylation, and a marker specific for sialylation or fucosylation, such as lectin SNA, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, i.e. compared to a reference extracellular vesicle. In some embodiments, the modified glycosylation is modified sialylation or fucosylation, and a marker specific for sialylation or fucosylation, such as a carbohydrate-binding molecule, e.g. an antibody, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell. If the marker binds to proteins which are internal to extracellular vesicles, it may be necessary to extract the proteins from the extracellular vesicles as is known in the art.

[0113] In some instances, the modified glycosylation profile may not be detectable using a marker; other glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof can then be used to compare the glycosylation profile to the profile of a reference extracellular vesicle. These methods can be used in addition to or in combination with marker detection when available.

[0114] The term “modified glycosylation” in relation to any of the glycosylation types can refer to an increased glycosylation or to a decreased glycosylation. In some embodiments, binding of said marker to the glycoengineered extracellular vesicle compared to the binding of said marker to a reference extracellular vesicle obtained from a reference cell is modified by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%. In some embodiments, binding of said marker to the glycoengineered extracellular vesicle compared to the binding of said marker to a reference extracellular vesicle obtained from a reference cell is increased by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 100%, such as at least 300%, such as at least 500% or more. In some embodiments, binding of said marker to the glycoengineered extracellular vesicle compared to the binding of said marker to a reference extracellular vesicle obtained from a reference cell is decreased by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%.

[0115] In an aspect, the present disclosure is directed to a method to identify an isolated glycoengineered extracellular vesicle, as defined herein, with a glycosylation pattern of interest comprising the steps of: a. providing an isolated glycoengineered extracellular vesicle, wherein the extracellular vesicle has been obtained from a cell, wherein the expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as defined herein; b. optionally, determining the extracellular surface glycosylation pattern, c. loading the isolated extracellular vesicle with a molecule of interest d. providing a target cell e. contacting said isolated extracellular vesicles with said target cell f. analyzing target cellular properties of interest, such as target specificity or tropism, total uptake of a molecule, uptake of a molecule per second, delivery efficiency or expression of a nucleic acid or polypeptide.

[0116] Modifying glycosylation of extracellular vesicles

[0117] The glycoengineered extracellular vesicles of the present disclosure are obtained from a cell, in which either the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified, and / or in which a nucleic acid has been introduced. Modification of the cell results in a modified glycosylation of the proteins, glycolipids, protegolycans or GAGs of the cell, including proteins, glycolipids, protegolycans or GAGs of the extracellular vesicles of the cell.

[0118] It is worth noting that the glycosylation pattern of a glycoengineered EV originating from an engineered cell does not necessarily have the same glycosylation pattern as the cell from which it originates. This opens up for a wider range of applications as one modification can give rise to several EV glycosylation patterns and hence functions.

[0119] In an aspect, the present disclosure is directed to a method of obtaining a glycoengineered extracellular vesicle, preferably a glycoengineered small extracellular vesicle, with a modified property of interest, said method comprising the steps of: a. providing a cell wherein i. at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or wherein the activity of a product encoded by said at least one nucleic acid has been modified; and / or ii. wherein at least one nucleic acid has been introduced, resulting in a modified glycosylation of extracellular vesicles produced by the cell, thereby obtaining a glycoengineered extracellular vesicle with a modified property of interest; and b. isolating the extracellular vesicle from said cell, wherein the property of interest is: i. modified nucleic acid transfer, such as gene, RNA or DNA transfer; ii. modified expression of a transferred nucleic acid; iii. increased editing of a target nucleic acid such as a gene; iv. modified target specificity or tropism; v. total uptake of a molecule by the glycoengineered extracellular vesicle; and / or vi. increased delivery of a molecule to a target, and wherein the property of interest is modified compared to an extracellular vesicle obtained from a reference cell in which said at least one nucleic acid, said expression of said at least one nucleic acid sequence and / or said activity of the product encoded by said at least one nucleic acid sequence has not been modified.

[0120] In some embodiments, the property of interest is modified nucleic acid transfer.

[0121] In some embodiments, the property of interest is modified expression of a transferred nucleic acid.

[0122] In some embodiments, the property of interest is increased editing of a target nucleic acid.

[0123] In some embodiments, the property of interest is modified target specificity or tropism.

[0124] In some embodiments, the property of interest is total uptake of a molecule by the glycoengineered extracellular vesicle.

[0125] In some embodiments, the property of interest is increased delivery of a molecule to a target. n some embodiments, the property of interest is modified nucleic acid transfer and modified expression of a transferred nucleic acid. In some embodiments, the property of interest is modified nucleic acid transfer and increased editing of a target nucleic acid.

[0126] In some embodiments, the property of interest is modified nucleic acid transfer and modified target specificity or tropism.

[0127] In some embodiments, the property of interest is modified nucleic acid transfer and total uptake of a molecule by the glycoengineered extracellular vesicle.

[0128] In some embodiments, the property of interest is modified nucleic acid transfer and increased delivery of a molecule to a target.

[0129] In some embodiments, the property of interest is modified expression of a transferred nucleic acid and increased editing of a target nucleic acid.

[0130] In some embodiments, the property of interest is modified expression of a transferred nucleic acid and modified target specificity or tropism.

[0131] In some embodiments, the property of interest is modified expression of a transferred nucleic acid and total uptake of a molecule by the glycoengineered extracellular vesicle.

[0132] In some embodiments, the property of interest is modified expression of a transferred nucleic acid and increased delivery of a molecule to a target.

[0133] In some embodiments, the property of interest is increased editing of a target nucleic acid and modified target specificity or tropism.

[0134] In some embodiments, the property of interest is increased editing of a target nucleic acid and total uptake of a molecule by the glycoengineered extracellular vesicle.

[0135] In some embodiments, the property of interest is increased editing of a target nucleic acid and increased delivery of a molecule to a target. In some embodiments, the property of interest is modified target specificity or tropism and total uptake of a molecule by the glycoengineered extracellular vesicle.

[0136] In some embodiments, the property of interest is modified target specificity or tropism and increased delivery of a molecule to a target.

[0137] In some embodiments, the property of interest is total uptake of a molecule by the glycoengineered extracellular vesicle and increased delivery of a molecule to a target.

[0138] In some embodiments, the property of interest is modified nucleic acid transfer, modified expression of a transferred nucleic acid, and increased editing of a target nucleic acid.

[0139] In some embodiments, the property of interest is modified nucleic acid transfer, modified expression of a transferred nucleic acid, and modified target specificity or tropism.

[0140] In some embodiments, the property of interest is modified nucleic acid transfer, modified expression of a transferred nucleic acid, and total uptake of a molecule by the glycoengineered extracellular vesicle.

[0141] In some embodiments, the property of interest is modified nucleic acid transfer, modified expression of a transferred nucleic acid, and increased delivery of a molecule to a target.

[0142] In some embodiments, the property of interest is modified nucleic acid transfer, increased editing of a target nucleic acid, and modified target specificity or tropism.

[0143] In some embodiments, the nucleic acid comprises or consist of a gene, such as a glycosylation gene, i.e. a gene encoding a product directly or indirectly involved in glycosylation. In some embodiments, the nucleic acid encodes a polypeptide selected from the group consisting of: a glycosyltransferase, such as a sialyltransferase; a glycosidase, such as a glycosyl hydrolase; an enzyme in the sugar precursor pathway; a transporter; and a chaperone. The expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by a method selected from the group consisting of: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of an mRNA encoded by the target nucleic acid; e. inhibition of the activity of a protein encoded by the target nucleic acid; f. knock-in of an additional sequence encoding the product of the target nucleic acid; g. upregulation of the transcription of the target nucleic acid; h. upregulation of the translation of the mRNA encoded by the target nucleic acid; i. stimulation of the activity of the protein encoded by the target nucleic acid; and j. introduction of another nucleic acid in the cell.

[0144] In some embodiments, the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by a method selected from the group consisting of: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of the mRNA encoded by the target nucleic acid; and e. inhibition of the activity of the protein encoded by the target nucleic acid.

[0145] In some embodiments, the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by a method selected from the group consisting of: a. knock-in of an additional sequence encoding the product of the target nucleic acid; b. upregulation of the transcription of the target nucleic acid; c. upregulation of the translation of the mRNA encoded by the target nucleic acid; and d. stimulation of the activity of the protein encoded by the target nucleic acid.

[0146] In preferred embodiments, the expression of the at least one nucleic acid has been modified, for example the nucleic acid has been knocked out or knocked down.

[0147] In preferred embodiments, the expression of the at least one nucleic acid has been modified by introducing another nucleic acid.

[0148] In some embodiments, the glycoengineered extracellular vesicle and the cell from which said extracellular vesicle is obtained have a different glycosylation pattern.

[0149] In some embodiments, the nucleic acid may not be present in the reference cell before glycoengineering. In some embodiments, the nucleic acid is present in the reference cell before glycoengineering

[0150] In preferred embodiments, the activity of the product encoded by the at least one nucleic acid has been modified, for example the product is a protein, the activity of which has been modified by modifying the nucleic acid, for instance the nucleic acid encodes a truncated protein which has less activity than the native protein.

[0151] In some embodiments, both the expression of said at least one nucleic acid of the cell and / or the activity of the product encoded by said nucleic acid have been modified, resulting in a modified glycosylation of the surface of an extracellular vesicle produced by the cell.

[0152] In some embodiments, the at least one nucleic acid is at least two nucleic acids, such as at least three nucleic acids, such as at least four nucleic acids, such as at least five nucleic acids, or more.

[0153] When more than one nucleic acid is modified, the nucleic acids may be involved in different types of glycosylation, as will be evident to the skilled person. In some embodiments, the cell has a total or partial reduction in the expression of said at least one nucleic acid. It will be understood by a skilled person in the art that any method known to cause total or partial reduction of the expression of a nucleic acid might be used, such as e.g. mutations, such as frame-shift mutations, deletions, etc.

[0154] In some embodiments, the expression of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell, has been modified.

[0155] In some embodiments, the activity of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell have been modified.

[0156] In some embodiments, the cell has a total or partial reduction in the expression of GNE. In some embodiments, the cell has a total or partial reduction in the activity of the product, namely UDP-GIcNAc 2-epimerase / ManNAc kinase, encoded by GNE.

[0157] Modifying the expression of GNE and / or modifying the activity of the UDP-GIcNAc 2- epimerase / ManNAc kinase encoded by GNE leads to modified sialylation. Modified sialylation may in addition indirectly result in modifications of any molecule containing a sialic acid, such as one or more of N-glycosylation, O-linked glycosylation, glycolipids such as glycosphingolipids, glycosaminoglycans, and fucosylation.

[0158] In some embodiments, the cell has a total or partial reduction of expression of GNE and / or total or partial reduction of the activity of GNE, and / or the cell has a total or partial reduction of expression of B4GALT7 and / or total or partial reduction of the activity of B4GALT7.

[0159] In some embodiments, the cell has a total or partial reduction of expression of B4GALT7 and / or total or partial reduction of the activity of B4GALT7.

[0160] In some embodiments, the cell has a total or partial reduction of expression of GNE and / or total or partial reduction of the activity of GNE.

[0161] In some embodiments, the cell has a total or partial reduction of expression of MGAT1 and / or total or partial reduction of the activity of MGAT 1. In some embodiments, the cell has a total or partial reduction of expression of C1GALT1C1 and / or total or partial reduction of the activity of C1GALT1C1.

[0162] In some embodiments, the cell has a total or partial reduction of expression of C1 GALT 1 and / or total or partial reduction of the activity of C1 GALT 1.

[0163] In some embodiments, the cell has a total or partial reduction of expression of B4GALT5 and / or total or partial reduction of the activity of B4GALT5.

[0164] In some embodiments, the cell has a total or partial reduction of expression of B4GALT6 and / or total or partial reduction of the activity of B4GALT6.

[0165] In some embodiments, the nucleic acid encodes a glycosyltransferase, and the expression of the nucleic acid encoding a glycosyltransferase has been modified, or the activity of the glycosyltransferase has been modified, or both. In some embodiments, the nucleic acid encodes a glycosyltransferase selected from the group consisting of: B4GALT5, (SEQ ID NO: 1); B4GALT6, (SEQ ID NO: 3); B4GALT7, (SEQ ID NO: 5); C1GALT1, (SEQ ID NO: 7); C1GALT1C1, (SEQ ID NO: 9); CHSY1, (SEQ ID NO: 11); CHSY3, (SEQ ID NO: 13); CMAS, (SEQ ID NO: 15); EXTL3, (SEQ ID NO: 17); FUT8, (SEQ ID NO: 19); GFUS, (SEQ ID NO: 21); GMDS, (SEQ ID NO: 23); GNE, (SEQ ID NO: 25); MGAT1, (SEQ ID NO: 27); MOGS, (SEQ ID NO: 29); ST3GAL1, (SEQ ID NO: 31); ST3GAL3, (SEQ ID NO: 33); ST3GAL4, (SEQ ID NO: 35);

[0166] ST3GAL6, (SEQ ID NO: 37); ST6GAL1, (SEQ ID NO: 39); A4GALT, (SEQ ID NO: 41); A4GNT, (SEQ ID NO: 42); ABO, (SEQ ID NO: 43); ALG1, (SEQ ID NO: 44); ALG10, (SEQ ID NO: 45); ALG10B, (SEQ ID NO: 46); ALG11, (SEQ ID NO: 47); ALG12, (SEQ ID NO: 48); ALG13, (SEQ ID NO: 49); ALG14, (SEQ ID NO: 50); ALG2, (SEQ ID NO: 51); ALG3, (SEQ ID NO: 52); ALG5, (SEQ ID NO: 53); ALG6, (SEQ ID NO: 55); ALG8, (SEQ ID NO: 56); ALG9, (SEQ ID NO: 57); ASGR1, (SEQ ID NO: 58); ASGR2, (SEQ ID NO: 59); B3GALNT1, (SEQ ID NO: 60); B3GALNT2, (SEQ ID NO: 61); B3GALT1, (SEQ ID NO: 62); B3GALT2, (SEQ ID NO: 63); B3GALT4, (SEQ ID NO: 64);

[0167] B3GALT5, (SEQ ID NO: 65); B3GALT6, (SEQ ID NO: 66); B3GAT1, (SEQ ID NO: 67); B3GAT2, (SEQ ID NO: 68); B3GAT3, (SEQ ID NO: 69); B3GLCT, (SEQ ID NO: 70); B3GNT2, (SEQ ID NO: 71); B3GNT3, (SEQ ID NO: 72); B3GNT4, (SEQ ID NO: 73); B3GNT5, (SEQ ID NO: 74); B4GAT1, (SEQ ID NO: 75); B3GNT6, (SEQ ID NO: 76); B3GNT7, (SEQ ID NO: 77); B3GNTL1, (SEQ ID NO: 78); B3GNT8, (SEQ ID NO: 79); B3GNT9, (SEQ ID NO: 80); B4GALNT1, (SEQ ID NO: 81); B4GALNT2, (SEQ ID NO: 82); B4GALNT3, (SEQ ID NO: 83); B4GALNT4, (SEQ ID NO: 84); B4GALT1, (SEQ ID NO: 85); B4GALT2, (SEQ ID NO: 86); B4GALT3, (SEQ ID NO: 87); B4GALT4, (SEQ ID NO: 88); CALR, (SEQ ID NO: 89); CANX, (SEQ ID NO: 90); CD207, (SEQ ID NO: 91); CD209, (SEQ ID NO: 92); CHPF, (SEQ ID NO: 93); CHPF2, (SEQ ID NO: 94); CHST1, (SEQ ID NO: 95); CHST10, (SEQ ID NO: 96); CHST11, (SEQ ID NO: 97); CHST12, (SEQ ID NO: 98); CHST13, (SEQ ID NO: 99); CHST14, (SEQ ID NO: 100); CHST15, (SEQ ID NO: 101); CHST2, (SEQ ID NO: 102); CHST3, (SEQ ID NO: 103); CHST4, (SEQ ID NO: 104); CHST5, (SEQ ID NO: 105); CHST6, (SEQ ID NO: 106); CHST7, (SEQ ID NO: 107); CHST8, (SEQ ID NO: 108); CHST9, (SEQ ID NO: 109); CLEC4E, (SEQ ID NO: 110); CLEC7A, (SEQ ID NO: 111); CLEC12A, (SEQ ID NO: 112); COLGALT1, (SEQ ID NO: 113); COLGALT2, (SEQ ID NO: 114); CSGALNACT1, (SEQ ID NO: 115); CSGALNACT2, (SEQ ID NO: 116); DCIR, (SEQ ID NO: 117);

[0168] DPAGT1, (SEQ ID NO: 118); DPMI, (SEQ ID NO: 119); DPY19L1, (SEQ ID NO: 120); DPY19L2, (SEQ ID NO: 121); DPY19L3, (SEQ ID NO: 122); DPY19L4, (SEQ ID NO: 123); DSE, (SEQ ID NO: 124); DSEL, (SEQ ID NO: 125); EDEM1, (SEQ ID NO: 126); EDEM2, (SEQ ID NO: 127); EDEM3, (SEQ ID NO: 128); EOGT, (SEQ ID NO: 129); EXT1, (SEQ ID NO: 130); EXT2, (SEQ ID NO: 131); EXTL1, (SEQ ID NO: 132); EXTL2, (SEQ ID NO: 133); FKRP, (SEQ ID NO: 134); FKTN, (SEQ ID NO: 135); FUCA 1, (SEQ ID NO: 136); FUCA2, (SEQ ID NO: 137); FUT1, (SEQ ID NO: 138); FUT10, (SEQ ID NO: 139); FUT11, (SEQ ID NO: 140); FUT2, (SEQ ID NO: 141); FUT3, (SEQ ID NO: 142); FUT4, (SEQ ID NO: 143); FUT5, (SEQ ID NO: 144); FUT6, (SEQ ID NO: 145); FUT7, (SEQ ID NO: 146); FUT9, (SEQ ID NO: 147); GAL3ST1, (SEQ ID NO: 148); GAL3ST2, (SEQ ID NO: 149); GAL3ST3, (SEQ ID NO: 150);

[0169] GAL3ST4, (SEQ ID NO: 151); GALNT1, (SEQ ID NO: 152); GALNT10, (SEQ ID NO: 153); GALNT11, (SEQ ID NO: 154); GALNT12, (SEQ ID NO: 155); GALNT13, (SEQ ID NO: 156); GALNT14, (SEQ ID NO: 157); GALNTL5, (SEQ ID NO: 158); GALNT15, (SEQ ID NO: 159); GALNT16, (SEQ ID NO: 160); GALNT17, (SEQ ID NO: 161); GALNT18, (SEQ ID NO: 162); GALNT2, (SEQ ID NO: 163); GALNT3, (SEQ ID NO: 164); GALNT4, (SEQ ID NO: 165); GALNT5, (SEQ ID NO: 166); GALNT6, (SEQ ID NO: 167); GALNT7, (SEQ ID NO: 168); GALNT8, (SEQ ID NO: 169); GALNT9, (SEQ ID NO: 170); GALNTL6, (SEQ ID NO: 171); GAN AB, (SEQ ID NO: 172); GBA1, (SEQ ID NO: 173); GBA2, (SEQ ID NO: 174); GBA3, (SEQ ID NO: 175); GCNT1, (SEQ ID NO: 176); GCNT2, (SEQ ID NO: 177); GCNT3, (SEQ ID NO: 178); GCNT4, (SEQ ID NO: 179); GCNT7, (SEQ ID NO: 180); GLA, (SEQ ID NO: 181); GLB1, (SEQ ID NO: 182); GLB1L, (SEQ ID NO: 183); GLB1L2, (SEQ ID NO: 184); GLB1L3, (SEQ ID NO: 185); GLCE, (SEQ ID NO: 186); GNPTAB, (SEQ ID NO: 187); GNPTG, (SEQ ID NO: 188); GXYLT1, (SEQ ID NO: 189); GXYLT2, (SEQ ID NO: 190); GAA, (SEQ ID NO: 191); HEXA, (SEQ ID NO: 192); HEXB, (SEQ ID NO: 193); HEXD, (SEQ ID NO: 194); HPSE, (SEQ ID NO: 195); HPSE2, (SEQ ID NO: 196); HS2ST1, (SEQ ID NO: 197); HS3ST1, (SEQ ID NO: 198); HS3ST2, (SEQ ID NO: 199); HS3ST3A1, (SEQ ID NO: 200); HS3ST3B1, (SEQ ID NO: 201); HS3ST4, (SEQ ID NO: 202); HS3ST5, (SEQ ID NO: 203); HS3ST6, (SEQ ID NO: 204); HS6ST1, (SEQ ID NO: 205); HS6ST2, (SEQ ID NO: 206); HS6ST3, (SEQ ID NO: 207); HYAL1, (SEQ ID NO: 208); HYAL2, (SEQ ID NO: 209); HYAL3, (SEQ ID NO: 210); SPAM1, (SEQ ID NO: 211); HYAL4, (SEQ ID NO: 212); IGF2R, (SEQ ID NO: 213); LARGE1, (SEQ ID NO: 214); LARGE2, (SEQ ID NO: 215); LENG, (SEQ ID NO: 216); LGALS1, (SEQ ID NO: 217); LGALS10, (SEQ ID NO: 218); LGALS12, (SEQ ID NO: 219); LGALS13, (SEQ ID NO: 220); LGALS14, (SEQ ID NO: 221); LGALS16, (SEQ ID NO: 222); LGALS2, (SEQ ID NO: 223);

