Method for manufacturing 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 vaccine production or as compound delivery vehicles. More specifically, the present invention provides steps of providing recombinant Trypanosoma brucei cells expressing sortaggable VSG, treating the cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells lyse, isolating cell membranes from the solution, suspending the isolated membranes already obtained in an isotonic solution, treating the suspended cell membranes obtained in the previous step using sonication to obtain a vesicle suspension, removing aggregated membranous residues from the already obtained vesicle suspension, separating the vesicle suspension into a population of vesicles, and providing vesicles from a population of vesicles characterized by the following parameters: (i) having a single dominant protein that becomes apparent 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 homogeneous surface structure in electron micrographs. The present invention also relates to a method for preparing vesicles. Furthermore, the present invention also relates to vesicles containing sortaggable VSG characterized by the above parameters and their use in the treatment and / or prevention of diseases or medical conditions or as compound delivery vehicles, preferably drug delivery vehicles, more preferably nucleic acid delivery vehicles. Finally, the present invention contemplates a kit for performing the method of the present invention, comprising recombinant Trypanosoma brucei cells expressing sortaggable VSG and at least one agent for performing the method of the present invention, or a kit comprising the vesicles of the present invention.

Background Art

[0002] Despite its potential, the field of gene therapy has not overcome the critical dilemma of how to deliver targeted therapies 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 their poor pharmacokinetics, sensitivity to enzymatic disruption, and large size. Novel tools are needed to realize the potential of gene therapy.

[0003] Adenoviruses or AAVs: Efficient targeting, but inadequate loading (also immunogenic). The vast majority of delivery systems utilized in this field involve viruses, which are essentially nucleic acid packaging systems coated with proteins responsible for driving the uptake of their genetic information into specific target cells through binding to surface receptors. Several viruses have been tested for gene therapy purposes, often with adverse effects (e.g., retroviruses with "off-target" carcinogenicity). Adeno-associated viruses (AAVs) engineered to be non-replicating have a particular advantage in that virions can acquire different directivity based on the precise composition of their capsids (Kuzmin et al., 2021). Combining AAV serotypes (and therefore tissue targeting) with specific promoters that drive expression (e.g., of capsidized RNA) is desirable to achieve tissue-specific delivery. Substantial interest in this approach has been amplified by the recent approval by the US FDA of gene therapies based on two types of AAVs.

[0004] However, despite its potential, several problems are associated with AAV-based gene therapy approaches. Firstly, 30–60 percent of humans have pre-existing antibodies against certain AAV serotypes, which can lead to a lack of efficacy even with first-dose formulations (Kruzik et al., 2019). Secondly, the packaging efficiency of recombinant AAV is limited to nucleic acids smaller than 5 kb (Wu et al., 2010). Thirdly, AAV is usually produced recombinantly by transfection, which presents scalability issues (in particular, clinical trials have reported administering 10¹²–10¹³ recombinant(r)AAV genome copies per kg of body weight for hepatic transduction gene therapy (Miesbach et al., 2018), thus reaching the acceptable cost limits of current production technologies). Finally, while some argue that approximately 90% of rAAV capsids are "empty," and that empty particles can also be used as decoys to suppress existing immunities, it should be noted that, in addition to their ineffectiveness, they can also function as a potential source of toxicity (Gao et al., 2014).

[0005] LNPs: Highly efficient loading and delivery, but insufficient targeting. These problems, along with the additional difficulties associated 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 in their structure and properties depending on the synthesis of their backbone (e.g., polyamide or polypropyleneimine backbone) and the subsequent selection of size, charge, and composition. LNPs can be formulated by combining various biomaterials with cholesterol, lipid-PEG compounds, and other lipids, and are currently widely used, for example, in the context of SARS-CoV-2 vaccines. In both cases, libraries of resulting nanoparticles (generated by varying the molar ratios of different components) have demonstrated some degree of tissue selectivity (Lokugamage et al., 2018). While promising, this approach relies on (unknown and not understood) similarities between humans and mice that could determine the reinterpretation of targeting, 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 [Overview of the project] [Problems that the invention aims to solve]

