Methods for Producing Delivery Vesicles

Abstract

The present invention relates to the development of vesicles that could be used for the production of vaccines or as compound delivery vehicles. More specifically, the present invention relates to a method for preparing vesicles comprising the steps of providing recombinant Trypanosoma brucei cells expressing a sortagable VSG, treating said cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed, isolating cell membranes from the solution, suspending the isolated membranes already obtained in an isotonic solution, treating the suspended cell membranes obtained in the preceding step with sonication to obtain a vesicle suspension, removing aggregated membranous debris from the vesicle suspension already obtained, separating the vesicle suspension into a population of vesicles, and providing a vesicle from the population of vesicles characterized by the following parameters: (i) having a single predominant protein, revealed after Coomassie staining of SDS-PAGE, with an apparent molecular weight of 55-60 kDa, (ii) having a spherical appearance in electron micrographs, and (iii) showing a homogenous surface structure in electron micrographs. Furthermore, the present invention also relates to vesicles comprising sortagable VSG characterized by the above-mentioned parameters and to such vesicles for use in the treatment and / or prevention of a disease or medical condition or as a compound delivery vesicle, preferably a drug delivery vehicle, more preferably a nucleic acid delivery vesicle.Finally, the present invention contemplates a kit for performing the method of the present invention, comprising recombinant Trypanosoma brucei cells expressing a sortagable VSG and at least one agent for performing the method of the present invention or a kit comprising a vesicle of the present invention.

Description

[Technical field]

[0001] The present invention relates to the development of vesicles that could be used for the production of vaccines or as compound delivery vehicles. More specifically, the present invention relates to a method for the development of a vesicle that could be used for the production of vaccines or as a compound delivery vehicle, comprising the steps of providing recombinant Trypanosoma brucei cells expressing a sortaggable VSG, treating said cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed, isolating cell membranes from the solution, suspending the isolated membranes already obtained in an isotonic solution, treating the suspended cell membranes obtained in the preceding step with sonication to obtain a vesicle suspension, removing aggregated membranous debris from the vesicle suspension already obtained, separating the vesicle suspension into a population of vesicles, and satisfying the following parameters: (i) having a single predominant protein, revealed after Coomassie staining of SDS-PAGE, with an apparent molecular weight of 55-60 kDa, (ii) having a spherical appearance in electron micrographs, and (iii) The present invention also relates to a method for preparing a vesicle, comprising the step of providing a vesicle from a population of vesicles characterized by exhibiting a homogenous surface structure in electron micrographs. Furthermore, the present invention also relates to vesicles comprising a sortagable VSG characterized by the above-mentioned parameters and to such vesicles for use in the treatment and / or prevention of a disease or medical condition or as a compound delivery vesicle, preferably a drug delivery vehicle, more preferably a nucleic acid delivery vesicle. Finally, the present invention contemplates a kit for performing the method of the present invention, comprising recombinant Trypanosoma brucei cells expressing a sortagable VSG and at least one agent for performing the method of the present invention, or a kit comprising a vesicle of the present invention. [Background technology]

[0002] Despite its promise, the field of gene therapy has not overcome the crucial dilemma of how to deliver targeted therapy to specific target sites with efficacy.Conventional drugs can be fused to cell type specific ligands or antibodies.However, this unfortunately does not apply to nucleic acids due to poor pharmacokinetics, susceptibility to enzymatic destruction, and large size.To realize the promise of gene therapy, new tools are needed.

[0003] Adenoviruses or AAV: Efficient targeting, but poor loading (also immunogenic). The majority of delivery systems utilized by the field include viruses, which are essentially nucleic acid packaging systems coated in proteins that are responsible for driving the uptake of their genetic information into specific target cells by binding to surface receptors. Several viruses are in clinical trials for gene therapy purposes, often with adverse effects (e.g., retroviruses with "off-target" oncogenic effects). Adeno-associated viruses (AAVs), engineered to be non-replicative, have the particular advantage that virions can acquire different tropisms based on the exact composition of their capsid (Kuzmin et al., 2021). The combination of AAV serotypes (and therefore tissue targeting) with specific promoters driving expression (e.g., of encapsidated RNA) is desired to achieve tissue-restricted delivery. Substantial interest in this approach has been boosted by the recent approval of two AAV-based gene therapies by the US FDA.

[0004] However, despite its promise, there are several problems associated with AAV-based gene therapy approaches. First, 30–60 percent of humans have pre-existing antibodies against some AAV serotypes, leading to lack of efficacy even with the first dose (Kruzik et al., 2019). Second, the packaging efficiency of recombinant AAV is limited to nucleic acids smaller than 5 kb in size (Wu et al., 2010). Third, AAV is usually produced recombinantly by transfection, which poses scalability issues (notably, clinical trials report the administration of 1012–1013 recombinant (r)AAV genome copies per kg body weight for liver transduction gene therapy (Miesbach et al., 2018), thus reaching the acceptable cost limit of current production technologies). Finally, it is noted that approximately 90% of rAAV capsids are “empty” and, in addition to being ineffective, may serve as a possible source of toxicity, although some argue that empty particles could also be used as decoys to suppress existing immunity (Gao et al., 2014).

[0005] LNPs: Highly efficient loading and delivery, but poor targeting. These issues and additional difficulties with other viruses have led many to propose nanoparticle-based methods for gene therapy delivery. Nanoparticles can be broadly distinguished into polymer-based and lipid-based nanoparticles (LNPs). Polymer-based nanoparticles vary by the synthesis of the backbone (e.g., polyamide or polypropyleneimine backbone) and the subsequent selection of size, charge, and composition, which makes them diverse in terms of structure and properties. LNPs can be formulated by combining various biomaterials with cholesterol, lipid-PEG compounds, and other lipids, and are now widely used, for example, in the context of Sars-CoV-2 vaccines. In both cases, the resulting libraries of nanoparticles (generated by varying the molar ratios of the different components) have demonstrated some degree of tissue selectivity (Lokugamage et al., 2018). While promising, this approach relies on (unknown and not understood) similarities that could determine the translatability of targeting between humans and mice, and therefore the possibility of using model systems to understand nanoparticle targeting in humans. The conclusion is that LNPs are not targetable. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Kuzmin et al., 2021 [Non-Patent Document 2] Kruzik et al., 2019 [Non-Patent Document 3] Wu et al., 2010 [Non-Patent Document 4] Miesbach et al., 2018 [Non-Patent Document 5] Gao et al., 2014 [Non-Patent Document 6] Lokugamage et al., 2018 Summary of the Invention [Problem to be solved by the invention]

[0007] However, since many pharmaceutical or biotechnological applications require both, there is a need for delivery systems that can be efficiently loaded and that are highly efficient in terms of targeting as well. [Means for solving the problem]

[0008] The technical problem underlying the present invention is seen to be the provision of means and methods for meeting the above-mentioned needs. The technical problem is solved by the embodiments characterized hereinbelow and in the claims. That is, the present invention comprises the following steps: (a) providing a recombinant Trypanosoma brucei cell expressing a VSG, preferably a sortagable VSG; (b) treating the cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed; (c) isolating the cell membranes from the solution of step (b); (d) suspending the isolated membranes obtained in step (c) in an isotonic solution; (e) treating the suspended cell membranes obtained in step (d) using sonication to obtain a vesicle suspension; (f) removing aggregated membranous residue from the vesicle suspension obtained in step (e); (g) separating the vesicle suspension into populations; and (h) providing a vesicle from a population of vesicles characterized by the following parameters: (i) having a single predominant protein with an apparent molecular weight of 55-60 kDa, as revealed after Coomassie staining of SDS-PAGE; (ii) having a spherical appearance in electron micrographs; and (iii) exhibiting a homogenous surface structure in electron micrographs. The present invention relates to a method for preparing a vesicle, comprising: [Brief description of the drawings]

