Novel scaffold proteins for engineering extracellular vesicles

Abstract

The present invention relates to a genetically engineered extracellular vesicle comprising an improved scaffold protein and a protein of interest. Polypeptide constructs, polynucleotide constructs, cells, pharmaceutical compositions, and methods related thereto are also disclosed.

Description

[0001] Citation Electronic Sequence Listing

[0002] The electronic sequence listing (EVOX_028_001WO_SeqList_ST26.xml; size: 8,927 bytes; creation date: April 28, 2023) is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to a newly identified extracellular vesicle (EV) scaffold protein, which can be advantageously used in genetically engineered extracellular vesicles. The present invention also relates to the use of the genetically engineered EV in therapy, methods for producing and purifying the EV, the EV population, and polypeptides and polynucleotide constructs encoding the new scaffold protein, as well as cells containing those polypeptides or polynucleotide constructs. Background Art

[0004] In general, EVs (e.g., exosomes) are nanosized vesicles produced by most cell types that serve as a natural transport system for proteins, nucleic acids, peptides, lipids, and various other molecules between cells of the body. EVs have a variety of potential therapeutic uses and are expected to serve as natural delivery vehicles for proteins, nucleic acids, and small molecule therapeutics. Engineering EVs to contain a protein of interest (POI) is critical to the utility of EVs in delivering therapeutic cargo; however, identifying suitable EV proteins as fusion partners to transport therapeutic cargo into EVs has proven to be difficult and unpredictable.

[0005] Loading of target therapeutic molecules can be achieved in a variety of ways. In the simplest case, small EVs from producer cells that have therapeutic potential themselves (such as immunomodulatory mesenchymal stem cells and chimeric antigen receptor T cells) can be passively loaded with therapeutic molecules, which can be directly used for disease management. In addition, physical and chemical methods such as incubation, sonication, and electroporation are also widely used for loading exogenous therapeutic molecules. However, this strategy is limited to small RNAs (e.g., siRNA, miRNA, shRNA) and low molecular weight chemicals, and has a considerable risk of technical artifacts (e.g., RNA precipitation). On the other hand, POIs are usually loaded into small EVs by genetic engineering. In a typical workflow, engineered producer cells are used to overexpress the desired protein fused to the EV sorting / carrier domain, which promotes the sorting of POIs into EVs during their biological production. In addition, potential RNA therapeutics can be loaded indirectly via the same strategy using compatible RNA binding proteins.

[0006] Depending on the topology and subcellular localization of the EV proteins (so-called “scaffold domains” or “scaffold proteins”) used to carry the POI into the engineered EVs, as well as the termini used for fusion, cargo molecules can be displayed on the EV surface or loaded into the vesicles. In contrast to surface display, intravesicular loading can protect cargo molecules from premature release and rapid degradation. Previously reported EV scaffold domains for intravesicular loading can be divided into three major categories: cytoplasmic proteins (SDCB-1, ARRDC1, and BASP1), monospanins (LAMP2B and PTGFRN), and tetraspanins (CD9, CD63, and CD81).

[0007] Traditionally, it is believed that "common" or "classical" EV proteins that are naturally highly expressed in EVs are the best EV proteins to serve as "scaffolds" to carry POIs into engineered EVs. Generating EV protein-POI fusion proteins has been shown to be an effective method to load selected POIs into EVs. As mentioned above, classical EV proteins such as CD63, CD9, and CD81, as well as single-spanning EV proteins such as Lamp2, and even membrane-associated EV proteins such as BASP have previously been shown to be useful as fusion partners for POIs.

[0008] However, the efficiency of EV sorting of the above domains depends on whether the producer cells can continuously and stably express a specific scaffold domain, as well as the specific type and size of the cargo molecule carried by the domain, which have previously led to highly variable results.

[0009] For example, it is known that expression of the transgenic CD63-POI can significantly decline over time in producer cells, thus affecting the yield of EV product. In other words, when using CD63 as an EV scaffold protein, even if a stable cell line is generated, the stability of that cell line is highly variable and gradually declines over time, resulting in fewer CD63-POI fusion constructs on any EV, as well as fewer EVs containing any transgenic fusion protein constructs.

[0010] Furthermore, when CD63 was engineered to be fused to more than one POI (e.g., different POIs were fused to the N-terminus, C-terminus, first loop, and / or second loop), the number of EVs expressing the CD63 fusion construct and the number of CD63 fusion constructs expressed on each EV were reduced.

[0011] Therefore, there remains a need for improved methods of introducing POIs into EVs. Summary of the invention

[0012] In addition to the known limitations mentioned above, the inventors have also found that problems arise when using these classic EV proteins when additional proteins or fusion protein constructs need to be added to therapeutic EVs. For example, it is impossible to construct a dual-stable cell line that always expresses more than one POI that will be expressed on or within the same EV population. The inventors have found that if a "classic EV protein" such as CD63, CD9 or CD81 is used to produce a dual-stable cell line, the expression of the second construct will quickly decrease (for example, co-expression of CD63-POI with another POI (such as VSVG) will quickly lead to loss of VSVG expression); as a result, the resulting EV population will soon contain only protein constructs containing "classic EV proteins". Due to the impossibility of producing consistent EV production cell lines, this is of course problematic for the need for therapeutic EVs to contain more than one POI. The reason for this is unclear.

[0013] Thus, although it was found that classical EV proteins (such as CD63, CD81 and CD9) were engineered to contain POIs for high expression on native EVs / exosomes, they were also found to hinder the introduction of subsequent (different) proteins into the same EV, thus preventing the co-localization of multiple protein constructs on the same EV. This severely limits the protein combinations that can be introduced into EVs, and therefore does not allow for multiplexing of functions. This also limits the development of therapeutic EVs as a platform technology and greatly reduces the utility of engineered EVs.

[0014] Furthermore, although it is theoretically possible to engineer classical tetraspanin EV proteins (such as CD63) to contain multiple POIs (i.e., at the N-terminus, C-terminus, first loop, and / or second loop), it has been found that engineering these classical tetraspanin EV proteins to fuse multiple POIs (e.g., different POIs are fused to the N-terminus, C-terminus, first loop, and / or second loop, respectively) results in a decrease in the number of EVs expressing these fusion constructs, as well as a decrease in the number of EVs expressing these fusion constructs per EV.

[0015] Therefore, new methods for introducing multiple POIs into EVs are still needed.

[0016] To address these limitations and meet future challenges of genetic engineering, identification of novel EV sorting domains is required. Therefore, the present invention stems from a study aimed at discovering novel EV sorting domains and investigated the differences between several producer cells and cargo molecules.

[0017] One object of the present invention is to overcome the above-mentioned problems associated with finding new EV carrier / scaffold proteins that can be used as fusion partners to transport POI into EVs with high efficiency and reliability over a long period of time. The inventors hope to identify new EV carrier / scaffold proteins that can serve as "universal EV carrier / scaffold proteins", i.e., effective for multiple cell types and capable of carrying a variety of cargoes.

[0018] Secondly, it is an object of the present invention to identify EV scaffold / carrier proteins that are able to maintain expression of an EV-protein-POI fusion construct and that do not prevent co-localization of a second or subsequent POI on the same EV. The inventors sought to identify an alternative scaffold protein that could be engineered to contain a POI while allowing engineering of a second or more proteins to be within / on the same EV, i.e. allowing co-localization.

[0019] Furthermore, the inventors sought to identify EV scaffold / carrier proteins that could be engineered to contain multiple POIs and, once engineered in this manner, were consistently expressed at high levels in EVs.

[0020] In addition, the inventors have been working on identifying scaffold proteins that can increase the loading of cargo, including RNA, into EVs.

[0021] To this end, the inventors conducted a large-scale screening of EV proteins and identified a new and highly flexible scaffold protein TSPAN2, which, unlike classical EV proteins, enables the development of a highly flexible and therefore diverse technology platform. Compared with classical EV proteins, TSPAN2 has significantly improved function as an EV scaffold protein (up to 10 times) and significantly improves the modularity of this therapeutic exosome platform technology by enabling other protein constructs to co-localize in the same EV.

[0022] Furthermore, both TSPAN2 and TSPAN3 increased mRNA loading compared to the classical EV tetraspanin CD63. Furthermore, expression of TSPAN2 fused to a POI resulted in an increased proportion of EVs expressing the fusion construct, as well as an increased number of these fusion constructs expressed per EV, compared to expression of the classical EV tetraspanin CD63 fused to a POI. This was also the case when TSPAN2 was fused to multiple POIs.