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

[0008] The underlying technical problem of the present invention is considered to be the provision of means and methods to address the above-mentioned needs. The technical problem is solved by the embodiments characterized in the claims and below herein. In other words, the present invention involves the following steps: (a) A step of providing recombinant Trypanosoma bruseyi cells expressing VSG, preferably a solutagging-capable VSG; (b) The step of treating the cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed; (c) A step to isolate the cell membrane from the solution in step (b); (d) A step of suspending the isolated membrane obtained in step (c) in an isotonic solution; (e) A step of processing the suspended cell membrane obtained in step (d) using sonication to obtain a vesicle suspension; (f) A step of removing aggregated membrane residue from the vesicle suspension obtained in step (e); (g) A step of separating the vesicle suspension into a group; and (h) A step of providing vesicles from a population of vesicles characterized by the following parameters: (i) having a single dominant 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 homogeneous surface structure in electron micrographs. This relates to a method for preparing vesicles, including [specific details omitted]. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic diagram illustrating the generation and utility of nanoVAST. (A) nanoVAST is generated by sonication of an osmotically lysed cell preparation of the single-cell eukaryote Trypanosoma burseyi. After sonication and a series of filtration and centrifugation steps, the sonicated membrane is purified (e.g., loaded onto a size exclusion column for purification). (B) nanoVAST is a cargo-carrying vesicle coated with T. burseyi surface protein VSG, which 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 target cells; in this case, B cells. (C) Since nanoVAST is coated with a soltagging-capable VSG protein, it can be coupled to a soltagging-capable ligand. The coupling of nanoVAST to a nanobody specific to B cell proteins is shown, which subsequently promotes an increased uptake rate compared to (B). (D) Attachment of cell type-specific ligands to nanoVAST not only increases the rate of delivery to cells, but can also specifically promote delivery to that cell type within an additional population of off-target cell types. [Figure 2]This figure shows nanoVAST vesicle generation and characterization. (A-B) Gel filtration chromatograms after separation based on Speros 6 Increase resin and Cefakryl S-500 resin of sonicated Trypanosoma b. brucei membrane lysates isolated from material equivalent to 5 billion cells. The Y axis shows the total amount of protein eluted at any point throughout the run, via absorbance at 280 nanometers. (C) SDS-PAGE and Coomassie staining of elution peaks from A. VSG is dominant as the major protein present in nanoVAST. (C) Dynamic light scattering of purified nanoVAST reveals that they are homogeneous and have an average diameter of approximately 250 nM. [Figure 3] This figure shows cryo-electron micrographs of nanoVAST vesicles. (A) Images were acquired on a Talos Arctica after embedding nanoVAST preparations on an UltrAuFoil grid. This two-dimensional representation of nanoVAST reinforces the hypothesis that the phospholipid bilayer is very densely covered by VSG proteins (hair-like structures around the circle of the entire membrane), similar to the density present on the natural membranes of living organisms. Furthermore, the scale bar (50 nM) supports the finding that nanoVAST has a diameter of approximately 200-250 nM. (B) NanoVAST derived from purified preparations was adsorbed onto a glow-discharge carbon-coated copper grid (mesh size 300), washed twice with distilled water, negatively stained with 1% aqueous uranyl acetate, and then images were acquired on a Zeiss (Zeiss EM912 microscope, 80 kV (Carl Zeiss, Oberkochen, Germany)). The scale bar (100 nm) supports prior findings on vesicles in the 200-250 nm range. [Figure 4]This figure shows the loading of nanoVAST vesicles by electroporation. (A) Flow cytometry data collected from fluorescently loaded nanoVASTs after electroporation using five different program settings on the Amaxa Nucleofector. Program U033 is the most effective for nanoVAST loading. (B) Charts from both (A), showing only the U033 setting and unstained nanoVASTs. [Figure 5] This figure shows the delivery of nanoVAST cargo to target cells. Flow cytometry data collected from HEK cells incubated with cargo-loaded nanoVAST. In (A), the fluorescent molecule FAM, representing a small therapeutic drug, was loaded onto the nanoVAST by electroporation. In (B), the fluorescent (Cy5)-labeled short RNA oligomer, representing, for example, CRISPR guide RNA, was loaded onto the nanoVAST. [Figure 6]This figure shows that VSG is detectable on the surface of nanoVAST-targeted cells. Flow cytometry data collected from (A) HEK cells and (B) Ramos B cells incubated with nanoVAST. Subsequently, the cells were stained with an anti-VSG antibody, which revealed that the VSG protein could be found on the surface of the treated cells for at least a certain period of time after treatment. This supports the hypothesis that the nanoVAST cargo can be delivered to target cells via membrane fusion (as opposed to endocytosis, which would not deposit VSG on the cell surface). (C) Possible pathways of nanoVAST entry and delivery into cells. Fusion to the cell membrane via (i) endocytosis with simultaneous release of the cargo or (ii) endocytosis with delayed release of the cargo. The finally fused vesicles are picked off (iii) and fused to the multiplasmic reticulum (iv). Release of the cargo via endocytosis can also occur in the multiplasmic reticulum via retrofusion (v). (D) Immunofluorescence microscopy observation of fixed and permeabilized nanoVAST-treated HEK cells. Multichannel synthesized images show co-localization 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. The nucleus and membrane were visualized using DAPI (4',6-diamidino-2-phenylindole dihydrochloride) and WGA (Alexa Flour 594 conjugated wheat germ agglutinin), respectively. Recycling endosomes were stained with rabbit monoclonal anti-RAB11 (Cell Signaling Technology, #5589T) and anti-rabbit IgG Alexa Fluor 488 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]This figure shows that targeting of nanoVAST to specific cell types via sol-tagging enhances delivery. (A) nanoVAST was sol-tagged to anti-CD19 nanobodies, incubated with Ramos B cells, and then stained with an anti-VSG antibody. The amount of VSG deposited on the target cell membrane increased compared to non-specific nanoVAST delivery. This experiment is similar to the schematic diagram shown in Example 4, Figure 6B. (B) nanoVAST sol-tagging was validated through FACS analysis. Flow cytometry data of nanoVAST prepared from VSG3-expressing T. brusey, which was sol-tagged using the fluorescent molecule TAMRA to track and measure vesicle sol-tagging. [Figure 8-1] This figure shows the generation and characterization of nanoVAST vesicles by fractional centrifugation. (A) Method for isolating nanoVAST from sonicated T. brusey membranes via repeated centrifugation steps. NanoVAST is expected to remain in the supernatant at rates less than 20,000 g. All centrifugation is performed at 4°C. (B) Dynamic light scattering of purified pelletized nanoVAST compared to purified nanoVAST from FPLC or gel filtration column reveals that they are also uniform but smaller, with an average diameter of approximately 150 nm. (C) Transmission electron microscopy reveals spherical structures with a high electron density coating; nanoVAST. (D) SDS-PAGE analysis by Coomassie staining of pelletized nanoVAST reveals that VSG is predominant as the major protein present in the preparation. [Figure 8-2] Continuation of Figure 8-1. [Figure 9-1]This figure shows a schematic diagram and readouts of an auxiliary polishing step during nanoVAST delivery vesicle preparation. (A) The insertion of a polishing step using CaptoCore resin may be at one or more time points during the preparation of the final nanoVAST delivery particles, for example, before (i) or after (ii) purification, after (iii) soltagging, and before administration (iv). (B) FPLC chromatogram of nanoVAST preparation after polishing with CaptoCore resin (step ii after purification). The y-axis shows the total amount of protein eluted at any point throughout the run via absorbance at 280 nanometers. (C) Electron microscope image of nanoVAST elution peaks from (B) after adsorption onto a glow-discharge carbon-coated copper grid (mesh size 300) washed twice with distilled water and negatively stained with 1% aqueous uranyl acetate. The vesicles remain at an appropriate size after CaptoCore purification. (D) Dynamic light scattering of the nanoVAST peak eluted from (B) reveals that nanoVAST is uniform and has an average diameter of approximately 150 nm. (E) SDS-PAGE and Coomassie staining of the eluted peak from (B). VSG is predominant as the main protein present in nanoVAST. [Figure 9-2] Continuation of Figure 9-1. [Figure 10-1]Figure showing freeze-drying as an alternative method of loading onto the nanoVAST vehicle. (A) To load RNA cargo onto nanoVAST using the freeze-drying method, 9 μg of VSG protein corresponding to nanoVAST was mixed with 42 pmol of Cy5-tagged RNA in an RNase-free centrifuge tube. The amount of nanoVAST used was determined by SDS-PAGE quantification referring to the VSG3 protein standard. The sample was loaded into a freeze dryer (Alpha 1-2 LSCbasic-Martin Christ), and the required drying time depends on the volume of the sample. Subsequently, the freeze-dried sample was rehydrated with HEPES buffer (20 mM HEPES, 150 mM NaCl), mixed by pipetting up and down, and gently vortexed. Then, the sample was centrifuged at 20,000 g for 30 minutes at 4°C to remove any unloaded RNA cargo. The obtained nanoVAST pellet was resuspended in 1×PBS for evaluation of RNA loading and for HEK cell delivery experiments. (B) Dynamic light scattering graph showing the size distribution of freeze-dried-rehydrated nanoVAST vehicles. The vehicles are uniform and have an average diameter of approximately 200 nm. (C) Transmission electron microscopy image of nanoVAST vehicles. The image confirms that the structure of nanoVAST is preserved after freeze-drying. (D) SDS-PAGE analysis of freeze-dried-hydrated nanoVAST showing VSG3 as the dominant protein. (E) Western blot verifying that the nanoVAST surface protein VSG3 is preserved after the freeze-drying process. (F) Flow cytometry data collected from freeze-dried-hydrated nanoVAST loaded with Cy5-tagged RNA by freeze-drying and subsequent rehydration. This results in efficient RNA loading. [Figure 10-2] Continuation of Figure 10-1. [Figure 11A]Figure showing the uptake of cargo by HEK cells through electroporation and lyophilization. (A) HEK cells supplied with nanoVAST loaded with Cy5-tagged RNA by electroporation. The chart shows HEK cells that took up nanoVAST as indicated by the positive VSG3 signal (i). VSG3-negative (ii) and VSG3-positive (iii) were selected and evaluated 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 showing the uptake of cargo by HEK cells through electroporation and lyophilization. (B) As a comparison, HEK cells supplied with 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 the positive VSG3 signal (i). VSG3-negative (ii) and VSG3-positive (iii) were selected and evaluated 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] Figure showing that nanoVAST classification can be changed through VSG modification or selection. The nanoVAST type can be changed through (i) the use of peptide sorting, such as peptide ligands, antibodies, or nanobodies; (ii) the use of VSG gene modification through the addition of a coded targeting peptide tag, such as those listed in Table 1; and (iii) complete VSG switching through selection from approximately 2000 different VSG genes encoded in the T. brucei genome. Examples of a part of the VSG protein of the exposed region as the molecular surface, shaded according to polarity (from white to gray: from polar to hydrophobic). Differences in surface topography, polarity, or other physical properties can affect nanoVAST cell targeting and physical properties. [Figure 13]This figure shows the purification of nanoVAST from T. bursey expressing the Iltat (ILTat1.24) protein as a substitute for VSG3. (A) Dynamic light scattering of Iltat nanoVASTs purified using the fractionation centrifugation method described in Figure 8 reveals that they are uniform and have an average diameter of approximately 150 nm, similar to VSG3 nanoVASTs. (B) Electron microscope images of ILTat nanoVASTs after adsorption onto a glow-discharge carbon-coated copper grid (mesh size 300) after being washed twice with distilled water and negatively stained with 1% aqueous uranyl acetate. The scale bar (250 nm) supports prior findings of vesicles in the 200-250 nm range. (C) SDS-PAGE and Coomassie staining of Iltat nanoVASTs. Iltat is predominant as the main protein present in nanoVASTs, demonstrating the possibility of successful purification of different nanoVAST types. (D) NanoVAST sol-tagging was validated through FACS analysis. Flow cytometry data of nanoVAST prepared from ILtat-expressing T. brusey, which was desoltagged using the fluorescent molecule TAMRA to track and measure vesicle desoltagging. [Figure 14] This figure shows that RAW264.7 macrophages do not produce TNF-α in response to nanoVAST stimulation. Cells were stimulated with inactivated Escherichia coli (E. coli), 100 nM monophosphoryl lipid A (MPLA), and 10 nM nanoVAST. For ELISA analysis, the culture medium was collected from the wells at the indicated time. Each data point represents the mean ± SD of the triple-repeat wells. The Y-axis shows the ELISA signal based on raw absorbance from the plate reader. [Modes for carrying out the invention]

[0010] In this specification and in the claims, it should be understood that "a" and "an" may mean one or more of the following items, depending on the context in which they are used. That is, for example, a reference to an item "an" may mean that at least one item is available.