[0009] [Figure 1] Schematic diagram showing the generation and utility of nanoVAST. (A) nanoVAST is generated by sonication of an osmotically lysed cell preparation of the unicellular eukaryote Trypanosoma brucei. After sonication and a series of filtration and centrifugation steps, the sonicated membranes are purified (e.g., loaded onto a size-exclusion column for purification). (B) nanoVAST are cargo-bearing vesicles coated with the T. brucei surface protein VSG that can deliver cargo to target cells. An example of nanoVAST carrying a nucleic acid cargo is shown here, and the nanoVAST is subsequently delivered to the target cell; in this case, B cells. (C) Because nanoVAST is coated with a sortagable VSG protein, it can be coupled to a sortagable ligand. Conjugation of nanoVAST to a nanobody specific for a B cell protein is shown, which subsequently promotes an increased uptake rate compared to (B). (D) Attachment of a cell type-specific ligand to nanoVAST can not only increase the rate of delivery to a cell, but can also specifically promote delivery to that cell type among a population of additional off-target cell types. [Diagram 2]Figure 2. NanoVAST vesicle generation and characterization. (A-B) Gel filtration chromatograms of sonicated T. b. brucei membrane lysate isolated from material equivalent to 5 billion cells after separation on Superose 6 Increase and Sephacryl S-500 resins. The Y-axis indicates the total amount of protein eluted at any time throughout the run via absorbance at 280 nanometers. (C) SDS-PAGE and Coomassie staining of the elution peak from A. VSG predominates as the major protein present in nanoVAST. (C) Dynamic light scattering of purified nanoVAST reveals that they are uniform and average approximately 250 nM in diameter. [Diagram 3] Cryo-electron micrographs of nanoVAST vesicles. (A) Images were acquired on a Talos Arctica after embedding of nanoVAST preparations onto UltrAuFoil grids. This two-dimensional representation of nanoVAST strengthens the hypothesis that the phospholipid bilayer is very densely covered by VSG proteins (hair-like structures around a circle across the membrane), similar to the density present on native membranes of living organisms. Furthermore, the scale bar (50 nM) supports the finding that nanoVAST is approximately 200-250 nM in diameter. (B) Images were acquired on a Zeiss (Zeiss EM912 microscope, 80 kV (Carl Zeiss, Oberkochen, Germany)) after adsorption of nanoVAST from purified preparations onto glow-discharged carbon-coated copper grids (mesh size 300), washing twice in distilled water, and negatively staining with 1% aqueous uranyl acetate. Scale bar (100 nm) supports previous findings of vesicles in the 200-250 nm range. [Figure 4]Figure 1. Loading of nanoVAST vesicles by electroporation. (A) Flow cytometry data collected from fluorescent cargo-loaded nanoVAST after electroporation using five different program settings on an Amaxa Nucleofector. Program U033 is the most effective for loading of nanoVAST. (B) Chart depicting the U033 setting and only unstained nanoVAST, both from (A). [Diagram 5] Figure 1 shows nanoVAST cargo delivery to target cells. Flow cytometry data collected from HEK cells incubated with cargo-loaded nanoVAST. In (A), nanoVAST were loaded by electroporation with the fluorescent molecule FAM as a representative of therapeutic small molecule drugs. In (B), nanoVAST were loaded with fluorescent (Cy5)-labeled short RNA oligomers as a representative for, e.g., CRISPR guide RNAs. [Figure 6]Figure 1: VSG is detectable on the surface of nanoVAST-targeted cells. Flow cytometry data collected from (A) HEK cells and (B) RamosB cells incubated with nanoVAST. Cells were subsequently stained with anti-VSG antibodies, which revealed that VSG protein can be found on the surface of treated cells for at least some time after treatment. This supports the hypothesis that nanoVAST cargo can be delivered to target cells through membrane fusion (as opposed to endocytosis, which would not deposit VSG on the cell surface). (C) Possible routes of nanoVAST entry and delivery into cells. Fusion to the cell membrane with simultaneous release of cargo (i) or via endocytosis with delayed release of cargo (ii). The fused vesicles are eventually pinched off (iii) and fuse to multivesicular bodies (iv). Release of cargo via endocytosis can also occur in multivesicular bodies through retrofusion (v). (D) Immunofluorescence microscopy of fixed and permeabilized nanoVAST-treated HEK cells. Multichannel composite images show colocalization of nanoVAST with membrane-derived Rab11 endosomes adjacent to the cell nucleus. Cells were fixed, permeabilized, and stained 8 hours after addition of nanoVAST particles. Nuclei and membranes were visualized with DAPI (4',6-diamidino-2-phenylindole dihydrochloride) and WGA (Alexa Flour594-conjugated wheat germ agglutinin), respectively. Recycling endosomes were stained with rabbit monoclonal anti-RAB11 (Cell Signaling Technology, #5589T) and anti-rabbit IgG Alexa Fluor488-conjugated (Invitrogen, #A-11008). nanoVAST was stained with mouse monoclonal anti-VSG3 and anti-mouse IgG-Abberior® STAR RED (Sigma, 52283). Images were acquired using a confocal microscope Leica TCS SP5 II. [Figure 7]Figure 3: Targeting nanoVAST to specific cell types through sortagging enhances delivery. (A) nanoVAST was sortagged to anti-CD19 nanobody and incubated with RamosB cells, then stained with anti-VSG antibody. The amount of VSG deposited on the target cell membrane was increased compared to non-specific nanoVAST delivery. This experiment is similar to the schematic shown in Example 4, Figure 6B. (B) nanoVAST sortagging was verified through FACS analysis. Flow cytometry data of nanoVAST prepared from VSG3-expressing T. brucei sortagged with the fluorescent molecule TAMRA to track and measure vesicle sortagging. [Figure 8-1] Figure 1. NanoVAST vesicle production and characterization by differential centrifugation. (A) Method for isolating nanoVAST from sonicated T. brucei membranes via repeated centrifugation steps. NanoVAST is expected to remain in the supernatant at speeds below 20,000g. All centrifugations are performed at 4°C. (B) Dynamic light scattering of purified pelleted nanoVAST compared to purified nanoVAST from FPLC or gel filtration columns reveals that they are also uniform but smaller, averaging approximately 150 nm in diameter. (C) Transmission electron microscopy reveals spherical structures with an electron-dense coating; nanoVAST. (D) SDS-PAGE analysis by Coomassie staining of pelleted nanoVAST reveals that VSG dominates as the major protein present in the preparation. [Figure 8-2] Continued from Figure 8-1. [Figure 9-1]Schematic diagram and readouts of supplementary polishing steps during nanoVAST delivery vesicle preparation. (A) Insertion of polishing steps using CaptoCore resin can be at single or multiple time points during preparation of the final nanoVAST delivery particles, e.g., before (i) or after (ii), after sortagging (iii) and before administration (iv). (B) FPLC chromatogram after CaptoCore resin-based polishing (post-purification step ii) of nanoVAST preparation. The y-axis indicates the total amount of protein eluting at any time point throughout the entire run via absorbance at 280 nanometers. (C) Electron microscopy images were acquired of the nanoVAST elution peak from (B) after adsorption onto glow-discharged carbon-coated copper grids (mesh size 300), washed in double-distilled water and negatively stained with 1% aqueous uranyl acetate. Vesicles remain the appropriate size after CaptoCore purification. (D) Dynamic light scattering of the eluted nanoVAST peak from (B) reveals that nanoVAST is uniform and averages approximately 150 nm in diameter. (E) SDS-PAGE and Coomassie staining of the eluted peak from (B). VSG predominates as the major protein present in nanoVAST. [Figure 9-2] Continued from Figure 9-1. [Figure 10-1]Figure 1: Freeze-drying as an alternative method of loading nanoVAST vesicles. (A) To load RNA cargo into nanoVAST using the freeze-drying method, 9 μg VSG protein corresponding to nanoVAST was mixed with 42 pmol Cy5-tagged RNA in an RNase-free centrifuge tube. The amount of nanoVAST used was determined by SDS-PAGE quantification with reference to a VSG3 protein standard. Samples were loaded into a freeze-dryer (Alpha 1-2 LSCbasic-Martin Christ) and the drying time required was dependent on the volume of the sample. The freeze-dried samples were subsequently rehydrated with HEPES buffer (20 mM HEPES, 150 mM NaCl), mixed by pipetting up and down, and vortexed gently. Samples were then centrifuged at 20,000 g for 30 min at 4° C. to remove any unloaded RNA cargo. The resulting nanoVAST pellet was resuspended in 1x PBS to assess RNA loading and for HEK cell feeding experiments. (B) Dynamic light scattering graph showing the size distribution of lyophilized-rehydrated nanoVAST vesicles. The vesicles are uniform and have an average diameter of approximately 200 nm. (C) Transmission electron microscopy image of nanoVAST vesicles. The image confirms that the structure of nanoVAST is preserved after lyophilization. (D) SDS-PAGE analysis of lyophilized-hydrated nanoVAST showing VSG3 as the predominant protein. (E) Western blot verifying that the nanoVAST surface protein VSG3 is preserved after the lyophilization process. (F) Flow cytometry data collected from lyophilized-hydrated nanoVAST loaded with Cy5-tagged RNA by lyophilization and subsequent rehydration, resulting in efficient RNA loading. [Figure 10-2] Continued from Figure 10-1. [Figure 11A]Cargo uptake by HEK cells through electroporation and lyophilization. (A) HEK cells delivered nanoVAST loaded with Cy5-tagged RNA by electroporation. The chart shows HEK cells that took up nanoVAST as indicated by a positive VSG3 signal (i). VSG3-negative (ii) and VSG3-positive (iii) were selected and assessed for the presence of Cy5 fluorescence as an indicator of RNA uptake. VSG3-positive cells showed significant uptake of Cy5-tagged RNA (iii). [Figure 11B] Figure 10 shows cargo uptake by HEK cells through electroporation and lyophilization. (B) In comparison, HEK cells were fed nanoVAST loaded with Cy5-tagged RNA by lyophilization as described in Figure 10A. The chart shows HEK cells that took up nanoVAST as indicated by a positive VSG3 signal (i). VSG3-negative (ii) and VSG3-positive (iii) were selected and assessed for the presence of Cy5 fluorescence as an indicator of RNA uptake. VSG3-positive cells showed significant uptake of Cy5-tagged RNA (iii). [Figure 12] FIG. 1 shows that nanoVAST classification can be altered through VSG modification or selection. nanoVAST types can be altered through (i) the use of peptide, e.g., peptide ligand, antibody, or nanobody sortagging; (ii) the use of VSG gene modification through the addition of encoded targeting peptide tags, e.g., those listed in Table 1; and (iii) complete VSG switching through selection from the approximately 2000 different VSG genes encoded in the T. brucei genome. Examples of portions of VSG proteins (top view) with exposed regions as molecular surfaces shaded according to polarity (white to gray: polar to hydrophobic). Differences in surface topography, polarity, or other physical properties can affect nanoVAST cell tropism and physical properties. [Figure 13]Purification of nanoVAST from T. brucei expressing the Iltat (ILTat1.24) protein as an alternative to VSG3. (A) Dynamic light scattering of Iltat nanoVAST purified using the differential centrifugation method described in Figure 8 reveals that they are uniform and on average about 150 nm in diameter, similar to VSG3 nanoVAST. (B) Electron microscopy images of Iltat nanoVAST were acquired after adsorption onto glow-discharged carbon-coated copper grids (mesh size 300), washed in double-distilled water and negatively stained with 1% aqueous uranyl acetate. The scale bar (250 nm) supports previous findings of vesicles in the 200-250 nm range. (C) SDS-PAGE and Coomassie staining of Iltat nanoVAST. Iltat predominates as the major protein present in nanoVAST, indicating that successful purification of different nanoVAST types is possible. (D) nanoVAST sortagging was verified through FACS analysis. Flow cytometry data from nanoVAST prepared from ILtat-expressing T. brucei sortagged with the fluorescent molecule TAMRA to track and measure vesicle sortagging. [Figure 14] Figure 1 shows that RAW264.7 macrophages do not produce TNF-α in response to stimulation with nanoVAST. Cells were stimulated with inactivated E. coli, 100 nM monophosphoryl lipid A (MPLA), and 10 nM nanoVAST. Media was collected from wells at the indicated time points for ELISA analysis. Each data point represents the mean ± SD of triplicate wells. The Y-axis shows the raw absorbance ELISA signal from the plate reader. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] It should be understood that, throughout this specification and the claims, "a" and "an" can refer to one or more of the subsequently referenced items, depending on the context in which it is used. Thus, for example, a reference to "an" item can mean that at least one item is available.

[0011] When used below, the terms "having", "including" or "including" are meant to have an open-ended or closed-ended meaning. That is, when they have a closed-ended meaning, they may refer to a situation in which there are no other features in the described embodiment other than the features introduced by these terms, i.e., they have a closed-ended meaning in the sense of "consisting of" or "consisting essentially of". When they have a closed-ended meaning, they refer to a situation in which there are one or more other features in the described embodiment other than the features introduced by these terms.

[0012] Furthermore, as used below, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "typically" and "more typically" are used in conjunction with features to indicate that these features are preferred features, i.e., these terms will indicate that alternative features may also be contemplated in accordance with the present invention.

[0013] Furthermore, it will be understood that the term "at least one" as used herein means that one or more of the items referenced following this term can be used according to the present invention. For example, when this term indicates that at least one item will be used, this can be understood as one item or two or more items, i.e., two, three, four, five or any other number. Depending on the item to which this term refers, a person skilled in the art will understand what upper limit, if any, this term can refer to.