[0170] LGALS3, (SEQ ID NO: 224); LGALS4, (SEQ ID NO: 225); LGALS7, (SEQ ID NO: 226); LGALS8, (SEQ ID NO: 227); LGALS9, (SEQ ID NO: 228); LGALS9B, (SEQ ID NO: 229); M6PR, (SEQ ID NO: 230); MAG, (SEQ ID NO: 231); MAN1A 1, (SEQ ID NO: 232); MAN1A2, (SEQ ID NO: 233); MAN1B1, (SEQ ID NO: 234); MAN1C1, (SEQ ID NO: 235); MAN2A 1, (SEQ ID NO: 236); MAN2A2, (SEQ ID NO: 237); MAN2B1, (SEQ ID NO: 238); MAN2B2, (SEQ ID NO: 239); MAN2C1, (SEQ ID NO: 240); MANEA, (SEQ ID NO: 241); MANEAL, (SEQ ID NO: 242); MENG, (SEQ ID NO: 243); MGAT2, (SEQ ID NO: 244); MGAT3, (SEQ ID NO: 245); MGAT4A, (SEQ ID NO: 246);

[0171] MGAT4B, (SEQ ID NO: 247); MGAT4C, (SEQ ID NO: 248); MGAT4D, (SEQ ID NO: 249); MGAT5, (SEQ ID NO: 250); MGAT5B, (SEQ ID NO: 251); MRC1, (SEQ ID NO: 252); NAG A, (SEQ ID NO: 253); NAGPA, (SEQ ID NO: 254); NANP, (SEQ ID NO: 255); NANS, (SEQ ID NO: 256); NDST1, (SEQ ID NO: 257); NDST2, (SEQ ID NO: 258); NDST3, (SEQ ID NO: 259); NDST4, (SEQ ID NO: 260); NEU1, (SEQ ID NO: 261); NEU2, (SEQ ID NO: 262); NEU3, (SEQ ID NO: 263); NEU4, (SEQ ID NO: 264); OGA, (SEQ ID NO: 265); OGT, (SEQ ID NO: 266); PDIA3, (SEQ ID NO: 267); PGAP4, (SEQ ID NO: 268); PIGA, (SEQ ID NO: 269); PIGB, (SEQ ID NO: 270); PIGM, (SEQ ID NO: 271); PIGV, (SEQ ID NO: 272); PIGZ, (SEQ ID NO: 273); POFUT1, (SEQ ID NO: 274); POFUT2, (SEQ ID NO: 275); POGLUT1, (SEQ ID NO: 276); POGLUT2, (SEQ ID NO: 277); POGLUT3, (SEQ ID NO: 278); POMGNT1, (SEQ ID NO: 279); POMGNT2, (SEQ ID NO: 280); POMT1, (SEQ ID NO: 281); POMT2, (SEQ ID NO: 282); RFNG, (SEQ ID NO: 283); RXYLT1, (SEQ ID NO: 284); SELF, (SEQ ID NO: 285); SELL, (SEQ ID NO: 286); SELP, (SEQ ID NO: 287); SIGLEC1, (SEQ ID NO: 288); SIGLEC10, (SEQ ID NO: 289); SIGLEC11, (SEQ ID NO: 290); SIGLEC12, (SEQ ID NO: 291); SIGLEC14, (SEQ ID NO: 292); SIGLEC15, (SEQ ID NO: 293); SIGLEC16, (SEQ ID NO: 294); SIGLEC2, (SEQ ID NO: 295); SIGLEC3, (SEQ ID NO: 296); SIGLEC5, (SEQ ID NO: 297); SIGLEC6, (SEQ ID NO: 298); SIGLEC7, (SEQ ID NO: 299); SIGLEC8, (SEQ ID NO: 300); SIGLEC9, (SEQ ID NO: 301); SLC35A1, (SEQ ID NO: 302); SLC35C2, (SEQ ID NO: 303); ST3GAL2, (SEQ ID NO: 304); ST3GAL5, (SEQ ID NO: 305); ST6GAL2, (SEQ ID NO: 306); ST6GALNAC1, (SEQ ID NO: 307); ST6GALNAC2, (SEQ ID NO: 308); ST6GALNAC3, (SEQ ID NO: 309); ST6GALNAC4, (SEQ ID NO: 310); ST6GALNAC5, (SEQ ID NO: 311); ST6GALNAC6, (SEQ ID NO: 312); ST8SIA1, (SEQ ID NO: 313); ST8SIA2, (SEQ ID NO: 314); ST8SIA3, (SEQ ID NO: 315); ST8SIA4, (SEQ ID NO: 316); ST8SIA5, (SEQ ID NO: 317); ST8SIA6, (SEQ ID NO: 318); STT3A, (SEQ ID NO: 319); STT3B, (SEQ ID NO: 320); TMTC1, (SEQ ID NO: 321);TMTC2, (SEQ ID NO: 322); TMTC3, (SEQ ID NO: 323); TMTC4, (SEQ ID NO: 324); TPST1, (SEQ ID NO: 325); TPST2, (SEQ ID NO: 326); UGCG, (SEQ ID NO: 327); UGGT1, (SEQ ID NO: 328); UGGT2, (SEQ ID NO: 329); UGT8, (SEQ ID NO: 330); UST, (SEQ ID NO: 331); XXYLT1, (SEQ ID NO: 332); XYLT1, (SEQ ID NO: 333); XYLT2, (SEQ ID NO: 334); or homologues thereof having at least 70% identity thereto, such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.

[0172] In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 2 and encodes B4GALT5 (SEQ ID NO: 1), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 4 and encodes B4GALT6 (SEQ ID NO: 3), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 6 and encodes B4GALT7 (SEQ ID NO: 5), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 8 and encodes C1GALT 1 (SEQ ID NO: 7), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 10 and encodes C1GALT1C1 (SEQ ID NO: 9), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 12 and encodes CHSY1 (SEQ ID NO: 11), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 14 and encodes CHSY3 (SEQ ID NO: 13), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 16 and encodes CMAS (SEQ ID NO: 15), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 54 and encodes ALG5 (SEQ ID NO: 53), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 18 and encodes EXTL3 (SEQ ID NO: 17), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 20 and encodes FLIT8 (SEQ ID NO: 19), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 22 and encodes GFLIS (SEQ ID NO: 21), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 24 and encodes GMDS (SEQ ID NO: 23), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 26 and encodes GNE (SEQ ID NO: 25), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 28 and encodes MGAT1 (SEQ ID NO: 27), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 30 and encodes MOGS (SEQ ID NO: 29), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 31 and encodes ST3GAL1 (SEQ ID NO: 32), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 34 and encodes ST3GAL3 (SEQ ID NO: 33), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 36 and encodes ST3GAL4 (SEQ ID NO: 35), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 38 and encodes ST3GAL6 (SEQ ID NO: 37), or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of SEQ ID NO: 40 and encodes ST6GAL1 (SEQ ID NO: 39), or a homologue thereof having at least 70% identity thereto.

[0173] In some embodiments, the at least one nucleic acid is two nucleic acids, such as B4GALT5 and B4GALT6. In some embodiments, the at least one nucleic acid is B4GALT7; GNE; B4GALT5;

[0174] B4GALT6; C1GALT1C1 ; C1GALT1 ; or MGATI .

[0175] In some embodiments, the at least one nucleic acid is: B4GALT7 and / or GNE.

[0176] Modifying the expression of a nucleic acid encoding a glycosyltransferase and / or modifying the activity of a glycosyltransferase leads to modified O-linked glycosylation. This may include one or more of modified O-GalNAc, modified O-GIcNAc, modified O- Man, modified O-Gal, modified O-Fuc, and / or modified O-GIc. Modified sialylation may in addition indirectly result in modifications of any molecule containing a sialic acid, such as one or more of N-glycosylation, glycolipids such as glycosphingolipids, glycosaminoglycans, sialylation and fucosylation.

[0177] In some embodiments, the nucleic acid encodes a glycosidase or a glycosyl hydrolase, such as a fucosidase, in particular an a-L fucosidase; a galactosidase; a glucosylceramidase P; a glucosidase; a heparanase; a hexosaminidase; a hyaluronidase; a mannosidase a; or a neuraminidase. In some embodiments, the glycosyl hydrolase is an a-L fucosidase selected from FUCA 1 and FUCA2, or homologues thereof having at least 70% identity thereto. In some embodiments, the glycosyl hydrolase is a glucosylceramidase p, such as selected from GBA 1, GBA2 and GBA3, or homologues thereof having at least 70% identity thereto. In some embodiments, the glycosyl hydrolase is a glucosidase, such as selected from MOGS, GANAB and GAA, or homologues thereof having at least 70% identity thereto. In some embodiments, the glycosyl hydrolase is a heparanase, such as selected from HPSE and HPSE2, or homologues thereof having at least 70% identity thereto. In some embodiments, the glycosyl hydrolase is a hexosaminidase, such as selected from HEXA, HEXB, HEXD and OGA, or homologues thereof having at least 70% identity thereto. In some embodiments, the glycosyl hydrolase is a hyaluronidase, such as selected from HYAL1, HYAL2, HYAL3 and HYAL4, or homologues thereof having at least 70% identity thereto. In some embodiments, the glycosyl hydrolase is a mannosidase a, such as selected from EDEM1, EDEM2, EDEM3, MAN1A 1, MAN1A2, MAN1B1, MAN1C1, MAN2A1, MAN2A2, MAN2B1, MAN2B2, MAN2C1, MANEA, and MANEAL, or homologues thereof having at least 70% identity thereto. In some embodiments, the glycosyl hydrolase is a neuraminidase, such as selected from NEU1, NEU2, NEU3 and NEU4, or homologues thereof having at least 70% identity thereto.

[0178] In some embodiments, the nucleic acid encodes an enzyme in the sugar precursor pathway. In some embodiments, said enzyme is selected from the group consisting of: GNE; NANS; NANP; CMAS; GMDS; and GFUS, or homologues thereof having at least 70% identity thereto. Modifying the expression of either of the nucleic acids encoding GNE, NANS, NANP or CMAS, or modifying the activity of said enzymes, leads to modified sialylation, which in turn may result in modifications of other types of glycosylation. Modifying the expression of the nucleic acids encoding GMDS or GFUS, or modifying the activity of said enzymes, leads to modified fucosylation, which in turn may result in modifications of other types of glycosylation.

[0179] In some embodiments, the nucleic acid encodes a transporter and the expression of the nucleic acid is modified, or the activity of a transporter is modified in the cell. In some embodiments, the transporter is a GDP-fucose transporter. In other embodiments, the transporter is a CMP-sialic acid transporter.

[0180] In some embodiments, the nucleic acid encodes a carbohydrate-binding protein transporter and the expression of the nucleic acid is modified, or the activity of the carbohydrate-binding protein encoded by the nucleic acid is modified in the cell. Preferably the carbohydrate-binding protein is endogenous. In some embodiments, the carbohydrate-binding protein is a lectin, a galectin or a siglec.

[0181] In some embodiments, the nucleic acid encodes a chaperone and the expression of the nucleic acid is modified, or the activity of the chaperone encoded by the nucleic acid is modified in the cell. In some embodiments, the chaperone is C1GALT1C1, CANX, CALR and / or PDIA3 or homologues thereof having at least 70% identity thereto. Modifying C1GALT1C1, or modifying the activity of the product encoded by C1GALT1C1, leads to modification of O-linked glycosylation, in particular modification of O-GalNAc glycosylation.

[0182] In some embodiments, the nucleic acid encodes a product, such as a protein or a polypeptide, which is a part of a glycosylation pathway, more particularly the product is involved in one or more of: biosynthesis and / or transport of precursor sugars; initiation of glycosylation; core extension; elongation; trimming; branching, capping and transport

[0183] By modifying the expression of a nucleic acid involved, directly or indirectly, in the glycosylation machinery, or by modifying the activity of the product encoded by the nucleic acid, different steps or parts of the glycosylation pathways can be affected.

[0184] The modification of the expression of the nucleic acid or of the activity of the product it encodes may affect one or more steps of the glycosylation pathway selected from: biosynthesis and / or transport of precursor sugars; initiation of glycosylation; core extension; elongation; trimming; branching, capping and transport.

[0185] In some embodiments, the modification of the expression of the nucleic acid and / or the modification of the activity of the product encoded by the nucleic acid affects the biosynthesis of at least one group of carbohydrates selected from the group consisting of: N-glycans; O-glycans; glycolipids such as glycosphingolipids; glycosaminoglycans; proteoglycans; GPI anchored glycoproteins; fucose, and sialic acid.

[0186] In some embodiments, the nucleic acid encodes a polypeptide which is part of the biosynthesis of at least one group of carbohydrates selected from the group consisting of: N-glycans; O-glycans; glycolipids such as glycosphingolipids; glycosaminoglycans; proteoglycans; GPI anchored glycoproteins; fucose, and sialic acid. In some embodiments, the modification of the expression of the nucleic acid and / or the modification of the activity of the product encoded by the nucleic acid affects the biosynthesis of at least one group of carbohydrates selected from the group consisting of: N-glycans; O-glycans; glycolipids such as glycosphingolipids; glycosaminoglycans; proteoglycans; GPI anchored glycoproteins; fucose, and sialic acid.

[0187] Importantly, the modification of the expression of a nucleic acid or of the activity of the product encoded by the nucleic acid leads to an altered phosphorylation of a carbohydrate, to an altered sulfation of a carbohydrate, or both.

[0188] In some embodiments, the nucleic acid comprises or consists of a gene selected from the group consisting of: SELE; SELP; SELL; CD207; CD209; MRC1; CLEC7A;

[0189] CLEC4E; CLECL12A; ASGR1; ASGR2; DCIR', B4GALT5; B4GALT6; B4GALT7; MGAT1; FUT8; MGAT2; MGAT3; MGAT5; MGAT4A; MGAT4B; MGAT4C; MGAT4D; GALNT1;GALNT2; GALNT3; GALNT4; GALNT5; GALNT6; GALNT7; GALNT8; GALNT9; GALNT10; GALNT11; GALNT12; GALNT13; GALNT14; GALNT15;

[0190] GALNT16; GALNTL6; GALNT18; GALNT17; GALNTL5; C1GALT1; FUT1; FUT10; FUT11; FUT2; FUT3; FUT4; FUT5; FUT6; FUT7; FUT9; ST3GAL1; ST3GAL2; ST3GAL3; ST3GAL4; ST3GAL5; ST3GAL6; ST6GAL1; ST6GAL2; ST6GALNAC1; ST6GALNAC2; ST6GALNAC3; ST6GALNAC4; ST6GALNAC5; ST6GALNAC6; ST8SIA1; ST8SIA2; ST8SIA3; ST8SIA4; ST8SIA5; ST8SIA6; MAN1A1; MAN1A2; MAN1B1; MAN1C1; MAN2A1; MAN2A2; GNE; GMDS; GFUS; and C1GALT1C1, or homologues thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of B4GALT7, or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of GNE, or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of B4GALT5, or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of B4GALT6, or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of C1GALT1C1, or a homologue thereof having at least 70% identity thereto. In some embodiments, the nucleic acid comprises or consists of MGAT1, or a homologue thereof having at least 70% identity thereto.

[0191] In some embodiments, the modification of the expression of said at least one nucleic acid of the cell and / or the activity of said product encoded by said at least one nucleic acid sequence results in the modification, such as in the inhibition and / or the block of, or such as in increase or stimulation of the synthesis of proteoglycans and / or glycosaminoglycans.

[0192] In some embodiments, the cell producing the extracellular vesicles has been genetically engineered. In other embodiments, the cell producing the extracellular vesicles has not been genetically engineered, but expression of the nucleic acid or activity of the product encoded by the nucleic acid are modified by methods known in the art, e.g. by using siRNA to prevent translation of the mRNA encoded by the nucleic acid, or by contacting the cell with a molecule which directly affects the activity of the product encoded by the nucleic acid. In some embodiments, the product encoded by the nucleic acid is a polypeptide, and the modification of the activity of that polypeptide is a stimulation of the activity. This can be achieved by contacting the extracellular vesicle or the cell with a compound stimulating the activity of the polypeptide as is known in the art. In other embodiments, the modification of the activity of that polypeptide is an inhibition of the activity. This can be achieved by contacting the extracellular vesicle or the cell with a compound inhibiting the activity of the polypeptide as is known in the art. Stimulatory or inhibitory compounds may be a small molecule stimulator or a small molecule inhibitor, respectively.

[0193] In some embodiments, the modified glycosylation comprises or consists of at least one modified glycosaminoglycan, and preferably a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, such as an anti-chondroitin sulfate antibody, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

[0194] In some embodiments, the modified glycosylation is a modification in the length of glycan chains resulting from modified sialylation or fucosylation, and preferably a marker specific for sialylation or fucosylation, such as lectin SNA or LCA, shows modified binding to the extracellular vesicle compared to the binding of same marker to an extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

[0195] In some embodiments, the binding of said marker specific for glycosaminoglycans, sialylation or fucosylation to the extracellular vesicle compared to the binding of said marker to the extracellular vesicle obtained from the reference cell is modified by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%.

[0196] In some embodiments, the at least one nucleic acid is PIGA; PIGM; PIGV; PIGB; PIGZ;

[0197] PGAP4; UGT8; UGCG; B4GALT5; B4GALT6; B3GALNT1; A4GALT; B4GALNT1;

[0198] B3GNT5; DPAGT1; ALG13; ALG14; ALG1; ALG2; ALG11; DPMI; UGGT1; UGGT2;

[0199] ALG3; ALG5; ALG9; ALG12; ALG6; ALG8; ALG10; ALG10B; STT3A; STT3B; MGAT1;

[0200] FUT8; MGAT2; MGAT3; MGAT5; MGAT4A; MGAT4B; MGAT4C; MGAT4D;

[0201] GALNT1;GALNT2; GALNT3; GALNT4; GALNT5; GALNT6; GALNT7; GALNT8;

[0202] GALNT9; GALNT10; GALNT11; GALNT12; GALNT13; GALNT14; GALNT15;

[0203] GALNT16; GALNTL6 (T17); GALNT18; GALNT17 (T19); GALNTL5 (T20); C1GALT1;

[0204] GCNT1; GCNT3; GCNT4; B3GNT6; POFUT1; POFUT2; B3GLCT; LFNG; MFNG;

[0205] RFNG; POMT1; POMT2; POMGNT1; MGAT5B; POMGNT2; B3GALNT2; FKRP;

[0206] FKTN; B4GAT1; RXYLT1; LARGE1; LARGE2; TMTC1; TMTC2; TMTC3; TMTC4;

[0207] DPY19L1; DPY19L2; DPY19L3; DPY19L4; POGLUT1; POGLUT2; POGLUT3;

[0208] GXYLT1; GXYLT2; XXYLT1; XYLT1; XYLT2; B4GALT7; B3GALT6; B3GAT3;

[0209] CSGALNACT1; CSGALNACT2; CHPF; CHPF2; CHSY1; CHSY3; EXT1; EXT2;

[0210] EXTL1; EXTL2; EXTL3; COLGALT1; COLGALT2; OGT; EOGT; B3GALT1; B3GALT2;

[0211] B3GALT4; B3GALT5; B3GNT2; B3GNT3; B3GNT4; B3GNT7; B3GNT8; B3GNT9;

[0212] B4GALNT3; B4GALNT4; B4GALT1; B4GALT2; B4GALT3; B4GALT4; GCNT2;

[0213] GCNT7; A4GNT; ABO; B3GAT1; B3GAT2; B4GALNT2; FUT1; FUT10; FUT11; FUT2;

[0214] FUT3; FUT4; FUT5; FUT6; FUT7; FUT9; ST3GAL1; ST3GAL2; ST3GAL3; ST3GAL4;

[0215] ST3GAL5; ST3GAL6; ST6GAL1; ST6GAL2; ST6GALNAC1; ST6GALNAC2;

[0216] ST6GALNAC3; ST6GALNAC4; ST6GALNAC5; ST6GALNAC6; ST8SIA1; ST8SIA2;

[0217] ST8SIA3; ST8SIA4; ST8SIA5; ST8SIA6; CHST1; CHST10; CHST11; CHST12;

[0218] CHST13; CHST14; CHST15; CHST2; CHST3; CHST4; CHST5; CHST6; CHST7;

[0219] CHST8; CHST9; GAL3ST1; GAL3ST2; GAL3ST3; GAL3ST4; HS2ST1; HS3ST1;

[0220] HS3ST2; HS3ST3A1; HS3ST3B1; HS3ST4; HS3ST5; HS3ST6; HS6ST1; HS6ST2;

[0221] HS6ST3; NDST1; NDST2; NDST3; NDST4; UST; DSEL; DSE; GLCE; TPST1; TPST2;

[0222] GNPTAB; GNPTG; NAGPA; M6PR; IGF2R; FUCA1; FUCA2; GLA; NAGA; GLB1;

[0223] GLB1L; GLB1L2; GLB1L3; GBA1; GBA2; GBA3; MOGS; GANAB; GAA; HPSE;

[0224] HPSE2; HEXA; HEXB; HEXD; OGA; HYAL1; HYAL2; HYAL3; HYAL4; EDEM1;

[0225] EDEM2; EDEM3; MAN1A1; MAN1A2; MAN1B1; MAN1C1; MAN2A1; MAN2A2;

[0226] MAN2B1; MAN2B2; MAN2C1; MANEA; MANEAL; NEU1; NEU2; NEU3; NEU4; GNE;

[0227] GMDS; GFUS; C1GALT1C1; CANX; CALR and PDIA3; SELE; SELP; SELL; CD207; CD209; MRC1; CLEC7A; CLEC4E; CLEC12A; ASGR1; ASGR2; DCIR; SIGLEC1; SIGLEC2; SIGLEC3; SIGLEC4; SIGLEC5; SIGLEC6; SIGLEC7; SIGLEC8; SIGLEC9; SIGLEC10; SIGLEC11 ; SIGLEC12; SIGLEC14; SIGLEC15; SIGLEC16; LGALS1 ; LGALS2; LGALS3; LGALS4; LGALS7; LGALS8; LGALS9; LGALS9B; LGALS10; LGALS12; LGALS13; LGALS14; LGALS16; SLC35A1 ; or SLC35C2.