[0023] Therefore, in a first aspect, the present invention provides an EV comprising a fusion protein, wherein the fusion protein comprises TSPAN2 fused to a POI.

[0024] In the second and third aspects, the present invention provides a polypeptide construct comprising TSPAN2 fused to a POI and a polynucleotide construct encoding the polypeptide construct.

[0025] In a fourth aspect, the present invention provides a cell comprising the polypeptide construct of the second aspect or the polynucleotide construct of the third aspect. Optionally, the cell may further comprise a second polypeptide construct or polynucleotide construct capable of expressing the POI.

[0026] In a fifth aspect, the present invention provides a pharmaceutical composition comprising the EV of the first aspect or the cell of the fourth aspect, and a pharmaceutically acceptable excipient or carrier.

[0027] In a sixth aspect, the present invention provides a method for producing the EV of the first aspect. The method for producing EVs comprises: (i) introducing a polynucleotide construct encoding a TSPAN2-POI fusion construct into an EV production cell; and (ii) expressing the construct in an EV production cell, thereby producing an EV comprising a TSPAN2-POI fusion protein. The method may also optionally include an intermediate step of introducing a second polynucleotide construct encoding a second POI (optionally, wherein the second POI is in the form of a fusion protein with a classical EV protein) into the same EV production cell before continuing to express the two constructs in the EV production cell, thereby producing an EV comprising a TSPAN2-POI fusion protein and a second POI.

[0028] In a seventh aspect, the present invention provides the EV of the first aspect for use in treatment. In an eighth aspect, the present invention also provides a method of treatment, comprising administering an effective amount of the EV of the first aspect or the pharmaceutical composition of the fifth aspect of the present invention to a patient in need thereof.

[0029] It is clear from this example that without the aforementioned large-scale screening method and the significant creative efforts of the present inventors, the discovery of TSPAN2 as a novel scaffold protein would not have been possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Schematic diagram of high-throughput screening protocol and results.

[0031] Figure 2 : Results of screening the ThermoLuc (Tluc) in the HEK-293T cell secretome. (AE shows correlations, indicating the reliability of the screening protocol and identifying the top 36 candidate proteins).

[0032] Figure 3 : Results of further screening to evaluate the ability of candidate EV scaffold proteins to sort into small extracellular vesicles.

[0033] Figure 4: Quantitative analysis of the number of engineered EVs produced using different EV scaffold candidate proteins using a dual-function bioluminescence and fluorescence reporter gene approach showed that TSPAN2 and TSPAN3 produced 3.5 times more engineered EVs than CD63.

[0034] Figure 5 : Evaluation of the best candidate proteins for different EV-producing cell lines.

[0035] Figure 6 : TSPAN2 and TSPAN3 engineered EVs were characterized by nanoparticle tracking analysis (NTA), western blotting, cryo-electron microscopy, confocal microscopy, and quantitative analysis of the in vivo biodistribution of Tluc-labeled EVs.

[0036] Figure 7 : Schematic diagram depicting the co-culture system versus the EV addition system used to screen for the functional activity of novel EV scaffold proteins.

[0037] Figure 8: Scaffold screening results.

[0038] Fig. 9 : Summary of screening results displayed as a heat map.

[0039] Figure 10: Further validation of the top 13 EV candidate proteins screened using other difficult-to-transfect reporter cells (mesenchymal stromal cells (MSC), THP1 Raw264.7, and K562 cells).

[0040] Fig.11 : Graph showing TSPAN2 versus classical EV scaffold proteins to assess the ability to co-express multiple constructs in the same EV.

[0041] Fig.12 : Cell sorting data demonstrating that TSPAN2 enables co-localization of two constructs on the same EV.

[0042] Fig.13 : In vivo data using TSPAN2-engineered EVs to deliver Cre protein using the B16F10 tumor model showed that TSPAN2 is capable of delivering functional POIs.

[0043] Figure 14: TSPAN2-albumin binding domain fusion EVs showed albumin binding function in vitro, and improved TSPAN2 engineered EVs had an in vivo circulation half-life, further confirming that TSPAN2 was able to deliver functional POIs.

[0044] Fig.15 : Data show that TSPAN2 can deliver nucleic acid cargo (Ago2 RNA binding protein serves as POI for loading shRNA cargo).

[0045] Fig.16 : Data show that TSPAN2 can deliver mRNA by attaching to nucleic acid binding proteins.

[0046] Figure 17: Data show that TSPAN2 and TSPAN3 can deliver mRNA via attachment to nucleic acid binding proteins.

[0047] Figures 18 and 19: Data show that compared with classic EV scaffold proteins, TSPAN2 can improve the efficiency of EV engineering in different cell lines and with different scaffold protein modifications.

[0048] Figure 20: Data show the temporal stability of TSPAN2 constructs in EV producing cells.

[0049] Figure 21: Data show the temporal stability of TSPAN2 constructs in EVs secreted from producer cells.

[0050] Description of Sequence Listing

[0051] SEQ ID NO: 1: TSPAN2 protein

[0052] SEQ ID NO:2: TSPAN2 nucleic acid

[0053] SEQ ID NO:3: TSPAN3 protein

[0054] SEQ ID NO:4: TSPAN3 nucleic acid

[0055] SEQ ID NO:5: TSPAN18 protein

[0056] SEQ ID NO:6: TSPAN18 nucleic acid DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention relates to EVs comprising a fusion protein, wherein the fusion protein comprises TSPAN2 fused to a POI. The present invention also relates to cells capable of producing these EVs, methods for preparing and purifying these EVs and their use in therapy. Due to the use of TSPAN2 in the fusion protein, the EVs of the present invention have many significant advantages.

[0059] In principle, the use of TSPAN2 enables improved stability of cell lines producing engineered EVs and higher levels of expression of TSPAN2-POI constructs on or within EVs. Furthermore, the use of TSPAN2 enables co-localization of other protein constructs in the same EV, solving the challenge of generating cell lines that stably express multiple fusion constructs, and this significantly improves the modularity of this therapeutic exosome platform technology.

[0060] The inventors have demonstrated that TSPAN2 is a scaffold protein that is superior to classical scaffold proteins because it exhibits consistently high expression across a wide range of cell sources, allowing consistent expression of a wide range of different types of POIs, up to 10-fold better expression, and EVs expressing TSPAN2 as a scaffold protein also exhibit better bioavailability compared to those expressing classical EV proteins. Surprisingly, TSPAN2 was found to be a highly manipulable scaffold protein that enables significant engineering without the expression or co-localization issues common in classical EV protein scaffolds. TSPAN2 consistently performed best in very large-scale screens of scaffold proteins. Importantly, only 36 of the 244 candidate proteins were found to be effective, indicating a high degree of heterogeneity in EV sorting activity even between closely related proteins. As shown in the examples provided herein, the inventors found, through a novel functional screening approach, a low correlation between hierarchical encapsulation and total secretion reporter proteins, indicating that the results obtained herein are highly unpredictable.

[0061] In a first aspect, the present invention provides an EV comprising a fusion protein, wherein the fusion protein comprises TSPAN2 fused to a POI.

[0062] In one embodiment, the POI is engineered into loop 1, loop 2, or both loops of TSPAN2. Advantageously, this allows the POI to be displayed on the surface of the EV.

[0063] In another embodiment, the POI is fused to the N-terminal domain (NTD) or C-terminal domain (CTD) or other luminal portion of TPSAN2. Advantageously, this allows the POI to be loaded into the EV via the lumen, thereby preventing degradation and / or reducing the immunogenicity associated with the POI.

[0064] In yet another embodiment, TSPAN2 is fused to at least two POIs. In a preferred embodiment, one POI is engineered into loop 1, loop 2, or both loops of TSPAN2, and a different POI is fused to the N-terminal domain (NTD) and / or the C-terminal domain (CTD). In a preferred embodiment, TSPAN2 is fused to one POI engineered into loop 2 and to a second POI engineered onto the C-terminal domain (CTD).