[0014] The term "about" as used herein means that there is an accuracy of an interval within which the technical effect can be achieved for any number listed after the term. Thus, about as referred to herein preferably means the exact numerical value or a range around that exact numerical value of ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5%.

[0015] The term "comprising" as used herein shall not be understood in a limiting sense. Rather, the term indicates that there may be more than the actual item referenced; for example, when referring to a method comprising a step, the presence of further steps shall not be excluded. However, the term also encompasses embodiments in which only the referenced item is present, i.e., it has a limiting meaning in the sense of "consisting of."

[0016] The method of the invention may consist of the steps mentioned above or may include further steps such as a step for generating recombinant Trypanosoma brucei cells expressing a VSG, preferably a sortagable VSG, or a step aimed at sortagging or coupling a desired compound to the VSG by a sortase or chemical reaction. Furthermore, there could be additional steps within or between the above steps. For example, step (a) could also include a step of sortagging or coupling a desired compound to the VSG by a sortase or chemical reaction. Alternatively, the sortagging or coupling could be performed after the step of providing the vesicles, i.e. after step (g).

[0017] In step (a) of the method of the invention, a recombinant Trypanosoma brucei cell expressing a VSG, preferably a sortagable VSG, is provided. The term "recombinant Trypanosoma brucei cells" refers to a single-cell parasite. Trypanosoma brucei is the pathogenic organism that causes sleeping sickness. Trypanosoma brucei, as referred to herein, encompasses all subspecies such as Trypanosoma brucei brucei, Trypanosoma brucei gambiense, and Trypanosoma brucei rhodesiense. Trypanosoma brucei cells can be genetically modified, thus allowing for the generation of recombinant organisms. Trypanosoma brucei displays a glycoprotein coat on its surface that includes a variant surface glycoprotein (VSG).

[0018] The term "VSG" as used herein refers to a glycoprotein belonging to a class of glycoproteins of about 60 kDa as monomers that can be found as monomers, dimers, trimers or multimers that are densely arranged on the outer membrane to form the surface coat. VSG dimers constitute about 90% of all cell surface proteins in trypanosomes. VSG is highly immunogenic, and an immune response raised against a specific VSG coat would rapidly kill trypanosomes expressing this variant. However, with each cell division, there is an opportunity for the progeny to switch from expressing one VSG gene to another, thereby altering the VSG expressed and thus evading immunity. Only one type of VSG gene is expressed at a given time. VSG expression is "switched" from the array to the active telomere-located expression site (directed by homology) by homologous recombination induced by double-strand breaks in the silent base copy gene. VSG annotations, protein sequences and gene sequences can be derived from public databases such as www.ensemble.org. A collection of VSGs from Trypanosoma brucei (strain Lister427) has been published in Cross 2014, Mol. Biochem. Parasitol. 195(1):59-73. A preferred VSG in the context of the present invention is Trypanosoma brucei VSG, more preferably VSG2, VSG3 ILTat1.24, VSG11, VSG13, VSGsur, VSG1954 or VSG531. A preferred VSG according to the present invention is characterized by an N-terminus that is three-dimensionally positioned within the VSG, in a position that is positioned sufficiently close to an accessible envelope surface such that, when the VSG is present within the VSG envelope on a trypanosome cell, the addition of a linker sequence, preferably 100 amino acids long or less, preferably 50 amino acids long or less, most preferably 20 amino acids long or less, allows modification of the so-extended or non-extended N-terminus of the VSG protein. Preferably, the insertion may be immediately downstream of the signal peptide cleavage site.

[0019] Furthermore, recombinant Trypanosoma brucei cells can be genetically engineered to express more than one type of VSG, such that vesicles obtained from such engineered Trypanosoma brucei cells by the methods of the invention will contain two or more different types of VSG on the surface of the vesicle.

[0020] Furthermore, recombinant Trypanosoma brucei cells are preferably deficient in GPI-phospholipase C. Genetic deletion of endogenous glycophosphatidylinositol phospholipase C (GPI-PLC), the enzyme that "sheds" VSG from the surface of dying cells, is crucial for the generation of T. brucei that can be used as an antigen-presenting platform, for example, since any form of inactivation of trypanosoma cells (e.g., UV irradiation) would lead to the disintegration of the VSG coat and also the cell itself unless GPI-PLC is removed from the genome. If VSG is shed due to the action of GPI-PLC, the VSG coat will disintegrate and the cell will lyse.

[0021] The term "sortagable" as used herein refers to a VSG that can be linked to another polypeptide or peptide by a sortase activity. Preferably, the VSG will comprise a sortase donor or a sortase acceptor amino acid sequence. Typically, the sortase donor or acceptor sequence can be introduced into the VSG by genetic engineering. For example, the sortase donor or acceptor sequence can be included in a peptide that is to be fused to the VSG. Alternatively, a suitable endogenous sequence of the VSG can be genetically modified to become a sortase donor or acceptor sequence.

[0022] Sortase, as used herein, refers to a protein with sortase activity, i.e., an enzyme capable of carrying out a transpeptidation reaction that conjugates the C-terminus of a protein to the N-terminus of a protein via transamination. The term includes full-length sortase proteins, e.g., full-length naturally occurring sortase proteins, fragments of such sortase proteins that have sortase activity, modified (e.g., mutant) variants or derivatives of such sortase proteins or fragments thereof, as well as proteins that are not derived from naturally occurring sortase proteins but that exhibit sortase activity. One of skill in the art would be readily able to determine whether a given protein or protein fragment exhibits sortase activity, for example, by contacting the protein or protein fragment of interest with a suitable sortase substrate under conditions that allow for transpeptidation and determining whether the relevant transpeptidation reaction product is formed. Suitable sortases will be apparent to one of skill in the art and include, but are not limited to, sortase A, sortase B, sortase C, and sortase D-type sortases. Suitable sortases are described, for example, in Dramsi 2005, Res. Microbiol. 156(3):289-97, Comfort 2004, Infect Immun., 72(5):2710-22, Chen 2011, Proc Natl Acad. Sci. USA. Jul. 12; 108(28):11399, and Pallen 2001, TRENDS in Microbiology, 2001, 9(3), 97-101. Additionally, the present invention encompasses embodiments relating to sortase A from any bacterial species or strain. One of skill in the art will appreciate that any sortase and any sortase recognition motif may be used in some embodiments of the invention, including, but not limited to, the sortases and sortase recognition motifs described in WO 2010 / 087994, WO 2011 / 133704, and WO 2020 / 84072.

[0023] A sortase substrate is an amino acid sequence that can be utilized in a transpeptidation reaction mediated by a sortase. Typically, a sortase utilizes two types of substrates, a substrate containing a C-terminal sortase recognition motif and a second substrate containing an N-terminal sortase recognition motif, and the transpeptidation reaction results in the conjugation of both substrates via a covalent bond. In the context of the present invention, the "C-terminal sortase recognition motif" is also referred to as the "sortagging donor sequence", while the term "N-terminal sortase recognition motif" is referred to as the "sortagging acceptor sequence". Preferably, the C-terminal and N-terminal recognition motifs are included in different amino acid sequences, for example, one at the N-terminus of the VSG and the other linked to the immunogen, thereby resulting in a free carboxyl group at the end of the sortagging donor site. Some sortase recognition motifs are described herein, and additional suitable sortase recognition motifs are well known to those skilled in the art. Sortase recognition motifs will be apparent to those skilled in the art. A sortase substrate may comprise additional moieties or entities apart from the peptidic sortase recognition motif.

[0024] For example, a sortase substrate can comprise an LPXTG / A motif, the N-terminus of which is conjugated to any agent (e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label). Similarly, a sortase substrate can comprise an oligoglycine (G1-5) or oligoalanine motif, preferably G3 or G5, the C-terminus of which is conjugated to any agent, e.g., a peptide or protein, a small molecule, a binding agent, a lipid, a carbohydrate, or a detectable label. Thus, a sortase substrate is not limited to a protein or peptide but includes any moiety or entity conjugated to a sortase recognition motif.

[0025] The VSG will preferably be "sortagable", i.e. a sortase substrate, i.e. comprising a sortase donor sequence or a sortase acceptor sequence. Examples of sortagable VSGs can be derived from Pinger 2017, Nat Commun. 8(1):828. Depending on whether the VSG comprises a sortase acceptor or a donor, the molecule that is linked to the VSG by sortagging will comprise the complement, i.e. in the case of a sortase acceptor included by the VSG, the molecule will comprise a sortase donor, or in the case of a sortase donor included by the VSG, the molecule will comprise a sortase acceptor.

[0026] It will be understood that the VSG can also be chemically modified. Preferably, the VSG can be modified by chemical coupling reactions to couple a desired molecule, such as a targeting compound as specified elsewhere herein. Typically, the targeting compound can be covalently attached to the N-terminus of the VSG, preferably via a linker. The targeting compound can be linked to the VSG by any technique known in the art, including any chemical reaction. Preferably, click chemistry or other crosslinking can be used. In the context of the present invention, click chemistry refers to chemical structures designed to rapidly and reliably create covalent bonds by linking together small units that contain reactive groups. Any variation of such approaches can be used to link the targeting compound to the VSG according to the present invention. In general, linking the VSG to the targeting compound can include the use of any linking means or linker, which in the context of the present invention disclosed herein means by which the VSG and the targeting compound are linked or connected to form a modified VSG. The one or more linkers or linking means for linking the VSG and the targeting compound can be any structurally suitable means for connecting the two. Exemplary linkers include the use of one or more amino acids, small chemical scaffolds, biotin-streptavidin, organic or inorganic nanoparticles, polynucleotide sequences, peptide nucleic acids, organic polymers, or immunoglobulin Fc domains, which in some embodiments can be used to form peptides with modified peptide backbones. Means for linking can include covalent and / or non-covalent bonds. One or more linkers can include different sequences or other structural features that provide different functions or properties. For example, one or more linkers can include structural elements to allow derivatization of VSG.

[0027] Preferably, the recombinant Trypanosoma brucei cells expressing a VSG, preferably a sortagable VSG, are provided in a purified or partially purified form. Typically, the cells will be free or substantially free of any culture medium. Preferably, such a preparation of recombinant Trypanosoma brucei cells expressing a VSG, preferably a sortagable VSG, can be obtained by centrifugation allowing the separation of the medium and the cells and resuspension of the cells in a suitable solvent, such as a hypotonic solution to be used to perform the subsequent step (b). If the preparation of recombinant Trypanosoma brucei cells expressing a sortagable VSG is to be preserved, an isotonic solution can be used that allows the preservation of the cells. Suitable solutions are well known to the skilled person, particularly preferred techniques for providing recombinant T. brucei cells are described in the accompanying examples below. These techniques can include centrifugation of the cells to obtain a pellet of cells as well as performing one or more washing steps.