[0228] Genetic engineering

[0229] Methods to genetically engineer a cell to produce glycoengineered extracellular vesicles with a modified glycosylation as described above are available to the skilled person. Targeted strategies aiming at modifying a specific locus or modifying / introducing a nucleic acid in the cell can be used, such as CRISPR-Cas9, ZFNs and TALENs. However the cell may also be engineered by untargeted strategies, for example by mutagenesis (induced or spontaneous) and / or selection of cells under selection pressure. For example, in some embodiments cells harbouring modified expression of a nucleic acid and / or modified activity of the product encoded by said nucleic acid have been obtained by cultivating isolated cells in a medium comprising a molecule, such as a toxic lectin, which inhibits the growth and / or kills cells comprising a specific carbohydrate and / or a specific modification of a carbohydrate.

[0230] In some embodiments, the modified glycosylation is modified glycosaminoglycans and / or modified sialylation on the surface of the glycoengineered extracellular vesicle, and / or the expression of said at least one nucleic is modified using CRISPR-associated nucleases, such as CRISPR-Cas9, zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), and / or said at least one nucleic acid is introduced using CRISPR-associated nucleases, such as CRISPR-Cas9, zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs).

[0231] For both types of targeting, once cells have been obtained which are suspected to produce glycoengineered vesicles, assays can be performed as described herein elsewhere, such as determining whether the obtained glycoengineered extracellular vesicles are recognized by specific markers, or performing other glycoanalytical assays such as HPLC and / or mass spectrometry. Thus in some embodiments the method comprises a step of genetically engineering a cell producing glycoengineered extracellular vesicles. In some embodiments the genetic engineering is performed using CRISPR-CAS. In some embodiments the genetic engineering is performed using TALENS. In some embodiments the genetic engineering is performed using ZFNs.

[0232] While the modifications to the expression of the nucleic acid or to the activity of the product encoded by the nucleic acid may be stable or permanent, i.e. are not lost upon propagation of the genetically engineered cell, it may be of interest to modify the glycosylation profile only transiently. This is within the skilled person’s reach. In some embodiments the expression of the nucleic acid is modified transiently. In some embodiments the activity of the product encoded by the nucleic acid is modified transiently.

[0233] It will be clear to the skilled person that modifying the expression of a nucleic acid in the cell means that a genomic nucleic acid can be modified, or a nucleic acid present on a vector comprised in the cell can be modified.

[0234] Cell

[0235] The glycoengineered extracellular vesicles described herein are obtained by methods which involve either genetically modifying the cell from which they are produced, or modifying the activity of a product encoded by a nucleic acid present in the cell, as described herein.

[0236] In some embodiments, the cell is a eukaryotic cell. For example, the cell is an animal cell, a plant cell, a fungus cell or a yeast cell. In some embodiments, the cell is an animal cell selected from the group consisting of: a mammalian cell; an avian cell; and an invertebrate cell, such as an insect cell. For example, the cell is a mammalian cell selected from the group consisting of: an cell of a Hominidae, such as a human cell; a cell of a Cricetidae, such as a hamster cell, such as a Chinese Hamster Ovary (CHO) cell; and a cell of a Muridae, such as a mouse cell, such as a rat cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a hamster cell such as a CHO cell. In some embodiments, the cell is a mouse cell or a rat cell. In some embodiments, the cell is selected from the group consisting of: a stem cell; such as a pluripotent cell, such as a hematopoietic stem cell, such as a neural stem cell, such as a mesenchymal stem cell, such as an embryonic stem cell; a precursor cell; a skin cell; a brain cell; and an immune cell. In some embodiments, the cell is a stem cell, such as an adult stem cell, which may be an artificially induced stem cell, such as an induced pluripotent stem cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a natural killer cell. In some embodiments, the cell is a chimeric antigen receptor-T (CAR-T) cell, a chimeric antigen receptor natural killer cell (CAR-NK), or a CAR-NKT cell. The cell may be a T cell, such as a CD8 T cell or a CD4 T cell. The cell may be a dendritic cell.

[0237] In some embodiments, the cell is a cell derived from anyone of the cells described herein, such as a cell derived from a stem cell, or a stem like cell.

[0238] In some embodiments, the stem cell is an adult stem cell, and / or is an artificially induced stem cell, such as an induced pluripotent stem cell, or a cell derived therefrom.

[0239] Non-limiting examples of suitable cells include HEK293 cells, such as HEK293F cells; CHO cells; MUTZ3 cells; K562 cells; NK92 cells; KHYG-1 cells; and KHYK cells.

[0240] Isolation of glycoengineered extracellular vesicles

[0241] The methods of the present disclosure provide glycoengineered extracellular vesicles with modified glycosylation, i.e. glycoengineered extracellular vesicles. In some embodiments, the method of obtaining a glycoengineered extracellular vesicle further comprises recovering the glycoengineered extracellular vesicle. The skilled person knows how to recover the glycoengineered extracellular vesicle. For instance, the extracellular vesicles can be isolated as described in example 5, example 10 or example 11. In some embodiments, the glycoengineered extracellular vesicles are isolated by centrifugation or ultracentrifugation. Beads such as aldehyde / sulphate latex beads can be employed to capture extracellular vesicles.

[0242] In some embodiments, the step of isolating the glycoengineered extracellular vesicle of the method described herein comprises the steps of: a. growing the cell in a medium supporting its growth and the formation of glycoengineered extracellular vesicles; and e. recovering the glycoengineered extracellular vesicles from the medium; thereby isolating the extracellular vesicles.

[0243] In some cases, the step of isolating the glycoengineered extracellular vesicle of the method described herein further comprises a step of recovering the medium. Thus in some embodiments, the step of isolating the glycoengineered extracellular vesicle comprises the steps of (preferably in this order): a. growing the cell in a medium supporting its growth and the formation of glycoengineered extracellular vesicles; b. Recovering the medium; and e. recovering the glycoengineered extracellular vesicles from the medium; thereby isolating the glycoengineered extracellular vesicles.

[0244] The method may further comprise a step c. following step b., wherein step c. consists of removing cells, dead and / or alive, from the medium. Cells can be removed by methods known in the art, such as by filtering and / or centrifugating the medium comprising the cells. The methods may further comprise a step d. of removing cell debris from the medium, such as by centrifugation and / or filtration, or removal of proteins from the host cell.

[0245] Any of the methods described herein may further comprise a final step f. of washing the recovered extracellular vesicles, for example in a saline solution such as PBS.

[0246] Glycoengineered extracellular vesicles

[0247] The extracellular vesicles obtained by the present methods, i.e. the glycoengineered extracellular vesicles obtained by the present methods, will harbour characteristics which distinguish them from reference extracellular vesicles, in that at least the glycosylation of such extracellular vesicles will be different from the glycosylation of reference extracellular vesicles.

[0248] Thus, in an aspect, the present disclosure is directed to a glycoengineered extracellular vesicle obtainable by any method described herein.

[0249] In some embodiments, the glycoengineered extracellular vesicle and the cell from which said extracellular vesicle is obtained have a different glycosylation pattern. In some embodiments, the glycoengineered extracellular vesicle harbours a modified glycosylation selected from: a. N-glycosylation, such as modified high-mannose N-glycans, modified hybrid N- glycans and / or modified complex N-glycans; b. O-linked glycosylation, such as modified O-GalNAc, modified O-GIcNAc, modified O-Man, modified O-Gal, modified O-Fuc, and / or modified O-GIc; c. glycolipids such as glycosphingolipids; d. proteoglycans and / or glycosaminoglycans; and e. sialylation and fucosylation; and combinations thereof.

[0250] How to determine the glycosylation pattern of the glycoengineered extracellular vesicles has been described herein, in particular in the section “Assays and markers for determining glycosylation profile”.

[0251] Modified properties

[0252] The methods described herein allow production of isolated extracellular vesicles, i.e., isolated glycoengineered extracellular vesicles, which have a modified property of interest, in addition to a modified glycosylation pattern. The property of interest may be for example modified target specificity or tropism compared to a reference extracellular vesicle. The term “reference extracellular vesicle” refers to the extracellular vesicles obtained from a reference cell, which is identical to the cell from which the glycoengineered extracellular vesicles are obtained, but in which neither the expression of a nucleic acid implicated in glycosylation, as described herein above, nor the activity of a product encoded by said nucleic acid, have been modified.

[0253] Without being bound by theory, the structural characteristics of the glycoengineered extracellular vesicles are expected to be identical, or at least similar, to reference extracellular vesicles, while physicochemical properties may be altered. Thus, in some embodiments, the glycoengineered extracellular vesicle has a diameter of less than 300 nm, such as less than 290 nm, such as less than 280 nm, such as less than 270 nm, such as less than 260 nm, such as less than 250 nm.

[0254] In some embodiments, the property of interest is: i. modified nucleic acid transfer, such as gene, RNA or DNA transfer; ii. modified expression of a transferred nucleic acid; iii. increased editing of a target nucleic acid such as a gene; iv. modified target specificity or tropism; v. total uptake of a molecule by the glycoengineered extracellular vesicle; and / or vi. increased delivery of a molecule to a target, and wherein the property of interest is modified compared to an extracellular vesicle obtained from a reference cell in which said at least one nucleic acid, said expression of said at least one nucleic acid sequence and / or said activity of the product encoded by said at least one nucleic acid sequence has not been modified.

[0255] The skilled person will understand that any combination of such properties might be displayed by said vesicle.

[0256] In some embodiments, the glycoengineered extracellular vesicle has a modified target specificity or tropism to a cell of interest or target cell compared to the target specific or tropism of a reference extracellular vesicle produced by a reference cell. In some embodiments, the target specific or tropism of the glycoengineered extracellular vesicle is modified by at least 1%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%. In some embodiments, the target specificity or the tropism to a target cell is at least 1% increased, such as at least 5% increased, such as at least 10% increased, or is at least 1% increased, such as at least 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased or more. In some embodiments, the target specificity or the tropism to a target cell is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, or is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, such as at least 20% decreased, such as at least 30% decreased, such as at least 40% decreased, such as at least 50% decreased, such as at least 75% decreased, such as at least 100% decreased, such as at least 150% decreased, such as at least 200% decreased or more. In some embodiments, the property of interest is the total uptake of a molecule of interest in a target cell compared to the total uptake of the molecule of interest by a reference extracellular vesicle. In some embodiments, the total uptake of the molecule of interest in the target cell compared to the total uptake of the molecule of interest by a reference extracellular vesicle is modified by at least 1%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%. In some embodiments, the total uptake of the molecule of interest in the target cell is at least 1% increased, such as at least 5% increased, such as at least 10% increased, or is at least 1% increased, such as at least 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased or more. In some embodiments, the total uptake of the molecule of interest in the target cell s at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, or is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, such as at least 20% decreased, such as at least 30% decreased, such as at least 40% decreased, such as at least 50% decreased, such as at least 75% decreased, such as at least 100% decreased, such as at least 150% decreased, such as at least 200% decreased or more.

[0257] A modified total uptake of a molecule of interest may be absolute, i.e. refers to the total amount of the molecule of interest taken up, or it may be relative, i.e. the speed of uptake may be modified. In other words, in some embodiments the total amount of the molecule of interest which is taken up per second using a glycoengineered extracellular vesicle is modified compared to the total amount of the molecule of interest which is taken up per second using a reference extracellular vesicle. In some embodiments, the total uptake of the molecule of interest in the target cell per second compared to the total uptake of the molecule of interest by a reference extracellular vesicle per second is modified by at least 1%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%. In some embodiments, the total uptake of the molecule of interest in the target cell per second is at least 1% increased, such as at least 5% increased, such as at least 10% increased, or is at least 1% increased, such as at least 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased or more. In some embodiments, the total uptake of the molecule of interest in the target cell per second is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, or is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, such as at least 20% decreased, such as at least 30% decreased, such as at least 40% decreased, such as at least 50% decreased, such as at least 75% decreased, such as at least 100% decreased, such as at least 150% decreased, such as at least 200% decreased or more.

[0258] In some embodiments, the property of interest the delivery efficiency of a molecule of interest to a target cell. In some embodiments the delivery efficiency of the molecule of interest to the target cell is modified compared to the delivery efficiency of the molecule of interest to the target cell using a reference extracellular vesicle. In some embodiments, the delivery efficiency is modified by at least 1%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%. In some embodiments, the delivery efficiency is at least 1% increased, such as at least 5% increased, such as at least 10% increased, or is at least 1% increased, such as at least 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased or more. In some embodiments, the delivery efficiency is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, or is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, such as at least 20% decreased, such as at least 30% decreased, such as at least 40% decreased, such as at least 50% decreased, such as at least 75% decreased, such as at least 100% decreased, such as at least 150% decreased, such as at least 200% decreased or more. In some embodiments, the property of interest is expression of a molecule of interest in a target cell contacted with the glycoengineered extracellular vesicle compared to the expression of said molecule of interest in said target cell contacted with a reference extracellular vesicle produced by a reference cell. In some embodiments, the expression of the molecule of interest is modified by at least 1%, such as at least 5%, such as at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 75%, such as at least 100%, such as at least 150%, such as at least 200%. In some embodiments, the expression is at least 1% increased, such as at least 5% increased, such as at least 10% increased, or is at least 1% increased, such as at least 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased or more. In some embodiments, the expression is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, or is at least 1% decreased, such as at least 5% decreased, such as at least 10% decreased, such as at least 20% decreased, such as at least 30% decreased, such as at least 40% decreased, such as at least 50% decreased, such as at least 75% decreased, such as at least 100% decreased, such as at least 150% decreased, such as at least 200% decreased or more.

[0259] In some embodiments, the property of interest is increased expression of a molecule of interest in a target cell contacted with said glycoengineered extracellular vesicle compared to expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by a reference cell, and / or the glycoengineered extracellular vesicle leads to an increased expression of a molecule of interest in a target cell contacted with said extracellular vesicle compared to the expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by said reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified.

[0260] In some embodiments, the property of interest is increased expression of a molecule of interest in a target cell contacted with said glycoengineered extracellular vesicle compared to expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by a reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified.

[0261] In some embodiments, the glycoengineered extracellular vesicle leads to an increased expression of a molecule of interest in a target cell contacted with said glycoengineered extracellular vesicle compared to the expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by said reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified.

[0262] The molecule of interest, of which it is desirable to modify the uptake in a target cell, or of which it is desirable to modify the expression, may be for example a small molecule, such as an inhibitor, such as a toxin; a nucleic acid sequence, such as a RNA sequence, such as a DNA sequence; a polypeptide, such as an antigen; a lipid; a metabolite; or an imaging agent, such as a fluorescent dye.

[0263] The skilled person will understand that the present methods can be used to simultaneously modify more than one property of interest. For instance, it may be desirable to modify both expression of a molecule of interest and uptake of the same or of another molecule of interest into a target cell.

[0264] In some embodiments, the therapeutic molecule, or the active ingredient, is a large nucleic acid and / or a large polypeptide.

[0265] In some embodiments, said large nucleic acid is a nucleic acid, such as a DNA, for example a gene, or an RNA, having a size of at least 1500 bp, such as at least 2000 bp, such as at least 3000 bp, such as at least 4000 bp, such as at least 4700 bp. While the present methods can be used for delivery of nucleic acids of any size, they are particularly advantageous for delivery of large genes which cannot be packaged in adeno-associated viruses. In some embodiments, the large polypeptide is a polypeptide such as a protein having a size of at least 500 amino acids, such as at least 600, at least 800, at least 1000, at least 1200, at least 1500 or at least 1700 amino acids.

[0266] In some embodiments, the molecule or active ingredient is a DNA molecule, or an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule, a gRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide, such as a peptide or a protein; or a dye.

[0267] Compositions and kits

[0268] Also provided herein is a composition comprising an isolated glycoengineered extracellular vesicle obtained via the methods disclosed herein, or a composition comprising the isolated, glycoengineered extracellular vesicle disclosed herein. The composition may be a pharmaceutical composition. The extracellular vesicle may be loaded with a molecule as described herein elsewhere.

[0269] Thus, in an aspect, the present disclosure is directed to a composition comprising an isolated glycoengineered extracellular vesicles obtainable by any method described herein.

[0270] The composition may further comprise an acceptable carrier. In some embodiments, the composition may comprise a liposome, and optionally an acceptable carrier. In some embodiments, the composition may comprise a nanoparticle, and optionally an acceptable carrier. In some embodiments, the composition may comprise a liposome and a nanoparticle, and optionally an acceptable carrier. The composition may further comprise a stabilizer and / or an additive, such as an additive for improving shell-life and stability as known in the art.

[0271] Also provided herein is a kit comprising an isolated, glycoengineered extracellular vesicle as described herein, preferably wherein said extracellular vesicle is obtainable via any of the methods described herein, and optionally further comprises instructions for use. Identification of glycoengineered extracellular vesicles

[0272] The glycoengineered extracellular vesicles obtained by the present methods have a modified property of interest compared to reference extracellular vesicles. However, it can be difficult to predict which effect the modification of the expression of at least one nucleic acid in the cell or the modification of the activity of the product encoded by said nucleic acid will actually have.

[0273] Herein is thus also provided a method to identify a glycoengineered extracellular vesicle with a glycosylation pattern of interest comprising the steps of: a. providing a glycoengineered extracellular vesicle, wherein the glycoengineered extracellular vesicle has been obtained by a cell, wherein expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as described herein; b. optionally, determining whether the glycoengineered extracellular vesicle has the glycosylation pattern of interest, such as by contacting the glycoengineered extracellular vesicle with a polypeptide, such as a lectin, such as an antibody, specific for the glycosylation pattern of interest, and / or such as by performing glycoanalytics of the extracellular vesicle, and / or by testing the extracellular vesicle in an assay.

[0274] The glycoengineered extracellular vesicle provided in step a. is preferably obtained by the methods described herein, i.e. by a. providing a cell wherein at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or the activity of a product encoded by said at least one nucleic acid has been modified, resulting in a modified glycosylation of extracellular vesicles produced by the cell; and isolating the glycoengineered extracellular vesicle from said cell.

[0275] Thus, if a specific property of the glycoengineered extracellular vesicle is sought, the cell producing the extracellular vesicles can be genetically modified, following which an appropriate assay is carried out to determine whether the extracellular vesicles have been glycoengineered in a way that confers that specific property. For instance, if extracellular vesicles are sought, which can load an increased amount of a molecule of interest, the cells can be genetically modified as described herein and the amount of a molecule of interest which can be loaded in the extracellular vesicle can be determined for each genetic modification, thus enabling to determine which genetic modification confers increased loading of the molecule of interest. While it may be of interest to determine the glycosylation pattern of the glycoengineered extracellular vesicles, this is not required to identify useful glycoengineered extracellular vesicles.

[0276] In an aspect, the present disclosure is directed to a method to identify an isolated glycoengineered extracellular vesicle, as defined herein, with a glycosylation pattern of interest comprising the steps of: a. providing an isolated glycoengineered extracellular vesicle, wherein the extracellular vesicle has been obtained from a cell, wherein the expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as defined herein; b. optionally, determining the extracellular surface glycosylation pattern, c. loading the isolated extracellular vesicle with a molecule of interest d. providing a target cell e. contacting said isolated extracellular vesicles with said target cell f. analyzing target cellular properties of interest, such as target specificity or tropism, total uptake of a molecule, uptake of a molecule per second, delivery efficiency or expression of a nucleic acid or polypeptide

[0277] Uses of glycoengineered extracellular vesicles

[0278] The present extracellular vesicles, i.e. the present glycoengineered extracellular vesicles, have a wide range of applications.

[0279] Herein is provided an isolated extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle, wherein the isolated glycoengineered extracellular vesicle has been obtained by the methods described herein, for use in medicine.

[0280] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in medicine, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified glycosylation; and contacting said glycoengineered extracellular vesicle with a therapeutic molecule or active ingredient, thereby obtaining an isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0281] Also provided herein is an isolated extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle, wherein the isolated glycoengineered extracellular vesicle has been obtained by the methods described herein, for use in a method of treating and / or preventing a disease and / or a medical condition in a subject in need thereof.

[0282] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in the treatment of a disease and / or a medical condition, in wound healing, in regenerative medicine or in gene therapy in a subject in need thereof, comprising the steps of: providing an isolated glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a therapeutic molecule or active ingredient,

[0283] -optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; and administering said isolated glycoengineered extracellular vesicle or composition to a subject in need thereof; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0284] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in medicine, the use comprising the steps of:

[0285] - providing a glycoengineered extracellular vesicle having a modified glycosylation and comprising a therapeutic molecule or active ingredient;

[0286] - optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0287] Also provided herein is an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in the treatment of a disease and / or a medical condition, in wound healing, in regenerative medicine or in gene therapy in a subject in need thereof, comprising the steps of: providing an isolated glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a therapeutic molecule or active ingredient,

[0288] - optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; and preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0289] Also provided herein is the use of an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for the delivery of a molecule to a cell, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a molecule, such as a therapeutic molecule, or active ingredient

[0290] - optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0291] The disease may be selected from the group consisting of: cancer; neurodegenerative diseases; cardiovascular disease; autoimmune diseases; metabolic disorders; rare genetic disorders; and skin diseases.

[0292] The isolated glycoengineered extracellular vesicle or composition comprising such can also be used for wound healing, regenerative medicine or gene therapy.