[0011] In their use below, the terms “have,” “include,” or “listed” have either a non-restrictive or restrictive meaning. That is, when restrictive, these terms may refer to a situation where there are no other features in the described embodiment besides those introduced by these terms; i.e., they have a restrictive meaning in the sense of “consisting of” or “essentially consisting of.” When non-restrictive, these terms refer to a situation where there is one or more other features in the described embodiment besides those introduced by these terms.

[0012] Furthermore, where used below, the terms “preferred,” “more preferably,” “most preferably,” “particularly,” “more specifically,” “typically,” and “more typically” are used in conjunction with features to indicate that these features are preferred features; that is, these terms indicate that alternative features may also be expected according to the present invention.

[0013] Furthermore, it will be understood that the term "at least one," when used herein, means that one or more of the items referred to following this term may be used in accordance with the invention. For example, where 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 referred to by this term, a person skilled in the art will understand what upper limit this term may refer to, if any.

[0014] When used herein, the term "about" means that, with respect to any number following the term, there exists an accuracy in the interval that can achieve the technical effect. Thus, as used herein, "about" preferably means the exact number or a range of ±20%, preferably ±15%, more preferably ±10%, or even more preferably ±5% of the exact number.

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

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

[0017] Step (a) of the method of the present invention provides recombinant Trypanosoma bruseyi cells expressing VSG, preferably a soltagging-enabled VSG. The term “recombinant Trypanosoma brucei cells” refers to single-celled parasites. Trypanosoma brucei is a pathogenic organism that causes sleeping sickness. When referenced herein, Trypanosoma brucei encompasses all subspecies, including Trypanosoma brucei brucei, Trypanosoma brucei gambiense, and Trypanosoma brucei rhodesiense. Trypanosoma brucei cells can be genetically modified, thus enabling the creation of recombinant organisms. Trypanosoma brucei exhibits a glycoprotein coat on its surface containing mutant surface glycoproteins (VSGs).

[0018] The term "VSG," as used herein, refers to a class of glycoproteins with a monomeric size of approximately 60 kDa that can be found as monomers, dimers, trimers, or multimers, densely arranged on the outer membrane to form the surface coat. VSG dimers constitute approximately 90% of all cell surface proteins in trypanosomes. VSG is highly immunogenic, and an immune response against a specific VSG coat will rapidly kill trypanosomes expressing this variant. However, with each cell division, offspring have the opportunity to switch from expressing one VSG gene to another, thereby altering the expressed VSG and thus escaping immunity. At any given time, only one type of VSG gene is expressed. VSG expression is "switched" from an array to an expression site located at an active telomere (directed by homology) by homologous recombination induced by silent double-strand breaks of the basic copy gene. VSG annotations, protein sequences, and gene sequences can be derived from public databases such as www.ensemble.org. A collection of VSGs for Trypanosoma bruisei (Lister 427 strain) is published in Cross 2014, Mol. Biochem. Parasitol. 195(1):59-73. Preferred VSGs in the context of the present invention are Trypanosoma bruisei VSG, more preferably VSG2, VSG3 ILTat1.24, VSG11, VSG13, VSGsur, VSG1954, or VSG531. For example, a preferred VSG according to the present invention is characterized by an N-terminus that is three-dimensionally positioned within the VSG, preferably at a position sufficiently close to the accessible coat surface, such that the addition of a linker sequence of preferably 100 amino acids or less in length, preferably 50 amino acids or less, and most preferably 20 amino acids or less, allows for modification of the thus elongated or unelongated N-terminus of the VSG protein. Preferably, the insertion may be immediately downstream of the signal peptide cleavage site.

[0019] Furthermore, recombinant Trypanosoma bruseyi cells can be genetically engineered to express two or more types of VSGs, thereby allowing vesicles obtained from such genetically engineered Trypanosoma bruseyi cells by the method of the present invention to contain two or more different types of VSGs on the surface of the vesicles.

[0020] Furthermore, recombinant Trypanosoma bruseyi cells are preferably deficient in GPI-phospholipase C. Since inactivation of any form of trypanosoma cell (e.g., UV irradiation) will also lead to the breakdown of the VSG coat and the cell itself unless GPI-PLC is removed from the genome, gene 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. bruseyi, which can be used, for example, as an antigen-presenting platform. If VSG is shed due to the action of GPI-PLC, the VSG coat breaks down and the cell lyses.

[0021] The term "soltagging-capable," as used herein, means a VSG that can be ligated to another polypeptide or peptide by saltase activity. Preferably, the VSG will contain a saltase donor or saltase acceptor amino acid sequence. Typically, such saltase donor or acceptor sequence can be introduced into the VSG by genetic engineering. For example, such saltase donor or acceptor sequence can be included in the peptide to be fused to the VSG. Alternatively, a suitable endogenous sequence of the VSG can be genetically modified to become a saltase donor or acceptor sequence.

[0022] As used herein, "soltase" refers to a protein having saltase activity, i.e., an enzyme capable of carrying out a peptide transfer reaction that conjugates the C-terminus of a protein to the N-terminus of a protein via aminotransfer. This term includes full-length saltase proteins, e.g., full-length naturally occurring saltase proteins, fragments of such saltase proteins having saltase activity, modified (e.g., mutant) variants or derivatives of such saltase proteins or fragments thereof, and proteins that do not originate from naturally occurring saltase proteins but exhibit saltase activity. Those skilled in the art can easily determine whether a given protein or protein fragment exhibits saltase activity by, for example, contacting the protein or protein fragment with a suitable saltase substrate under conditions that enable peptide transfer and determining whether the corresponding peptide transfer reaction product is formed. Suitable saltases, as will be apparent to those skilled in the art and are not limited to, include saltase A, saltase B, saltase C, and saltase D type saltases. Suitable saltases 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. Furthermore, the present invention encompasses embodiments relating to saltase A derived from any of the bacterial species or strains. Those skilled in the art will understand that, without limitation, any of the saltases and saltase recognition motifs described in International Publication Nos. 2010 / 087994, 2011 / 133704, and 2020 / 84072 may be used in some embodiments of the present invention.

[0023] A saltase substrate is an amino acid sequence that can be utilized in a saltase-mediated peptide transfer reaction. Typically, saltase utilizes two types of substrates: a substrate containing a C-terminal saltase recognition motif and a second substrate containing an N-terminal saltase recognition motif, and the peptide transfer reaction results in the conjugate of both substrates via covalent bonding. In the context of this invention, the “C-terminal saltase recognition motif” is also referred to as the “solutagging donor sequence,” while the term “N-terminal saltase recognition motif” is referred to as the “solutagging acceptor sequence.” Preferably, the C-terminal and N-terminal recognition motifs are contained in different amino acid sequences, for example, one of the N-terminuses of a VSG and another linked to an immunogen, thereby generating a free carboxyl group at the end of the solutagging donor site. Some saltase recognition motifs are described herein, and additional preferred saltase recognition motifs are well known to those skilled in the art. The saltase recognition motifs will be obvious to those skilled in the art. The saltase substrate may contain additional parts or entities separate from the peptidolytic saltase recognition motif.

[0024] For example, a saltase substrate may include an LPXTG / A motif whose N-terminus is conjugated to any of the agents (e.g., a peptide or protein, a small molecule, a binder, a lipid, a carbohydrate, or a detectable label). Similarly, a saltase substrate may include an oligoglycine (G1-5) motif or an oligoalanine motif, preferably G3 or G5, whose C-terminus is conjugated to any of the agents (e.g., a peptide or protein, a small molecule, a binder, a lipid, a carbohydrate, or a detectable label). Therefore, a saltase substrate is not limited to a protein or peptide, but includes any portion or entity conjugated to a saltase recognition motif.