[0028] More preferably, T. brucei cells when grown in cell culture medium, preferably standard HMI9 with 10% FBS, are centrifuged at about 2,000 g for about 20 minutes at room temperature. Preferably, the pelleted cells are washed twice in phosphate buffered saline (PBS). Such washed cells are then subjected to lysis according to step (b) of the method of the present invention. Typically, about 5 billion cells are provided for further processing.

[0029] In step (b) of the method of the invention, the cells, i.e. recombinant Trypanosoma brucei cells expressing a VSG, preferably a sortagable VSG, are treated in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed.

[0030] The term "hypotonic solution" as used herein means a solution that will be hypotonic with respect to the cytoplasm of trypanosome cells. Suitable hypotonic solutions are well known to those skilled in the art. Typically, the hypotonic solution may be deionized water or a buffer solution with low salt content. Water molecules in the hypotonic solution will move due to osmosis to the interior of the cell, i.e., into the cytoplasm. As a result, the volume of the cytoplasm will increase and disrupt the cell membrane, thereby causing the cell membrane to tear and form several small pieces of isolated cell membrane. Preferably, deionized water is used as the hypotonic solution.

[0031] The treatment will be carried out for a time and under conditions sufficient to allow osmotic disruption of the cell membrane of the trypanosome cells as described above. Preferably, the treatment is carried out for a time period of about 30 minutes to about 90 minutes, more preferably about 45 minutes to about 60 minutes, and most preferably about 60 minutes. More specifically, the cells are treated for 10 minutes, centrifuged, and the pellet is resuspended. These steps can be repeated several times to total up to the above-mentioned treatment time. Furthermore, the treatment can also include gentle or ridged shaking of the cells. The temperature applied during the treatment with the hypotonic solution is typically a temperature of about 4°C to about 37°C, more typically a temperature of about 4°C.

[0032] The treatment is also carried out in the presence of at least one protease inhibitor, which will prevent protease activity that may degrade the VSG proteins of the VSG envelope present on the membrane. Suitable protease inhibitors are known to those skilled in the art. In particular, it is envisaged to use a mixture of several different protease inhibitors. Preferably, the HALT protease inhibitor composition as referred to herein comprises at least a serine protease inhibitor, an aminopeptidase inhibitor, a cysteine ​​protease inhibitor, a metalloprotease inhibitor, a serine and cysteine ​​protease inhibitor and / or an aspartic acid protease inhibitor. More preferably, it comprises AEBSF, aprotinin, bestatin, E-64, EDTA, leupeotin and pepstatin A. More preferably, the mixture of protease inhibitors is a commercially available HALT protease inhibitor composition (ThermoFisher Scientific).

[0033] Lysis of the cells typically results in a torn cell membrane, also referred to as the "cell membrane", of the recombinant Trypanosoma brucei cells expressing a sortagable VSG. The membrane may be of various sizes and will preferably contain an intact VSG. The extent of lysis can be visualized by application of a dye to viable cells. Particularly preferred lysis conditions are described in the accompanying Examples below.

[0034] More preferably, the pelleted T. brucei cells provided in step (a) of the method of the present invention are suspended in ice-cold deionized water for lysis in the presence of a HALT protease composition.

[0035] In step (c) of the method of the invention, cell membranes are isolated from the solution obtained when performing step (b). Isolation of cell membranes from the remaining material of lysed cells can be achieved by techniques well known in the art. Preferably, cell membranes are isolated by centrifugation, more preferably as described in the accompanying examples below. Alternatively, cell membranes can be obtained in isolated form by filtration, for example affinity purification using antibodies against VSG, size exclusion chromatography, size / density-based differential centrifugation, ion exchange chromatography, gel filtration chromatography, osmotic enrichment using semipermeable membranes or electrophoretic separation techniques. The above techniques for isolating cell membranes can be used alone or in combination to obtain cell membranes of higher purity. Furthermore, cell membranes can be subjected to one or more washing steps. Typically, isolation of cell membranes is performed at a temperature as described above for step (b), more preferably at about 4°C. More preferably, cell membranes are isolated by centrifugation at about 10,000g for about 10 minutes at about 4°C. The pellet is then preferably resuspended in ice-cold deionized water and the centrifugation is repeated under the same conditions.

[0036] In step (d) of the method of the present invention, the isolated membranes obtained in step (c) are suspended in an isotonic solution. The term "isotonic solution" as used herein means a solution that will be isotonic with respect to the cytoplasm of trypanosome cells. Suitable isotonic solutions are well known to those skilled in the art. Typically, an isotonic solution may be a buffer solution with intermediate (physiological) salt content, such as a phosphate, HEPES or Tris-based buffer. Preferably, 20 mM HEPES, 150 mM NaCl2, pH 8.0 is used as the hypotonic solution.

[0037] In step (e) of the method of the present invention, the suspended cell membranes obtained in step (d) are treated using sonication to obtain a vesicle suspension. Sonication can be performed by applying constant or pulsar ultrasound for a sufficient time to allow the formation of vesicle structures in the solution. Typically, the time is about 3 minutes to about 10 minutes, more preferably about 4 minutes to about 6 minutes, and most preferably about 5 minutes. More preferably, sonication is performed as described in the examples below, i.e., on ice using a Bandelin Sonoplus machine with a pulsar at 40% load for 5 minutes. This treatment preferably results in the shearing of large cell membranes, thereby obtaining smaller vesicle structures, including vesicles with an average diameter of about 250 nm.

[0038] In step (f) of the method of the present invention, aggregated membranous residues are removed from the vesicle suspension obtained in step (e). Preferably, the aggregated membranous debris is removed from the vesicle suspension obtained in step (e) by centrifugation and filtration. Preferably, the filtration is performed by using a 0.45 μM filter.

[0039] The aggregated membrane debris, typically having a size of about 750 nm or more, is removed by centrifugation, preferably performed at about 2,000 g for about 5 minutes at about 4° C. A filtration step will then be performed. Preferably, a filter with sieves in the micrometer range is used. Pressure can be applied by known methods, and typically by using a syringe. Preferably, the aggregated membranous debris is removed from the vesicle suspension obtained in step (e) by centrifugation and filtration, as described in the attached examples below. More preferably, the suspension containing vesicles and aggregated membrane debris is centrifuged at 2,000 g for 5 minutes at 4° C., thereby pelleting the remaining large aggregated pelletable membrane debris and, if present, intact cells. The supernatant will then be filtered through a 0.45 μM filter using a 10 mL syringe. Each of the above steps of sonication, centrifugation and / or filtration may preferably be repeated at least once in accordance with the present invention.

[0040] In step (g) of the method of the present invention, the vesicle suspension is separated into populations. Separation into populations of vesicles of roughly similar size can be achieved by a variety of means. Preferably, the separation referred to herein is size-based separation. More preferably, size-exclusion chromatography, such as gel filtration or CaptoCore chromatography, can be used. Alternatively, centrifugation-based techniques, such as differential centrifugation, centrifugation using cesium or sucrose gradients, or ultracentrifugation, can be applied. Those skilled in the art are well aware of how such size-based separation can be achieved.

[0041] Gel filtration of the vesicle suspension according to the invention as referred to in the context of the present invention can be performed by a chromatographic method in which particles or molecules in a solution are separated according to their size. Typically, an aqueous solution, i.e., a mobile phase, is used to transport the sample through a stationary phase (gel), which may be present, for example, in a column. The chromatographic column is filled with fine porous beads composed of a polymer, preferably a dextran polymer (Sephadex), agarose (Sepharose), or polyacrylamide (Sephacryl or Biogel P). The polymer allows larger particles or molecules to pass through the column somewhat quickly, while smaller molecules can penetrate the space formed by the polymer and therefore may take longer to pass through the column. Preferably, for gel filtration, Sephacryl S-500 26 / 60 or Superose 6 columns can be used. Preferably, gel filtration can be performed as described in the attached examples below.

[0042] CaptoCore chromatography as referred to in the context of the present invention refers to a purification method for purifying and polishing viruses and other large biomolecules in a flow-through mode. Typically, the chromatography medium (resin) comprises a non-functionalized outer layer (ligand-free) and a bead (core bead) with a functionalized core with ligands attached. The inert outer layer of cross-linked agarose prevents viruses and other large entities with molecular weights above about 400.000 (CaptoCore400) or 700.000 (CaptoCore700) from penetrating the core of the bead, while small molecules penetrate the bead and are captured there. Excluded target molecules are collected in the flow-through. The functionalized core comprises an octylamine ligand in the core of the bead, which is multimodal, i.e., both hydrophobic and positively charged. This ligand binds strongly to various contaminants over a wide range of pH and salt concentrations. CaptoCore chromatography can be used at a single or multiple points during the preparation of the delivery particles of the present invention. For example, CaptoCore chromatography can be applied before or after purification of the vesicles, after sortagging, or before administration of the vesicles.

[0043] More preferably, the vesicle suspension obtained after the filtration step in step (f) can be subjected to gel filtration chromatography on a Sephacryl S-500 26 / 60 column (FIG. 1A) equilibrated with 20 mM HEPES, 150 mM NaCl2, pH 8, in order to remove small debris and isolate the largest vesicles, the latter of which represent the majority of the contents of the preparation, and these vesicles are also referred to hereinafter as "nanoVAST".

[0044] Preferably, the population of vesicles can be obtained by using a chromatographic technique other than that based on size exclusion. More preferably, ion exchange or immunoaffinity chromatography can be applied. A skielled person is well aware how such techniques can be cartied out.