[0293] In some embodiments, the use as described herein comprises the steps of: delivering a molecule of interest as described herein to a target cell; and delivering said target cell to a subject in need thereof. In some embodiments, the disease is a monogenic disease.

[0294] In some embodiments, the disease is a pulmonary disease, such as cystic fibrosis.

[0295] In some embodiments, the therapeutic molecule is a nucleic acid comprising or consisting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, such as the genomic DNA, cDNA, RNA, or fragments thereof; or a polypeptide comprising or consisting of the CFTR protein, or fragments thereof.

[0296] In some embodiments, the therapeutic molecule is a nucleic acid comprising or consisting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, or fragments thereof.

[0297] In some embodiments, the therapeutic molecule is a polypeptide comprising or consisting of the CFTR protein, or fragments thereof.

[0298] The glycoengineered extracellular vesicles may in such embodiments be loaded with a molecule of interest, for instance a therapeutic molecule or active ingredient, which may be selected from the group consisting of: a nucleic acid sequence, such as a DNA molecule, such as an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule, a gRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide; and a dye.

[0299] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to deliver a molecule of interest to a target cell, wherein the molecule of interest is preferably selected from the group consisting of: a nucleic acid sequence, such as a DNA molecule, such as an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule, a gRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide; and a dye. Also provided herein is the use of an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for the delivery of a molecule to a cell, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified extracellular glycosylation; and contacting said glycoengineered extracellular vesicle with a molecule or active ingredient, thereby obtaining an isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

[0300] In some embodiments, providing the isolated glycoengineered extracellular vesicle comprises the method of obtaining a isolated glycoengineered extracellular vesicle described herein.

[0301] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to modulate the expression of a target nucleic acid, such as to increase the expression of a target nucleic acid, such as to decrease the expression of a target nucleic acid, and / or to modulate the activity of a product encoded by a target nucleic acid, such as to increase the activity of a polypeptide encoded by a target nucleic acid, or such as to decrease the activity of a product encoded by a target nucleic acid.

[0302] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to modulate the expression of a target nucleic acid, such as to increase the expression of a target nucleic acid, or such as to decrease the expression of a target nucleic acid.

[0303] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to modulate the activity of a product encoded by a target nucleic acid, such as to increase the activity of a product encoded by a target nucleic acid, such as to decrease the activity of a product encoded by a target nucleic acid.

[0304] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to edit a target nucleic acid in a target cell.

[0305] Also provided herein is the use of an isolated glycoengineered extracellular vesicle as described herein, use of a composition as described herein, or use of a kit as described herein, to deliver a compound, to improve transfer of a nucleic acid of interest to a target cell compared to the transfer of said nucleic acid of interest to said target cell by another extracellular vesicle, wherein the other extracellular vesicle is an extracellular vesicle from a reference cell in which expression of the at least one nucleic acid and / or the activity of the product encoded by the at least one nucleic acid has not been modified, preferably wherein the improved transfer leads to increased expression of the nucleic acid of interest compared to the expression obtained when using the other extracellular vesicle.

[0306] Also provided herein is a method of modulating the tropism and / or target specificity of a glycoengineered extracellular vesicle, wherein the method comprises or consists of the method of obtaining an isolated extracellular vesicle as described herein, wherein said extracellular vesicle is glycoengineered and has a modified glycosylation on its surface.

[0307] Also provided herein is a method of modifying, such as increasing or decreasing, the total uptake of a target molecule in a target cell, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as described herein.

[0308] Also provided herein is a method of modifying, such as increasing or decreasing, the delivery efficiency of a target molecule to a target cell, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as described herein, and wherein the method further comprises loading said target molecule in the glycoengineered extracellular vesicle and contacting said extracellular vesicle with the target cell. Also provided herein is a method of modifying, such as increasing or decreasing, the loading of a target molecule to a glycoengineered extracellular vesicle, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as defined herein, and further comprises loading said target molecule in the glycoengineered extracellular vesicle.

[0309] Also provided herein is a method of increasing the transfer of a target nucleic acid to a target cell or a method of increasing the expression of a product encoded by a target nucleic acid in a target cell, wherein the method comprises the method of obtaining a glycoengineered extracellular vesicle as defined herein, and further comprises: a. loading said target nucleic acid in the glycoengineered extracellular vesicle; and b. contacting the extracellular vesicle with the target cell, whereby the target nucleic acid is transferred to the target cell and / or whereby the product encoded by the target nucleic acid is expressed.

[0310] Glycoengineered extracellular vesicles which are particularly well suited for this preferably display modified glycosaminoglycans and / or modified sialylation, preferably of modified glycosaminoglycans. For instance, such glycoengineered extracellular vesicles have been obtained from a cell in which the expression of GNE and / or the activity of the product encoded by GNE have been modified. Another example is a glycoengineered vesicle obtained from a cell in which the expression of B4GALT7 and / pr the activity of the product encoded by B4GALT7 have been modified.

[0311] Also disclosed herein is a method to identify a nucleic acid of interest, which, when modified in a cell, or when the activity of the product it encodes is modified in a cell, results in modified glycosylation of the glycoengineered extracellular vesicles produced by the cell.

[0312] Also provided is a method to identify a nucleic acid of interest, wherein modification of the expression of the nucleic acid of interest in a cell and / or modification of the activity of a product encoded by the nucleic acid of interest in said cell results in a modified glycosylation on the surface of an extracellular vesicle produced by the cell, said method comprising the steps of: a. obtaining a glycoengineered extracellular vesicle from a cell, wherein expression of a nucleic acid sequence in the cell and / or the activity of a product encoded by the nucleic acid has been modified as described herein; b. optionally, determining whether the glycoengineered extracellular vesicle has the glycosylation pattern of interest, such as by contacting the glycoengineered extracellular vesicle with a polypeptide, such as a lectin, such as an antibody, specific for the glycosylation pattern of interest; and / or such as by performing glycoanalytics of the glycoengineered extracellular vesicle; and / or by testing the extracellular vesicle in an assay; and c. concluding that the nucleic acid sequence is a nucleic acid sequence of interest.

[0313] The glycoengineered extracellular vesicles can thus be used to test whether a specific nucleic acid can, when modified, or when the activity of the product it encodes is modified, result in glycoengineered extracellular vesicles having not only modified glycosylation, but also potentially different properties of interest than reference extracellular vesicles. The actual step of determining the glycosylation pattern of the glycoengineered extracellular vesicles may be redundant. In some embodiments, it may be sufficient to test in a suitable assay whether the obtained extracellular vesicles have the modified property of interest.

[0314] If it is desirable to get insight into the modified glycosylation as such, any of the markers or methods described herein, e.g. in “Assays and markers for determining glycosylation profiles”. For instance, the extracellular vesicle may be contacted with a polypeptide, such as described herein earlier, which is specific for a glycosylation feature of interest. In particular, the polypeptide may be a carbohydrate-binding polypeptide or a polypeptide comprising a carbohydrate-binding domain. Such polypeptide may be a lectin, a galectin, a siglec or an antibody.

[0315] In some embodiments, the polypeptide is a lectin, preferably selected from the group consisting of: an antibody, a Sambucus Nigra Lectin (Lectin SNA), lectin VVA and a Cholera Toxin Subunit B. In some embodiments, the antibody is selected from the group consisting of: an anti-syntenin-1 antibody, and an anti-chondroitin sulfate antibody. It will be evident that in order to properly compare the features of the glycoengineered extracellular vesicles to reference extracellular vesicles, the cells producing the glycoengineered extracellular vesicles and the reference cells producing the reference extracellular vesicles should be grown under similar or identical conditions.

[0316] Examples

[0317] Example 1

[0318] Parental cells were glycoengineered through cell-based glycoengineering through CRISPR / Cas9. Major glycosylation pathways were modified by knocking-out essential genes for N-glycosylation (MGAT1), O-GalNAc glycosylation (C1GALT1C1), glycosphingolipids (B4GALT5 / 6), glycosaminoglycans (B4GALT7) and sialylation (GNE) involved in the elongation or biosynthesis of the according glycans in HEK293F cells. The modified extracellular vesicles were isolated from these cells, purified and analysed as described in examples 2-16. The various knock-outs represents the knockout of various types of enzymes in the glycosylation machinery including glycosyltransferases (MGAT1 , B4GALT5, B4GALT6, B4GALT7), enzymes of sugar precursor pathways (GNE), and chaperones (C1GALT1C1), and double knock-out (B4GALT5 / 6).

[0319] Example 2 - Cell cultures

[0320] HEK293F cells (Freestyle™ 293-F Cells, Thermo Fisher Scientific) were cultured according to the manufacturer's instructions. The cells were propagated using Freestyle™ 293 Expression Medium (Thermo Fisher Scientific), supplemented with 100 U / rnL penicillin / streptomycin (Thermo Fisher Scientific), and maintained at 37°C and 5% CO2 in either static culture in flasks or shake culture in 50 mL TPP TubeSpin® Bioreactors (TPP Techno) with constant agitation (150 rpm). Passages were performed at a density of 0.25x106 cells / ml 2-3 times per week for cell line maintenance.

[0321] Example 3 - CRISPR / Cas9 targeted knock-out in HEK293F cells

[0322] The gRNA targets in table 1 used were sourced either from the previously published and validated GlycoCRISPR gRNA library (Narimatsu et al., 2018), or were designed through CHOPCHOP (https: / / chopchop.cbu.uib.no / ). HEK293F cells were transfected using Lipofectamine 3000 reagents (Thermo Fisher Scientific), similar as described in Narimatsu et al., 2019. Specifically, one million HEK293F cells were seeded in 6-well cell culture plate and co-transfected with 1 pg of gRNA plasmid (backbone vector is Addgene#68370) and 1 pg of Cas9PBKS plasmid (Addgene# #68371). Following day, transfection efficiency was examined using the EVOS FL Auto 2 fluorescent microscope (Invitrogen). Two days after transfection, cells positive for GFP fluorescence were enriched by fluorescence-activated cell sorting (FACS) using MA900 Multi-Application Cell Sorter (Sony Biotechnology). After 7-10 days of culture, the enriched cell pool was further single-sorted into 384-well plates containing 50ul of culture medium supplemented with 5% Solentim InstiGRO™ HEK (Advanced Instruments). The successful knock-outs were validated using lectin and antibody staining. Selected clones were further verified through Sanger sequencing and Synthego ICE analysis.

[0323] Table 1. CRISPR gRNA sequence and corresponding PCR primers for target site amplification

[0324] Gene gRNA Forward primer Reverse primer

[0325] TTCGGAGTGCTTATGCC TGC H I L I GCAGTGAACA ACCTCTCTTGGGAGGAGA

[0326] B4GALT5 AAG (SEQ ID NO: 335) CC (SEQ ID NO: 336) CA (SEQ ID NO: 337)

[0327] CTCTTTATGGTACAAGCT CGTTTGGACGTGCTGATT GGACGCTTAGGGGAAGA

[0328] B4GALT6 CG (SEQ ID NO: 338) GAG (SEQ ID NO: 339) TACC (SEQ ID NO: 340)

[0329] TGACCTGCTCCCTCTCAA GGCTGAGTGAAGTCAGTG CCCAAGTTCTCCTGCTAGG

[0330] B4GALT7 CG (SEQ ID NO: 341) CT (SEQ ID NO: 342) C (SEQ ID NO: 343)

[0331] GTAGGTGATGATGCTCA TGAAGGGTGTGATGCTTG ACTGCAGCCCAAAGACTC

[0332] C1GALT1 d TGG (SEQ ID NO: 344) GAA (SEQ ID NO: 345) AC (SEQ ID NO: 346)

[0333] GCGATCATGGTCCTCAC AAGCTGCCAGATGTCCTT CA I I I I L I GACAAAAACA

[0334] GNEACA (SEQ ID NO: 347) AATC (SEQ ID NO: 348) GCCA (SEQ ID NO: 349)

[0335] CCCTCAGTCAGCGCTCT GGGCGCTATCCTCTTTGT TGGCTAACGATGATGGGG

[0336] MGAT1 CGA (SEQ ID NO: 350) GG (SEQ ID NO: 351) AAG (SEQ ID NO: 352) Example 4 - Analysis of cell surface glycosylation features using Flow cytometry-based cell binding assays

[0337] Cell binding assays were conducted using lectins or antibody molecules that were specific to certain glycan features (table 2).

[0338]

[0339] To perform the assays, biotinylated lectins (Vector Laboratories), biotinylated Cholera Toxin Subunit B (Thermo Fisher Scientific), and anti-CS antibody (Abeam) were incubated at two different concentrations (0.3ug / ml or 1.0 ug / ml) for 30 minutes. Following incubation, the cells were washed and incubated with Streptavidin-Alexa Fluor488 (obtained from Invitrogen) or Rabbit Anti-Mouse Immunoglobulins / FITC (Agilent Technologies) for an additional 30 minutes. Washing was carried out using PBA buffer (0.1% BSA in DPBS buffer), and the cells were subsequently resuspended in 100ul of IC Fixation buffer (Thermo Fisher Scientific) until flow cytometry analysis using a MACSQuant® Analyzer 16 (Miltenyi Biotec). All steps were conducted on ice to ensure the stability of the binding interactions. The LIVE / DEAD™ Fixable Yellow Dead Cell Stain Kit was used to preclude the dead cells.

[0340] Example 5 - Extracellular vesicle production and isolation

[0341] In order to collect conditioned medium from conventional cultures, HEK293F cells were seeded at a density of 0.25 million cells / mL in Corning® Erlenmeyer Shake Flasks containing Freestyle™ 293 Expression Medium supplemented with 100 U / rnL penicillin / streptomycin and 0.2% Anti-Clumping Agent (Thermo Fisher Scientific). The cultures were incubated at 37°C and 5% CO2 with constant agitation (150 rpm) for 6 days. After incubation, the cells underwent two rounds of centrifugation - first at 300 x g for 10 minutes and then at 2,000 x g for 10 minutes - to eliminate any living or dead cells. The resulting supernatants were moved to Labcon 50 mL SuperClear Centrifuge Tubes and frozen at -80°C until further processing. To begin the extracellular vesicle isolation, the frozen supernatants were transferred to 4°C the day before and left to thaw overnight. Next, the samples were spun for 30 minutes at 10,000 x g (using Sorvall LYNX 4000 Superspeed Centrifuge, Thermo Fisher Scientific) to remove cell debris. The extracellular vesicles were pelleted for 90 minutes at 100,000 x g with a Optima XE-90 Ultracentrifuge (Beckman Coulter). To eliminate any potentially contaminating soluble proteins, the resulting pellets were washed with 70ml PBS and pelleted again for 90 minutes at 100,000 x g. The extracellular vesicle pellets were resuspended in 1-2 mL of PBS, aliquoted, and stored at -80°C for downstream applications. All purification steps were conducted at 4°C or on ice.

[0342] Example 6 - Extracellular vesicle quantification through total protein determination A small aliquot of 2 pL of resuspended extracellular vesicles was combined with 8 pL of RIPA lysis buffer containing complete™ Protease Inhibitor Cocktail. The extracellular vesicles were then subjected to lysis on ice for a duration of 30 minutes to release their protein content. The quantity of extracellular vesicle proteins released was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) following manufacture’s instruction and using BSA proteins serving as the standard. Example 7 - Extracellular vesicle characterization using the Particle Metrix Zeta View® The optimal conditions for measuring extracellular vesicles were tested beforehand by diluting samples to different concentrations and determining the appropriate number of particles per frame (50-150). To prepare the extracellular vesicle samples for analysis, they were finally diluted to 0.02ug / ml based on extracellular vesicle protein content in a final volume of 1 ml using DPBS. The default software settings for analyses of liposomes were used for extracellular vesicles. Each measurement consisted of three cycles, each cycle scanned 11 positions with each position 30 frames captured using the following parameters: autofocus mode, 100 for shutter and 70 for camera sensitivity. The captured videos were analyzed using ZetaView Software 8.05.11 with parameters: Maximum area of 100, minimum area of 5, and minimum brightness of 30. Data in fig 3A show that genetically modifying the parental cell does not affect the size of isolated extracellular vesicles, the size is comparable to those from the WT parental cell. Similarly fig 3B does not show a significant difference in the number of isolated extracellular vesicles decrease except for ASA (GNE knock-out) where a slight reduction can be observed.

[0343] Example 8 - Extracellular vesicle characterization using Western blot analysis Extracellular vesicle samples and cells were lysed using RIPA lysis buffer containing complete™ Protease Inhibitor Cocktail. Equal amounts of protein (based on BCA assay) were loaded followed by electrophoresis. After SDS-gel separation, proteins were transferred from the gel to PVDF membranes according to Tian et al., 2019. The following primary antibodies were used: p-Actin (13E5) Rabbit mAb (Cell Signaling Technology #4970, 1 :1000), Anti-human syntenin (EPR8102) (Abeam# ab133267, 1 :1 ,000) were diluted in 5% skim milk in TBST and coated overnight at 4C. Goat AntiRabbit IgG H&L HRP-conjugated Abeam #ab 205718, 1:4,000) secondary antibodies were incubated for 1 h at room temperature in 5% skim milk in TBST. Visualization of immunolabels was performed using Clarity Max Western ECL Substrate (Bio-Rad) according to the manufacturer’s instructions. Chemiluminescent signals were captured using ChemiDoc Imaging System membrane reader (Bio-Rad).

[0344] To evaluate the expression of inclusion and exclusion markers in the glycoengineered extracellular vesicles, Western blot analysis was conducted using specific antibodies, fig 3C. Anti- -Actin was included to ensure equal protein loading across the samples among cell pellets and extracellular vesicles. The exosome biomarker, Syntenin-1 (Kugeratski et al., 2021), showed compared intense bands for all extracellular vesicles from WT and all knock-outs and is strongly increased in the extracellular vesicles compared to the parental cell lysates. This finding confirms the purity of the isolated extracellular vesicles and supports their resemblance to exosomes. These results also confirm that glycoengineering did not significantly alter the characteristics of extracellular vesicles in respect to the biomarker Syntenin-1.

[0345] Example 9 - Analysis of cell and extracellular vesicle surface glycosylation features using latex beads and flow cytometry-based binding assays

[0346] The protocol for latex beads based lectin staining of extracellular vesicle surface glycans was modified from previous publication (Kugeratski et al., 2021). Briefly, 2.5 pg of extracellular vesicles were resuspended in 20 pl of phosphate-buffered saline (PBS) and mixed with 0.125 pl of aldehyde / sulfate beads (Invitrogen, A37304) followed by a 15-min incubation at room temperature. Subsequently, 40 pl of PBS was added to each staining, and the extracellular vesicles were allowed to bind to the beads by overnight rotation at 4°C. The following day, 30 pl of 1 M glycine (dissolved in PBS) was added to each tube and incubated at room temperature for 1 h with rotation. After incubation, the bead-bound extracellular vesicles were pelleted at 4816g for 10 min, and the supernatant was discarded. The precipitate was then resuspended in 16 pl of 10% bovine serum albumin (BSA, Sigma) for blocking, followed by another 1h incubation at room temperature with rotation. Finally, the samples were centrifuged again, and the bead-bound-exosome precipitate was resuspended in biotinylated lectins (Vector Laboratories), biotinylated Cholera Toxin Subunit B (Thermo Fisher Scientific), or anti- CS antibody (obtained from Abeam) and incubated at two different concentrations (0.3ug / ml or 1ug / ml) for 30 minutes. Following incubation, the extracellular vesicle captured beads were washed and incubated with Streptavidin-Alexa Fluor488 (Thermo Fisher Scientific) or Rabbit Anti-Mouse Immunoglobulins / FITC (Agilent Technologies) for an additional 30 minutes. Extracellular vesicles captured beads were subsequently resuspended in 100ul of PBA buffer for flow cytometry analysis using a MACS Quant 16 Analyzer.

[0347] Example 10 - Glycoengineering does not alter the characteristics of the extracellular vesicles.

[0348] An advantage of using HEK293F cells is that they can be cultured in animal origin-free and protein-free medium (Freestyle™ 293 Expression Medium, Thermo), which eliminates the risk of extracellular vesicles contamination from the culture environment. Ultracentrifugation was used for isolating extracellular vesicles because it is most commonly used and currently considered the gold standard for obtaining extracellular vesicles with relatively homogenous size (Lobb et al., 2015). To avoid any disruption from potential unforeseen alteration of extracellular vesicles caused by cell-based glycoengineering, the isolated extracellular vesicles were first normalized based on total protein content measured by BCA assay before further analysis. The size distribution and particle numbers of extracellular vesicles were measured using Nanoparticle Tracking Analysis (NTA). As seen in figure 3A, the majority of extracellular vesicles are smaller than 250 nm and the size is similar for WT extracellular vesicles and all different glycoengineered extracellular vesicles. The number of extracellular vesicle particles is also comparable, with slightly fewer present in A SA EV (figure 3B). To evaluate the expression of inclusion and exclusion markers in the glycoengineered extracellular vesicles, Western blot analysis was conducted using specific antibodies. Anti- -Actin was included to ensure equal protein loading across the samples among cell pellets and extracellular vesicle (figure 3C). The newly identified biomarker of exosomes, Syntenin-1 (Kugeratski et al., 2021), showed compared intense bands for all extracellular vesicles from WT and all knock-outs and is strongly increased in the extracellular vesicles compared to the parental cell lysates. This finding confirms the purity of the isolated extracellular vesicles and supports their resemblance to exosomes. These results also confirm that glycoengineering did not significantly alter the characteristics of extracellular vesicles in respect to size distribution, vesicle numbers and the biomarker Syntenin-1. Although there are clonal variations in the yield of extracellular vesicles among different clones including clones from the same knockout, no direct impact was noticed due to specific glycoengineering (figure 6A, 6B).