[0025] The VSG is preferably “soltagging-able,” that is, it is a saltase substrate, i.e., it will contain a saltase donor sequence or a saltase acceptor sequence. An example of a solderagging-able VSG can be derived from Pinger 2017, Nat Commun. 8(1):828. Depending on whether the VSG contains a saltase acceptor or a donor, the molecule linked to the VSG by solderagging will contain a complement, i.e., if the VSG contains a saltase acceptor, the molecule will contain a saltase donor, or if the VSG contains a saltase donor, the molecule will contain a saltase acceptor.

[0026] It will be understood that the VSG can also be chemically modified. Preferably, the VSG can be modified by a chemical coupling reaction to couple a desired molecule, such as a targeted compound as specified elsewhere in this specification. Typically, the targeted compound can be covalently bonded to the N-terminus of the VSG, preferably via a linker. The targeted 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 this invention, click chemistry means a chemical structure created to rapidly and reliably produce a covalent bond by linking together small units containing reactive groups. Any variation of such an approach can be used to link the targeted compound to the VSG according to this invention. Generally, linking of the VSG to the targeted compound may include the use of any linking means or linker, which in the context of this invention as disclosed herein means by which the VSG and the targeted compound are linked or connected to form a modified VSG. One or more linkers or linking means for linking the VSG and the targeted compound may be any structurally suitable means for connecting the two. Examples of 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 having a modified peptide backbone. The means for linking may include covalent and / or non-covalent bonds. One or more linkers may include various sequences or other structural features that provide various functions or properties. For example, one or more linkers may include structural elements that enable VSG derivatization.

[0027] Preferably, recombinant Trypanosoma bruseyi cells expressing VSG, preferably sol-tagging VSG, are provided in a purified or partially purified form. Typically, the cells will contain or substantially no culture medium. Preferably, such preparations of recombinant Trypanosoma bruseyi cells expressing VSG, preferably sol-tagging VSG, can be obtained by centrifugation to allow separation of the medium from the cells and resuspension of the cells in a suitable solvent, such as a hypotonic solution to be used for the subsequent step (b). If the preparations of recombinant Trypanosoma bruseyi cells expressing sol-tagging VSG are to be stored, an isotonic solution can be used to allow storage of the cells. Suitable solutions are well known to those skilled in the art. Particularly preferred techniques for providing recombinant T. bruseyi cells are described in the appended examples below. These techniques may include centrifugation of the cells to obtain a cell pellet and one or more washing steps.

[0028] More preferably, T. brusey cells grown in a cell culture medium, preferably standard HMI9 containing 10% FBS, are centrifuged at approximately 2,000 g for approximately 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 execution.

[0029] In step (b) of the method of the present invention, the cells, i.e., recombinant Trypanosoma brusey cells expressing VSG, preferably soltagging-capable 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 a trypanosoma cell. Suitable hypotonic solutions are well known to those skilled in the art. Typically, a hypotonic solution may be deionized water or a buffer solution with a low salt content. Water molecules in a hypotonic solution will move into the cell, i.e., into the cytoplasm, due to osmosis. As a result, the volume of the cytoplasm will increase, disrupting the cell membrane, thereby tearing it and forming several small fragments of isolated cell membrane. Preferably, deionized water is used as the hypotonic solution.

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

[0032] The process is also carried out in the presence of at least one protease inhibitor. This at least one protease inhibitor will inhibit the protease activity that can degrade the VSG protein of the VSG coat present on the membrane. Suitable protease inhibitors are known to those skilled in the art. In particular, it is expected that a mixture of several different protease inhibitors will be used. Preferably, the HALT protease inhibitor composition as referred 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 aspartate protease inhibitor. More preferably, this 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] Cell lysis typically results in a torn cell membrane of recombinant Trypanosoma bruseyi cells expressing soltagging-capable VSG, also referred to as the "cell membrane." This membrane can vary in size and preferably contains intact VSG. The degree of lysis can be visualized by applying a dye to the viable cells. Particularly preferred lysis conditions are described in the attached examples below.

[0034] More preferably, the pelleted T. brusey 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 present invention, the cell membrane is isolated from the solution obtained in step (b). Isolation of the cell membrane from the residual material of lysed cells can be achieved by techniques well known in the art. Preferably, the cell membrane is isolated by centrifugation, more preferably as described in the appended examples below. Alternatively, the cell membrane can be obtained in an isolated form by filtration, affinity purification using antibodies against VSG, size exclusion chromatography, size / density-based fractionation centrifugation, ion exchange chromatography, gel filtration chromatography, osmotic enrichment using semipermeable membranes, or electrophoretic separation techniques. The above techniques for isolating the cell membrane can be used alone or in combination to obtain a cell membrane of higher purity. Furthermore, the cell membrane can be subjected to one or more washing steps. Typically, the isolation of the cell membrane is carried out at the temperature described above with respect to step (b), more preferably at about 4°C. More preferably, the cell membrane is isolated by centrifugation at about 10,000 g for about 10 minutes at about 4°C. Subsequently, the pellet is preferably resuspended in ice-cold deionized water and centrifugation is repeated under the same conditions.

[0036] In step (d) of the method of the present invention, the isolated membrane obtained in step (c) is suspended in an isotonic solution. The term "isotonic solution," as used herein, means a solution that would be isotonic with respect to the cytoplasm of trypanosoma cells. Suitable isotonic solutions are well known to those skilled in the art. Typically, an isotonic solution may be a buffer solution having an 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 a hypotonic solution.

[0037] In step (e) of the method of the present invention, the suspended cell membrane obtained in step (d) is treated with sonication to obtain a vesicle suspension. Ultrasonic disruption can be performed by applying constant or pulser ultrasound for a sufficient amount of time to allow the formation of vesicle structures in solution. Typically, this time is about 3 to 10 minutes, more preferably about 4 to 6 minutes, and most preferably about 5 minutes. More preferably, ultrasonic disruption is performed for 5 minutes at 40% load using a Bandelin Sonoplus machine with pulser, as described in the examples below. This process preferably results in shearing of larger cell membranes, thereby yielding 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 membrane residue is removed from the vesicle suspension obtained in step (e). Preferably, the aggregated membrane residue is removed from the vesicle suspension obtained in step (e) by centrifugation and filtration. Preferably, filtration is performed using a 0.45 μM filter.

[0039] Aggregated membrane residue, typically having a size of about 750 nm or larger, is removed by centrifugation, preferably at about 2,000 g for about 5 minutes at about 4°C. A filtration step will then be performed. Preferably, a filter having a sieve in the micrometer range is used. Pressure can be applied by known methods, and typically by using a syringe. Preferably, the aggregated membrane residue is removed from the vesicle suspension obtained in step (e) by centrifugation and filtration as described in the appended examples below. More preferably, the suspension containing vesicles and aggregated membrane residue is centrifuged at 2,000 g for 5 minutes at 4°C, thereby pelletizing any remaining large aggregated pelletizable membrane residue and intact cells, if present. The supernatant will then be filtered through a 0.45 μM filter using a 10 mL syringe. Each of the above steps relating to ultrasonic fracturing, centrifugation, and / or filtration can 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 aggregates. The separation of vesicles into groups of roughly similar size can be achieved by various means. Preferably, the separation as referred to herein is size separation. More preferably, size exclusion chromatography such as gel filtration or CaptoCore chromatography can be used. Alternatively, centrifugation-based techniques such as fractional centrifugation, centrifugation using a cesium or sucrose gradient, or ultracentrifugation can be applied. Those skilled in the art will be well aware of how such size separation can be achieved.

[0041] Gel filtration of vesicle suspensions according to the present invention, as referred to in the context of the present invention, can be carried out by a chromatographic method in which particles or molecules in a solution are separated by their size. Typically, an aqueous solution, i.e., the 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 packed with fine porous beads composed of a polymer, preferably a dextran polymer (Cefadex), agarose (Cefalose), or polyacrylamide (Cefakryl or BioGel P). The polymer allows larger particles or molecules to pass through the column somewhat quickly, while relatively smaller molecules can enter the spaces formed by the polymer and therefore may take longer to pass through the column. Preferably, for gel filtration, a Cefakryl S-500 26 / 60 or Superose 6 column can be used. Preferably, gel filtration can be carried out as described in the appendix examples below.