[0045] In step (h) of the method of the invention, vesicles are provided from the population of vesicles obtained in step (g). The vesicles obtained by the method of the invention (i.e., nanoVAST) can be subjected to various characterizations, including SDS-PAGE for protein purity and dynamic light scattering for vesicle size and uniformity determination (FIG. 1B and C). The provided vesicles are characterized by the following parameters: (i) having a single predominant protein with an apparent molecular weight of 55-60 kDa, revealed after Coomassie staining of SDS-PAGE, (ii) having a spherical appearance in electron micrographs, and (iii) exhibiting a homogenous surface structure in electron micrographs. SDS-PAGE as referred to herein is preferably performed as described in the accompanying examples below. In particular, nanoVAST samples are diluted in a standard SDS-PAGE denaturing sample buffer prior to separation on a 10% Tris-glycine polyacrylamide gel. Subsequently, the gel is either Coomassie stained to observe the various proteinaceous components or subjected to Western blot analysis to specifically identify the VSG bands. Standard transmission electron micrographs are preferably obtained using the following settings and conditions: vesicles from purified preparations are adsorbed onto glow-discharged carbon-coated copper grids (mesh size 300), washed twice in distilled water, and negatively stained with 1% aqueous uranyl acetate. Micrographs are obtained using a Zeiss EM912 (Carl Zeiss, Oberkochen, Germany) at 80 kV, using a slow-scan CCD camera (TRS, Moorenweis, Germany) and producing a pixel size of 1.2 or 0.6 nm (depending on the nominal magnification of 10kx or 20kx, respectively). Cryo-electron micrographs are preferably acquired using the following settings and conditions: samples are loaded onto plasma-cleaned UltrAuFoil grids (200 mesh; pore size 2 μM×2 μM), followed by blotting for 2 seconds and plunge-freezing using an FEI Vitrobot Mark IV. Grids are imaged using a Talos Artica microscope.

[0046] Preferably, the population of vesicles is further characterized by an average diameter in the range of about 50 nm to about 500 nm, preferably about 150 nm to about 250 nm, as determined by dynamic light scattering analysis. Dynamic light scattering analysis is preferably performed as described in Example 1 below. In particular, a ZetaSizer Nano (Malvern Platical) was used for the measurements, and the accompanying Zetasizer software was used for the analysis of the vesicles. 20 μL of the sample was diluted in 1 mL of PBS and placed in a disposable polystyrene cuvette, followed by measurement.

[0047] Advantageously, it was found in the research underlying the present invention that artificial vesicles can be generated from T. brucei cell membranes, which allow efficient loading as well as excellent targeting, thus avoiding the drawbacks of other delivery systems known in the prior art. NanoVAST vesicles would consist of a phospholipid bilayer (which would protect the cargo, for example, from nucleases or proteases) and could be decorated with targeting molecules such as surface receptors or ligands. Such "synthetic extracellular vesicles" (or EVs) would essentially mimic naturally occurring exosomes in shape and function: (i) they would be innately capable of shuttling genetic information between distant cells (Bobis-Wozowicz et al., 2015), (ii) they would be stable in body fluids, and (iii) they would be preserved for long periods of time.

[0048] Currently, natural EVs are not produced in sufficient quantities to be useful. However, the loading, targeting and overall efficiency of prior art synthetic EVs remain low (Almeida et al., 2020). nanoVAST vesicles are centered around semi-synthetic EVs, whose main backbone can be produced in large quantities and can be efficiently loaded with protein as well as nucleic acid cargo. In addition, nanoVAST vesicles can be enzymatically fused to receptor-binding ligands specific for individual cell types, ensuring targeted delivery from the outset. nanoVAST vesicles are inexpensive to produce, scalable, and retain the best qualities of viral systems (cell type-specific targeting and convenient packaging) while avoiding excessive immunogenicity or oncogenic potential.

[0049] The similarity of nanoVAST technology to naturally occurring exosomes (nature's own tissue-to-tissue long-distance delivery service), the feasibility of loading nanoVAST vesicles with targeted payloads, and the ability to derivatize them with rationally designed "tags" (e.g., antibodies) that target specific cell types would be a major improvement to the field of precise payload delivery.

[0050] The following embodiments are preferred embodiments of the method of the present invention, and the explanations of terms given above apply mutatis mutandis. In a preferred embodiment of the method of the present invention, the recombinant Trypanosoma brucei cells are GPI-phospholipase C deficient. In a preferred embodiment of the method of the present invention, said at least one protease inhibitor is a HALT protease inhibitor composition.

[0051] In a preferred embodiment of the method of the present invention, said method further comprises, after step (c) and before step (d), the following step: - treating the cells in a deionized water solution; and - isolating the cell membranes from said solution. Further includes: In a preferred embodiment of the method of the present invention, said isotonic solution is a buffer comprising 20 mM HEPES, 150 mM NaCl2, pH 8. In a preferred embodiment of the method of the present invention, said sonication in step (e) is carried out with a pulser at 40% load for 5 minutes.

[0052] In a preferred embodiment of the method of the present invention, said step of removing aggregated membranous debris from the vesicle suspension in step (f) is carried out by filtration in step (f) performed by using a 0.45 μM filter.

[0053] In a preferred embodiment of the method of the present invention, said separation in step (g) comprises gel filtration performed through gel filtration chromatography on a Sephacryl S-500 column equilibrated with 20 mM HEPES, 150 mM NaCl2, pH 8 or through a Superose 6 column.

[0054] In a preferred embodiment of the method of the present invention, said method further comprises the step of introducing a cargo drug of interest into the vesicles provided in step (h). Preferably, said introducing step comprises the following steps: (a) suspending the vesicles in a transfection buffer containing an excess of the cargo drug of interest; (b) performing electroporation; and (c) Purifying the loaded vesicles after electroporation Includes.

[0055] The cargo drug of interest can also be loaded into the vesicles by lyophilization, the loading step comprising the following steps: (a) freeze-drying the vesicles in the presence of the cargo; (b) resuspending the lyophilized vesicles and cargo in a resuspension buffer; (c) mixing the resuspension mixture; and (d) Purifying the loaded vesicles Includes. More preferably, said cargo agent of interest is selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

[0056] The term "cargo molecule" as used herein means any compound that needs to be transported by the vesicle of the present invention. Typically, such compounds are molecules that need to be transported in encapsulated form, such as compounds that will enter cells but do not interact with extracellular structures in organisms or are unstable in culture medium. Preferably, the cargo molecule according to the present invention is selected from the group consisting of therapeutic proteins and peptides, such as small molecule drugs, antibodies, aptamers, cytokines, growth factors, hormones, etc., nucleic acid molecules, such as RNA or DNA, and detectable labels, such as dyes used to stain cells.

[0057] In a preferred embodiment, the cargo molecule that needs to be transported by the vesicle of the present invention is an RNA editing molecule.More preferably, the RNA editing molecule is an antisense nucleic acid, preferably an antisense oligonucleotide.As used herein, "antisense oligonucleotide" (ASO) refers to a single-stranded DNA and / or RNA molecule that can interfere with DNA and / or RNA processing.Antisense oligonucleotides comprise a nucleic acid sequence that is complementary to a specific RNA or DNA sequence.

[0058] Typically, antisense oligonucleotides will bind to their respective complementary oligonucleotides, DNA or RNA in a sequence-specific manner, thereby interfering with DNA and / or RNA processing. It is known to those skilled in the art that antisense oligonucleotides can interfere with mRNA processing through RNase H-mediated degradation, translation termination, regulation of splicing, or act through steric hindrance of proteins. Means and methods for designing and synthesizing antisense oligonucleotides are well known in the art, and include, for example, rational design, chemical modification and design of antisense oligonucleotides including locked nucleic acid (LNA) and solid-phase chemical synthesis. Antisense oligonucleotides can be chemically synthesized or expressed in cells, for example, by introducing respective recombinant DNA constructs. It will be understood by those skilled in the art that such DNA constructs can include regulatory elements such as enhancers, constitutive or inducible promoters or terminators in addition to the nucleic acid sequence encoding the antisense oligonucleotide.

[0059] Preferably, the cargo molecule according to the present invention is an antisense oligonucleotide that can recruit RNA editing enzyme. More preferably, the RNA editing enzyme is adenosine deaminase acting on RNA (ADAR). The term "ADAR" refers to double-stranded RNA-specific adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA), also referred to as AtoI editing. Preferably, the ADAR is of human origin. The ADAR can be ADAR1, ADAR2 or ADAR3. The ADAR can be endogenous ADAR, which is recombinantly expressed in target cell or delivered to target cell.

[0060] The antisense oligonucleotide that needs to be transported by the vesicle of the present invention can be part of an expression cassette. The term "expression cassette" refers to a separate component of vector DNA or its isolated fragment, consisting of genes and regulatory sequences that allow expression in prokaryotic or eukaryotic cells. For example, antisense oligonucleotides can be ligated into a nucleic acid expression construct (i.e., vector) under the transcriptional control of suitable cis-regulatory sequences to direct the constitutive or inducible transcription of nucleotide sequences in cells. Typically, an expression cassette includes a promoter sequence, an open reading frame, and a 3' untranslated region (3'UTR). The untranslated region can also include a polyadenylation site to increase the efficiency of translation.

[0061] In particular, the antisense oligonucleotides referred to above can be used to generate neoepitopes in immune peptides associated with cancer, and are preferably suitable for RNA editing, preferably for generating neoepitopes in immunogenic peptides in cancer cells.The target antisense oligonucleotides delivered by the vesicles of the present invention as cargo agents can therefore preferably be capable of specifically hybridizing to the nucleic acid sequence of mRNA encoding at least one tumor epitope, thereby being capable of recruiting ADAR and converting said tumor epitope into a neoepitope.The target neoepitopes generated by RNA editing will preferably be tumor-specific and presented via HLA.For this purpose, the antisense oligonucleotides can be expressed from an expression construct, such as a vector.Preferably, said expression construct can also comprise an expression cassette for expressing ADAR as described above.More preferably, said vectors used as expression constructs can be viral vectors, such as lentiviral vectors.It will be understood that the vesicles in such cases can target cancer cells in which neoepitopes will be generated.

[0062] In a preferred embodiment of the method of the present invention, said method further comprises the step of sortagging a targeting compound to the sortagable VSG on the vesicle provided in step (h). Preferably, the sortagging step comprises the following steps: (a) treating the vesicles with a sortase in the presence of a targeting compound; and (b) purifying the sortagged vesicles with a targeting compound Includes.

[0063] The term "targeting compound" as used herein means a compound that allows the binding of a vesicle to a target molecule present on a target cell. Thus, the targeting compound can be any compound capable of interacting with the target molecule. Typically, the targeting compound and the target molecule interact specifically. Preferably, the targeting compound is selected from the group consisting of antibodies, nanobodies, aptamers, peptides, proteins, and small molecules. It will be understood that the sortagable VSG present on the surface of the vesicle of the present invention can be coupled to one or more different targeting compounds as well. Further details regarding targeting compounds can also be found in WO2020 / 84072. More preferably, the targeting compound is an antibody or a nanobody that recognizes a target molecule on a target cell.

[0064] The present invention also relates to vesicles comprising VSGs, preferably sortagable VSGs, characterized by the following parameters: (i) having a single predominant protein, as revealed after Coomassie staining of SDS-PAGE, with an apparent molecular weight of 55-60 kDa, (ii) having a spherical appearance in electron micrographs, and (iii) exhibiting a homogenous surface structure in electron micrographs. Preferably, the vesicles have an average diameter of about 50 nm to about 500 nm, more preferably about 150 nm to about 250 nm, as determined by dynamic light scattering.