[0349] Three clones from each gene modification were chosen to address potential clonal variation. PHA-L staining, which binds to pi,6-GlcNAc branched N-glycans, showed no binding in the MGAT1 knock-out clones, while GNE knock-out clones which is devoid of sialylation had higher binding (fig. 2A). Lectin VVA which binds to terminal GalNAc structures increased significantly in both COSMC and GNE knock-out clones, while on the other hand, a decrease in VVA binding was observed in MGAT1 knock-out clones (fig. 2B). PNA specifically binds to Gaipi-3 GalNAc residues (core-1 structure of O- glycans; Thomsen-Friedenreich TF antigen) found in O-GalNAc glycans, and the binding is abolished upon sialylation (Merant et al., 2005). The binding of PNA significantly increased in GNE knock-outs clones while a decrease was observed in COSMC and MGAT1 KO clones (fig. 2C). GM1, a sialic acid-containing glycosphingolipid and known receptor for the B subunit of cholera toxin (CTB), was used to indicate the status of cell surface glycosphingolipids. A clean removal of binding in B4GALT5 / 6 knock-out clones and a significant decrease in GNE knock-out clones were observed, confirming that sialic acid is required for CTB binding (Kuziemko et al., 1996) (fig. 2D). Removal of GAGs in B4GALT7 knock-out clones was indicated by staining with an antibody that specifically target Chondroitin sulfate, while other knock-outs showed no consistent change among clones in binding (fig. 2E). Lectin SNA, which specifically bind a2-6 sialylated LacNAc or LacdiNAc structures, was used to stain all glycoengineered cells and a decrease in binding was observed in cells with GNE knock-out and MGAT1 knock-out (fig. 2F). These results confirmed the targeted modification and display of “simple cell” glycans on HEK293F cells through cell-based glycoengineering, and no significant impact on cell growth was evidenced due to gene modification.

[0350] Example 11 - Glycan characterisation of glycoengineered extracellular vesicles To characterize the glycan modifications on isolated extracellular vesicles, the FACS- based analysis was performed with glycan binding molecules in order to facilitate quantification. Since extracellular vesicles cannot be directly measured using conventional flow cytometers due to the detection limit of forward scattered light (FSC), Aldehyde / Sulphate Latex beads were employed to capture extracellular vesicles and then stained them with binding molecules (Kugeratski et al., 2021). Similar to parental cells, PHA-L staining had no binding with ANG EV and higher binding with ASA EV (fig. 4A). VVA staining of extracellular vesicles increased considerably in AOG EV (fig.4B), but surprisingly no increase in ASA EV and no decrease in ANG EV which is not consistent with the cell staining (fig.3B). PNA binding decreased significantly in AOG EV but increased significantly in ASA EV (fig. 4C). The binding of B subunit of cholera toxin was low in all extracellular vesicles, but a complete removal of binding was observed with AGSL EV and a significant decrease of binding with ASA EV (fig. 4D). The anti-chondroitin sulphate binding had higher binding on extracellular vesicles compared to parental cells, suggesting enrichment of GAGs on extracellular vesicles, while B4GALT7 knock-out completely removed the binding (fig. 4E). The SNA staining is surprising and puzzling, a slightly increased binding in ASA / AOG / AGAG EV was observed (fig 4F). Overall, the results indicate that the investigated four main types of glycoconjugates as well as sialylation are present on extracellular vesicles and can be specifically manipulated through cell-based glycoengineering.

[0351] Example 12 - Modulation and improvement of cellular uptake of exosomes through cellular glycoengineering.

[0352] Extracellular vesicles were labelled with a fluorescence dye using the standard technology for exosome uptake and targeting assays. The labelled glycoengineered extracellular vesicles were tested for various time intervals for the uptake into target cells. The data show that glycoengineering can modulate and improve the up-take of the exosomes substantially. The data also points to a kinetic effect showing that the speed of uptake can be improved and modulated as well as the total effect of uptake. The data clearly show that the knockout of especially sialylation (GNE = ASA) and glycosaminoglycans (B4GALT7 = AGAG), but also to lesser extend O-GalNAc glycosylation (C1GALT1C1 = AOG) can largely improve the targeting and uptake of exosomes (fig. 7A, 7B).

[0353] Example 13 - The speed of uptake can be improved and modulated showing also a kinetic effect.

[0354] During an initial five-hour period, the labelled A SA extracellular vesicles and AOG extracellular vesicles displayed a faster uptake by the recipient cells compared to other extracellular vesicles, as evident from the presence of positively labelled cells or higher MFI at various time points. The labelled WT extracellular vesicles exhibited minimal presence of positively labelled cells during this period.

[0355] However, as time progressed, their uptake increased and eventually reached a level comparable to that of AOG extracellular vesicles and AGSL extracellular vesicles, though still lower than ASA extracellular vesicles. The labelled AGAG extracellular vesicles showed moderate improvement in being taken up by the recipient cells in the first five-hour period, however, both the percentage of positively labelled cells and MFI reach the highest level among all genetically modified extracellular vesicles at the twenty-four hour time point. The MFI and percentage of positive cells are shown in (fig. 8A, 8B). Uptake studies using not only isogenic target cells but also different target cells indicate that the effect is also applicable for targeting other cell types as shown in fig. 8C, 8D, 8E and 8F.

[0356] Example 14 - Improved DNA oligonucleotides-based genetic transfer

[0357] DNA oligonucleotides conjugated with FAM (fluorescence label) was loaded onto various genetically modified extracellular vesicles via standard exosome genetic loading technologies (Exo-Fect™, SBI). Loaded genetically modified extracellular vesicles were incubated with target cells for a 16-hour incubation period and the FAM signal in the target cells was determined through FACS fluorescence intensity analysis. The FAM positive cells were gated by comparing the FAM signal with negative control. The data showed that glycoengineering could substantially change the ability of extracellular vesicles to deliver DNA oligonucleotides, similar with the results using exosome labelling dye. The modulated delivery ability through glycoengineering not only show large improvement (AGAG EV 58,5% and ASA EV 29,5%) but also decrease for certain design (ANG EV 3,88%) compared to WT control (WT EV 14,3%). The data indicate that genetically modified extracellular vesicles from knock-outs of glycosaminoglycans (B4GALT7 = AGAG) are most potent in increasing DNA oligonucleotide transfer, followed by knock-out of especially sialylation (GNE = ASA). Knock-out of complex- N-linked glycans (MGAT1) significantly decreased the percentage of FAM positive cells. The flow cytometry dot-plots data as shown in fig. 9.

[0358] Example 15 - Improved plasmid DNA-based genetic transfer and improved expression of DNA-encoded proteins in target cells.

[0359] A DNA plasmid encoding green fluorescence protein (GFP) was loaded onto various genetically modified extracellular vesicles using same loading technologies (Exo- Fect™, SBI). Loaded genetically modified extracellular vesicles were incubated with target cells and the expression of GFP in each cell through FACS fluorescence intensity analysis was determined after 24 hours. The data show that glycoengineering can substantially improve gene delivery to target cells via extracellular vesicles. The assay not only measures the transfer of the gene but also the expression of the gene product through transcription and translation processes, mRNA expression and subsequent protein expression, which is measured through the fluorescence of the encoded GFP protein when successfully expressed. The amount of fluorescence is a measure for the amount of genes transferred, transcribed into mRNA, and translated into protein via exosome targeting and delivery. The improvement of using genetically modified extracellular vesicles not only results in much larger number of transformed cells but also to much higher expression of the protein in transfected cells including cells which have a very high expression not achievable with non genetically modified extracellular vesicles. The data indicate that genetically modified extracellular vesicles from knock-outs of glycosaminoglycans (B4GALT7 = AGAG 21 ,2%) are most potent in increasing gene transfer, followed by knockout of especially sialylation (GNE = ASA 13,1%), and knock-out of glycosphingolipids (B4GALT5 / 6 = AGSL 7,34%) compared to WT control (WT EV 3,41%). The percentage of GFP positive cells achieved through different genetically modified extracellular vesicles are shown in figure 10A. The fold change of GFP positive cells normalized to WT EV are shown in figure 10B.

[0360] Example 16 - Improved RNA-based genetic transfer.

[0361] The data in figure 11 indicate that genetically modified extracellular vesicles have a substantially improved ability for delivery of RNA; such as mRNA, siRNA and RNA based therapeutics, to target cells via extracellular vesicles.

[0362] Using Texas Red-labelled siRNA shows that delivery of the fluorescently labelled siRNA can be increased using AGAG extracellular vesicles and ASA extracellular vesicles whereby AGAG extracellular vesicles show the highest effect. Therefore it has to be expected that delivery of various types of RNA molecules can be improved by glycoengineering of extracellular vesicles and can improve the effects mediated via those RNA molecules such as inhibition of certain gene expression (e.g. siRNA), expression of certain RNA coded genes (e.g. mRNA) and that exosome-based delivery of RNA based therapeutics can also be improved. The fold change of Texas Red positive cells normalized to WT EV are shown in figure 11.

[0363] Example 17

[0364] Evaluation of the Insertions and Deletions (InDeis) percentage caused by different EVs encapsulated with Cas9 and guide RNA (gRNA) plasmids. Plasmids encoding CRISPR-Cas9 and gRNA were encapsulated into HEK WT EV, HEK ASA EV and HEK AGAG EV using a commercial kit. These EVs containing plasmids were applied to KHYG-1 cells, a cell line resistant to all known non-viral transfection methods. After 48 hours, GFP-positive cells were enriched using Sony MA900 under identical gating conditions. Subsequently, genotyping was performed through PCR and Sanger sequencing, and the percentage of InDeis was determined using Synthego ICE (fig 12).

[0365] Example 18

[0366] A selected glycoengineered sEV, the AGAG EV, WT EV and standard liposome were compared in an experiment for their ability to deliver GFP-encoding DNA in an air- liquid-interphase epithelial lung cell culture.

[0367] The data show that AGAG EV was manifold better than WT EV and liposome in delivering GFP-encoding DNA when delivered apical on the surface exposed to the air. This includes successful DNA delivery as well as transcription and translation. Measurement was performed via fluorescence microscopy. This mimics inhalation administration and drug delivery of the sEV for lung delivery.

[0368] The AGAG EV also performed better than liposome in direct delivery of GFP-protein. This was tested by applying it to the basolateral compartment. This mimics a parenteral administration and drug delivery of the sEV.

[0369] The AGAG EV ability to deliver GFP-proteins to a cell was more efficient than what four times the amount of WT EV could achieve in the same time span.

[0370] Microscope images illustrating the GFP expression as well as the quantification of GFP fluorescence intensity using Imaged software are shown in figure 13.

[0371] Example 19

[0372] Trans-Epithelial Electrical Resistance (TEER) is a method used to evaluate the integrity of cell layers, focusing on the tight junctions between cells. By measuring TEER it can be determine how “tight” the junctions are within a monolayer of cells. Tight junctions play a vital role in maintaining the barrier function of epithelial and endothelial layers, which control the movement of ions, molecules, and cells. This measurement provides an indication of the overall health and functionality of the cells. TEER measurements of electrical resistance from the apical to the basolateral side suggest that all sEVs are gentle delivery tools and exhibit low or no cytotoxicit. Data is shown in figure 14. Sequence overview

[0373] References

[0374] Kugeratski, F. G., Hodge, K., Lilia, S., McAndrews, K. M., Zhou, X., Hwang, R. F., Zanivan, S., & Kalluri, R. (2021). Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker. Nature Cell Biology 2021 23:6, 23(6), 631-641. https: / / doi . org / 10.1038 / s41556-021 -00693-y

[0375] Kuziemko, G. M., Stroh, M., & Stevens, R. C. (1996). Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance. Biochemistry, 35(20), 6375-6384. https: / / doi.org / 10.1021 / BI952314l

[0376] Lobb, R. J., Becker, M., Wen, S. W., Wong, C. S. F., Wiegmans, A. P., Leimgruber, A., & Moller, A. (2015). Optimized exosome isolation protocol for cell culture supernatant and human plasma. Journal of Extracellular Vesicles, 4(1). https: / / doi.org / 10.3402 / JEV.V4.27031

[0377] Martins, A. M., Ramos, C. C., Freitas, D., & Reis, C. A. (2021). Glycosylation of Cancer Extracellular Vesicles: Capture Strategies, Functional Roles and Potential Clinical Applications. Cells, 10(1), 1-26. https: / / doi.org / 10.3390 / CELLS100101Q9

[0378] Narimatsu, Y., Joshi, H. J., Nason, R., Van Coillie, J., Karlsson, R., Sun, L., Ye, Z., Chen, Y. H., Schjoldager, K. T., Steentoft, C., Furukawa, S., Bensing, B. A., Sullam, P. M., Thompson, A. J., Paulson, J. C., Bull, C., Adema, G. J., Mandel, U., Hansen, L., ... Clausen, H. (2019). An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells. Molecular Cell, 75(2), 394-407. e5. https: / / doi.Org / 10.1016 / J.MQLCEL.2019.05.017

[0379] Narimatsu, Y., Joshi, H. J., Yang, Z., Gomes, C., Chen, Y. H., Lorenzetti, F. C., Furukawa, S., Schjoldager, K. T., Hansen, L., Clausen, H., Bennett, E. P., & Wandall, H. H. (2018). A validated gRNA library for CRISPR / Cas9 targeting of the human glycosyltransferase genome. Glycobiology, 28(5), 295-305. https: / / doi.Org / 10.1093 / GLYCQB / CWX101 Tian, W., Ye, Z., Wang, S., Schulz, M. A., Van Coillie, J., Sun, L., Chen, Y. H., Narimatsu, Y., Hansen, L., Kristensen, C., Mandel, II., Bennett, E. P., Jabbarzadeh- Tabrizi, S., Schiffmann, R., Shen, J. S., Vakhrushev, S. Y., Clausen, H., & Yang, Z. (2019). The glycosylation design space for recombinant lysosomal replacement enzymes produced in CHO cells. Nature Communications 2019 10:1, 10(1), 1-13. https: / / doi.org / 10.1038 / s41467-019-098Q9-3

[0380] Zheng, W., He, R., Liang, X., Roudi, S., Bost, J., Coly, P. M., Niel, G. van, & Andaloussi, S. E. L. (2022). Cell-specific targeting of extracellular vesicles though engineering the glycocalyx. Journal of Extracellular Vesicles, 11(12), 12290. https: / / doi.org / 10.1002 / JEV2.12290

[0381] Items

[0382] 1. A method of obtaining an extracellular vesicle, such as a glycoengineered extracellular vesicle, said method comprising the steps of: a. providing a cell comprising wherein at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or the activity of a product encoded by said at least one nucleic acid has been modified, resulting in a modified glycosylation of extracellular vesicles produced by the cell; and b. isolating the extracellular vesicle from said cell.

[0383] 2. The method according to item 1, wherein the modified glycosylation is selected from: a. N-glycosylation, such as modified high-mannose N-glycans, modified hybrid N-glycans and / or modified complex N-glycans; b. O-linked glycosylation, such as modified O-GalNAc, modified O-GIcNAc, modified O-Man, modified O-Gal, modified O-Fuc, and / or modified O- Glc; c. glycolipids such as glycosphingolipids; d. glycosaminoglycans; e. proteoglycans; and f. sialylation and / or fucosylation; and combinations thereof.

[0384] 3. The method according to any one of the preceding items, wherein the modified glycosylation is modified N-glycosylation, wherein a marker specific for N- glycosylation, such as PHA-L, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified N-glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. The method according to any one of the preceding items, wherein the modified glycosylation is O-linked glycosylation, wherein a marker specific for O-linked glycosylation, such as lectin VVA, core-1 structure antibodies such as A78- G / A7, an anti-Tn antibody or PNA, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified O-linked glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. The method according to any one of the preceding items, wherein the modified glycosylation is modified glycolipids, such as modified glycosphingolipids, preferably wherein a marker specific for glycolipids, such as a marker specific for glycosphingolipids, such as GM1 , shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. The method according to any one of the preceding items, wherein the modified glycosylation is modified glycosaminoglycans, preferably wherein a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, such as an anti-chondroitin sulfate antibody, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. 7. The method according to any one of the preceding items, wherein the modified glycosylation is a modification in the length of glycan chains resulting from modified sialylation or fucosylation, preferably wherein a marker specific for sialylation or fucosylation, such as lectin SNA or LCA, shows modified binding to the extracellular vesicle compared to the binding of same marker to an extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

[0385] 8. The method according to any one of the preceding items, wherein binding of said marker to the extracellular vesicle compared to the binding of said marker to the extracellular vesicle obtained from the reference cell is modified by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%.

[0386] 9. The method according to any one of the preceding items, wherein the extracellular vesicle binds to markers specific for extracellular vesicles, such as syntenin-1 , ALIX and / or CD63.

[0387] 10. The method according to any one of the preceding items, wherein the modified glycosylation is two modified glycosylations selected from: a. N-glycosylation and O-linked glycosylation; b. N-glycosylation and glycolipids; c. N-glycosylation and glycosaminoglycans; d. N-glycosylation and proteoglycans; e. N-glycosylation and modified glycan chains resulting from modified sialylation f. O-linked glycosylation and glycolipids; g. O-linked glycosylation and glycosaminoglycans; h. O-linked glycosylation and proteoglycans; i. O-linked glycosylation and modified glycan chains resulting from modified sialylation; j. glycolipids and glycosaminoglycans; k. glycolipids and proteoglycans; l. glycolipids and modified glycan chains resulting from modified sialylation; and m. glycosaminoglycans and modified glycan chains resulting from modified sialylation; n. glycosaminoglycans and proteoglycans; o. glycosaminoglycans and sialylation. The method according to any one of the preceding items, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by a method selected from the group consisting of: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of an mRNA encoded by the target nucleic acid; e. inhibition of the activity of a protein encoded by the target nucleic acid; f. knock-in of an additional sequence encoding the product of the target nucleic acid; g. upregulation of the transcription of the target nucleic acid; h. upregulation of the translation of the mRNA encoded by the target nucleic acid; and i. stimulation of the activity of the protein encoded by the target nucleic acid. The method according to any one of the preceding items, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by a method selected from the group consisting of: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of the mRNA encoded by the target nucleic acid; and e. inhibition of the activity of the protein encoded by the target nucleic acid. The method according to any one of the preceding items, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by a method selected from the group consisting of: a. knock-in of an additional sequence encoding the product of the target nucleic acid; b. upregulation of the transcription of the target nucleic acid; c. upregulation of the translation of the mRNA encoded by the target nucleic acid; and d. stimulation of the activity of the protein encoded by the target nucleic acid. The method according to any one of the preceding items, wherein the expression of said at least one nucleic acid has been modified. The method according to any one of the preceding items, wherein the activity of said product encoded by said at least one nucleic acid has been modified. The method according to any one of the preceding items, wherein expression of said at least one nucleic acid of the cell and the activity of said product encoded by said at least one nucleic acid have been modified, resulting in a modified glycosylation of the surface of an extracellular vesicle produced by the cell. The method according to any one of the preceding items, wherein the nucleic acid comprises or consists of a gene. The method according to any one of the preceding items, wherein the nucleic acid comprises or consists of a glycosylation gene. The method according to any one of the preceding items, wherein the nucleic acid encodes a polypeptide selected from the group consisting of: a glycosyltransferase, such as a sialyltransferase; a glycosidase, such as a glycosyl hydrolase; an enzyme in the sugar precursor pathway; a transporter; and a chaperone. The method according to any one of the preceding items, wherein the cell has a total or partial reduction of expression of GNE and / or total or partial reduction of the activity of GNE, and / or wherein the cell has a total or partial reduction of expression of B4GALT7 and / or total or partial reduction of the activity of B4GALT7. The method according to any one of the preceding items, wherein the nucleic acid encodes a glycosyltransferase. The method according to any one of the preceding items, wherein the nucleic acid encodes a glycosyltransferase selected from the group consisting of:

[0388] PIG A; PIGM; PIGV; PIGB; PIGZ; PGAP4; UGT8; UGCG; B4GALT5; B4GALT6; B3GALNT1; A4GALT; B4GALNT1; B3GNT5; DPAGT1; ALG13; ALG14; ALG1; ALG2; ALG11; DPMI; UGGT1; UGGT2; ALG3; ALG5; ALG9; ALG12; ALG6; ALG8; ALG10; ALG10B; STT3A; STT3B; MGAT1; FUT8; MGAT2; MGAT3; MGAT5; MGAT4A; MGAT4B; MGAT4C; MGAT4D; GALNT1;GALNT2;

[0389] GALNT3; GALNT4; GALNT5; GALNT6; GALNT7; GALNT8; GALNT9; GALNT10; GALNT11; GALNT12; GALNT13; GALNT14; GALNT15; GALNT16; GALNTL6 (T17); GALNT18; GALNT17 (T19); GALNTL5 (T20); C1GALT1; GCNT1; GCNT3; GCNT4; B3GNT6; POFUT1; POFUT2; B3GLCT; LFNG; MFNG; RFNG; P0MT1; P0MT2; P0MGNT1; MGAT5B; P0MGNT2;

[0390] B3GALNT2; FKRP; FKTN; B4GAT1; RXYLT1; LARGE1; LARGE2; TMTC1; TMTC2; TMTC3; TMTC4; DPY19L1; DPY19L2; DPY19L3; DPY19L4; POGLUT1; POGLUT2; POGLUT3; GXYLT1; GXYLT2; XXYLT1; XYLT1; XYLT2; B4GALT7; B3GALT6; B3GAT3; CSGALNACT1; CSGALNACT2; CHPF; CHPF2; CHSY1; CHSY3; EXT1; EXT2; EXTL1; EXTL2; EXTL3; COLGALT1; COLGALT2; OGT; EOGT; B3GALT1; B3GALT2; B3GALT4; B3GALT5; B3GNT2; B3GNT3; B3GNT4; B3GNT7; B3GNT8; B3GNT9; B4GALNT3;

[0391] B4GALNT4; B4GALT1; B4GALT2; B4GALT3; B4GALT4; GCNT2; GCNT7; A4GNT; ABO; B3GAT1; B3GAT2; B4GALNT2; FUT1; FUT10; FUT11; FUT2; FUT3; FUT4; FUT5; FUT6; FUT7; FUT9; ST3GAL1; ST3GAL2; ST3GAL3;

[0392] ST3GAL4; ST3GAL5; ST3GAL6; ST6GAL1; ST6GAL2; ST6GALNAC1; ST6GALNAC2; ST6GALNAC3; ST6GALNAC4; ST6GALNAC5; ST6GALNAC6; ST8SIA1; ST8SIA2; ST8SIA3; ST8SIA4; ST8SIA5; ST8SIA6; CHST1; CHST10;

[0393] CHST11; CHST12; CHST13; CHST14; CHST15; CHST2; CHST3; CHST4; CHST5; CHST6; CHST7; CHST8; CHST9; GAL3ST1; GAL3ST2; GAL3ST3; GAL3ST4; HS2ST1; HS3ST1; HS3ST2; HS3ST3A1; HS3ST3B1; HS3ST4;

[0394] HS3ST5; HS3ST6; HS6ST1; HS6ST2; HS6ST3; NDST1; NDST2; NDST3; NDST4; UST; DSEL; DSE; GLCE; TPST1; TPST2; GNPTAB; GNPTG; NAGPA; M6PR; IGF2R; FUCA1; FUCA2; GLA; NAG A; GLB1; GLB1L; GLB1L2;

[0395] GLB1L3; GBA1; GBA2; GBA3; MOGS; GAN AB; GAA; HPSE; HPSE2; HEXA;

[0396] HEXB; HEXD; OGA; HYAL1; HYAL2; HYAL3; HYAL4; EDEM1; EDEM2; EDEM3; MAN1A1; MAN1A2; MAN1B1; MAN 1 C1; MAN2A1; MAN2A2;

[0397] MAN2B1; MAN2B2; MAN2C1; MANEA; MANEAL; NEU1; NEU2; NEU3; NEU4; GNE; GMDS; GFUS; C1GALT1C1; CANX; CALR and PDIA3; SELF; SELP;

[0398] SELL; CD207; CD209; MRC1; CLEC7A; CLEC4E; CLEC12A; ASGR1; ASGR2;

[0399] DCIR; SIGLEC1; SIGLEC2; SIGLEC3; SIGLEC4; SIGLEC5; SIGLEC6;

[0400] SIGLEC7; SIGLEC8; SIGLEC9; SIGLEC10; SIGLEC11; SIGLEC12;

[0401] SIGLEC14; SIGLEC15; SIGLEC16; LGALS1; LGALS2; LGALS3; LGALS4; LGALS7; LGALS8; LGALS9; LGALS9B; LGALS10; LGALS12; LGALS13;

[0402] LGALS14; LGALS16; SLC35A1; and SLC35C2.