[0042] In the context of this invention, CaptoCore chromatography refers to a purification method for purifying and polishing viruses and other large biomolecules in a flow-through manner. Typically, the chromatography medium (resin) comprises beads (core beads) having an unfunctionalized outer layer (ligand-free) and a functionalized core to which the ligand is attached. The inert outer layer of cross-linked agarose prevents viruses and other large entities with molecular weights greater than approximately 400,000 (CaptoCore400) or 700,000 (CaptoCore700) from entering the core of the beads, while small molecules penetrate the beads and are captured there. The excluded target molecules are collected during flow-through. The functionalized core contains an octylamine ligand within the core of the beads, 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 time point during the preparation of the delivery particles of this invention. For example, CaptoCore chromatography can be applied before or after vesicle purification, after soltagging, or before vesicle administration.

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

[0044] Preferably, the vesicle clusters can be obtained by using chromatographic techniques other than those based on size exclusion. More preferably, ion exchange or immunoaffinity chromatography can be applied. A person skilled in the art will be well aware of how such techniques can be performed.

[0045] In step (h) of the method of the present invention, vesicles are provided from the population of vesicles obtained in step (g). The vesicles obtained by the method of the present invention (i.e., nanoVAST) can be subjected to various characterizations, including SDS-PAGE for protein purity and dynamic light scattering for vesicle size and homogeneity determination (Figures 1B and C). The provided vesicles are characterized by the following parameters: (i) having a single dominant 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) exhibiting a homogeneous surface structure in electron micrographs. SDS-PAGE as referred to herein is preferably carried out as described in the appendix examples below. In particular, the nanoVAST sample is diluted in a standard SDS-PAGE denaturing sample buffer prior to separation on a 10% Tris-glycine polyacrylamide gel. Next, the gel is either stained Coomassie to observe various protein components or subjected to Western blot analysis to specifically identify VSG bands. Standard transmission electron micrographs are preferably acquired using the following settings and conditions, where vesicles from the purified preparation are adsorbed onto a glow-discharge carbon-coated copper grid (mesh size 300), washed twice with distilled water, and negatively stained with 1% aqueous uranyl acetate. Micrographs were acquired using a slow-scan CCD camera (TRS, Moorenweis, Germany) and a Zeiss EM912 (Carl Zeiss, Oberkochen, Germany) at 80kV, producing a pixel size of 1.2 or 0.6 nm (depending on the nominal magnification of 10k× or 20k×, respectively). Cryo-electron micrographs are preferably obtained using the following settings and conditions: the sample is placed on a plasma-washed UltrAuFoil grid (200 mesh; pore size 2 μM × 2 μM), followed by blotting for 2 seconds and plunge-freezing using an FEI Vitrobot Mark IV. The grid is imaged using a Talos Artica microscope.

[0046] Preferably, the vesicle population 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 Playtical) was used for measurement, and the accompanying ZetaSizer software was used for vesicle analysis. 20 μL of sample was diluted in 1 mL of PBS, placed in a disposable polystyrene cuvette, and subsequently measured.

[0047] Advantageously, it has been found in the foundational research of this invention that artificial vesicles can be generated from T. brusey cell membranes, enabling efficient loading and excellent targeting, and thus avoiding the shortcomings of other delivery systems known in the prior art. nanoVAST vesicles consist of a phospholipid bilayer (which would protect the cargo from, for example, nucleases or proteases) and may 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 inherently capable of transporting genetic information back and forth between distant cells (Bobis-Wozowicz et al., 2015), (ii) they would be stable in body fluids, and (iii) they would be preserved over long periods.

[0048] Currently, natural extracellular vehicles (EVs) are not produced in sufficiently large quantities to be useful. However, the loading, targeting, and overall efficiency of synthetic EVs from prior art remain low (Almeida et al., 2020). nanoVAST vesicles are centered around semi-synthetic EVs, whose main skeleton can be mass-produced and which can efficiently load protein and nucleic acid cargoes. In addition, nanoVAST vesicles can be enzymatically fused to receptor-binding ligands specific to individual cell types, which ensures targeted delivery from the outset. nanoVAST vesicles are inexpensive to manufacture, scalable, and avoid excessive immunogenicity or oncogenicity while retaining the best qualities of the viral system (cell-type specific targeting and convenient packaging).

[0049] The similarity of nanoVAST technology to naturally occurring exosomes (a long-distance delivery service between tissues in nature itself), the feasibility of nanoVAST vesicle loading with target payloads, and the ability to derivatize them using rationally designed "tags" (e.g., antibodies) that target specific cell types represent a significant improvement in the field of precise payload delivery.

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

[0051] In a preferred embodiment of the method of the present invention, the method includes the following steps after step (c) and before step (d): - The step of treating the cells in a deionized aqueous solution; and - Step of isolating the cell membrane from the solution. It also includes. In a preferred embodiment of the method of the present invention, the isotonic solution is a buffer containing 20 mM HEPES, 150 mM NaCl2, and pH 8. In a preferred embodiment of the method of the present invention, the ultrasonic fracturing in step (e) is performed using a pulser at a 40% load for 5 minutes.

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

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

[0054] In a preferred embodiment of the method of the present invention, the method further includes the step of introducing the target cargo agent into the vesicle provided in step (h). Preferably, the introduction step is the following steps: (a) The step of suspending the vesicles in a transfection buffer containing the excess cargo agent to be targeted; (b) the step of performing electroporation; and (c) Step of purifying the loaded vesicles after electroporation. Includes.

[0055] The cargo chemicals to be introduced into the vesicles can also be introduced by freeze-drying, and the introduction step is as follows: (a) A step of freeze-drying the vesicles in the presence of the load; (b) Resuspending the lyophilized vesicles and cargo in a resuspension buffer; (c) the step of mixing the resuspended mixture; and (d) Step of purifying the loaded vesicles Includes. More preferably, the target cargo drug 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 a capsule-encapsulated form, such as compounds that will enter cells but do not interact with extracellular structures in the organism or are unstable in culture media. Preferably, cargo molecules according to the present invention are selected from the group consisting of small molecule drugs, antibodies, aptamers, cytokines, growth factors, hormones and other therapeutic proteins and peptides, nucleic acid molecules such as RNA or DNA, and detectable labels such as dyes used to stain cells.

[0057] In preferred embodiments, the cargo molecule 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) means a single-stranded DNA and / or RNA molecule capable of interfering with DNA and / or RNA processing. An antisense oligonucleotide contains a nucleic acid sequence 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 may interfere with mRNA processing through RNase H-mediated degradation, translational arrest, or splicing regulation, or through protein steric hindrance. Means and methods for the design and synthesis of antisense oligonucleotides are well known in the art and include, for example, rational design, chemical modification, and solid-phase chemical synthesis of antisense oligonucleotides including locked nucleic acids (LNAs). Antisense oligonucleotides can be chemically synthesized or expressed in cells, for example, by introducing their respective recombinant DNA constructs. It will be understood by those skilled in the art that such DNA constructs may 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 capable of recruiting an RNA editing enzyme. More preferably, the RNA editing enzyme is an adenosine deaminase (ADAR) that acts on RNA. The term "ADAR" refers to a double-stranded RNA-specific adenosine deaminase that catalyzes hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA), also known as AtoI editing. Preferably, the ADAR is of human origin. The ADAR may be ADAR1, ADAR2, or ADAR3. The ADAR may be an endogenous ADAR that is recombinantly expressed in target cells or delivered to target cells.

[0060] The antisense oligonucleotides to be transported by the vesicles of the present invention may be part of an expression cassette. The term “expression cassette” means a separate component or isolated fragment of vector DNA consisting of genes and regulatory sequences that enable expression in prokaryotic or eukaryotic cells. For example, antisense oligonucleotides can be ligated into a nucleic acid expression construct (i.e., a vector) under the transcriptional control of cis-regulatory sequences suitable for directing 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 may also include polyadenylation sites to increase translation efficiency.