[0065] In a preferred embodiment of the vesicle of the present invention, the vesicle is loaded with a cargo agent of interest, preferably selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

[0066] In a preferred embodiment of the vesicles of the invention, said vesicles are sortagged with a targeting compound, preferably an antibody or a nanobody, which recognizes a target molecule on a target cell. In a preferred embodiment of the vesicle of the invention, said vesicle is obtainable by the method of the invention.

[0067] The present invention also relates to a vesicle according to the invention as defined above for use in the treatment and / or prevention of a disease or medical condition.Furthermore, the present invention also relates to a vesicle according to the invention as defined above for use as a compound delivery vesicle, preferably as a drug delivery vehicle.

[0068] The vesicles of the present invention can be used as carriers for drugs, i.e. to treat and / or prevent diseases. Suitable drugs are preferably selected from the group consisting of small molecule drugs, antibodies, aptamers, therapeutic proteins and peptides such as cytokines, growth factors, hormones, etc., and nucleic acid molecules such as RNA or DNA. The skilled artisan is well aware of which diseases can be treated and / or prevented by administering encapsulated drugs, and thus which are suitable diseases to be treated and / or prevented by using drugs encapsulated in the vesicles of the present invention. Furthermore, depending on the disease, the vesicles can be modified to allow specific targeting of cells related to the disease, for example by sortagging of targeting antibodies or aptamers, due to the presence of sortagable VSGs on their surface.

[0069] The vesicles of the present invention can also be used as vaccines. For this purpose, the sortagable VSG on its surface can be coupled to an antigen or hapten by sortagging. The vesicles thus modified can be administered to a subject and induce an immune response. Depending on the immunization scheme, such vesicles can be used for priming and / or boosting, which are applied once or several times. Preferably, immunization with such vesicles can be performed as described in WO2021 / 214043.

[0070] Furthermore, the vesicles of the present invention can be used as an in vitro delivery tool for drugs as specified above or other compounds to be introduced into cultured cells. Transfection techniques can also be applied to the vesicles of the present invention, for example, to introduce nucleic acid molecules into cultured cells.

[0071] The present invention also relates to a kit for carrying out the method of the invention, comprising recombinant Trypanosoma brucei cells expressing a sortagable VSG and at least one agent for carrying out the method of the invention as defined above.

[0072] In a preferred embodiment of the kit of the present invention, the at least one agent is selected from the group consisting of deionized water, at least one protease inhibitor, preferably a HALT protease inhibitor composition, a buffer comprising 20 mM HEPES, 150 mM NaCl2, pH 8, a 0.45 μM filter, 20 mM HEPES, 150 mM NaCl2, and a sortase. The present invention also relates to a kit comprising a vesicle of the invention.

[0073] The following are certain preferred embodiments of the present invention. Embodiment 1: The method comprises the steps of: (a) providing a recombinant Trypanosoma brucei cell expressing a VSG, preferably a sortagable VSG; (b) treating the cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed; (c) isolating the cell membranes from the solution of step (b); (d) suspending the isolated membranes obtained in step (c) in an isotonic solution; (e) treating the suspended cell membranes obtained in step (d) using sonication to obtain a vesicle suspension; (f) removing aggregated membranous residue from the vesicle suspension obtained in step (e); (g) separating the vesicle suspension into a population of vesicles; and (h) providing a vesicle from a population of vesicles characterized by the following parameters: (i) having a single predominant protein with an apparent molecular weight of 55-60 kDa, as revealed after Coomassie staining of SDS-PAGE; (ii) having a spherical appearance in electron micrographs; and (iii) exhibiting a homogenous surface structure in electron micrographs. A method for preparing a vesicle comprising:

[0074] Embodiment 2: The method of embodiment 1, wherein the population of vesicles is further characterized by an average diameter in the range of about 50 nm to about 500 nm, preferably about 150 nm to about 250 nm, as determined by dynamic light scattering analysis. Embodiment 3: The method of embodiment 1 or 2, wherein said recombinant Trypanosoma brucei cells are GPI-phospholipase C deficient. Embodiment 4: The method of any one of embodiments 1 to 3, wherein said at least one protease inhibitor is a HALT protease inhibitor composition.

[0075] Embodiment 5: After step (c) and before step (d), the following steps: - treating the cells in a deionized water solution; and - isolating the cell membranes from said solution. 5. The method of any one of embodiments 1 to 4, further comprising: Embodiment 6: The method of any one of embodiments 1 to 5, wherein the isotonic solution is a buffer comprising 20 mM HEPES, 150 mM NaCl2, pH 8. Embodiment 7: The method of any one of embodiments 1 to 6, wherein the sonication in step (e) is performed using a pulser at 40% load for 5 minutes.

[0076] Embodiment 8: The method of any one of embodiments 1 to 7, wherein the step of removing aggregated membranous debris from the vesicle suspension in step (f) is carried out by filtration in step (f) using a 0.45 μM filter. Embodiment 9: The method of any one of embodiments 1 to 8, wherein the separation in step (g) comprises gel filtration performed through gel filtration chromatography on a Sephacryl S-500 26 / 60 column, a Superose 6 column or a CaptoCore column equilibrated with 20 mM HEPES, 150 mM NaCl2, pH 8. Embodiment 10: The method of any one of embodiments 1 to 9, further comprising the step of introducing a cargo agent of interest into the vesicle provided in step (h).

[0077] Embodiment 11: The introducing step comprises the steps of: (a) suspending the vesicles in a transfection buffer containing an excess of the cargo drug of interest; (b) performing electroporation; and (c) Purifying the loaded vesicles after electroporation 11. The method of embodiment 10, comprising: Embodiment 12: The introducing step comprises the steps of: (a) freeze-drying the vesicles in the presence of the cargo; (b) resuspending the lyophilized vesicles and cargo in a resuspension buffer; (c) mixing the resuspension mixture; and (d) Purifying the loaded vesicles 11. The method of claim 10, comprising: Embodiment 13: The method of any one of embodiments 10 to 12, wherein the cargo agent of interest is selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

[0078] Embodiment 14: The method of any one of embodiments 1 to 13, further comprising the step of sortagging a targeting compound to the sortagable VSG on the vesicle provided in step (h). Embodiment 15: The salt tagging step comprises the following steps: (a) treating the vesicles with a sortase in the presence of a targeting compound; and (b) purifying the sortagged vesicles with a targeting compound 15. The method of embodiment 14, comprising: Embodiment 16: The method of embodiment 14 or 15, wherein the targeting compound is an antibody or nanobody that recognizes a target molecule on a target cell.

[0079] Embodiment 17: A vesicle comprising VSG, preferably sortagable VSG, characterized by the following parameters: (i) having a single predominant protein with an apparent molecular weight of 55-60 kDa, as revealed after Coomassie staining of SDS-PAGE, (ii) having a spherical appearance in electron micrographs, and (iii) exhibiting a homogenous surface structure in electron micrographs. Embodiment 18: The vesicle of embodiment 17, further characterized by an average diameter in the range of about 50 nm to about 500 nm, preferably about 150 nm to about 250 nm, as determined by dynamic light scattering analysis. Embodiment 19: The vesicle of embodiment 17 or 18, which is loaded with a cargo agent of interest, preferably selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

[0080] Embodiment 20: The vesicle of embodiment 19, wherein the nucleic acid molecule is an antisense oligonucleotoide or an expression construct encoding same. Embodiment 21: The vesicle of embodiment 20, wherein said antisense oligonucleotide is suitable for RNA editing, preferably for the generation of neoepitopes in immunogenic peptides in cancer cells. Embodiment 22: A vesicle of any one of embodiments 17 to 21, which is sortagged with a targeting compound, preferably an antibody or nanobody that recognizes a target molecule on a target cell.

[0081] Embodiment 23: A vesicle according to any one of embodiments 17 to 22, obtainable by a method according to any one of embodiments 1 to 16. Embodiment 24: A vesicle as defined in any one of embodiments 17 to 23 for use in the treatment and / or prevention of a disease or medical condition. Embodiment 25: A vesicle as defined in any one of claims 17 to 23 for use as a compound delivery vehicle, preferably a drug delivery vehicle.

[0082] Embodiment 26: A kit for performing the method of any one of embodiments 1 to 16, comprising a recombinant Trypanosoma brucei cell expressing a sortagable VSG and at least one agent for performing the method as defined in any one of embodiments 1 to 16. Embodiment 27: The kit of embodiment 26, wherein the at least one agent is selected from the group consisting of deionized water, at least one protease inhibitor, preferably a HALT protease inhibitor composition, a buffer comprising 20 mM HEPES, 150 mM NaCl2, pH 8, a 0.45 μM filter, 20 mM HEPES, 150 mM NaCl2, and a sortase. Embodiment 28: A kit comprising a vesicle according to any one of embodiments 17 to 23.

[0083] All references cited throughout this specification are incorporated herein by reference in their entirety as well as with respect to the disclosure content specifically mentioned. EXAMPLES

[0084] The examples are merely illustrative of the invention and are not to be construed as limiting the invention in any way.

[0085] Example 1: nanoVAST vesicle preparation Approximately 10–10 billion T. brucei cells expressing sortagable VSG and lacking the GPI-phospholipase C gene were grown in standard HMI-9 (containing 10% fetal bovine serum) and isolated by centrifugation (2,000 g for 20 min at room temperature).

[0086] Cells were washed once with phosphate-buffered saline (PBS) to remove residual extracellular proteins, and the resulting pellet was then lysed for 10 min on ice via suspension in 5 mL of ice-cold deionized HO containing HALT protease inhibitors for lysis.

[0087] The lysed suspension was centrifuged (10,000 g for 10 min at 4° C.) to isolate membranous material and remove cytoplasmic contents. The ice-cold deionized H2O extraction and centrifugation process was repeated twice.

[0088] The membrane pellet was then suspended in 2 mL of 20 mM HEPES, 150 mM NaCl2, pH 8, and sonicated on ice (5 min at 40% load using a pulser) to shear the larger membranes into smaller vesicles. The suspension was then centrifuged (2,000 g, 4° C. for 5 min) to remove any remaining large aggregated pelletable debris (e.g., cells that remained intact) and filtered through a 0.45 μM filter using a 2.5 mL syringe. The filtered supernatant was then sonicated and centrifuged again using the same process, and the new supernatant was filtered once more to obtain a purer solution free of aggregated cell debris and suitable for gel filtration.