[0065] In some embodiments, the fusion protein further comprises: (i) a release domain capable of being cleaved to release the POI; (ii) a linker or spacer; (iii) a multimerization domain; or (iv) at least one additional POI. The inventors have found that TSPAN2 is highly amenable to significant engineering, such as the addition of any of the additional domains listed above without losing expression of the TSPAN2 scaffold. This clearly makes TSPAN2 an efficient and reliable scaffold protein for developing platform EV technologies, in which the ability to reliably add and exchange different protein domains within the scaffold protein is highly advantageous for the flexibility and modularity of the technology.

[0066] In some embodiments, the EV of the first aspect further comprises a second POI expressed on a separate construct. Optionally, the second POI exists as a fusion protein with TSPAN2 or alternatively any classic exosomal polypeptide. It is very advantageous that TSPAN2 has been found to not only tolerate important engineering, but also to be stably co-localized with other protein constructs. This ability to allow another construct to be well expressed, so that the two constructs can be co-localized on the same EV population, is something that classic EV proteins cannot do, which also enables TSPAN2 to be used as a scaffold protein for EV platform engineering, with great versatility. It should also be understood that the ability to consistently engineer more than one construct into an EV allows functional multiplexing, which is another significant benefit of the EV of the present invention compared to EVs using classic EV proteins as scaffold proteins. The inventors unexpectedly found that TSPAN2 can be used as an efficient EV protein scaffold, engineering POIs thereon while allowing the second construct to be positioned on the same EV. This finding was highly unexpected, as TSPAN2 is a paralog of CD9, which, along with other canonical EV proteins such as CD63 and CD81, has been shown to prevent colocalization with the second construct once it has been engineered to include a POI.

[0067] In certain embodiments of the present invention, the POI may be: (i) a therapeutic protein, (ii) a binding protein of a therapeutic agent, such as an RNA binding protein, a virus binding protein, an Fc binding protein or a small molecule binding protein, (iii) an endosomal escape moiety, (iv) a targeting moiety, (v) an albumin binding domain or (vi) a purification moiety. In more detail, the therapeutic protein may be selected from: an enzyme, a receptor such as a decoy receptor, a membrane protein, a transporter, a cytokine, an antigen, a neoantigen, an immune effector molecule, a ribonucleoprotein, a nucleic acid binding protein, an antibody, a nanobody, an antibody fragment, an antibody-drug conjugate, a gene editing protein such as a CRISPR effector protein, including Cas proteins, TALENs, meganucleases. As can be seen from the examples, it is highly advantageous that TSPAN2 has been found to be useful not only for loading a wide range of different POIs, but has also been surprisingly found to be up to 10 times more effective than classical EV scaffold proteins. TSPAN2 has never been previously identified as a useful scaffold protein for loading POIs into exosomes, let alone that it would be more effective than currently preferred EV scaffold proteins. TSPAN2 has no known function and is therefore little studied or understood. TSPAN2 (and TSPAN3, which was also identified as a useful scaffold protein in the Examples) interacted very poorly with CD63 or any other EV protein, further indicating the surprising nature of this finding.

[0068] In another embodiment of the present invention, the EVs comprising TSPAN2 are further exogenously loaded with a therapeutic cargo. The exogenous loading method can be electroporation, use of a transfection reagent, co-incubation or by contact with a cell penetrating peptide (CPP) or any combination of the above. The cargo that can be exogenously loaded into the EV expressing TSPAN2 can be a protein, a nucleic acid, a virus, a viral genome, an antigen or a small molecule. When the cargo is a nucleic acid cargo, this can be: an RNA molecule, a DNA molecule or a mixture, messenger mRNA (mRNA), an antisense or splice switching oligonucleotide, gRNA, siRNA, shRNA, miRNA, DNA Plasmid DNA (pDNA), supercoiled or non-supercoiled plasmids, minicircles, etc. Exogenous addition of therapeutic cargo has the additional advantage that it allows for greater versatility of EVs as a platform technology, i.e., EVs expressing one or more constructs, which can, for example, serve as a brain targeting moiety plus an endosomal escape moiety, can then be used as general brain-targeting EVs to which any number of different therapeutic cargoes can be added.

[0069] Another embodiment of the invention relates to a population of EVs according to the first aspect, wherein the TSPAN2-POI fusion protein and a second POI construct are expressed on the same EV. Such a population of EVs that reliably express two constructs co-localized to the same EV is advantageous for the same reasons detailed above, namely that existing EV scaffold proteins are unable to achieve such co-localization, coupled with the ease of use as a platform tool, providing the ability to allow POI multiplexing.

[0070] In the second and third aspects, the present invention provides a polypeptide construct comprising TSPAN2 fused to a POI and a polynucleotide construct encoding the polypeptide construct.

[0071] In a fourth aspect, the present invention also relates to a cell comprising a polypeptide construct comprising TSPAN2 fused to a POI, or a polynucleotide construct encoding such a fusion protein. Optionally, in a further embodiment, the cell may also comprise a second polypeptide construct or polynucleotide construct capable of expressing the POI. These cells may be transiently modified to comprise these polynucleotide or polypeptide constructs, or they may be stably modified to comprise at least one monocistronic, bicistronic or multicistronic polynucleotide construct. A significant advantage of using TSPAN2 as a scaffold protein is that it allows for the development of cell lines for the first time, whether transiently transfected or stable cell lines, which reliably express engineered fusion protein constructs, particularly those in which more than one construct needs to be expressed. This makes TSPAN2 uniquely useful in developing clinical-grade cell lines for the production of therapeutic EVs / exosomes, because it is critical that clinical-grade cell lines reliably express one or more POIs of interest over a sustained period of time. This is actually a requirement for clinical-grade cell lines due to quality control and batch release standards to ensure the consistency of EV products. In the specific case of establishing stable cell lines, this bypasses transfection of parental cells, thus greatly simplifying the EV production procedure. Furthermore, compared with transient transfection, the expression level of the transgene in stable production cells is more controllable, thereby reducing the variability of EV production.

[0072] The term "stably modified" refers to a cell line that can pass the introduced polynucleotide construct to its offspring (i.e., daughter cells), either because the transfected DNA has been incorporated into endogenous chromosomes, or by stable inheritance of exogenous chromosomes. A stable cell line can be defined as a homogenous cell population that retains 70% or more, 80% or more, or even 90% or more volumetric productivity titers for at least 50 generations, optionally more than 60 generations, more than 70 generations, more than 85 generations, or even more than 100 generations. As determined by structure, function, purity, chemical properties, and / or biological activity, "no clinically significant differences" can be demonstrated in at least 50 generations, optionally more than 60 generations, more than 70 generations, more than 85 generations, or even more than 100 generations. Ideally, the production titer of the cell can show a deviation of less than 15%, less than 10% or even less than 5% (within the measurement limit) at the end of 100 doubling cycles.

[0073] As demonstrated in the Examples, the inventors also identified TSPAN3 and TSPAN18 as improved EV scaffold proteins. Therefore, the present invention also relates to EVs comprising fusion proteins, wherein the fusion protein comprises TSPAN3 or TSPAN18 fused to a POI. Optionally, the POI can be engineered into loop 1, loop 2, or both loops of TSPAN3 or TSPAN18 to facilitate surface display of the POI. Alternatively, the POI can be engineered into the NTD and / or CTD or other luminal portions of TSPAN3 or TSPAN18 to facilitate luminal loading of the POI. In other embodiments, the TSPAN3 or TSPAN18 fusion protein further comprises: a release domain capable of being cleaved to release the POI; a linker or spacer; a multimerization domain; and / or at least one further POI. In certain embodiments, the EV containing TSPAN3 or TSPAN18 also comprises a second POI expressed on a separate construct, optionally wherein the second POI is present as a fusion protein with an exosomal polypeptide. Similar to the case described above for TSPAN2, TSPAN3 or TSPAN18EVs can be exogenously loaded with therapeutic cargo (such as nucleic acids, viruses or small molecule cargoes) by any known exogenous loading mechanism, including: electroporation, transfection reagents, co-incubation or by contact with CPP, or any combination of these methods. The present invention also relates to a pharmaceutical composition comprising TSPAN3-POI or TSPAN18-POI EV. In addition, the present invention relates to a method of treatment comprising administering an effective amount of TSPAN3-POI or TSPAN18-POI EV to a patient in need, and similarly, the use of TSPAN3-POI or TSPAN18-POI EV for medicine.