[0061] In particular, the antisense oligonucleotides referenced above can be used to generate neoepitopes in immunopeptides associated with cancer, preferably for RNA editing, and more preferably for the generation of neoepitopes in immunogenic peptides in cancer cells. The target antisense oligonucleotide delivered as a cargo agent by the vesicle of the present invention may therefore preferably be specifically hybridized to the nucleic acid sequence of mRNA encoding at least one tumor epitope, thereby enabling the mobilization of ADARs and the conversion of the tumor epitope into a neoepitope. The target neoepitope generated by RNA editing will preferably be tumor-specific and presented via HLA. For this purpose, the antisense oligonucleotide can be expressed from an expression construct such as a vector. Preferably, the expression construct may also include an expression cassette for expressing ADARs as described above. More preferably, the vector used as the expression construct may be a viral vector such as a lentiviral vector. In such cases, it will be understood that the vesicle can target cancer cells in which neoepitopes will be generated.

[0062] In a preferred embodiment of the method of the present invention, the method further comprises the step of soltagging a targeted compound to a soltagging-able VSG on a vesicle provided in step (h). Preferably, the soltagging step includes the following steps: (a) The step of treating the vesicle with saltase in the presence of a targeted compound; and (b) A step of purifying the soltagged vesicles using a targeted compound. Includes.

[0063] The term "targeting compound," as used herein, means a compound that enables the binding of a vesicle to a target molecule present on a target cell. Therefore, a targeting compound may 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 sol-tagging VSG present on the surface of the vesicle of the present invention can similarly be coupled to one or more different targeting compounds. Further details regarding targeting compounds can also be found in International Publication No. 2020 / 84072. More preferably, the targeted compound is an antibody or nanobody that recognizes a target molecule on a target cell.

[0064] The present invention also relates to vesicles containing VSGs, preferably sol-tagging VSGs, characterized by the following parameters: (i) having a single dominant protein revealed after Coomassie staining of SDS-PAGE having an apparent molecular weight of 55-60 kDa; (ii) having a spherical appearance in electron micrographs; and (iii) exhibiting a homogeneous 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 target cargo drug, preferably selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

[0066] In a preferred embodiment of the vesicle of the present invention, the vesicle is soltagged with a targeted compound, preferably an antibody or nanobody that recognizes a target molecule on a target cell. In a preferred embodiment of the vesicle of the present invention, the vesicle can be obtained by the method of the present invention.

[0067] The present invention also relates to vesicles according to the present invention as defined above for use in the treatment and / or prevention of diseases or medical conditions. Furthermore, the present invention also relates to vesicles according to the present invention as defined above for use as compound delivery vesicles, preferably drug delivery vehicles.

[0068] The vesicles of the present invention can be used as carriers for drugs, that is, they can be used to treat and / or prevent diseases. Suitable drugs are preferably selected from the group consisting of small molecule drugs, therapeutic proteins and peptides such as antibodies, aptamers, cytokines, growth factors, hormones, etc., and nucleic acid molecules such as RNA or DNA. Those skilled in the art will be well aware of what diseases can be treated and / or prevented by administering encapsulated drugs, and therefore 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 enable specific targeting of disease-related cells, for example, by soltagging of targeted antibodies or aptamers, due to the presence of soltagging-capable VSGs on their surface.

[0069] The vesicles of the present invention can also be used as vaccines. For this purpose, the solutagging-capable VSG on their surface can be coupled to an antigen or hapten by solutagging. The vesicles thus modified can be administered to a subject and elicit an immune response. Depending on the immunization scheme, such vesicles can be used for primary and / or booster immunizations, which may be applied once or several times. Preferably, immunization using such vesicles can be carried out as described in International Publication No. 2021 / 214043.

[0070] Furthermore, the vesicles of the present invention can be used as in vitro delivery tools for drugs or other compounds intended to be introduced into cultured cells as specified above. 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 present invention, comprising recombinant Trypanosoma brusey cells expressing a soltagging-capable VSG and at least one agent for carrying out the method of the present 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 containing 20 mM HEPES, 150 mM NaCl2, and pH 8, a 0.45 μM filter, 20 mM HEPES, 150 mM NaCl2, and saltase. The present invention also relates to a kit containing the vesicles of the present invention.

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

[0074] Embodiment 2: The method of Embodiment 1, wherein the group 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, when determined by dynamic light scattering analysis. Embodiment 3: The method of Embodiment 1 or 2, wherein the recombinant Trypanosoma bruseyi cells are deficient in GPI-phospholipase C. Embodiment 4: Any one of Embodiments 1 to 3, wherein the at least one protease inhibitor is a HALT protease inhibitor composition.

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

[0076] Embodiment 8: A method according to any one of Embodiments 1 to 7, wherein step (f), in which the aggregated membrane residue is removed from the vesicle suspension, is performed by filtration in step (f) using a 0.45 μM filter. Embodiment 9: Any one of Embodiments 1 to 8, wherein the separation in step (g) is carried out by gel filtration via gel filtration chromatography on a Sephacryl S-500 26 / 60 column, Sperose 6 column or CaptoCore column equilibrated with 20 mM HEPES, 150 mM NaCl2, pH 8. Embodiment 10: Any one of Embodiments 1 to 9, further comprising the step of introducing the target cargo agent into the vesicle provided in step (h).

[0077] Embodiment 11: The introduction step is as follows: (a) The step of suspending the vesicles in a transfection buffer containing the excess cargo agent to be targeted; (b) the step of performing electroporation; and (c) Step of purifying the loaded vesicles after electroporation. A method of embodiment 10, including the method of embodiment 10. Embodiment 12: The introduction step is as follows: (a) A step of freeze-drying the vesicles in the presence of the load; (b) Resuspending the lyophilized vesicles and cargo in a resuspension buffer; (c) the step of mixing the resuspended mixture; and (d) Step of purifying the loaded vesicles The method of claim 10, including the method of claim 10. Embodiment 13: Any one of Embodiments 10 to 12, wherein the target cargo drug is selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

[0078] Embodiment 14: Any one of Embodiments 1 to 13, further comprising the step of soltagging a targeting compound to a soltagging-able VSG on a vesicle provided in step (h). Embodiment 15: The soltagging step is the following: (a) The step of treating the vesicle with saltase in the presence of a targeted compound; and (b) A step of purifying the soltagged vesicles using a targeted compound. The method of Embodiment 14, including the method of Embodiment 14. 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 containing VSG, preferably a soltagging-capable VSG, characterized by the following parameters: (i) having a single dominant protein having 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 homogeneous 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: A vesicle of Embodiment 17 or 18, on which a target cargo drug is loaded, 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 oligonucleotide (oligonucleotoide) or an expression construct encoding it. Embodiment 21: The vesicle of Embodiment 20, wherein the antisense oligonucleotide is suitable for RNA editing, preferably for the generation of neoepitope in immunogenic peptides in cancer cells. Embodiment 22: A vesicle from any one of Embodiments 17 to 21, which is soltagged with a targeted compound, preferably an antibody or nanobody that recognizes a target molecule on a target cell.

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

[0082] Embodiment 26: A kit for performing any one of Embodiments 1 to 16, comprising recombinant Trypanosoma brusey cells expressing a soltagging-capable 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 drug is selected from the group consisting of deionized water, at least one protease inhibitor, preferably a HALT protease inhibitor composition, a buffer containing 20 mM HEPES, 150 mM NaCl2, pH 8, a 0.45 μM filter, 20 mM HEPES, 150 mM NaCl2, and saltase. Embodiment 28: A kit comprising one of the vesicles from Embodiments 17 to 23.

[0083] All references cited throughout this specification are incorporated herein by reference, both in their entirety and with respect to the disclosures specifically mentioned. [Examples]

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

[0085] Example 1: nanoVAST vesicle preparation Approximately 1 to 10 billion T. brusey cells expressing soltagging-capable 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, room temperature for 20 minutes).

[0086] The cells were washed once with phosphate-buffered saline (PBS) to remove residual extracellular proteins, and then the resulting pellet was dissolved on ice for 10 minutes by suspending it in 5 mL of ice-cold deionized H2O containing a HALT protease inhibitor for lysis.

[0087] To isolate membrane-bound substances and remove cytoplasmic content, the dissolved suspension was centrifuged (10,000 g, 4°C for 10 minutes). The ice-cold deionization H2O extraction and centrifugation process was repeated twice.