[0089] The filtered vesicle suspension was then separated by gel filtration chromatography on a Sephacryl S-500 26 / 60 column equilibrated with 20 mM HEPES, 150 mM NaCl, pH 8 to remove small debris and isolate the largest vesicles, which constituted the majority of the preparation's contents and are henceforth referred to as "nanoVAST" (Figures 1A, 2A).

[0090] The nanoVASTs were then subjected to various characterizations including SDS-PAGE for protein purity and dynamic light scattering for vesicle size and homogeneity determination (Figure 1B and C, Figure 2). Specifically for dynamic light scattering, a ZetaSizer Nano (Malvern Playtical) was used for measurements and the accompanying Zetasizer software was used for vesicle analysis. 20 μL of the samples were diluted in 1 mL of PBS and placed in a disposable polystyrene cuvette and subsequently measured. The following parameters were found to be characteristic for nanoVAST vesicles: (i) have a single predominant protein, revealed after Coomassie staining of SDS-PAGE, with an apparent molecular weight of 55-60 kDa; (ii) have a spherical appearance in electron micrographs; and (iii) exhibit a homogeneous surface structure in electron micrographs; Furthermore, the vesicles typically had an average diameter of 250 nm as determined by dynamic light scattering.

[0091] Example 2: nanoVAST loading Subsequently, to load nanoVAST with cargo, 0.5 mg of protein equivalent to nanoVAST (determined by colorimetric BCA protein quantification assay and by SDS-PAGE comparative quantification against protein standards) was suspended in homemade transfection buffer (90 mM Na2HPO4, pH 7.3, 5 mM KCl, 0.15 mM CaCl2, 50 mM HEPES, pH 7.3) and electroporated in the presence of a molar excess of the cargo of interest (concentration of cargo is molecule dependent).

[0092] After evaluating nanoVAST transfection efficiency using a wide range of program options, electroporation was performed using the Amaxa Nucleofector 2b with the U033 program (e.g., Figure 4). Cargo-loaded nanoVAST were isolated from free cargo by centrifugation (20,000g, 4°C, 30 min) and washed multiple times.

[0093] The generated nanoVAST could be detected by flow cytometry. When the selected cargo was fluorescently labeled, both cargo-loaded and cargo-free nanoVAST could be measured for their fluorescence intensity through flow cytometry (shown in FIG. 4). This facilitated the determination of loading efficiency. Using the U033 program for electroporation, nanoVAST loading with an efficiency of over 99% was seen (FIG. 4A). For the experimental setup described, a BD FACSCalibur TM was used.

[0094] Example 3: nanoVAST Delivery Cells treated with cargo-loaded nanoVASTs will incorporate them, thereby taking up molecules of interest, which hypothetically could be small enough to fit into the nanoVASTs, including small molecule drugs (represented by the fluorescent molecule FAM, FIG. 5A) and nucleic acids (represented by fluorescent RNA molecules, FIG. 5B).

[0095] For treatment, cells were plated in 24-well plates at the appropriate density. A mixture of fluorescent cargo-loaded nanoVAST and the appropriate cell culture medium was prepared. The amount of nanoVAST used could vary from 100 to 5000:1 ratio of vesicles:cells, depending on the setup.

[0096] The above mixture of nanoVAST and medium was then added dropwise to each cell culture well, and the cells were incubated in an incubator (37° C., 5% CO 2 ) for at least 1.5 hours before further analysis.

[0097] The cells were harvested and washed three times with PBS (100 g, 7 min at room temperature). After flow cytometry, it was determined that the treated cells had an increase in their fluorescence, suggesting that they had taken up the cargo delivered by nanoVAST ( FIG. 5 ).

[0098] Example 4: Membrane fusion-facilitated nanoVAST delivery The predominant protein of nanoVAST (VSG3) was present on the surface of both cell lines tested (RamosB and HEK293T cells) after nanoVAST treatment, suggesting membrane fusion as a mechanism of delivery.

[0099] We determined this through the following experimental setup: Cells were plated in 24-well plates at the appropriate density for each cell line, as described in Example 3. A mixture of nanoVAST and the appropriate cell medium (RPMI1640 for RamosB cell line and DMEM for HEK293T cells, both supplemented with 10% FBS) was prepared. This was added dropwise to each well, and the cells were then incubated for at least 1.5 hours.

[0100] Cells were harvested and washed three times with PBS (100 g, 7 min at room temperature) and subsequently stained with anti-VSG3-FITC conjugated antibody for 10 min on ice. After washing three times with PBS (100 g, 7 min at room temperature), flow cytometry results showed a clear increase in the presence of VSG3 on the surface of the cells (Figure 6A-B). Based on the fact that VSG3 is usually completely absent from the surface of these cell lines, we concluded that its presence was a direct result of interaction with VSG3-coated nanoVAST via membrane fusion. If nanoVAST had been taken up instead via endocytosis, we would not have seen any surface detectable VSG, as nanoVAST would have also been internalized along with the cargo.

[0101] Figure 6C illustrates the cellular mechanisms of nanoVAST cargo delivery into cells, either via membrane fusion and direct release of cargo (Figure 6C, i) or via endocytosis (Figure 6C, ii) and subsequent release of cargo (Figure 6C, v). Both delivery mechanisms result in the release of cargo into cells, as well as the internalization and accumulation of VSG3 in endocytic vesicles (Figure 6C, iv). Internalized VSG3 could be detected in RAB11-positive endocytic vesicles 8 hours after nanoVAST treatment of cells, in this case HEK cells (Figure 6D).

[0102] Example 5: nanoVAST Targeted Delivery To subsequently sortag the nanoVAST with a targeting moiety of interest (e.g., an antibody or nanobody that binds to a specific target cell surface marker), the loaded nanoVAST was then incubated for 3 hours at 37°C with gentle shaking in the presence of purified recombinant Streptococcus pyogenes sortase A (100 μM), 30 mM CaCl2, and the sortagable molecule / targeting moiety of interest (300 μM).

[0103] The sortagged nanoVAST was then reisolated from the free sortagging reagent by centrifugation (20,000 g, 4° C., 30 min) and stored in 20 mM HEPES, 150 mM NaCl2, pH 8. The nanoVAST sortagging potential was assessed using the sortagable fluorescent molecule TAMRA. Vesicles were sortagged using the protocol described above and subsequently analyzed using flow cytometry. Figure 7B illustrates that nanoVAST can be sortagged efficiently. We then proceeded to sortag functionally important targeting moieties.

[0104] In this example, the targeting molecule sortagged under the conditions described above was an anti-CD19 nanobody that would target the CD19 receptor on the surface of Ramos B cells. Ramos B cells were treated with sortagged nanoVAST as described in Example 3, followed by anti-VSG3 staining of the cells as described in Example 4. As shown in Figure 7A, B cells treated with targeted nanoVAST incorporated more VSG3 into their membranes than individual cells treated with non-specific nanoVAST. This implies that the present invention can be targeted, resulting in increased delivery efficiency.

[0105] Example 6: nanoVAST vesicle preparation using alternative methods As an alternative method, the filtered supernatant from step f of the first embodiment of the invention can be subjected to several centrifugation steps to gradually remove more cell and membrane debris. It was observed that nanoVAST can be pelleted at speeds of 20,000 g and above. Thus, by gradually increasing the speed of the repeated centrifugations (Figure 8A), it is possible to progressively pellet the aggregated membranous debris prior to pelleting of nanoVAST, while leaving the soluble proteins in the final supernatant. Five different speeds ranging from 5,000 to 20,000 g were used. The supernatant after each centrifugation is collected and transferred to a clean test tube. Centrifugation at the selected speed is repeated until a pellet is no longer formed, indicating that the relatively heavy debris that could be removed at this speed has indeed been removed. This typically occurs after 2 to 5 rounds. Once the pellet is removed, the supernatant is centrifuged at a higher speed. All centrifugations below 20,000 g are performed for 5 minutes at 4°C. The resulting supernatant after centrifugation at 17,000 g is transferred to a new tube and centrifuged at 20,000 g for 30 min at 4° C. At this stage, nanoVAST is found in the pellet. The pellet is resuspended in 20 mM HEPES, 150 mM NaCl2, pH 8.0 and centrifuged again at 20,000 g for 30 min at 4° C. as an additional cleaning step. At this point the pellet contains nanoVAST and can be stored.

[0106] NanoVAST obtained from this alternative method was examined by dynamic light scattering, transmission electron microscopy, and SDS-PAGE as previously described (Figure 8B-D), indicating that this method can be used to purify nanoVAST populations. NanoVAST purified by differential centrifugation was a more homogenous population with a diameter of approximately 150 nM, but was otherwise similar to that purified by gel filtration / FPLC.

[0107] For the experiments, approximately 10-10 billion T. brucei expressing sortagable VSG and lacking the GPI-phospholipase C gene were grown in standard HMI-9 (containing 10% fetal bovine serum) and isolated by centrifugation (2,000 g, 20 min at room temperature). Cells were washed once with phosphate-buffered saline (PBS) to remove residual extracellular proteins, and the resulting pellet was subsequently lysed for 10 min on ice through suspension in 5 mL ice-cold deionized H2O containing HALT protease inhibitor for lysis. The lysed suspension was then centrifuged (10,000 g, 10 min at 4° C.) to isolate membranous material and remove cytoplasmic contents. The ice-cold deionized H2O extraction and centrifugation process was repeated twice. The membrane pellet was then suspended in 2 mL of 20 mM HEPES, 150 mM NaCl2, pH 8, and sonicated on ice (5 min at 40% load using a pulser) to shear the larger membranes into smaller vesicles. The suspension was then centrifuged (2,000 g for 5 min at 4° C.) and filtered through a 0.45 μM filter using a 2.5 mL syringe to remove any remaining large aggregated pelletable debris (e.g., cells that remained intact). The filtered vesicle suspension was then subjected to a differential centrifugation protocol as shown in FIG. 8A.

[0108] The 5,000g centrifugation step for 5 min at 4°C was repeated three times until no pellet was present. The resulting supernatant was centrifuged at 8,000g for 5 min at 4°C, and this step was repeated twice until no pellet was present. Similarly, the supernatant from this step was centrifuged at 12,000g for 5 min at 4°C. This step was repeated twice until no pellet was present. The supernatant was then centrifuged at 17,000g for 5 min at 4°C. After the second centrifugation, the supernatant was transferred to a new tube and recentrifuged at 20,000g for 30 min at 4°C. An additional washing step followed in which the pellet was resuspended in 20 mM HEPES, 150 mM NaCl2, pH 8, and centrifuged at 20,000g for 30 min at 4°C. The resulting pellet was examined by dynamic light scattering, by transmission electron microscopy, and by SDS-PAGE (FIG. 8B-D), revealing that spherical VSG-coated vesicles had been isolated.