[0074] The present invention also relates to polypeptide constructs comprising TSPAN3 or TSPAN19 fused to a POI or polynucleotide constructs encoding such polypeptide constructs. The present invention further relates to cells comprising such TSPAN3 or TSPAN18 polypeptide or polynucleotide constructs, optionally comprising a second polypeptide construct or polynucleotide construct capable of expressing a second POI.

[0075] Naturally, the present invention also relates to a method for producing EVs, the method comprising: (i) introducing a polynucleotide construct encoding a TSPAN3 / TSPAN18-POI fusion construct into a cell producing EVs; and (ii) expressing the construct in a cell producing EVs, thereby producing EVs comprising a TSPAN3 / 18-POI fusion protein. The method may also comprise an intermediate step of introducing a second polynucleotide construct encoding a second POI (optionally wherein the second POI is in the form of a fusion protein with an EV protein) into the same EV producing cell; allowing the two constructs to be expressed in the cell producing EVs, thereby producing EVs comprising a TSPAN3 / 18-POI fusion protein and a second POI.

[0076] definition

[0077] For convenience and clarity, certain terms used herein are collected and described below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0078] Where features, aspects, embodiments or alternatives of the invention are described in terms of Markush groups, the skilled artisan will recognize that the invention is also described in terms of any individual member or subgroup of members of the Markush group. The skilled artisan will further recognize that the invention is also described in terms of any combination of individual members or subgroups of members of the Markush group. In addition, it should be noted that the embodiments and features described in conjunction with one aspect and / or embodiment of the invention are also applicable mutatis mutandis to all other aspects and / or embodiments of the invention. For example, the TSPAN2 fusion protein described herein should be understood to be disclosed, related and compatible with all other aspects, teachings and embodiments herein, such as aspects and / or embodiments related to methods of producing or purifying EVs, or aspects or embodiments related to the corresponding polynucleotide constructs described herein or the engineered EV-producing cells from which the EVs are derived. In addition, certain embodiments related to certain aspects, such as the routes of administration of EVs comprising therapeutic cargo molecules and optionally fusion polypeptides described in aspects related to the treatment of certain medical indications, may naturally also be related to other aspects and / or embodiments, such as aspects and embodiments related to pharmaceutical compositions comprising such EVs. In addition, all polypeptides and proteins identified herein can be freely combined in fusion proteins using conventional strategies for fusion polypeptides. As a non-limiting example, the TSPAN2-POI fusion proteins described herein can be freely combined with one or more targeting moieties, endosomal escape moieties, albumin binding domains, additional POIs, optionally with all other polypeptide domains, regions, sequences, peptides, groups herein, such as any multimerization domains, linker sequences, release domains, etc. In addition, when the teachings herein relate to a single EV and / or to EVs as discrete natural nanoparticle-like vesicles, it should be understood that all such teachings are equally relevant and applicable to a variety of EVs and EV populations. As a general illustration, the therapeutic POI, binding protein of the therapeutic agent, the endosomal escape moiety, the albumin binding domain or the purification domain and all other aspects, embodiments and alternatives according to the present invention can be freely combined in any and all possible combinations without departing from the scope and spirit of the present invention. In addition, any polypeptide or polynucleotide or any polypeptide or polynucleotide sequence (amino acid sequence or nucleotide sequence, respectively) of the present invention can deviate significantly from the original polypeptide, polynucleotide and sequence, as long as any given molecule retains the ability to achieve the desired technical effect associated with it. As long as their biological properties are maintained, the polypeptide and / or polynucleotide sequences according to the present application may deviate by up to 50% (calculated using, for example, BLAST or ClustalW) compared to the native sequence, although as high a sequence identity or similarity as possible is preferred (e.g., 60%, 70%, 80%, or, for example, 90% or more). Standard methods in the art can be used to determine homology.For example, PILEUP and BLAST algorithms can be used to calculate homology or queuing sequences. For example, the combination (fusion) of several polypeptides means that certain fragments of the corresponding polypeptides can be replaced and / or modified and / or the sequence can be interrupted by the insertion of other amino acid sequence segments, which means that the deviation from the native sequence can be considerable, as long as the key properties (such as maintaining its therapeutic effect, or binding therapeutic cargo or binding albumin and thus extending the half-life, the ability to transport the fusion construct to EV, targeting ability, etc.) are conservative. Therefore, similar reasoning naturally applies to the polynucleotide sequences encoding these polypeptides. Any accession number or SEQ ID NO related to peptides, polypeptides and proteins mentioned herein should be regarded as an example only and for reference only, and all peptides, polypeptides and proteins should be given their ordinary meaning, as will be understood by those skilled in the art. Therefore, as described above, those skilled in the art will also understand that the present invention not only covers the specific SEQ ID NO and / or accession number mentioned herein, but also covers variants and derivatives thereof. All accession numbers mentioned herein are UniProtKB accession numbers, and all proteins, polypeptides, peptides, nucleotides and polynucleotides mentioned herein should be interpreted according to the conventional meaning understood by those skilled in the art.

[0079] EVs:

[0080] The terms "extracellular vesicle" or "EV" are used interchangeably herein and may be understood to refer to any type of vesicle that may be obtained from any form of cell. The size of EVs may vary significantly, but EVs typically comprise a volume defined by a double lipid membrane and have a hydrodynamic radius of nanometer size, i.e., a radius below 1000 nm. The volume may comprise a vesicular secretion body. Different types of EVs are defined by different morphologies, structures, messages, and functions. EVs may be broadly divided into two categories, (1) exosomes and (2) exosomes. Some examples of EVs include, for example, exosomes, apoptotic bodies, inhibitory domain-containing protein 1 [ARRDC1]-mediated microvesicles (ARMMs), exosomes (such as microparticles or microvesicles), or cardiosomes, etc. In essence, the terms 'extracellular vesicle' and / or 'EV' may refer to any type of lipid-based structure (having a vesicular morphology or having any other type of suitable morphology) that may serve as a delivery or transport vehicle or have a natural therapeutic or pharmacological effect.

[0081] Furthermore, in some embodiments, the term may be understood to also refer to extracellular vesicle mimics, cell membrane vesicles obtained by membrane extrusion, sonication or other techniques, and the like.

[0082] Obviously, EVs may be derived from any cell type, whether in vivo, ex vivo or in vitro (further details of suitable source or producer cells are described below).

[0083] Exosomes, microvesicles and ARMMs are just some examples of different subtypes that fall within the broader description of EVs above and represent particularly preferred EVs, but it will be appreciated that other EVs may also be advantageous in certain circumstances. Advantageously, the EVs are exosomes.

[0084] The term "apoptotic body" or "apoptotic bodies" are used interchangeably herein and may be understood to refer to any type of vesicle obtainable or derived from an apoptotic cell. Typically, an apoptotic body has a diameter of about 1 μm to about 5 μm.

[0085] The terms "cardiosome" or "cardiosomes" are used interchangeably herein and may be understood to refer to any type of vesicle obtainable or derived from cardiac cells.

[0086] The term "ectosome" or "ectosomes" are used interchangeably herein and may be understood to refer to any type of heterologous vesicle that can be obtained or derived from the plasma membrane and / or outward budding of the cell membrane, preferably from neutrophils and monocytes in serum. Examples of ectosomes include, but are not limited to, microvesicles, microparticles, and macrovesicles. Typically, the size range of an ectosome is about 50 nm to about 1 μm.

[0087] The terms "microparticle" or "microparticles" are used interchangeably herein and are understood to refer to any type of vesicle obtainable or derived from platelets.

[0088] The terms "microvesicle" and "microvesicles" are used interchangeably herein and may be understood to refer to any type of vesicle that may be obtained or derived or shed from the plasma membrane or cell membrane of a cell.

[0089] The term "ARMMs" is understood to refer to any type of vesicle that can be obtained or derived from the plasma membrane or cell membrane of a cell, from which they bud directly. Such microvesicles are mediated by ARRDC1 and generally lack known late endosomal markers. Therefore, ARMMs are different from exosomes as described herein.

[0090] The term "exosome" or "exosomes" are used interchangeably herein and may be understood to refer to any type of vesicle that may be obtained or derived from the endosomal, lysosomal and / or endolysosomal pathways and / or from inward budding of the plasma membrane and / or cell membrane. Exosomes typically have a size of about 30 to about 300 nm, typically in the range of about 40 to about 250 nm, and sometimes in the range of about 40 to about 160 nm, which is a highly suitable size range.