[0088] Next, the membrane pellet was suspended in 2 mL of 20 mM HEPES, 150 mM NaCl2, pH 8, and sonicated on ice (using a pulser at 40% load for 5 minutes) to shear the large membranes into relatively smaller vesicles. Subsequently, the suspension was centrifuged (2,000 g, 4°C for 5 minutes) to remove any remaining large aggregated pelletizable residue (e.g., intact cells), 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 fresh supernatant was filtered once more to obtain a higher purity solution free of aggregated cell residue and suitable for gel filtration.

[0089] Next, the filtered vesicle suspension was separated by gel filtration chromatography on a Sephacryl S-500 26 / 60 column equilibrated with 20 mM HEPES, 150 mM NaCl2, pH 8 to remove small residues and isolate the largest vesicles, which constitute the majority of the contents of the preparation and will be referred to as "nanoVAST" from now on (Figure 1A, Figure 2A).

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

[0091] Example 2: nanoVAST loading Next, to load the nanoVAST with cargo, 0.5 mg of protein equivalent to the nanoVAST (determined by a colorimetric BCA protein quantification assay and SDS-PAGE comparative quantification against a protein standard) was suspended in a custom-made 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 target cargo (the cargo concentration is molecularly 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). The loaded nanoVASTs were isolated from the free load by centrifugation (20,000 g, 4°C, 30 minutes) and washed multiple times.

[0093] The generated nanoVASTs could be detected by flow cytometry. When the selected cargo was fluorescently labeled, both cargo-loaded and cargo-free nanoVASTs could be measured for their fluorescence intensity via flow cytometry (shown in Figure 4). This facilitated the determination of loading efficiency. Using the U033 program for electroporation, nanoVAST loading efficiency exceeding 99% was observed (Figure 4A). Regarding the experimental setup described, see BD FACSCalibur. TM I used it.

[0094] Example 3: nanoVAST delivery Cells treated with a loaded nanoVAST will incorporate these materials, thereby taking up target molecules. These molecules can be hypothetically small enough to fit into the nanoVAST, including small molecule drugs (represented by the fluorescent molecule FAM, Figure 5A) and nucleic acids (represented by the fluorescent RNA molecule, Figure 5B).

[0095] For processing, cells were plated in 24-well plates at an appropriate density. A mixture of fluorescently loaded nanoVAST and appropriate cell medium was prepared. The amount of nanoVAST used could vary depending on the setting, ranging from 100 to 5000:1 vesicle:cell ratio.

[0096] The aforementioned mixture of nanoVAST and culture medium was then added dropwise to each cell culture well, and the cells were incubated in an incubator (37°C, 5% CO2) for at least 1.5 hours before further analysis.

[0097] The cells were harvested and washed three times with PBS (100g, room temperature for 7 minutes). After flow cytometry, it was determined that the treated cells had increased fluorescence, suggesting that they had taken up the cargo delivered by nanoVAST (Figure 5).

[0098] Example 4: nanoVAST delivery facilitated by membrane fusion The dominant nanoVAST protein (VSG3) was present on the surface of both cell lines (Ramos B cells and HEK293T cells) tested after nanoVAST treatment. This suggests membrane fusion as the delivery mechanism.

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

[0100] Cells were harvested, washed three times with PBS (100g, room temperature for 7 minutes), and then stained with anti-VSG3-FITC conjugated antibody on ice for 10 minutes. After three washes with PBS (100g, room temperature for 7 minutes), flow cytometry results showed a clear increase in the presence of VSG3 on the cell surface (Figure 6A-B). Based on the fact that VSG3 is normally 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 via endocytosis instead, no surface-detectable VSG would have been observed, as nanoVAST would also have 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 cargo release (Figure 6C, i) or via endocytosis (Figure 6C, ii) and subsequent cargo release (Figure 6C, v). Both delivery mechanisms result in cargo release into cells, as well as the internalization and accumulation of VSG3 in endocytic vesicles (Figure 6C, iv). Internalized VSG3 was detectable in RAB11-positive endocytic vesicles of cells, in this case HEK cells, 8 hours after nanoVAST treatment (Figure 6D).

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

[0103] Next, the soltagged nanoVAST was re-isolated from the free soltagging reagent by centrifugation (20,000 g, 4°C, 30 minutes) and stored in 20 mM HEPES, 150 mM NaCl2, pH 8. The soltagging potential of nanoVAST was evaluated using the soltagging-capable fluorescent molecule TAMRA. Vesicles were soltagged using the protocol described above and subsequently analyzed by flow cytometry. Figure 7B illustrates that nanoVAST can be efficiently soltagged. Subsequently, the inventors proceeded to soltagging of functionally important targeting regions.

[0104] In this example, the targeted molecule, soltagged 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 the soltagged 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 took up more VSG3 into their membranes than individual cells treated with non-specific nanoVAST. This suggests that the present invention can be targeted, thereby resulting in increased delivery efficiency.

[0105] Example 6: NanoVAST vesicle preparation using an alternative method As an alternative method, the filtered supernatant from step f of the first embodiment of the present invention can be subjected to several centrifugation steps to gradually remove more cell and membrane residue. It has been observed that nanoVAST can be pelletized at rates of 20,000 g or more. Therefore, by gradually increasing the rate of repeated centrifugation (Figure 8A), it is possible to progressively pelletize the aggregated membrane residue prior to the pelletization of nanoVAST while leaving soluble proteins in the final supernatant. Five different rates in the range of 5,000 to 20,000 g were used. The supernatant after each centrifugation was collected and transferred to a clean test tube. Centrifugation at the selected rate was repeated until no more pellets were formed, indicating that the relatively heavy residues that could be removed at this rate had indeed been removed. This typically occurs after 2 to 5 rounds. After the pellets were removed, the supernatant was centrifuged at a higher rate. All centrifugation below 20,000 g was performed at 4°C for 5 minutes. Transfer the supernatant obtained after centrifugation at 17,000 g to a new test tube and centrifuge at 20,000 g for 30 minutes at 4°C. At this stage, nanoVAST is found in the pellet. Resuspend the pellet in 20 mM HEPES, 150 mM NaCl2, pH 8.0, and as an additional purification step, centrifuge again at 20,000 g for 30 minutes at 4°C. At this point, the pellet contains nanoVAST and can be stored.

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

[0107] For the aforementioned experiment, approximately 1 to 10 billion T. brusey cells expressing a soltagging-capable 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, room temperature for 20 minutes). The cells were washed once with phosphate-buffered saline (PBS) to remove residual extracellular proteins, and the resulting pellet was then dissolved on ice for 10 minutes by suspension in 5 mL of ice-cold deionized H2O containing a HALT protease inhibitor. Subsequently, the dissolved suspension was centrifuged (10,000 g, 4°C for 10 minutes) to isolate membrane material and remove cytoplasmic content. The ice-cold deionized H2O extraction and centrifugation process was repeated twice. Next, the membrane pellet was suspended in 2 mL of 20 mM HEPES, 150 mM NaCl2, pH 8, and sonicated on ice (using a pulser at 40% load for 5 minutes) to shear the large membranes into relatively small vesicles. Subsequently, to remove any remaining large, aggregated pelletizable residue (e.g., intact cells), the suspension was centrifuged (2,000 g, 4°C for 5 minutes) and filtered through a 0.45 μM filter using a 2.5 mL syringe. The filtered vesicle suspension was then subjected to the fractionation centrifugation protocol shown in Figure 8A.

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

[0109] Regardless of the purification methodology employed, the nanoVAST preparation may subsequently be polished using CaptoCore resin. The polishing step is performed by passing the rough or semi-processed nanoVAST preparation through a CaptoCore 700 (Cytiva) resin column. This may be inserted as an auxiliary step in one or more steps during nanoVAST preparation, loading, or "soltagging" as needed (Figure 9A). This separates larger nanoVAST vesicles while retaining other particles smaller than the 700 kDa molecular weight cutoff; thus removing nucleic acids, non-nanoVAST proteins, and other cellular residues.

[0110] Next, the inventors investigated whether nanoVAST could also be loaded using alternative strategies to electroporation, which may be beneficial or necessary for specific applications. Here, freeze-drying is described as a novel alternative method for loading RNA cargoes into nanoVAST vesicles.