[0403] 23. The method according to any one of the preceding items, wherein the nucleic acid encodes a glycosidase.

[0404] 24. The method according to any one of the preceding items, wherein the nucleic acid encodes a glycosyl hydrolase.

[0405] 25. The method according to any one of the preceding items, wherein the nucleic acid encodes a glycosyl hydrolase selected from the group consisting of: FUCA1; FUCA2; GLA; NAG A; GLB1; GLB1L; GLB1L2; GLB1L3; GBA1; GBA2;

[0406] GBA3; MOGS; GANAB; GAA; HPSE; HPSE2; HEXA; HEXB; HEXD; OGA; HYAL1; HYAL2; HYAL3; HYAL4; EDEM1; EDEM2; EDEM3; MAN1A1; MAN1A2; MAN1B1; MAN1C1; MAN2A1; MAN2A2; MAN2B1; MAN2B2;

[0407] MAN2C1; MANEA; MANEAL; NEU1; NEU2; NEU3; and NEU4.

[0408] 26. The method according to any one of the preceding items, wherein the nucleic acid encodes an enzyme in the sugar precursor pathway.

[0409] 27. The method according to any one of the preceding items, wherein the nucleic acid encodes an enzyme in the sugar precursor pathway selected from the group consisting of: GNE; NANS; NANP; CMAS; GMDS; and GFUS.

[0410] 28. The method according to any one of the preceding items, wherein the nucleic acid encodes a transporter, such as a GDP-fucose transporter or a CMP-sialic acid transporter.

[0411] 29. The method according to any one of the preceding items, wherein the nucleic acid encodes a carbohydrate-binding protein such as a lectin, a galectin or a siglec, preferably wherein the carbohydrate-binding protein is endogenous.

[0412] 30. The method according to any one of the preceding items, wherein the nucleic acid encodes a chaperone.

[0413] 31. The method according to any one of the preceding items, wherein the nucleic acid encodes a chaperone, wherein the chaperone is C1GALT1C1, CANX, CALR or PDIA3.

[0414] 32. The method according to any one of the preceding items, wherein the modification of the expression of the nucleic acid affects a step in a glycosylation pathway selected from the group consisting of: a. biosynthesis and / or transport of a precursor sugar; b. initiation; c. core extension; d. elongation; e. trimming; f. branching; g. capping; and h. transport. The method according to any one of the preceding items, wherein the nucleic acid encodes a product, such as a polypeptide, which is part of a step in a glycosylation pathway selected from the group consisting of: a. biosynthesis and / or transport of a precursor sugar; b. initiation; c. core extension; d. elongation; e. trimming; f. branching; g. capping; and h. transport. The method according to any one of the preceding items, wherein modification of the expression of the nucleic acid affects the biosynthesis of at least one group of carbohydrates selected from the group consisting of: a. N-glycans; b. O-glycans; c. Glycolipids such as glycosphingolipids; d. glycosaminoglycans; e. proteoglycans; f. GPI anchored glycoproteins; g. glycolipids; h. fucose, and i. sialic acid. The method according to any one of the preceding items, wherein the nucleic acid encodes a polypeptide which is part of the biosynthesis pathway of at least one group of carbohydrates selected from the group consisting of: a. N-glycans; b. O-glycans; c. glycosphingolipids; d. glycosaminoglycans; e. proteoglycans; f. GPI anchored glycoproteins g. glycolipids; h. fucose, and i. sialic acid. The method according to any one of the preceding items, wherein the nucleic acid encodes a polypeptide which adds or removes a phosphorylation or a sulfation of a carbohydrate. The method according to any one of the preceding items, wherein the nucleic acid comprises or consists of a gene selected from the group consisting of: SELF; SELP; SELL; CD207; CD209; MRC1; CLEC7A; CLEC4E; CLEC12A; ASGR1; ASGR2; ; DCIR', B4GALT5; B4GALT6; B4GALT7; MGAT1; FUT8; MGAT2; MGAT3; MGAT5; MGAT4A; MGAT4B; MGAT4C; MGAT4D;

[0415] GALNT1;GALNT2; GALNT3; GALNT4; GALNT5; GALNT6; GALNT7; GALNT8; GALNT9; GALNT10; GALNT11; GALNT12; GALNT13; GALNT14; GALNT15; GALNT16; GALNTL6 ; GALNT18; GALNT17 ; GALNTL5; C1GALT1; FUT1; FUT10; FUT11; FUT2; FUT3; FUT4; FUT5; FUT6; FUT7; FUT9; ST3GAL1; ST3GAL2; ST3GAL3; ST3GAL4; ST3GAL5; ST3GAL6; ST6GAL1; ST6GAL2;

[0416] ST6GALNAC1; ST6GALNAC2; ST6GALNAC3; ST6GALNAC4; ST6GALNAC5; ST6GALNAC6; ST8SIA1; ST8SIA2; ST8SIA3; ST8SIA4; ST8SIA5; ST8SIA6;

[0417] MAN1A1; MAN1A2; MAN1B1; MAN1C1; MAN2A1; MAN2A2; GNE; GMDS; GFUS; and C1GALT1C1. The method according to any one of the preceding items, wherein the nucleic acid comprises or consists of a gene selected from the group consisting of: B4GALT7, GNE, B4GALT5, B4GALT6, C1GALT1C1 and MGAT1. The method according to any one of the preceding items, wherein modification of the expression of said at least one nucleic acid of the cell and / or the activity of said product encoded by said at least one nucleic acid sequence results in the modification, such as in the inhibition and / or the block of, or such as in increase or stimulation of the synthesis of protegolycans and / or glycosaminoglycans.

[0418] 40. The method according to any one of the preceding items, wherein the expression of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell, has been modified.

[0419] 41. The method according to any one of the preceding items, wherein the activity of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell have been modified.

[0420] 42. The method according to any one of the preceding items wherein the cell has been genetically engineered, or wherein the cell has been obtained by mutagenesis, such as induced or spontaneous mutagenesis, or selection of cells grown under selection pressure.

[0421] 43. The method according to any one of the preceding items wherein the cell has been obtained by selection of cells cultured in a medium comprising a molecule, such as a toxic lectin, which inhibits the growth and / or kills cells comprising a specific carbohydrate and / or a specific modification of a carbohydrate.

[0422] 44. The method according to any one of the preceding items, wherein the cells grown under selection pressure are isolated cells.

[0423] 45. The method according to any one of the preceding items, wherein the cell is a genetically engineered cell.

[0424] 46. The method according to any one of the preceding items, wherein the cell has been genetically engineered via a method selected from the group consisting of: CRISPR-associated nucleases, such as CRISPR-Cas9, zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs).

[0425] 47. The method according to any one of the preceding items, wherein the modification of the expression of the nucleic acid is transient. 48. The method according to any one of the preceding items, wherein the product encoded by the nucleic acid is a polypeptide, and wherein the modification, such as the inhibition or stimulation, of the expression of the nucleic acid is achieved by contacting said polypeptide with an inhibitory or stimulatory compound respectively, such as a small molecule inhibitor or such as a small molecule stimulator respectively, inhibiting or stimulating said polypeptide, respectively.

[0426] 49. The method according to any one of the preceding items, wherein the cell is a eukaryotic cell.

[0427] 50. The method according to any one of the preceding items, wherein the cell is selected from the group consisting of: an animal cell; a plant cell; a fungus cell and a yeast cell.

[0428] 51. The method according to any one of the preceding items wherein the animal cell is selected from the group consisting of: a mammalian cell; an avian cell; and an invertebrate cell, such as an insect cell.

[0429] 52. The method according to any one of the preceding items, wherein the animal cell is a mammalian cell selected from the group consisting of: an cell of a Hominidae, such as a human cell; a cell of a Cricetidae, such as a hamster cell, such as a CHO cell; and a cell of a Muridae, such as a mouse cell, such as a rat cell.

[0430] 53. The method according to any one of the preceding items, wherein the animal cell is a mammalian cell selected from the group consisting of: a human cell, a hamster cell, a mouse cell, a rat cell.

[0431] 54. The method according to any one of the preceding items, wherein the cell is a human cell.

[0432] 55. The method according to any one of the preceding items, wherein the cell is selected from the group consisting of: a stem cell; such as a pluripotent cell, such as a hematopoietic stem cell, such as a neural stem cell, such as a mesenchymal stem cell, such as an embryonic stem cell; a precursor cell; a skin cell; a brain cell; and an immune cell.

[0433] 56. The method according to the preceding item, wherein the stem cell is an adult stem cell, and / or is an artificially induced stem cell, such as an induced pluripotent stem cell.

[0434] 57. The method according to any one of the preceding items, wherein the cell is a cancer cell.

[0435] 58. The method according to any one of the preceding items, wherein the cell is selected from the group consisting of: a natural killer cell, a chimeric antigen receptor-T cell (CAR-T) or a chimeric antigen receptor natural killer cell (CAR- NK), a T cell, such as a CD8 T cell or a CD4 T cell, and a dendritic cell.

[0436] 59. The method according to any one of the preceding items, wherein the cell is selected from the group consisting of: HEK293, such as HEK293F; CHO; MUTZ3; K562; NK92; KHYG-1 ; and KHYK.

[0437] 60. The method according to any one of the preceding items, wherein isolating the extracellular vesicle comprises the steps consisting of: a. growing the cell in a medium; and e. recovering the extracellular vesicle from the medium; thereby isolating the extracellular vesicle.

[0438] 61. The method according to the preceding items, further comprising a step of: b. recovering the medium.

[0439] 62. The method according to any one of the preceding items, further comprising a step: c. removing dead and / or living cells from the medium.

[0440] 63. The method according to the preceding items, wherein step c. is performed by centrifugation or by filtering the medium. 64. The method according to the preceding item, further comprising a step of: d. removing cell debris from the medium, such as by centrifugation or filtration.

[0441] 65. The method according to the preceding items, further comprising a step of: f. washing the extracellular vesicle in a saline solution, such as PBS, and recovering the extracellular vesicle.

[0442] 66. An isolated extracellular vesicle with a modified property of interest, preferably wherein the isolated extracellular vesicle has been obtained by the method according to any one of the preceding items.

[0443] 67. The method according to any one of the preceding items, or the isolated extracellular vesicle according to item 66, wherein the extracellular vesicle has a diameter of less than 300 nm, such as less than 290 nm, such as less than 280 nm, such as less than 270 nm, such as less than 260 nm, such as less than 250 nm.

[0444] 68. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one preceding items, wherein the extracellular vesicle has a modified target specificity or tropism to a cell of interest compared to an extracellular vesicle produced by another reference cell, wherein the other reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified.

[0445] 69. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the property of interest is increased specificity or tropism to a target cell compared to an extracellular vesicle produced by said reference cell, and / or increased specificity or tropism to a target cell compared to an extracellular vesicle produced by said reference cell. The method according to the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the target specificity or the tropism to a target cell is at least 1% increased or decreased, such as at least 5% increased or decreased, such as at least 10% increased or decreased, or is at least 1% increased or decreased, such as at least 5% increased or decreased, such as at least 10% increased or decreased, such as at least 20% increased or decreased, such as at least 30% increased or decreased, such as at least 40% increased or decreased, such as at least 50% increased or decreased, such as at least 75% increased or decreased, such as at least 100% increased or decreased, such as at least 150% increased, such as at least 200% increased or more. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the property of interest is increased total uptake of a molecule of interest in a target cell compared to an extracellular vesicle produced by a reference cell, and / or wherein the extracellular vesicle cell has an increased total uptake of a molecule of interest to a target cell compared to an extracellular vesicle produced by said reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified. The method according to the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the total uptake of the molecule of interest is at least 1% increased, such as 5% increased, such as at least 10% increased, such as at least 20% increased decreased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased or more. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the property of interest is increased uptake of a molecule of interest in a target cell per second compared to the uptake of said molecule of interest in said target cell per second by another extracellular vesicle, wherein the other extracellular vesicle is produced by a reference cell wherein the expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified and / or wherein the extracellular vesicle cell has an increased uptake of a molecule of interest in the extracellular vesicle per second compared to an extracellular vesicle produced by said reference cell. The method according to the preceding items, or the isolated extracellular vesicle according to the preceding items, wherein the uptake of a molecule of interest per second is at least 1% increased, such as 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 125% increased, such as at least 150% increased, such as at least 200% increased, or more. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the property of interest is: increased delivery efficiency of a molecule of interest to a target cell compared to the delivery efficiency of said molecule of interest to said target cell by an extracellular vesicle produced by a reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the delivery efficiency is at least 1% increased, such as 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 125% increased, such as at least 150% increased, such as at least 200% increased, or more. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the property of interest is increased expression of a molecule of interest in a target cell contacted with said extracellular vesicle compared to expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by a reference cell, and / or wherein the extracellular vesicle leads to an increased expression of a molecule of interest in a target cell contacted with said extracellular vesicle compared to the expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by said reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the delivery efficiency of a molecule of interest is at least 1% increased, such as 5% increased, such as at least 10% increased, such as at least 20% increased, such as at least 30% increased, such as at least 40% increased, such as at least 50% increased, such as at least 75% increased, such as at least 100% increased, such as at least 125% increased, such as at least 150% increased, such as at least 200% increased, or more. The method according to any one of the preceding items, or the isolated extracellular vesicle according to any one of the preceding items, wherein the molecule of interest is selected from the group consisting of: a small molecule, such as an inhibitor, such as a toxin; a nucleic acid sequence, such as a RNA sequence, such as a DNA sequence; a polypeptide, such as an antigen; a lipid; a metabolite; and an imaging agent, such as a fluorescent dye. An isolated extracellular vesicle, such as a glycoengineered extracellular vesicle, having a modified glycosylation compared to another extracellular vesicle obtained from a reference cell, wherein the modified glycosylation and the reference cell are as defined in any one of the preceding items. The isolatedextracellular vesicle, such as a glycoengineered extracellular vesicle, according to item 80, wherein the modified glycosylation is selected from: a. N-glycosylation, such as modified high-mannose N-glycans, modified hybrid N-glycans and / or modified complex N-glycans; b. O-linked glycosylation, such as modified O-GalNAc, modified O-GIcNAc, modified O-Man, modified O-Gal, modified O-Fuc, and / or modified O- Glc; c. glycolipids such as glycosphingolipids; d. proteoglycans and / or glycosaminoglycans; and e. sialylation and fucosylation; and combinations thereof. The isolated extracellular vesicle according to any one of items 80 to 81 , wherein the modified glycosylation is one or more of: modified N-glycosylation; preferably wherein a marker specific for N- glycosylation, such as PHA-L, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, and / or wherein the modified N- glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof;

[0446] O-linked glycosylation; preferably wherein a marker specific for N- glycosylation, such as lectin VVA, core-1 structure antibodies such as A78- G / A7, an anti-Tn antibody or PNA, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, and / or wherein the modified O-linked glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof modified glycolipids such as modified glycosphingolipids; preferably wherein a marker specific for glycolipids, such as a marker specific for glycosphingolipids, such as GM1 , shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof; glycosaminoglycans; preferably wherein a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, such as an anti-chondroitin sulfate antibody, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell; and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof; and modification of the length of glycan chains resulting from modified sialylation or fucosylation; preferably wherein a marker specific for sialylation or fucosylation, such as lectin SNA, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

[0447] 83. The isolated extracellular vesicle according to any one of items 80 to 82, wherein the extracellular vesicle is obtained by the method according to any one of items 1 to 79.

[0448] 84. A composition comprising an isolated extracellular vesicle obtained via the method according to any one of the preceding items, or the isolated extracellular vesicle as defined in any one of the preceding items.

[0449] 85. The composition according to any one of the preceding items, wherein the composition further comprises an acceptable carrier.

[0450] 86. The composition according to any one of the preceding items, further comprising at least one of the following: a. a liposome; and / or b. a nanoparticle. The composition according to any one of the preceding items, wherein the composition further comprises a stabilizer and / or an additive. The composition according to any one of the preceding items, wherein the composition is a pharmaceutical composition. A kit comprising an isolated extracellular vesicle according to any one of items 80 to 83, preferably wherein said extracellular vesicle is obtained via the method according to any one of the preceding items, optionally further comprising instructions for use. An isolated extracellular vesicle, or a composition comprising an isolated extracellular vesicle, wherein the isolated extracellular vesicle has been obtained by the method according to any one of the preceding items, for use in medicine. An isolated extracellular vesicle, or a composition comprising an isolated extracellular vesicle, wherein the isolated extracellular vesicle has been obtained by the method according to any one of the preceding items, for use in a method of treating and / or preventing a disease and / or a medical condition in a subject in need thereof. The isolated extracellular vesicle for use according to any one of the preceding items, wherein the disease is selected from the group consisting of: cancer; neurodegenerative diseases; cardiovascular disease; autoimmune diseases; metabolic disorders; rare genetic disorders; and skin diseases. An isolated extracellular vesicle, or a composition comprising an isolated extracellular vesicle, wherein the isolated extracellular vesicle has been obtained by the method according to any one of the preceding items, for use in wound healing, regenerative medicine or gene therapy. The isolated extracellular vesicle for use according to any one of the preceding items, wherein the extracellular vesicle is loaded with an active ingredient. The isolated extracellular vesicle for use according to any one of the preceding items, wherein the extracellular vesicle is loaded with a molecule selected from the group consisting of: a nucleic acid sequence, such as a DNA molecule, such as an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide; and a dye. Use of an isolated extracellular vesicle according to any one of the preceding items, use of a composition according to any one of the preceding items, or use of a kit according to any one of the preceding items, to deliver a molecule of interest to a target cell, preferably wherein the molecule of interest is selected from the group consisting of: a nucleic acid sequence, such as a DNA molecule, such as an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule, a gRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide; and a dye. Use of an isolated extracellular vesicle according to any one of the preceding items, use of a composition according to any one of the preceding items, or use of a kit according to any one of the preceding items, to modulate the expression of a target nucleic acid, such as to increase the expression of a target nucleic acid, such as to decrease the expression of a target nucleic acid, and / or to modulate the activity of a product encoded by a target nucleic acid, such as to increase the activity of a polypeptide encoded by a target nucleic acid, or such as to decrease the activity of a product encoded by a target nucleic acid. Use of an isolated extracellular vesicle according to any one of the preceding items, use of a composition according to any one of the preceding items, or use of a kit according to any one of the preceding items, to modulate the expression of a target nucleic acid, such as to increase the expression of a target nucleic acid, such as to decrease the expression of a target nucleic acid. Use of an isolated extracellular vesicle according to any one of the preceding items, use of a composition according to any one of the preceding items, or use of a kit according to any one of the preceding items, to modulate the activity of a product encoded by a target nucleic acid, such as to increase the activity of a product encoded by a target nucleic acid, such as to decrease the activity of a product encoded by a target nucleic acid. . Use of an isolated extracellular vesicle according to any one of the preceding items, use of a composition according to any one of the preceding items, or use of a kit according to any one of the preceding items, to edit a target nucleic acid in a target cell. . Use of an isolated extracellular vesicle according to any one of the preceding items, use of a composition according to any one of the preceding items, or use of a kit according to any one of the preceding items, to deliver a compound, such as a small molecule, such as a toxin, to a target cell, such as an animal cell, a plant cell or a yeast cell. . Use of an isolated extracellular vesicle according to any one of the preceding items, use of a composition according to any one of the preceding items, or use of a kit according to any one of the preceding items, to improve transfer of a nucleic acid of interest to a target cell compared to the transfer of said nucleic acid of interest to said target cell by another extracellular vesicle, wherein the other extracellular vesicle is an extracellular vesicle from a reference cell in which expression of the at least one nucleic acid and / or the activity of the product encoded by the at least one nucleic acid has not been modified, preferably wherein the improved transfer leads to increased expression of the nucleic acid of interest compared to the expression obtained when using the other extracellular vesicle. . A method of modulating the tropism and / or target specificity of an extracellular vesicle, wherein the method comprises or consists of the method of obtaining an isolated extracellular vesicle according to any one of the previous items, wherein said extracellular vesicle has a modified glycosylation on its surface. 104. A method of increasing the total uptake of a target molecule in a target cell, wherein the method comprises the method of obtaining an extracellular vesicle according to any one of the previous items.