[0091] The term "modified" may refer to modification of a vesicle using genetic or chemical methods, such as by genetic engineering of EV-producing cells, preferably exosome-producing cells, or by, for example, chemical conjugation, e.g., attachment of a moiety to the surface of an EV, preferably an exosome.

[0092] The terms "genetically modified" and "genetically engineered" are used interchangeably herein and may mean that EVs (preferably exosomes) are derived from genetically modified / engineered cells or are otherwise genetically engineered to express and / or modify the expression of proteins in the lumen, outer membrane of vesicles and / or displayed on the surface of EVs (e.g., exosomes), which are typically incorporated into EVs (preferably exosomes) produced by those cells. Such genetically modified or genetically modified EVs do not exist in nature.

[0093] Furthermore, in some embodiments, the term should be understood to also refer to EV mimics, cell membrane vesicles obtained by membrane extrusion, sonication or other techniques, and the like.

[0094] It will be clear to those skilled in the art that when describing the medical and scientific uses and applications of EVs, the present invention generally relates to a plurality of EVs, i.e. a population of EVs that may contain thousands, millions, billions or even trillions of EVs. As can be seen in the experimental section below, EVs may be present in the form of, for example, 10 5 , 10 8 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 , 10 30EVs (often referred to as "particles") per unit volume (e.g. per ml or per liter), or any other number larger, smaller or in between. In the same vein, the term "population" (which may, for example, relate to EVs comprising a certain POI) is to be understood as covering a plurality of entities that together constitute such a population. In other words, a single EV when present in plurality constitutes an EV population. Therefore, naturally, the present invention relates to individual EVs and populations comprising EVs, as will be clear to a person skilled in the art. When administered in vivo, the dose of EVs may naturally vary significantly depending on the disease to be treated, the route of administration, the activity and action of the albumin binding domain (ABD), the therapeutic cargo, any targeting moieties present on the EVs, the pharmaceutical formulation, etc.

[0095] TSPAN2:

[0096] TSPAN2 is understood to mean tetraspanin-2 (also known as TSN2) or tetraspanin NET-3, as well as derivatives, domains, variants, mutants or regions thereof. (Database references for TSPAN2 include: HGNC:20659, NCBI Entrez Gene:10100, Ensembl:ENSG00000134198, 613133, UniProtKB / Swiss-Prot:O60636). The TSPAN2 protein is a multipass transmembrane protein with 4 transmembrane regions. TSPAN2 is a paralog of CD9 and is thought to play a role in signaling in oligodendrocytes during the early stages of their terminal differentiation into myelinating glial cells, and may also play a role in stabilizing the mature sheath. The sequence of TSPAN2 is provided in SEQ ID No:1 and 2. A derivative, domain, variant, mutant or region of TSPAN2 may be a protein having at least 10% sequence identity with SEQ ID No:1 and / or 2. For example, a derivative, domain, variant, mutant or region may have at least 20%, or even at least 30% sequence identity with SEQ ID No:1 and / or 2. A derivative, domain, variant, mutant or region may have "substantially the nucleotide and / or amino acid sequence" of SEQ ID No: 1 and / or 2, which may refer to a sequence having at least 40% sequence identity to the sequence of SEQ ID No: 1 and / or 2. Thus, in one embodiment, the derivative, domain, variant, mutant or region has at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID No: 1 and / or 2. Loop 1 of TSPAN2 is defined by residues 35-54 and loop 2 of TSPAN2 is defined by residues 112-188.

[0097] TSPAN3:

[0098] TSPAN3 is understood to mean: tetraspanin 3, also known as TSN3, transmembrane 4 superfamily member 8, TSPAN-3, TM4SF8, TM4-A, tetraspanin TM4-A, tetraspanin-3, and derivatives, domains, variants, mutants or regions thereof. (Database references for TSPAN3 include: HGNC: 17752, NCBI Entrez Gene: 10099, Ensembl: ENSG00000140391, 613134, UniProtKB / Swiss-Prot:060637). TSPAN3 is a member of the transmembrane 4 superfamily, also known as the tetraspanin superfamily. TSPAN3 is a paralog of CD82 and is thought to regulate the proliferation and migration of oligodendrocytes, processes necessary for normal myelination and repair. The sequence of TSPAN3 is given by SEQ ID No:3 and 4. A derivative, domain, variant, mutant or region of TSPAN3 may be a protein having at least 10% sequence identity with SEQ ID No:3 and / or 4. For example, a derivative, domain, variant, mutant or region may have at least 20%, or even at least 30% sequence identity with SEQ ID No:3 and / or 4. A derivative, domain, variant, mutant or region may have "substantially a nucleotide and / or amino acid sequence" of SEQ ID No:3 and / or 4, which may refer to a sequence having at least 40% sequence identity with the sequence of SEQ ID No:3 and / or 4. Thus, in one embodiment, the derivative, domain, variant, mutant or region has at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID No: 3 and / or 4.

[0099] TSPAN18:

[0100] TSPAN18 should be understood to refer to tetraspanin-18, as well as derivatives, domains, variants, mutants or regions thereof. (Database references for TSPAN18 include: HGNC:20660, NCBI Entrez Gene:90139, Ensembl:ENSG00000157570, 619399, UniProtKB / Swiss-Prot:Q96SJ8.) The function of TSPAN18 is poorly understood, but it is predicted to be plasma membrane and a paralog of TSPAN1. The sequence of TSPAN18 is given by SEQ ID No:5 and 6. A derivative, domain, variant, mutant or region of TSPAN18 may be a protein having at least 10% sequence identity with SEQ ID No:5 and / or 6. For example, a derivative, domain, variant, mutant or region may have at least 20%, or even at least 30% sequence identity with SEQ ID No:5 and / or 6. A derivative, domain, variant, mutant or region may have "substantially the nucleotide and / or amino acid sequence" of SEQ ID No:5 and / or 6, which may refer to a sequence having at least 40% sequence identity with the sequence of SEQ ID No:5 and / or 6. Thus, in one embodiment, the derivative, domain, variant, mutant or region has at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID No: 5 and / or 6.

[0101] POI:

[0102] The POI can be any protein or polypeptide that is desired to be loaded into or on an EV. Exemplary POIs can be i) a therapeutic protein, (ii) a binding protein of a therapeutic agent, such as an RNA binding protein, a virus binding protein, a small molecule binding protein, an Fc binding protein, (iii) an endosomal escape moiety, (iv) a targeting moiety, (v) an albumin binding domain, or (vi) a purification moiety.

[0103] In the specific embodiments described below, any particular POI may be included in the TSPAN2 fusion polypeptide. Alternatively, the POI may alternatively or also be included in the EV as a second POI expressed on a separate construct.

[0104] As previously mentioned, an advantage of using TSPAN2 as a scaffold protein is that it allows the development of cell lines that have undergone significant engineering while maintaining strong consistent expression of single or multiple protein constructs. As described above, this is particularly advantageous when engineering into the loop / NTD / CTD of TSPAN2, and is also very advantageous when more than one construct is expressed in the same EV, as TSPAN2 allows co-localization of constructs. Given below are some non-limiting exemplary advantageous constructs and pairings of constructs that can be engineered into EVs with greater certainty and stability when using TSPAN2:

[0105] Fused to a single POI of TSPAN2

[0106] -TSPAN2-surface displayed therapeutic proteins

[0107] -TSPAN2-luminal loading of therapeutic proteins

[0108] -TSPAN2-Binding Protein for Therapeutic Agents

[0109] -TSPAN2-surface displayed targeting moiety

[0110] -TSPAN2-surface displayed ABD part

[0111] -TSPAN2-endosome escape fraction

[0112] In addition, any of the fusion constructs listed above can be expressed in the EV / cell of the invention in combination with a second construct comprising any POI, such that the EV comprises at least two different protein constructs. Optionally, the second construct is a fusion protein of an EV protein-POI.