[0109] Regardless of which purification methodology is deployed, the nanoVAST preparation can also be subsequently polished using CaptoCore resin. The polishing step is performed by passing the crude or semi-processed nanoVAST preparation through a CaptoCore 700 (Cytiva) resin column. This could be inserted as an auxiliary step at one or several steps during nanoVAST preparation, loading or "sortagging" if necessary (Figure 9A). This separates larger nanoVAST vesicles and retains other particles smaller than the 700 kDa molecular weight cutoff; thus removing nucleic acids, non-nanoVAST proteins, and other cellular debris.

[0110] We next investigated whether nanoVASTs could also be loaded using alternative strategies to electroporation that might be beneficial / necessary for certain applications. Here, lyophilization is described as a novel alternative method to load RNA cargo into nanoVAST vesicles.

[0111] The freeze-drying loading process (FIG. 10A) involved mixing nanoVAST and fluorescently labeled RNA in HEPES buffer (20 mM HEPES, 150 mM NaCl). The nanoVAST-RNA mix was then flash frozen in liquid nitrogen. The frozen samples were then rapidly transferred to a freeze dryer (Alpha 1-2 LSCbasic-Martin Christ), where the samples were dehydrated at low pressure. The resulting product was a dried nanoVAST-RNA formulation.

[0112] After the freeze-drying step, the quality of nanoVAST was examined by checking the presence and quality of VSG3 protein and by evaluating the size and structure of nanoVAST. For this, dried nanoVAST that was not co-dried with RNA cargo was hydrated in HEPES buffer. SDS-PAGE analysis was then performed, and the results showed that the VSG3 protein was still intact after the freeze-drying step (FIG. 10D). The presence of VSG3 was further confirmed by Western blotting (FIG. 10E). The size and uniformity of the vesicles were determined by dynamic light scattering, which was performed as described in Example 1. As illustrated in FIG. 10B, the expected size and uniformity were maintained after the freeze-drying step. Finally, the rehydrated nanoVAST vesicles were analyzed by transmission electron microscopy, and the standard rounded appearance of the vesicles remained intact (FIG. 10C). Taken together, these analyses confirmed that the freeze-drying step did not alter the structure and quality of nanoVAST. Therefore, lyophilized nanoVAST can be used for loading and delivery of cargo.

[0113] In this example, lyophilized nanoVAST was loaded with fluorescently labeled RNA. The dried nanoVAST-RNA formulation was rehydrated with HEPES buffer, which resulted in the incorporation of RNA. RNA-loaded nanoVAST was isolated from unloaded RNA by centrifugation at 20,000 g for 30 min at 4° C. Since the RNA cargo was labeled with a fluorescent tag, loaded nanoVAST could be detected by flow cytometry, and RNA-loaded nanoVAST was distinguished from unloaded nanoVAST by evaluating the fluorescence intensity. As illustrated in FIG. 10F, nanoVAST was efficiently loaded with RNA (FIG. 10F), and the loading capacity was comparable to that of electroporation (FIG. 4B). This result demonstrates lyophilization as an alternative method of loading nanoVAST. The advantage of having an additional loading method to electroporation is that each approach may be suitable for loading specific cargo types. Thus, the present invention expands the capabilities of the nanoVAST payload platform.

[0114] Example 7: nanoVAST vesicle loading using alternative methods Having demonstrated that lyophilized-hydrated nanoVAST could be loaded with RNA cargo (FIG. 10F), we assessed whether the vesicles could deliver RNA to HEK293T cells. HEK cells at an appropriate density were plated in 24-well plates as described in Example 3. Then, RNA-loaded lyophilized nanoVAST in DMEM cell culture medium was slowly added dropwise to each cell culture well, followed by incubation at 37° C., 5% CO2 humidified environment for at least 1.5 hours.

[0115] After this incubation, cells were harvested and washed twice with 1×PBS by centrifugation at 100 g for 5 min at room temperature. To identify HEK cells that had taken up nanoVAST, washed cells were incubated with 1:100 FITC-conjugated anti-VSG3 antibody on ice for 30 min. To identify cells that had taken up nanoVAST and RNA cargo, samples were analyzed by flow cytometry. HEK cells that had taken up nanoVAST were VSG3 positive and represented 48.4% of the HEK cell population (FIG. 11B.i). This population was also loaded with RNA, as depicted by the increased intensity of fluorescently tagged RNA (FIG. 11B.iii). A proportion of HEK cells without VSG3 signal but loaded with RNA cargo was also seen (FIG. 11B.ii). This finding suggested that there is a proportion of nanoVAST that loses VSG3 surface protein upon uptake but still releases RNA cargo into HEK cells. Results from the lyophilized nanoVAST HEK feeding experiment were comparable to those of electroporated nanoVAST-fed HEK cells ( FIG. 11A ). From this data, it was concluded that lyophilized nanoVAST can deliver cargo to target cells. Here again, we highlight that this novel method of cargo delivery expands the application of our nanoVAST loading platform.

[0116] Example 8: Preparation of nanoVASTs from different VSG-expressing trypanosomes As previously described in Example 5, nanoVAST cell specificity or targeting can be achieved through modification of the VSG envelope of nanoVAST. In the case of Example 5, a "targeting moiety," an anti-CD19 targeting nanobody, was covalently attached to the VSG3 envelope of nanoVAST using a sortase. However, several additional methods could be used to modify the VSG envelope to switch nanoVAST targeting or cell specificity, for example, to add a different "targeting moiety" or to change the physicochemical properties of the VSG (Figure 12). In addition to direct nanoVAST "sortagging," the VSG gene of trypanosome from which nanoVAST will be generated can be genetically modified to directly encode a peptide-based "targeting moiety" (Figure 12ii). Such examples of peptide-based "targeting moieties" are listed, but are not limited to, in Table 1, which can target nanoVAST vesicles to specific cells based on their design. In addition, we were able to completely switch the VSG coat of trypanosomes to another genome-encoded VSG gene that translated into a VSG with a different cell tropism and therefore completely different electrochemical properties that affected nanoVAST cell targeting (Figure S12iii).

[0117] [Table 1]

[0118] We have already generated nanoVAST from trypanosomes expressing a different VSG than VSG3, namely ILtat 1.24 (so-called ILtat). Vesicles prepared from ILtat-expressing trypanosomes showed similar physical properties to their VSG3 counterparts (FIG. 13A-C). Their size and integrity as well as their purity were very similar to VSG3 nanoVAST. In addition, ILtat nanoVAST could be sortagged using the same sortagable fluorescent molecule TAMRA that was successfully used for VSG3 nanoVAST (described in Example 5, FIG. 7B). Using the sortagging method described in Example 5 and flow cytometry for validation, it was shown that ILtat vesicles could be efficiently sortagged and thus maintain their functional protein layer.

[0119] Example 9: nanoVAST immunogenicity nanoVAST is an in vivo delivery tool for specific delivery of cargo to specific cell types. All in vivo delivery tools must be analyzed for their immunogenicity to ensure that the treatment itself does not initiate several different inflammatory signaling pathways and therefore does not result in toxicity. The most important cell type to investigate would be macrophages, as they are the sentinels of the immune system and often coordinate these cytokine signaling pathways. Here, we evaluated immunogenicity using an in vitro macrophage system based on RAW cells. nanoVAST was not able to stimulate any cytokine production in these cells compared to inactivated E. coli or MPLA, which are well-understood positive controls for such experiments (Figure 14).

[0120] References TIFF2025509565000002.tif224165TIFF2025509565000003.tif167164

Claims

1. Steps below: (a) providing a recombinant Trypanosoma brucei cell expressing a VSG, preferably a sortagable VSG; (b) treating the cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed; (c) isolating the cell membranes from the solution of step (b); (d) suspending the isolated membranes obtained in step (c) in an isotonic solution; (e) treating the suspended cell membranes obtained in step (d) using sonication to obtain a vesicle suspension; (f) removing aggregated membranous debris from the vesicle suspension obtained in step (e); (g) separating the vesicle suspension into populations of vesicles; and (h) providing a vesicle from a population of vesicles characterized by the following parameters: (i) having a single predominant protein with an apparent molecular weight of 55-60 kDa, as revealed after Coomassie staining of SDS-PAGE; (ii) having a spherical appearance in electron micrographs; and (iii) exhibiting a homogenous surface structure in electron micrographs. A method for preparing a vesicle comprising:

2. After step (c) and before step (d), the following steps: - treating the cells in a deionized water solution; and - isolating the cell membranes from the solution. The method of claim 1, further comprising:

3. 3. The method of claim 1 or 2, wherein the step of removing aggregated membranous debris from the vesicle suspension of step (f) is carried out by filtration using a 0.45 μM filter.

4. 4. The method of any one of claims 1 to 3, further comprising the step of introducing a cargo drug of interest into the vesicles provided in step (h).

5. The step of introducing comprises the steps of: (a) suspending the vesicles in a transfection buffer containing an excess of the cargo drug of interest; (b) performing electroporation; and (c) Purifying the loaded vesicles after electroporation 5. The method of claim 4, comprising:

6. 6. The method of claim 4 or 5, wherein the cargo drug of interest is selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

7. 7. The method of any one of claims 1 to 6, further comprising the step of sortagging a targeting compound to the sortagable VSG on the vesicle provided in step (h).

8. The salt tagging step comprises the steps of: (a) treating the vesicles with a sortase in the presence of a targeting compound; and (b) purifying the sortagged vesicles with a targeting compound 8. The method of claim 7, comprising:

9. 9. The method of claim 7 or 8, wherein the targeting compound is an antibody or nanobody that recognizes a target molecule on a target cell.

10. Vesicles comprising VSG, preferably sortagable VSG, characterized by the following parameters: (i) having a single predominant protein with an apparent molecular weight of 55-60 kDa, as revealed after Coomassie staining of SDS-PAGE; (ii) having a spherical appearance in electron micrographs; and (iii) exhibiting a homogeous surface structure in electron micrographs.

11. The vesicle of claim 10, which is loaded with a cargo agent of interest, preferably selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

12. 12. A vesicle according to claim 10 or 11, which is sortagged with a targeting compound, preferably an antibody or a nanobody, which recognises a target molecule on a target cell.

13. A vesicle according to any one of claims 10 to 12 for use in the treatment and / or prevention of a disease or medical condition.

14. A vesicle according to any one of claims 10 to 12 for use as a compound delivery vehicle, preferably a drug delivery vehicle.

15. A kit for carrying out the method according to any one of claims 1 to 9, comprising a recombinant Trypanosoma brucei cell expressing a sortagable VSG and at least one agent for carrying out the method as defined in any one of claims 1 to 9.

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