[0451] 105. A method of increasing the uptake of a target molecule per second in a target cell, wherein the method comprises or consists of the method of obtaining an extracellular vesicle according to any one of the previous items.

[0452] 106. A method of increasing the delivery efficiency of a target molecule to a target cell, wherein the method comprises the method of obtaining an extracellular vesicle according to any one of the previous items, and wherein the method further comprises loading said target molecule in the extracellular vesicle and contacting said extracellular vesicle with the target cell.

[0453] 107. A method of increasing the loading of a target molecule to an extracellular vesicle, wherein the method comprises the method of obtaining an extracellular vesicle according to any one of the preceding items, and further comprises loading said target molecule in the extracellular vesicle.

[0454] 108. A method of increasing the transfer of a target nucleic acid to a target cell or a method of increasing the expression of a product encoded by a target nucleic acid in a target cell, wherein the method comprises the method of obtaining an extracellular vesicle according to any one of the preceding items, and further comprises: a. loading said target nucleic acid in the extracellular vesicle; and b. contacting the extracellular vesicle with the target cell, whereby the target nucleic acid is transferred to the target cell and / or whereby the product encoded by the target nucleic acid is expressed.

[0455] 109. The method according to item 108, wherein the modified glycosylation of the extracellular vesicle comprises or consists of modified glycosaminoglycans and / or modified sialylation, preferably of modified glycosaminoglycans.

[0456] 110. The method according to any one of items 108 to 109, wherein the transfer of said target nucleic acid to said target cell and / or the expression of the product encoded by said target nucleic acid in said target cell is increased by at least 10%, such as by at least 25%, such as by at least 50%, such as by at least, 75%, such as by at least 100%, such as by at least 150%, such as by at least 200%, such as by at least 300%, such as by at least 400%, such as by at least 500%, such as by at least 600%, or more, compared to the transfer of said target nucleic acid to said target cell when using another extracellular vesicle obtained from a reference cell, in which the expression of said target nucleic acid and / or the activity of the nucleic acid encoded by said target nucleic acid of interest has not been modified, wherein the target nucleic acid is a DNA or an RNA.

[0457] 111. The method according to any one of items 104 to 110, wherein the target molecule is a drug, a therapeutic agent, or a nucleic acid such as a DNA or an RNA.

[0458] 112. A method to identify an extracellular vesicle with a glycosylation pattern of interest comprising the steps of: a. providing an extracellular vesicle, wherein the extracellular vesicle has been obtained by a cell, wherein expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as defined in any one of the preceding items; b. optionally, determining whether the extracellular vesicle has the glycosylation pattern of interest, such as by contacting the extracellular vesicle with a polypeptide, such as a lectin, such as an antibody, specific for the glycosylation pattern of interest, and / or such as by performing glycoanalytics of the extracellular vesicle, and / or by testing the extracellular vesicle in an assay.

[0459] 113. A method to identify a nucleic acid of interest, wherein modification of the expression of the nucleic acid of interest in a cell and / or modification of the activity of a product encoded by the nucleic acid of interest in said cell results in a modified glycosylation on the surface of an extracellular vesicle produced by the cell, said method comprising the steps of: a. obtaining an extracellular vesicle from a cell, wherein expression of a nucleic acid sequence in the cell and / or the activity of a product encoded by the nucleic acid has been modified as defined in any one of the preceding items; b. optionally, determining whether the extracellular vesicle has the glycosylation pattern of interest, such as by contacting the extracellular, such as by contacting the extracellular vesicle with a polypeptide, such as a lectin, such as an antibody, specific for the glycosylation pattern of interest; and / or such as by performing glycoanalytics of the extracellular vesicle; and / or by testing the extracellular vesicle in an assay; and c. concluding that the nucleic acid sequence is a nucleic acid sequence of interest. . The method according to any one of the preceding items, wherein the polypeptide is a carbohydrate-binding polypeptide and / or a polypeptide comprising a carbohydrate-binding domain. . The method according to any one of the preceding items, wherein the polypeptide is selected from the group consisting of: a lectin, a galectin, a siglec and an antibody. . The method according to any one of the preceding items, wherein the lectin is selected from the group consisting of: an antibody, a Sambucus Nigra Lectin (Lectin SNA), lectin VVA and a Cholera Toxin Subunit B. . The method according to any one of the preceding items, wherein the polynucleotide specific for a glycosylation feature of interest is an antibody. . The method according to any one of the preceding items, wherein the antibody is selected from the group consisting of: an anti-syntenin-1 antibody, and an anti-chondroitin sulfate antibody. . The method according to any one of the preceding items, wherein the cell and the reference cell are grown in the same conditions. 120. The method according to any one of items 103 to 119, wherein the extracellular vesicle is as defined in any one of the preceding items.

[0460] Items 2

[0461] 1. A method of obtaining a glycoengineered extracellular vesicle, preferably a glycoengineered small extracellular vesicle, with a modified property of interest, said method comprising the steps of: a. providing a cell wherein i. at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or wherein the activity of a product encoded by said at least one nucleic acid has been modified; and / or ii. wherein at least one nucleic acid has been introduced, resulting in a modified glycosylation of extracellular vesicles produced by the cell, thereby obtaining a glycoengineered extracellular vesicle with a modified property of interest; and b. isolating the extracellular vesicle from said cell, wherein the property of interest is: i. modified nucleic acid transfer, such as gene, RNA or DNA transfer; ii. modified expression of a transferred nucleic acid; iii. increased editing of a target nucleic acid such as a gene; iv. modified target specificity or tropism; v. total uptake of a molecule by the glycoengineered extracellular vesicle; and / or vi. increased delivery of a molecule to a target, and wherein the property of interest is modified compared to an extracellular vesicle obtained from a reference cell in which said at least one nucleic acid, said expression of said at least one nucleic acid sequence and / or said activity of the product encoded by said at least one nucleic acid sequence has not been modified.

[0462] 2. The method according to item 1 , wherein the modified glycosylation is modified glycosaminoglycans and / or modified sialylation on the surface of the glycoengineered extracellular vesicle, and / or wherein the expression of said at least one nucleic is modified using CRISPR-associated nucleases, such as CRISPR-Cas9, zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), and / or wherein said at least one nucleic acid is introduced using CRISPR-associated nucleases, such as CRISPR-Cas9, zinc- finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs).

[0463] 3. The method according to any one of the preceding items, wherein the at least one nucleic acid is B4GALT7; GNE; B4GALT5; B4GALT6; C1GALT1C1 ; C1GALT1; or MGATI.

[0464] 4. The method according to any one of the preceding items, wherein the at least one nucleic acid is: B4GALT7 and / or GNE.

[0465] 5. The method according to any one of the preceding items, wherein the cell has a total or partial reduction of expression of GNE and / or total or partial reduction of the activity of GNE, and / or wherein the cell has a total or partial reduction of expression of B4GALT7 and / or total or partial reduction of the activity of B4GALT7.

[0466] 6. The method according to any one of the preceding items, wherein the cell has a total or partial reduction of expression of B4GALT7 and / or total or partial reduction of the activity of B4GALT7.

[0467] 7. The method according to any one of the preceding items, wherein the cell has a total or partial reduction of expression of GNE and / or total or partial reduction of the activity of GNE.

[0468] 8. The method according to any one of the preceding items, wherein the glycoengineered isolated extracellular vesicle binds to at least one marker specific for extracellular vesicles, such as syntenin-1 , ALIX and / or CD63.

[0469] 9. The method according to any one of the preceding items, wherein the glycoengineered isolated extracellular vesicle is a small extracellular vesicle and has a diameter of less than 300 nm, such as less than 290 nm, such as less than 280 nm, such as less than 270 nm, such as less than 260 nm, such as less than 250 nm.

[0470] 10. The method according to any one of the preceding items, wherein the modified glycosylation comprises or consist of at least one modified glycosaminoglycan, preferably wherein a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, such as an anti-chondroitin sulfate antibody, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. The method according to any one of the preceding items, wherein the modified glycosylation is a modification in the length of glycan chains resulting from modified sialylation or fucosylation, preferably wherein a marker specific for sialylation or fucosylation, such as lectin SNA or LCA, shows modified binding to the extracellular vesicle compared to the binding of same marker to an extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. The method according to any one of items 10-12, wherein binding of said marker specific for glycosaminoglycans, sialylation or fucosylation to the extracellular vesicle compared to the binding of said marker to the extracellular vesicle obtained from the reference cell is modified by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%. An isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in medicine, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified glycosylation and comprising a therapeutic molecule or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle. An isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in the treatment of a disease and / or a medical condition, in wound healing, in regenerative medicine or in gene therapy in a subject in need thereof, comprising the steps of: providing an isolated glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a therapeutic molecule or active ingredient, optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; and administering said isolated glycoengineered extracellular vesicle or composition to a subject in need thereof; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle. Use of an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for the delivery of a molecule to a cell, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a molecule or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of items 13-14, or the use according to item 15, wherein providing the isolated glycoengineered extracellular vesicle comprises the method according to any one of items 1-12. 17. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of items 13-14 or 16, or the use according to item 15- 16, wherein the modified glycosylation is modified glycosaminoglycans and / or modified sialylation on the surface of the glycoengineered extracellular vesicle.

[0471] 18. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of items 13-14 or 16-17, or the use according to item 15-17 wherein the molecule, the therapeutic molecule, or the active ingredient, is a large nucleic acid and / or a large polypeptide, wherein said large nucleic acid is a nucleic acid, such as a DNA, for example a gene, or an RNA, having a size of at least 1500 bp, such as at least 2000 bp, such as at least 3000 bp, such as at least 4000 bp, such as at least 4700 bp, and / or wherein the large polypeptide is a polypeptide such as a protein having a size of at least 500 amino acids, such as at least 600, at least 800, at least 1000, at least 1200, at least 1500 or at least 1700 amino acids.

[0472] 19. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of items 13-14 or 16-18, or the use according to item 15-18, wherein the molecule or active ingredient is a DNA molecule, or an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule, a gRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide; or a dye.

[0473] 20. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of items 16-19, or the use according to item 16-19, wherein the at least one nucleic acid is B4GALT7; GNE; B4GALT5; B4GALT6; C1GALT1 ; C1GALT1C1 ; or MGATI .

[0474] 21. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of items 16-20, or the use according to item 16-20, wherein the at least one nucleic acid is B4GALT7 and / or GNE.

[0475] 22. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of items 13-14 or 16-21 , wherein the disease and / or medical condition is a cancer; a neurodegenerative disease; a cardiovascular disease; an autoimmune disease; a metabolic disorder; a genetic disorder; or a skin disease. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of items 13-14 or 16-22, wherein the disease is a monogenic disease. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of items 13-14 or 16-23, wherein the disease is a pulmonary disease, such as cystic fibrosis. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of items 13-14 or 16-24, wherein the therapeutic molecule is a nucleic acid comprising or consisting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, or a polypeptide comprising or consisting of the CFTR protein, or fragments thereof. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 13-14 or 16-25, or the use according to anyone of items 15-21 wherein the modified glycosylation comprises or consist of at least one modified glycosaminoglycan, preferably wherein a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, such as an antichondroitin sulfate antibody, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 13-14 or 16-26, or the use according to anyone of items 15-21 or 26, wherein the modified glycosylation is a modification in the length of glycan chains resulting from modified sialylation or fucosylation, preferably wherein a marker specific for sialylation or fucosylation, such as lectin SNA or LCA, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to an extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of items 16-27, or the use according to anyone of items 16-21 or 26-27, wherein the binding of said marker to the extracellular vesicle obtained from the reference cell is modified by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%. The method according to anyone of items 1-12, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 16-28, or the use according to anyone of items 16-21 or 26-28, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of an mRNA encoded by the target nucleic acid; e. inhibition of the activity of a protein encoded by the target nucleic acid; f. knock-in of an additional sequence encoding the product of the target nucleic acid; g. upregulation of the transcription of the target nucleic acid; h. upregulation of the translation of the mRNA encoded by the target nucleic acid; i. stimulation of the activity of the protein encoded by the target nucleic acid; or j. introduction of another nucleic acid in the cell.

[0476] 30. The method according to anyone of items 1-12 or 29, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 16-29, or the use according to anyone of items 16-21 or 26-

[0477] 29, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of the mRNA encoded by the target nucleic acid; or e. inhibition of the activity of the protein encoded by the target nucleic acid.

[0478] 31. The method according to anyone of items 1-12 or 29-30, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of items 16-30, or the use according to anyone of items 16-21 or 26-

[0479] 30, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by: a. knock-in of an additional sequence encoding the product of the target nucleic acid; b. upregulation of the transcription of the target nucleic acid; c. upregulation of the translation of the mRNA encoded by the target nucleic acid; or d. stimulation of the activity of the protein encoded by the target nucleic acid.

[0480] 32. The method according to anyone of items 1-12 or 29-31 , the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of 16-31 , or the use according to anyone of items 16-21 or 26-31 , wherein the at least one nucleic acid is PIGA; PIGM; PIGV; PIGB; PIGZ; PGAP4; UGT8; UGCG; B4GALT5; B4GALT6; B3GALNT1; A4GALT;

[0481] B4GALNT1 ; B3GNT5; DPAGT1; ALG13; ALG14; ALG1 ; ALG2; ALG11; DPMI; UGGT1; UGGT2; ALG3; ALG5; ALG9; ALG12; ALG6; ALG8; ALG10; ALG10B; STT3A; STT3B; MGAT1; FUT8; MGAT2; MGAT3; MGAT5; MGAT4A; MGAT4B; MGAT4C; MGAT4D; GALNT1 ;GALNT2; GALNT3; GALNT4;

[0482] GALNT5; GALNT6; GALNT7; GALNT8; GALNT9; GALNT10; GALNT11;

[0483] GALNT12; GALNT13; GALNT14; GALNT15; GALNT16; GALNTL6 (T17);

[0484] GALNT18; GALNT17 (T19); GALNTL5 (T20); C1GALT1; GCNT1; GCNT3;

[0485] GCNT4; B3GNT6; POFUT1 ; POFUT2; B3GLCT; LFNG; MFNG; RFNG;

[0486] POMT1; POMT2; POMGNT1 ; MGAT5B; POMGNT2; B3GALNT2; FKRP;

[0487] FKTN; B4GAT1; RXYLT1; LARGE1 ; LARGE2; TMTC1 ; TMTC2; TMTC3;

[0488] TMTC4; DPY19L1 ; DPY19L2; DPY19L3; DPY19L4; POGLUT1 ; POGLUT2;

[0489] POGLUT3; GXYLT1; GXYLT2; XXYLT1 ; XYLT1; XYLT2; B4GALT7; B3GALT6;

[0490] B3GAT3; CSGALNACT1; CSGALNACT2; CHPF; CHPF2; CHSY1 ; CHSY3;

[0491] EXT1 ; EXT2; EXTL1; EXTL2; EXTL3; COLGALT1; COLGALT2; OGT; EOGT;

[0492] B3GALT1; B3GALT2; B3GALT4; B3GALT5; B3GNT2; B3GNT3; B3GNT4;

[0493] B3GNT7; B3GNT8; B3GNT9; B4GALNT3; B4GALNT4; B4GALT1 ; B4GALT2;

[0494] B4GALT3; B4GALT4; GCNT2; GCNT7; A4GNT; ABO; B3GAT1; B3GAT2;

[0495] B4GALNT2; FUT1 ; FUT10; FUT11 ; FUT2; FUT3; FUT4; FUT5; FUT6; FUT7;

[0496] FUT9; ST3GAL1; ST3GAL2; ST3GAL3; ST3GAL4; ST3GAL5; ST3GAL6;

[0497] ST6GAL1; ST6GAL2; ST6GALNAC1; ST6GALNAC2; ST6GALNAC3;

[0498] ST6GALNAC4; ST6GALNAC5; ST6GALNAC6; ST8SIA1; ST8SIA2; ST8SIA3;

[0499] ST8SIA4; ST8SIA5; ST8SIA6; CHST1; CHST10; CHST11 ; CHST12; CHST13;

[0500] CHST14; CHST15; CHST2; CHST3; CHST4; CHST5; CHST6; CHST7; CHST8;

[0501] CHST9; GAL3ST1; GAL3ST2; GAL3ST3; GAL3ST4; HS2ST1 ; HS3ST1;

[0502] HS3ST2; HS3ST3A1; HS3ST3B1; HS3ST4; HS3ST5; HS3ST6; HS6ST1;

[0503] HS6ST2; HS6ST3; NDST1 ; NDST2; NDST3; NDST4; UST; DSEL; DSE; GLCE;

[0504] TPST1 ; TPST2; GNPTAB; GNPTG; NAGPA; M6PR; IGF2R; FUCA1 ; FUCA2;

[0505] GLA; NAGA; GLB1; GLB1 L; GLB1L2; GLB1 L3; GBA1; GBA2; GBA3; MOGS;

[0506] GANAB; GAA; HPSE; HPSE2; HEXA; HEXB; HEXD; OGA; HYAL1 ; HYAL2;

[0507] HYAL3; HYAL4; EDEM1; EDEM2; EDEM3; MAN1A1; MAN1A2; MAN1B1;

[0508] MAN1C1; MAN2A1 ; MAN2A2; MAN2B1 ; MAN2B2; MAN2C1 ; MANEA;

[0509] MANEAL; NEU1; NEU2; NEU3; NEU4; GNE; GMDS; GFUS; C1GALT1C1;

[0510] CANX; CALR and PDIA3; SELE; SELP; SELL; CD207; CD209; MRC1;

[0511] CLEC7A; CLEC4E; CLEC12A; ASGR1 ; ASGR2; DCIR; SIGLEC1; SIGLEC2;

[0512] SIGLEC3; SIGLEC4; SIGLEC5; SIGLEC6; SIGLEC7; SIGLEC8; SIGLEC9;

[0513] SIGLEC10; SIGLEC11 ; SIGLEC12; SIGLEC14; SIGLEC15; SIGLEC16;

[0514] LGALS1; LGALS2; LGALS3; LGALS4; LGALS7; LGALS8; LGALS9; LGALS9B;

[0515] LGALS10; LGALS12; LGALS13; LGALS14; LGALS16; SLC35A1; or SLC35C2. 33. The method according to anyone of items 1-12 or 29-32, the isolated glycoengineered extracellular vesicle, or the composition for the use according to anyone of items 16-32, or the use according to anyone of items 16-21 or 26-

[0516] 32, wherein the cell has a total or partial reduction of expression of MGAT1 and / or total or partial reduction of the activity of MGAT1.

[0517] 34. The method according to anyone of items 1-12 or 29-33, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 16-33, or the use according to anyone of items 16-21 or 26-

[0518] 33, wherein the cell has a total or partial reduction of expression of C1GALT1C1 and / or total or partial reduction of the activity of C1GALT1C1.

[0519] 35. The method according to anyone items 1-12 or 29-34, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 16-34, or the use according to anyone of items 16-21 or 26-

[0520] 34, wherein the cell has a total or partial reduction of expression of B4GALT5 and / or total or partial reduction of the activity of B4GALT5.

[0521] 36. The method according to anyone of items 1-12 or 29-35, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 16-35, or the use according to anyone of items 16-21 or 26-

[0522] 35, wherein the cell has a total or partial reduction of expression of B4GALT6 and / or total or partial reduction of the activity of B4GALT6.

[0523] 37. The method according to anyone of items 1-12 or 29-36, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 16-36, or the use according to anyone of items 16-21 or 26-

[0524] 36, wherein the expression of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell, has been modified.

[0525] 38. The method according to anyone of items 1-12 or 29-37, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of item 16-37, or the use according to anyone of items 16-21 or 26-

[0526] 37, wherein the activity of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell have been modified. 39. The method according to anyone of items 1-12 or 29-38, wherein the modification of the expression of the nucleic acid is transient.

[0527] 40. The method according to anyone of items 1-12, or 29-39, wherein the cell is selected from: an animal cell; a plant cell; a fungal cell and a yeast cell.