[0113] Dual POI fused to TSPAN2

[0114] -TSPAN2 - therapeutic protein in loop 1 and loop 2

[0115] -TSPAN2 - Therapeutic protein on loop 1 / 2 and therapeutic protein on NTD / CTD

[0116] -TSPAN2 - Therapeutic protein in loop 1 / 2 and targeting moiety in loop 1 / 2

[0117] -TSPAN2 - Therapeutic proteins in loop 1 / 2 and endosomal escape moiety in loop 1 / 2

[0118] -TSPAN2 - Therapeutic protein in loop 1 / 2 and ABD in loop 1 / 2

[0119] -TSPAN2 - therapeutic protein on NTD and CTD

[0120] -TSPAN2 - About NTD / CTD (luminal) therapeutic proteins and targeting moieties in loop 1 / 2

[0121] -TSPAN2 - Therapeutic proteins with NTD / CTD (luminal) and ABD in loop 1 / 2

[0122] -TSPAN2 - on NTD / CTD (luminal) therapeutic proteins and endosomal escape moieties in loop 1 / 2

[0123] -TSPAN2-binding protein in loop 1 and loop 2

[0124] -TSPAN2-binding protein in loop 1 / 2 and targeting moiety in loop 1 / 2

[0125] -TSPAN2-binding protein in loop 1 / 2 and endosomal escape moiety in loop 1 / 2

[0126] -TSPAN2-binding protein in loop 1 / 2 and ABD in loop 1 / 2

[0127] - TSPAN2-binding proteins on the NTD and CTD

[0128] -TSPAN2 - Regarding the NTD / CTD (luminal) binding protein and the targeting moiety in loop 1 / 2

[0129] -TSPAN2 - for NTD / CTD (luminal) binding protein and ABD in loop 1 / 2

[0130] -TSPAN2 - NTD / CTD (luminal) binding protein and endosomal escape moiety in loop 1 / 2

[0131] -TSPAN2-targeting moiety in loop 1 / 2 and endosomal escape moiety in loop 1 / 2

[0132] -TSPAN2-targeting moiety in loop 1 / 2 and ABD in loop 1 / 2

[0133] -TSPAN2 - targeting moiety in loop 1 / 2 and purification moiety in loop 1 / 2

[0134] -TSPAN2-endosomal escape moiety in loop 1 / 2 and ABD in loop 1 / 2

[0135] -TSPAN2-endosomal escape fraction in loop 1 / 2 and purified fraction in loop 1 / 2

[0136] -TSPAN2-purified part in loop 1 / 2 and ABD in loop 1 / 2

[0137] In addition, any of the fusion constructs listed above can be expressed in the EV / cell of the invention in combination with a second construct comprising any POI, such that the EV comprises at least two different protein constructs. Optionally, the second construct is a fusion protein of an EV protein-POI.

[0138] Particularly preferred combinations include a therapeutic protein or binding protein of a therapeutic agent fused to TSPAN2 plus a second construct comprising the endosomal escape moiety VSVG, optionally wherein VSVG is fused to TSPAN2 or a classical EV protein.

[0139] In other embodiments, the TSPAN2 feature of the above constructs may be replaced with similarly acting TSPAN3 or TSPAN 18. The present invention includes polynucleotides encoding any of the polypeptide fusion constructs described above, whether comprising TSPAN3 or TSPAN 18 in place of TSPAN2.

[0140] In certain embodiments, the POI or at least one POI may be a therapeutic protein. In one embodiment, the therapeutic POI is included in a TSPAN2 fusion polypeptide. Alternatively, the POI fused to TSPAN2 may be any other exemplary POI, and the therapeutic POI may be expressed on a separate construct.

[0141] The therapeutic protein may be selected from: enzymes, receptors such as decoy receptors, membrane proteins, transport proteins, cytokines, antigens, neoantigens, immune effector molecules, ribonucleoproteins, nucleic acid binding proteins, antibodies, nanobodies, antibody fragments, antibody-drug conjugates, gene editing proteins such as CRISPR effector proteins, including Cas proteins, transcription activator-like effector nucleases (TALENs), and large-range nucleases.

[0142] In more detail, the therapeutic protein cargo (POI) according to the present invention includes: antibodies, intrabodies, nanobodies, single chain variable fragments (scFv), affibodies, bispecific and multispecific antibodies or binders, including bispecific T cell engagers (BiTE), receptors, ligands, transporters, enzymes for, for example, ERT or gene editing, tumor inhibitors, viral or bacterial inhibitors, cellular component proteins, DNA repair inhibitors, nucleases, proteases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins (e.g. Pseudomonas exotoxins), structural proteins, neurotrophic factors such as NT3 / 4, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and individual subunits thereof, such as the 2.5Sβ subunit, ion channels, Membrane transport proteins, protein homeostasis factors, proteins involved in cell signaling, translation and transcription-related proteins, nucleotide binding proteins, protein binding proteins, lipid binding proteins, glycosaminoglycans (GAGs) and GAG binding proteins, metabolic proteins, cell stress regulatory proteins, inflammation and immune system regulatory proteins, such as cytokines and inhibitors of such cytokines (cytokines may include: CXCL8, GMCSF, interleukins, including: IL-1 family, IL-2, IL-4, IL-6, IL-6-like, IL-9, IL-10, IL12, IL-13, IL-17, interferons, including INFα / β / γ, TNF family members, CD40 and CD40L, TRAIL and TGF-β family), mitochondrial proteins and heat shock proteins, etc. The cargo protein can be a reporter protein, such as green fluorescent protein (GFP) or nanoLuc.

[0143] In a preferred embodiment, the encoded protein is a CRISPR-associated (Cas) polypeptide (such as Cas9) with complete nuclease activity, which is associated with an RNA chain that enables the Cas polypeptide to achieve its nuclease activity in the target cell once delivered by a peptide (i.e., carries it). The CRISPR (clustered regularly spaced short palindromic repeats) components according to the present invention include CRISPR components derived from any bacterial source. The CRISPR components may be from Class 1 or Class 2, and specifically, the Cas type may be Cas type I, II, III, IV, V or VI. The specific Cas protein may be Cas9, Cas12 (Cas12a or Cas12b), C2c2, Cpf1, Cas10, Cas13 (cas13a, Cas13b or Cas13c), Cas3, Cas14 protein, CasX protein or CasY protein, CasMINI or SuperFi-Cas9. The CRISPR protein may be a CRISPR nuclease, a CRISPR nickase or a nuclease-deficient CRISPR variant. Alternatively, in another preferred embodiment, the Cas polypeptide may be catalytically inactive to achieve targeted genetic engineering. Another alternative may be any other type of CRISPR effector, such as a single RNA-guided endonuclease Cpf1. Including Cpf1 is a particularly preferred embodiment of the present invention because it cleaves target DNA by staggered double-strand breaks. Cpf1 can be obtained from species such as Acidaminococcus or Lachnospiraceae. In another exemplary embodiment, the Cas polypeptide can also be fused to a transcriptional activator (such as the P3330 core protein) to specifically induce gene expression.

[0144] Other preferred embodiments include therapeutic protein cargos selected from the group comprising enzymes or transporters for lysosomal storage diseases, such as glucocerebrosidases, such as imiglucerase, α-galactosidase, α-L-duroniase, iduronate-2-sulfatase and iduronate sulfatase, arylsulfatase, sulfadylase, acid α-glucosidase (GAA), sphingomyelinase, galactosylceramidase, galactosylceramidase, ceramidase, α-N-acetylgalactosamidase, β-galactosidase, lysosomal acid lipase, acid sphingomyelinase, NPC1, NPC2, heparin sulfonamides, N-acetyl sphingomyelinase, NPC2, sulphamidase, N-acetyl sphingomyelinase, NPC3, NPC4, NPC5, NPC6, NPC7, NPC8, NPC9, NPC10, NPC110, NPC120, NPC130, NPC140, NPC150, NPC160, NPC170, NPC180, NPC190, NPC210, NPC220, NPC230, NPC240, NPC250, NPC260, NPC270, NPC270, NPC280, NPC29 ... glucosaminidase, heparin-α-glucosaminidase-N-acetyltransferase, N-acetylglucosamine 6-sulfatase, galactose-6-sulfate sulfatase, galactose-6-sulfate sulfatase, hyaluronidase, αN-acetylneuraminidase, GlcNAc phosphotransferase, mucolipidase 1, palmitoyl protein thioesterase, tripeptidyl peptidase I, palmitoyl protein thioesterase 1, tripeptidyl peptidase 1, batin, linclin, α-D-mannosidase, β-mannosidase, aspartate glucosidase, α-L-fucosidase, cystinase, cathepsin K, sialic acid, LAMP2, and hexanoyl aminoacylase.