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

[0112] After the lyophilization step, the quality of nanoVAST was investigated by checking the presence and quality of the VSG3 protein and evaluating the size and structure of nanoVAST. For this purpose, dried nanoVAST that was not co-dried with the RNA load was hydrated in HEPES buffer. Subsequently, SDS-PAGE analysis was performed, and the results showed that the VSG3 protein was still intact after the lyophilization step (Figure 10D). The presence of VSG3 was further confirmed by Western blotting (Figure 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 Figure 10B, the expected size and uniformity were maintained after the lyophilization step. Finally, the rehydrated nanoVAST vesicles were analyzed by transmission electron microscopy, and the standard rounded appearance of the vesicles remained intact (Figure 10C). Taken together, these analyses confirmed that the lyophilization step does not alter the structure and quality of nanoVAST. Therefore, freeze-dried nanoVAST can be used for loading and delivering cargo.

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

[0114] Example 7: NanoVAST Vesicle Loading Using an Alternative Method Since it was demonstrated that RNA cargoes could be loaded onto lyophilized-hydrated nanoVAST (Figure 10F), we evaluated whether vesicles could deliver RNA to HEK293T cells. HEK cells of appropriate density were plated in 24-well plates as described in Example 3. Subsequently, RNA-loaded lyophilized nanoVAST in DMEM cell medium was slowly added dropwise to each cell culture well, followed by incubation at 37°C in a 5% CO2 humidified environment for at least 1.5 hours.

[0115] Following incubation, cells were harvested and washed twice with 1×PBS by centrifugation at 100g for 5 minutes at room temperature. To identify HEK cells that had taken up nanoVAST, the washed cells were incubated on ice for 30 minutes with 1:100 FITC-conjugated anti-VSG3 antibody. Samples were analyzed by flow cytometry to identify cells that had taken up nanoVAST and RNA cargo. HEK cells that had taken up nanoVAST were VSG3-positive and accounted for 48.4% of the HEK cell population (Figure 11B.i). This population also carried RNA, as depicted by the increased intensity of the fluorescently tagged RNA (Figure 11B.iii). A proportion of HEK cells that lacked VSG3 signaling but carried RNA cargo was also observed (Figure 11B.ii). This finding suggests that there is a proportion of nanoVAST that loses VSG3 surface protein upon uptake but still releases RNA cargo into HEK cells. Results from freeze-dried nanoVAST HEK supply experiments were comparable to those from electroporated nanoVAST-supplied HEK cells (Figure 11A). From this data, it was concluded that freeze-dried nanoVAST can deliver its cargo to target cells. Here again, we emphasize that this novel method of cargo delivery expands the applications of our nanoVAST loading platform.

[0116] Example 8: Preparation of nanoVAST from different VSG-expressing trypanosomes As previously described in Example 5, nanoVAST cell specificity or targeting can be achieved through modification of the nanoVAST VSG coat. In Example 5, the “targeting moiety,” which is an anti-CD19 targeted nanobody, was covalently bonded to the nanoVAST VSG3 coat using saltase. However, several additional methods could be used to modify the VSG coat to switch nanoVAST targeting or cell specificity, for example, by adding different “targeting moieties” or changing the physicochemical properties of the VSG (Figure 12). In addition to direct nanoVAST “soltagging,” the VSG gene of the trypanosome from which nanoVAST would be produced could be genetically modified to directly encode a peptide-based “targeting moiety” (Figure 12ii). Such examples of peptide-based “targeting moieties,” though not limited to these, are listed in Table 1, and these can direct nanoVAST vesicles to specific cells based on design. In addition, we were able to completely switch the VSG coat of trypanosomes to a different genomically coded VSG gene that translates to a VSG with completely different electrochemical properties that have different cell affinities and therefore affect nanoVAST cell targeting (Figure 12iii).

[0117] [Table 1]

[0118] The inventors have previously generated (form) nanoVASTs from trypanosomes expressing a VSG different from VSG3, namely ILtat 1.24 (so-called ILtat). Vesicles prepared from ILtat-expressing trypanosomes exhibited similar properties to their VSG3 counterparts (Figures 13A-C). Their size, integrity, and purity were very similar to those of VSG3 nanoVASTs. In addition, ILtat nanoVASTs could be deltagged using the same deltagging fluorescent molecule TAMRA that was successful for VSG3 nanoVASTs (described in Example 5, Figure 7B). Using the deltagging method described in Example 5 and flow cytometry for verification, it was demonstrated that ILtat vesicles could be efficiently deltagged, and thus their functional protein layer could be maintained.

[0119] Example 9: nanoVAST immunogenicity nanoVAST is an in vivo delivery tool for the 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 link these cytokine signaling pathways. Here, we evaluated immunogenicity using an in vitro macrophage system based on RAW cells. nanoVAST failed 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 TIFF0007880979000002.tif224165TIFF0007880979000003.tif167164

Claims

1. The following steps: (a) A step of providing recombinant Trypanosoma brusey cells expressing VSG; (b) The step of treating the cells in a hypotonic solution in the presence of at least one protease inhibitor until the cells are lysed; (c) A step to isolate the cell membrane from the solution in step (b); (d) A step of suspending the isolated membrane obtained in step (c) in an isotonic solution; (e) A step of processing the suspended cell membrane obtained in step (d) using sonication to obtain a vesicle suspension; (f) A step of removing aggregated membrane residue from the vesicle suspension obtained in step (e); (g) the step of separating the vesicle suspension into a group of vesicles; and (h) A step of providing vesicles from a population of vesicles characterized by the following parameters: (i) having a single dominant protein revealed after Coomassie staining of SDS-PAGE with an apparent molecular weight of 55–60 kDa, (ii) having a spherical appearance on electron micrographs, and (iii) showing a homogeneous surface structure on electron micrographs. A method for preparing vesicles, including [a specific term].

2. The method according to claim 1, wherein the VSG is a soltagging VSG.

3. After step (c) and before step (d), the following steps are taken: - The step of treating the cells in a deionized aqueous solution; and - A step of isolating the cell membrane from the solution. The method according to claim 1, further comprising:

4. The method according to claim 1, wherein the step of removing aggregated membrane residue from the vesicle suspension in step (f) is performed by filtration using a 0.45 μM filter.

5. The method according to claim 1, further comprising the step of introducing the target cargo agent into the vesicle provided in step (h).

6. The steps to be implemented are as follows: (a) The step of suspending the vesicles in a transfection buffer containing the excess cargo agent to be targeted; (b) the step of performing electroporation; and (c) Step of purifying the loaded vesicles after electroporation. The method according to claim 5, including the method described in claim 5.

7. The method according to claim 5, wherein the target cargo drug is selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

8. The method according to claim 1, further comprising the step of soltagging a targeted compound to a soltagging-able VSG on a vesicle provided in step (h).

9. The aforementioned soltagging step is as follows: (a) The step of treating the vesicle with saltase in the presence of a targeted compound; and (b) A step of purifying the soltagged vesicles using a targeted compound. The method according to claim 8, including the method described in claim 8.

10. The method according to claim 8, wherein the targeted compound is an antibody or nanobody that recognizes a target molecule on a target cell.

11. VSG-containing vesicles characterized by the following parameters: (i) having a single dominant protein with an apparent molecular weight of 55–60 kDa, revealed after Coomassie staining of SDS-PAGE; (ii) having a spherical appearance on electron micrographs; and (iii) exhibiting a homogeneous surface structure on electron micrographs.

12. The vesicle according to claim 11, wherein the VSG is a soltaging-capable VSG.

13. The vesicle according to claim 11, on which the target cargo drug is loaded.

14. The vesicle according to claim 13, wherein the cargo drug is selected from the group consisting of small molecule drugs, peptides, proteins, and nucleic acid molecules.

15. The vesicle according to claim 11, which is soltagged using a targeted compound.

16. The vesicle according to claim 15, wherein the targeting compound is an antibody or nanobody that recognizes a target molecule on a target cell.

17. A pharmaceutical composition comprising a vesicle according to any one of claims 11 to 16 for use in the treatment and / or prevention of a disease or medical condition.

18. A vesicle according to any one of claims 11 to 16 for use as a compound delivery vehicle.

19. The vesicle according to claim 18, wherein the compound delivery vehicle is a drug delivery vehicle.

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