[0528] 41. The method according to anyone of items 1-12, or 29-40, wherein the cell is a human cell.

[0529] 42. The method according to anyone of items 1-12, or 29-41 , wherein the human cell is selected from the group consisting of: a stem cell; such as a pluripotent cell, such as a hematopoietic stem cell, such as a neural stem cell, such as a mesenchymal stem cell, such as an embryonic stem cell; a precursor cell; a skin cell; a brain cell; and an immune cell.

[0530] 43. The method according to anyone of items 1-12, or 29-42, wherein the stem cell is an adult stem cell, and / or is an artificially induced stem cell, such as an induced pluripotent stem cell, or a cell derived therefrom.

[0531] 44. The method according to anyone of items 1-12, or 29-43, wherein the cell is selected from the group consisting of: a natural killer cell, a chimeric antigen receptor-T cell (CAR-T), a chimeric antigen receptor natural killer cell (CAR- NK), a CAR-NKT cell, a T cell, such as a CD8 T cell or a CD4 T cell, and a dendritic cell.

[0532] 45. The method according to anyone of items 1-12, or 29-44, wherein the cell is a cancer cell.

[0533] 46. The method according to anyone of items 1-12, or 29-45, wherein the cell is selected from the group consisting of: HEK293, such as HEK293F; CHO; MUTZ3; K562; NK92; KHYG-1 ; and KHYK.

[0534] 47. The method according to anyone of items 1-12, or 29-46, wherein the property of interest is increased expression of a molecule of interest in a target cell contacted with said extracellular vesicle compared to expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by a reference cell, and / or wherein the extracellular vesicle leads to an increased expression of a molecule of interest in a target cell contacted with said extracellular vesicle compared to the expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by said reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified. An isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle, obtainable by the method according to anyone of items 1-12, or 29-47. A method to identify an isolated glycoengineered extracellular vesicle, as defined in any one of the preceding items, with a glycosylation pattern of interest comprising the steps of: a. providing an isolated glycoengineered extracellular vesicle, wherein the extracellular vesicle has been obtained from a cell, wherein the expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as defined in any one of the preceding items; b. optionally, determining the extracellular surface glycosylation pattern, c. loading the isolated extracellular vesicle with a molecule of interest d. providing a target cell e. contacting said isolated extracellular vesicles with said target cell f. analyzing target cellular properties of interest, such as target specificity or tropism, total uptake of a molecule, uptake of a molecule per second, delivery efficiency or expression of a nucleic acid or polypeptide.

Claims

Claims1. A method of obtaining a glycoengineered extracellular vesicle, preferably a glycoengineered small extracellular vesicle, with a modified property of interest, said method comprising the steps of: a. providing a cell wherein i. at least one nucleic acid has been modified, wherein the expression of said at least one nucleic acid and / or wherein the activity of a product encoded by said at least one nucleic acid has been modified; and / or ii. wherein at least one nucleic acid has been introduced, resulting in a modified glycosylation of extracellular vesicles produced by the cell, thereby obtaining a glycoengineered extracellular vesicle with a modified property of interest; and b. isolating the extracellular vesicle from said cell, wherein the property of interest is: i. modified nucleic acid transfer, such as gene, RNA or DNA transfer; ii. modified expression of a transferred nucleic acid; iii. increased editing of a target nucleic acid such as a gene; iv. modified target specificity or tropism; v. total uptake of a molecule by the glycoengineered extracellular vesicle; and / or vi. increased delivery of a molecule to a target, and wherein the property of interest is modified compared to an extracellular vesicle obtained from a reference cell in which said at least one nucleic acid, said expression of said at least one nucleic acid sequence and / or said activity of the product encoded by said at least one nucleic acid sequence has not been modified.

2. The method according to claim 1, wherein the modified glycosylation is modified glycosaminoglycans and / or modified sialylation on the surface of the glycoengineered extracellular vesicle, and / or wherein the expression of said at least one nucleic is modified using CRISPR-associated nucleases, such as CRISPR-Cas9, zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), and / or wherein said at least one nucleic acid isintroduced using CRISPR-associated nucleases, such as CRISPR-Cas9, zinc- finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs).

3. The method according to any one of the preceding claims, wherein the at least one nucleic acid is B4GALT7; GNE; B4GALT5; B4GALT6; C1GALT1C1 ; C1GALT1; or MGATI.

4. The method according to any one of the preceding claims, wherein the at least one nucleic acid is: B4GALT7 and / or GNE.

5. The method according to any one of the preceding claims, wherein the cell has a total or partial reduction of expression of GNE and / or total or partial reduction of the activity of GNE, and / or wherein the cell has a total or partial reduction of expression of B4GALT7 and / or total or partial reduction of the activity of B4GALT7.

6. The method according to any one of the preceding claims, wherein the cell has a total or partial reduction of expression of B4GALT7 and / or total or partial reduction of the activity of B4GALT7.

7. The method according to any one of the preceding claims, wherein the cell has a total or partial reduction of expression of GNE and / or total or partial reduction of the activity of GNE.

8. The method according to any one of the preceding claims, wherein the glycoengineered isolated extracellular vesicle binds to at least one marker specific for extracellular vesicles, such as syntenin-1 , ALIX and / or CD63.

9. The method according to any one of the preceding claims, wherein the glycoengineered isolated extracellular vesicle is a small extracellular vesicle and has a diameter of less than 300 nm, such as less than 290 nm, such as less than 280 nm, such as less than 270 nm, such as less than 260 nm, such as less than 250 nm.

10. The method according to any one of the preceding claims, wherein the modified glycosylation comprises or consist of at least one modified glycosaminoglycan,preferably wherein a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, such as an anti-chondroitin sulfate antibody, shows modified binding to the extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

11. The method according to any one of the preceding claims, wherein the modified glycosylation is a modification in the length of glycan chains resulting from modified sialylation or fucosylation, preferably wherein a marker specific for sialylation or fucosylation, such as lectin SNA or LCA, shows modified binding to the extracellular vesicle compared to the binding of same marker to an extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

12. The method according to any one of claims 10-12, wherein binding of said marker specific for glycosaminoglycans, sialylation or fucosylation to the extracellular vesicle compared to the binding of said marker to the extracellular vesicle obtained from the reference cell is modified by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%.

13. An isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in medicine, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified glycosylation and comprising a therapeutic molecule or active ingredient;optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

14. An isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for use in the treatment of a disease and / or a medical condition, in wound healing, in regenerative medicine or in gene therapy in a subject in need thereof, comprising the steps of: providing an isolated glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a therapeutic molecule or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said therapeutic molecule or active ingredient in a composition; and administering said isolated glycoengineered extracellular vesicle or composition to a subject in need thereof; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

15. Use of an isolated glycoengineered extracellular vesicle, or a composition comprising an isolated glycoengineered extracellular vesicle for the delivery of a molecule to a cell, the use comprising the steps of: providing a glycoengineered extracellular vesicle having a modified extracellular glycosylation and comprising a molecule, such as a therapeutic molecule, or active ingredient; optionally, formulating said isolated glycoengineered extracellular vesicle loaded with said molecule or active ingredient in a composition; preferably wherein the glycoengineered extracellular vesicle is a small extracellular vesicle.

16. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of claims 13-14, or the use according to claim 15, wherein providing the isolated glycoengineered extracellular vesicle comprises the method according to any one of claims 1-12.

17. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of claims 13-14 or 16, or the use according to claim 15-16, wherein the modified glycosylation is modified glycosaminoglycans and / or modified sialylation on the surface of the glycoengineered extracellular vesicle.

18. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of claims 13-14 or 16-17, or the use according to claim 15-17 wherein the molecule, the therapeutic molecule, or the active ingredient, is a large nucleic acid and / or a large polypeptide, wherein said large nucleic acid is a nucleic acid, such as a DNA, for example a gene, or an RNA, having a size of at least 1500 bp, such as at least 2000 bp, such as at least 3000 bp, such as at least 4000 bp, such as at least 4700 bp, and / or wherein the large polypeptide is a polypeptide such as a protein having a size of at least 500 amino acids, such as at least 600, at least 800, at least 1000, at least 1200, at least 1500 or at least 1700 amino acids.

19. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of claims 13-14 or 16-18, or the use according to claim 15-18, wherein the molecule or active ingredient is a DNA molecule, or an RNA molecule, such as an mRNA molecule, an siRNA molecule, a miRNA molecule, a snoRNA molecule, an ncRNA molecule, a gRNA molecule; a small molecule, such as a drug, such as an inhibitor, such as a chemotherapeutic drug, such as a toxin; a polypeptide; or a dye.

20. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of claims 16-19, or the use according to claim 16-19, wherein the at least one nucleic acid is B4GALT7; GNE; B4GALT5; B4GALT6; C1GALT1; C1GALT1C1; or MGATI.

21. The isolated glycoengineered extracellular vesicle or the composition for the use according to any one of claims 16-20, or the use according to claim 16-20, wherein the at least one nucleic acid is B4GALT7 and / or GNE.

22. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claims 13-14 or 16-21, wherein the disease and / ormedical condition is a cancer; a neurodegenerative disease; a cardiovascular disease; an autoimmune disease; a metabolic disorder; a genetic disorder; or a skin disease.

23. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claims 13-14 or 16-22, wherein the disease is a monogenic disease.

24. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claims 13-14 or 16-23, wherein the disease is a pulmonary disease, such as cystic fibrosis.

25. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claims 13-14 or 16-24, wherein the therapeutic molecule is a nucleic acid comprising or consisting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, or a polypeptide comprising or consisting of the CFTR protein, or fragments thereof.

26. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 13-14 or 16-25, or the use according to anyone of claims 15-21 wherein the modified glycosylation comprises or consist of at least one modified glycosaminoglycan, preferably wherein a marker specific for glycosaminoglycans, such as a marker for chondroitin sulfate, such as an anti-chondroitin sulfate antibody, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to another extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

27. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 13-14 or 16-26, or the use according to anyone of claims 15-21 or 26, wherein the modified glycosylation is a modification in the length of glycan chains resulting from modified sialylation orfucosylation, preferably wherein a marker specific for sialylation or fucosylation, such as lectin SNA or LCA, shows modified binding to the glycoengineered extracellular vesicle compared to the binding of same marker to an extracellular vesicle obtained from a reference cell, wherein said reference cell is a cell in which the expression of said at least one nucleic acid and / or the activity of the product encoded by said at least one nucleic acid has not been modified, and / or wherein the modified glycosylation is determined by glycoanalytical methods such as mass spectrometry, HPLC and combinations thereof.

28. The isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-27, or the use according to anyone of claims 16-21 or 26-27, wherein the binding of said marker to the extracellular vesicle obtained from the reference cell is modified by at least 10%, such as at least 20%, such as at least 30%, such as at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, 96%, 97%, 98%, 99% or 100%.

29. The method according to anyone of claims 1-12, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-28, or the use according to anyone of claims 16-21 or 26-28, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of an mRNA encoded by the target nucleic acid; e. inhibition of the activity of a protein encoded by the target nucleic acid; f. knock-in of an additional sequence encoding the product of the target nucleic acid; g. upregulation of the transcription of the target nucleic acid; h. upregulation of the translation of the mRNA encoded by the target nucleic acid; i. stimulation of the activity of the protein encoded by the target nucleic acid; orj. introduction of another nucleic acid in the cell.

30. The method according to anyone of claims 1-12 or 29, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-29, or the use according to anyone of claims 16-21 or 26-29, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by: a. knock-out of the target nucleic acid; b. knock-down of the target nucleic acid; c. downregulation of the transcription of the target nucleic acid; d. downregulation of the translation of the mRNA encoded by the target nucleic acid; or e. inhibition of the activity of the protein encoded by the target nucleic acid.

31. The method according to anyone of claims 1-12 or 29-30, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-30, or the use according to anyone of claims 16-21 or 26-30, wherein the expression of said at least one nucleic acid and / or the activity of said product encoded by at least one nucleic acid has been modified by: a. knock-in of an additional sequence encoding the product of the target nucleic acid; b. upregulation of the transcription of the target nucleic acid; c. upregulation of the translation of the mRNA encoded by the target nucleic acid; or d. stimulation of the activity of the protein encoded by the target nucleic acid.

32. The method according to anyone of claims 1-12 or 29-31, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of 16-31 , or the use according to anyone of claims 16-21 or 26-31, wherein the at least one nucleic acid is PIGA; PIGM; PIGV; PIGB; PIGZ; PGAP4; UGT8; UGCG; B4GALT5; B4GALT6; B3GALNT1; A4GALT;B4GALNT1 ; B3GNT5; DPAGT1; ALG13; ALG14; ALG1 ; ALG2; ALG11; DPMI; UGGT1; UGGT2; ALG3; ALG5; ALG9; ALG12; ALG6; ALG8; ALG10; ALG10B; STT3A; STT3B; MGAT1; FUT8; MGAT2; MGAT3; MGAT5; MGAT4A;MGAT4B; MGAT4C; MGAT4D; GALNT1 ;GALNT2; GALNT3; GALNT4;GALNT5; GALNT6; GALNT7; GALNT8; GALNT9; GALNT10; GALNT11;GALNT12; GALNT13; GALNT14; GALNT15; GALNT16; GALNTL6 (T17);GALNT18; GALNT17 (T19); GALNTL5 (T20); C1GALT1; GCNT1; GCNT3;GCNT4; B3GNT6; POFUT1 ; POFUT2; B3GLCT; LFNG; MFNG; RFNG;POMT1; POMT2; POMGNT1 ; MGAT5B; POMGNT2; B3GALNT2; FKRP;FKTN; B4GAT1; RXYLT1; LARGE1 ; LARGE2; TMTC1 ; TMTC2; TMTC3;TMTC4; DPY19L1 ; DPY19L2; DPY19L3; DPY19L4; POGLUT1 ; POGLUT2;POGLUT3; GXYLT1; GXYLT2; XXYLT1 ; XYLT1; XYLT2; B4GALT7; B3GALT6;B3GAT3; CSGALNACT1; CSGALNACT2; CHPF; CHPF2; CHSY1 ; CHSY3;EXT1 ; EXT2; EXTL1; EXTL2; EXTL3; COLGALT1; COLGALT2; OGT; EOGT;B3GALT1; B3GALT2; B3GALT4; B3GALT5; B3GNT2; B3GNT3; B3GNT4;B3GNT7; B3GNT8; B3GNT9; B4GALNT3; B4GALNT4; B4GALT1 ; B4GALT2;B4GALT3; B4GALT4; GCNT2; GCNT7; A4GNT; ABO; B3GAT1; B3GAT2;B4GALNT2; FUT1 ; FUT10; FUT11 ; FUT2; FUT3; FUT4; FUT5; FUT6; FUT7;FUT9; ST3GAL1; ST3GAL2; ST3GAL3; ST3GAL4; ST3GAL5; ST3GAL6;ST6GAL1; ST6GAL2; ST6GALNAC1; ST6GALNAC2; ST6GALNAC3;ST6GALNAC4; ST6GALNAC5; ST6GALNAC6; ST8SIA1; ST8SIA2; ST8SIA3;ST8SIA4; ST8SIA5; ST8SIA6; CHST1; CHST10; CHST11 ; CHST12; CHST13;CHST14; CHST15; CHST2; CHST3; CHST4; CHST5; CHST6; CHST7; CHST8;CHST9; GAL3ST1; GAL3ST2; GAL3ST3; GAL3ST4; HS2ST1 ; HS3ST1;HS3ST2; HS3ST3A1; HS3ST3B1; HS3ST4; HS3ST5; HS3ST6; HS6ST1;HS6ST2; HS6ST3; NDST1 ; NDST2; NDST3; NDST4; UST; DSEL; DSE; GLCE;TPST1 ; TPST2; GNPTAB; GNPTG; NAGPA; M6PR; IGF2R; FUCA1 ; FUCA2;GLA; NAGA; GLB1; GLB1 L; GLB1L2; GLB1 L3; GBA1; GBA2; GBA3; MOGS;GANAB; GAA; HPSE; HPSE2; HEXA; HEXB; HEXD; OGA; HYAL1 ; HYAL2;HYAL3; HYAL4; EDEM1; EDEM2; EDEM3; MAN1A1; MAN1A2; MAN1B1;MAN1C1; MAN2A1 ; MAN2A2; MAN2B1 ; MAN2B2; MAN2C1 ; MANEA;MANEAL; NEU1; NEU2; NEU3; NEU4; GNE; GMDS; GFUS; C1GALT1C1;CANX; CALR and PDIA3; SELE; SELP; SELL; CD207; CD209; MRC1;CLEC7A; CLEC4E; CLEC12A; ASGR1 ; ASGR2; DCIR; SIGLEC1; SIGLEC2;SIGLEC3; SIGLEC4; SIGLEC5; SIGLEC6; SIGLEC7; SIGLEC8; SIGLEC9;SIGLEC10; SIGLEC11 ; SIGLEC12; SIGLEC14; SIGLEC15; SIGLEC16;LGALS1; LGALS2; LGALS3; LGALS4; LGALS7; LGALS8; LGALS9; LGALS9B;LGALS10; LGALS12; LGALS13; LGALS14; LGALS16; SLC35A1; or SLC35C2.

33. The method according to anyone of claims 1-12 or 29-32, the isolated glycoengineered extracellular vesicle, or the composition for the use according to anyone of claim 16-32, or the use according to anyone of claims 16-21 or 26-32, wherein the cell has a total or partial reduction of expression of MGAT1 and / or total or partial reduction of the activity of MGAT1.

34. The method according to anyone of claims 1-12 or 29-33, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-33, or the use according to anyone of claims 16-21 or 26-33, wherein the cell has a total or partial reduction of expression of C1GALT1C1 and / or total or partial reduction of the activity of C1GALT1C1.

35. The method according to anyone of claims 1-12 or 29-34, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-34, or the use according to anyone of claims 16-21 or 26-34, wherein the cell has a total or partial reduction of expression of B4GALT5 and / or total or partial reduction of the activity of B4GALT5.

36. The method according to anyone of claims 1-12 or 29-35, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-35, or the use according to anyone of claims 16-21 or 26-35, wherein the cell has a total or partial reduction of expression of B4GALT6 and / or total or partial reduction of the activity of B4GALT6.

37. The method according to anyone of claims 1-12 or 29-36, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-36, or the use according to anyone of claims 16-21 or 26-36, wherein the expression of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell, has been modified.

38. The method according to anyone of claims 1-12 or 29-37, the isolated glycoengineered extracellular vesicle or the composition for the use according to anyone of claim 16-37, or the use according to anyone of claims 16-21 or 26-37, wherein the activity of two or more nucleic acids, such as three or more, such as four or more nucleic acids of the cell have been modified.

39. The method according to anyone of claims 1-12 or 29-38, wherein the modification of the expression of the nucleic acid is transient.

40. The method according to anyone of claims 1-12, or 29-39, wherein the cell is selected from: an animal cell; a plant cell; a fungal cell and a yeast cell.

41. The method according to anyone of claims 1-12, or 29-40, wherein the cell is a human cell.

42. The method according to anyone of claims 1-12, or 29-41 , wherein the human cell is selected from the group consisting of: a stem cell; such as a pluripotent cell, such as a hematopoietic stem cell, such as a neural stem cell, such as a mesenchymal stem cell, such as an embryonic stem cell; a precursor cell; a skin cell; a brain cell; and an immune cell.

43. The method according to anyone of claims 1-12, or 29-42, wherein the stem cell is an adult stem cell, and / or is an artificially induced stem cell, such as an induced pluripotent stem cell, or a cell derived therefrom.

44. The method according to anyone of claims 1-12, or 29-43, wherein the cell is selected from the group consisting of: a natural killer cell, a chimeric antigen receptor-T cell (CAR-T), a chimeric antigen receptor natural killer cell (CAR- NK), a CAR-NKT cell, a T cell, such as a CD8 T cell or a CD4 T cell, and a dendritic cell.

45. The method according to anyone of claims 1-12, or 29-44, wherein the cell is a cancer cell.

46. The method according to anyone of claims 1-12, or 29-45, wherein the cell is selected from the group consisting of: HEK293, such as HEK293F; CHO; MUTZ3; K562; NK92; KHYG-1 ; and KHYK.

47. The method according to anyone of claims 1-12, or 29-46, wherein the property of interest is increased expression of a molecule of interest in a target cell contacted with said glycoengineered extracellular vesicle compared toexpression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by a reference cell, and / or wherein the extracellular vesicle leads to an increased expression of a molecule of interest in a target cell contacted with said glycoengineered extracellular vesicle compared to the expression of said molecule of interest in said target cell contacted with another extracellular vesicle produced by said reference cell, wherein the reference cell is a cell in which expression of the at least one nucleic acid sequence and / or the activity of the product encoded by the at least one nucleic acid sequence has not been modified.

48. An isolated glycoengineered extracellular vesicle or the composition obtainable by the method according to anyone of claims 1-12, or 29-47.

49. A method to identify an isolated glycoengineered extracellular vesicle, as defined in any one of the preceding claims, with a glycosylation pattern of interest comprising the steps of: a. providing an isolated glycoengineered extracellular vesicle, wherein the extracellular vesicle has been obtained from a cell, wherein the expression of at least one nucleic acid in the cell and / or the activity of a product encoded by the at least one nucleic acid in the cell has been modified as defined in any one of the preceding claims; b. optionally, determining the extracellular surface glycosylation pattern, c. loading the isolated extracellular vesicle with a molecule of interest d. providing a target cell e. contacting said isolated extracellular vesicles with said target cell f. analyzing target cellular properties of interest, such as target specificity or tropism, total uptake of a molecule, uptake of a molecule per second, delivery efficiency or expression of a nucleic acid or polypeptide.

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