[0145] Additional preferred embodiments include therapeutic protein cargoes selected from the group comprising enzymes associated with urea cycle disorders, including: N-acetylglutamate synthase, carbamoyl phosphate synthase, ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase, arginase, mitochondrial ornithine transporter, limonin, y+L amino acid transporter 1, uridine monophosphate synthase UMPS.

[0146] In other preferred embodiments, the PoI may be, for example, an intracellular protein that modifies the inflammatory response, e.g., an epigenetic protein such as a methylase and a bromodomain, or an intracellular protein that modifies muscle function, e.g., a transcription factor such as MyoD or Myf5, a protein that regulates muscle contractility, e.g., myosin, actin, a calcium / binding protein such as troponin, or a structural protein such as dystrophin, mini-dystrophin, micro-dystrophin, utrophin, titin, nebulin, dystrophin-related proteins such as dystrobrevin, syntrophin, syncoilin, desmin, sarcoglycan, dystroglycan, sarcoplasmic protein, agrin and / or fukutin. POIs are generally proteins or peptides of human origin unless otherwise indicated by their name, any other nomenclature, or known to those skilled in the art, and they can be found in various publicly available databases such as Uniprot, RCSB, etc.

[0147] In another preferred embodiment, the therapeutic cargo is an antigen / neoantigen, optionally wherein the antigen / neoantigen is suitable for cancer immunotherapy.

[0148] Any antigen / neoantigen may be incorporated into the EV of the present invention. The antigen may be suitable for eliciting an immune response against pathogens such as bacteria, viruses, fungi, or the antigen may be a tumor antigen that can be used to induce an immune response against a tumor for cancer immunotherapy. One or more antigens / neoantigens may be present in any EV according to the present invention. One or more antigens / neoantigens may be endogenous / autologous (from the subject himself) or exogenous / allogeneic (from another subject), or in the case where more antigens / neoantigens are incorporated into / on the EV, the antigen / neoantigen may be any mixture of autologous / allogeneic antigens. Preferably, the antigen is autologous. In addition, one or more antigens / neoantigens may be of any origin, such as, for example, a virus or a bacterium, or may be a tumor antigen, and may further be immunostimulatory or immunosuppressive or a combination thereof. The antigen / neoantigen may be used to treat any disease by immunotherapy. Treatment of cancer by immunotherapy is a particularly preferred embodiment. When the antigen is a neoantigen, it may be identified by identifying the neoantigen by tumor sequencing.

[0149] Exemplary tumor antigens can be: alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), WT-1, NY-ESO-1, LY6K, IMP3, DEPDC1, CDCA-1, abnormal products of ras, p53, KRAS or NRAS, CTAG1B, peptides derived from chromosomal translocations, such as BCR-ABL or ETV6-AML1, viral antigens, such as peptides from HPV-related cancers, peptides derived from proteins, such as tyrosinase, gp100 / pmel17, Melan-A / MART-1, gp75 / TRP1 or TRP2, and overexpressed antigens, such as MOK (RAGE-1), ERBB2 (HER2 / NEU).

[0150] When the therapeutic cargo is an antigen or a neoantigen, the EV or a pharmaceutical composition comprising the EV may optionally further comprise an adjuvant. When the antigen is administered with an adjuvant to stimulate an immune response, the adjuvant may be: an inorganic compound: such as aluminum hydroxide, aluminum phosphate, calcium hydroxide phosphate, a mineral oil such as paraffin oil, a bacterial product such as the inactivated bacteria Bordetella pertussis, Mycobacterium bovis, a toxoid, a non-bacterial organic substance such as squalene, a detergent such as Quil A, a plant saponin, a cytokine such as IL-1, IL-2, IL-12 or RIBI (muramyl dipeptide), or an ISCOM (immunostimulatory complex) such as a STING (stimulator of interferon genes) agonist, which may include a cyclic dinucleotide. Such adjuvants may protect the therapeutic EV from rapid dispersion by sequestering it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic to macrophages and other components of the immune system. Adjuvants that can be incorporated into the vaccine are well known to those skilled in the art and will be selected in such a way that they do not negatively affect the immunological activity of the EV. When the adjuvant is a protein (e.g., a cytokine), in specific embodiments, the adjuvant can be incorporated into the EV itself as part of a TSPAN2 fusion protein or a second POI.

[0151] In certain embodiments, the POI or at least one POI may be a binding protein of the therapeutic agent. Such a binding protein may be, for example, an RNA or DNA binding protein, a viral binding protein, a small molecule binding protein, or an Fc binding protein. In some embodiments, the binding protein capable of binding to the therapeutic agent may be a POI contained in a TSPAN2 fusion polypeptide. Alternatively, the binding protein may be present as a second POI expressed on a separate construct.

[0152] In one embodiment, the POI is a nucleic acid binding protein (NA-binding protein), such as an RNA or DNA binding protein. In these embodiments, the nucleic acid cargo is loaded into the EV by binding of the nucleic acid to the NA-binding protein.

[0153] Non-limiting examples of NA-binding proteins are Ago2, Dicer, Drosha, DGCR8, hnRNPA1, hnRNPA2B1, DDX4, ADAD1, DAZL, ELAVL4, IGF2BP3, SAMD4A, TDP43, FUS, FMR1, FXR1, FXR2, EIF4A13, MS2 coat protein, and any domain, portion or derivative thereof. More broadly, specific subclasses of RNA-binding proteins and domains, such as mRNA binding proteins (mRBPs), pre-rRNA-binding proteins, tRNA-binding proteins, small nuclear or nucleolar RNA-binding proteins, non-coding RNA-binding proteins, miRNA-binding proteins, shRNA-binding proteins, and transcription factors (TFs). In addition, various domains and derivatives may also be used as NA-binding domains to transport NA cargo into EVs. Non-limiting examples of RNA-binding domains include small RNA-binding domains (RBDs) (which can be both single-stranded and double-stranded RBDs (ssRBDs and dsRBDs), such as DEAD, KH, GTP_EFTU, dsrm, G-patch, IBN_N, SAP, TUDOR, RnaseA, MMR-HSR1, KOW, RnaseT, MIF4G, zf-RanBP, NTF2, PAZ, RBM1CTR, PAM2, Xpo1, Piwi, CSD, and Ribosomal_L7Ae. Such RNA-binding domains can be present in multiples, alone or in combination with other RNAs, and can also form part of larger RNA-binding protein constructs, as long as their key function (i.e., the ability to transport the NA cargo of interest, such as mRNA or short RNA) is maintained.

[0154] In a preferred embodiment, the present invention relates to two groups of NA-binding domains, namely PUF proteins and CRISPR-associated polypeptides (Cas), in particular Cas9, Cas6 and Cas13, and various types of NA-binding aptamers. The present invention uses the term PUF protein to cover all related proteins and domains of these proteins (also referred to as PUM proteins), such as human Pumilio homolog 1 (PUM1), PUMx2 or PUFx2, which are copies of PUM1, etc., or any NA binding domain that can be obtained from any PUF (PUM) protein. The typical feature of PUF proteins is the presence of 8 consecutive PUF repeats, each of approximately 40 amino acids, usually flanked by two related sequences Csp1 and Csp2. Each repeat has a "core consensus sequence" containing aromatic and basic residues. RNA binding requires the entire PUF repeat cluster. The PUF protein according to the present invention can be natural or engineered to bind anywhere in the RNA molecule, or alternatively, PUF proteins with different binding affinities for different sequences can be selected and RNA molecules can be engineered to contain the sequence. There are also engineered and / or replicated PUF domains that bind 16 nucleotides in a sequence-specific manner, which can also be used to further increase the specificity for NA cargo molecules. Thus, the PUF domain can be modified to bind any sequence with different affinities and sequence lengths, which makes the system highly modular and applicable to any RNA cargo molecule according to the present invention. PUF proteins and their regions and derivatives that can be used as NA-binding domains of the present invention include the following non-limiting list of PUF proteins: FBF, FBF / PUF-8 / PUF-6, -7, -10, all from C. elegans; Pumilio, from D. melanogaster; Puf5p / Mpt5p / Uth4p, Puf4p / Ygl014wp / Ygl023p, Puf5p / Mpt5p / Uth4p, Puf5p / Mpt5p / Uth4p, Puf3p, all from S. cerevisiae; PufA, from Dictyostelium; human PUM1 (Pumilio 1, sometimes also referred to as PUF-8R) and any domains thereof, a polypeptide comprising at least two NA-binding domains from PUM1, any truncated or modified or engineered PUF protein, such as PUF-6R, PUF-9R, PUF-10R, PUF-12R, and PUF-16R or derivatives thereof; and X-Puf1 from Xenopus laevis.Particularly suitable NA-binding PUFs according to the present invention include the following: PUF 531, PUF mRNA locus (sometimes referred to as PUF engineering or PUFeng), and / or PUFx2 (whose sequence is available in PCT / EP2018 / 080681) and any derivatives, domains and / or regions thereof. PUF / PUM proteins are very advantageous because they can be selected to be of human origin. In addition, as in the case of PUF proteins, Cas proteins, such as Cas 6, Cas 9 and Cas13, are very preferred examples of releasable NA-binding domains that bind to NA cargo molecules with suitable affinity, thereby enabling Cas proteins to releasably and reversibly attach to NA cargo. As with PUF-based NA-binding domains, Cas proteins represent releasable, irreversible NA-binding domains that have programmable, modifiable sequence specificity for target NA cargo molecules, resulting in higher specificity at lower overall affinity, thereby allowing NA cargo to be loaded into EVs and released at target locations.

[0155] "Nucleic acid" refers to polynucleotides, including polyribonucleotides and polydeoxyribonucleotides. Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, such as cytosine, thymine, and uracil, as well as adenine and guanine, respectively. (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) and G. Michael Blackburn, Michael J. Gait, David Loakes and David M. Williams, Nucleic Acids in Chemistry and Biology 3 rd The invention encompasses any deoxyribonucleotide or ribonucleotide component and any chemical variant thereof. The polymer or oligomer may be heterogeneous or homogeneous in composition and may be isolated from a naturally occurring source or may be artificially or synthetically produced. In addition, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded form, including homoduplexes, heteroduplexes, and hybrid states.

[0156] "Oligonucleotide" or "polynucleotide" is a nucleic acid of at least 2, at least 8, at least 15 or at least 25 nucleotides in length but may be at most 50, 100, 1000, 5000, 10000, 15000 or 20000 nucleotides in length, or a compound that specifically hybridizes to a polynucleotide. Polynucleotides include sequences of DNA or RNA or their mimetics, which may be isolated from natural sources, recombinantly produced or artificially synthesized. Another example of a polynucleotide used in the present invention may be a peptide nucleic acid (PNA; see U.S. Pat. No. 6,156,501, the entire contents of which are incorporated herein by reference.) The present invention also encompasses situations where non-traditional base pairing exists, such as Hoogsteen base pairing, which has been identified in certain tRNA molecules and is assumed to exist as a triple helix. "Polynucleotide" and "oligonucleotide" are used interchangeably herein. It should be understood that when a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G and C), this also includes a corresponding RNA sequence (e.g., A, U, G, C), wherein "U" replaces "T".

[0157] As used herein, "polynucleotides" include, for example, cDNA, RNA, DNA / RNA hybrids, antisense RNA, siRNA, mRNA, ribozymes, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derived, synthetic or semi-synthetic nucleotide bases. In addition, changes in wild-type or synthetic genes are contemplated, including but not limited to deletions, insertions, substitutions of one or more nucleotides, or fusions with other polynucleotide sequences.

[0158] The present invention specifically relates to ABD-EV, which is further loaded with nucleic acids targeting oncogenes known to be associated with cancer development, such as siRNA. The genes targeted by the nucleic acids according to the present invention can be ABL, AF4 / HRX, AKT-2, ALK, ALK / NPM, AML1, AML1 / MTG8, AXL, BCL-2, 3, 6, BCR / ABL, c-MYC, DBL, DEK / CAN, E2A / PBX1, EGFR, ENL / HRX, ERG / TLS, ERBB, ERBB-2, ETS-1, EWS / FLI-1, FMS, FOS, FPS, GLI, GSP, HER2 / neu, HOX11, HST, IL-3, INT-2, JUN, KIT, KS3, K-SAM, LBC, LCK , LMO1,LMO2, L-MYC, LYL-1, LYT-10, LYT-10 / Cα1, MAS, MDM-2, MLL, MOS, MTG8 / AML1, MYB, MYH11 / CBFB, NEU, N-MYC, OST, PAX-5, PBX1 / E2A, PIM- 1. PRAD-1, RAF, RAR / PML, RAS-H, RAS-K, RAS-N, REL / NRG, RET, RHOM1, RHOM2, ROS, SKI, SIS, SET / CAN, SRC, TAL1,TAL2, TAN-1, TIAM1, TSC2, TRK.

[0159] In more detail, the nucleic acid cargo molecule bound to the RNA / DNA binding protein (POI) of the present invention or exogenously loaded (described further herein) into the exosome can be selected from the group comprising: shRNA, siRNA, saRNA, miRNA, anti-miRNA, mRNA, modified mRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA, lncRNA, ribozymes, minicircle DNA, plasmid DNA, RNA / DNA vectors, trans-splicing oligonucleotides, splice switching oligonucleotides, CRISPR guide strands, morpholino (PMO) antisense oligonucleotides (ASOs), peptide-nucleic acids (PNAs), viral genomes, and viral genetic material (e.g., naked AAV genomes), but essentially any type of nucleic acid molecule can be delivered by the EVs of the present invention. Both single-stranded and double-stranded nucleic acid molecules are within the scope of the present invention, and the nucleic acid molecules can be naturally occurring (such as RNA or DNA) or can be chemically synthesized RNA and / or DNA molecules, which can contain chemically modified nucleotides, such as 2'-O-Me, 2'-O-allyl, 2'-O-MOE, 2'-F, 2'-CE, 2'-EA, 2'-FANA, LNA, CLNA, ENA, PNA, phosphorothioate, tricyclic DNA, thionucleotides, phosphoramidates, PNA, PMO, etc.

[0160] When the cargo is mRNA, the mRNA can be a natural or non-naturally occurring mRNA. The mRNA may include one or more modified nucleobases, nucleosides or nucleotides. The nucleobases of mRNA are organic bases, such as purines or pyrimidines or derivatives thereof. The nucleobases can be standard bases (e.g., adenine, guanine, uracil and cytosine) or non-standard or modified bases, including one or more substitutions or modifications, including but not limited to alkyl, aryl, halogen, oxo, hydroxyl, alkoxy and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction. Therefore, the nucleobase can be selected from the non-limiting group consisting of: adenine, guanine, uracil, cytosine, 7-methylguanine, 5-methylcytosine, 5-hydroxymethylcytosine, thymine, pseudouracil, dihydrouracil, hypoxanthine and xanthine. The nucleoside of mRNA is a compound comprising a combination of a sugar molecule (e.g., a 5-carbon or 6-carbon sugar, such as a pentose, ribose, arabinose, xylose, glucose, galactose, or a deoxy derivative thereof) and a nucleobase. The nucleoside can be a canonical nucleoside (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine) or an analog thereof, and can include one or more substitutions or modifications, including but not limited to alkyl, aryl, halogen, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction of nucleobases and / or sugar components. The nucleotide of mRNA is a compound containing a nucleoside and a phosphate group or an alternative group (e.g., borane phosphate, thiophosphate, selenophosphate, phosphonic acid, alkyl, amidate, and glycerol). Nucleotides can be canonical nucleotides (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine monophosphate) or analogs thereof, and can include one or more substitutions or modifications, including but not limited to alkyl, aryl, halogen, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction of nucleobases, sugars, and / or phosphates or alternative components. Nucleotides can include one or more phosphate groups or alternative groups. For example, a nucleotide can include a nucleoside and a triphosphate group. "Nucleoside triphosphates" (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) can refer to canonical nucleoside triphosphates or analogs or derivatives thereof, and can include one or more substitutions or modifications as described herein. For example, "guanosine triphosphate" should be understood to include canonical guanosine triphosphate, 7-methylguanosine triphosphate, or any other definition covered herein. The mRNA may include a 5' untranslated region, a 3' untranslated region and / or a coding or translation sequence that is translated to produce a fusion protein of the present invention. The mRNA may include any number of base pairs, including tens, hundreds or thousands of base pairs. Any number (e.g., all, some or none) of the nucleobases, nucleosides or nucleotides may be analogs of the canonical species, substituted, modified or non-naturally occurring.In certain embodiments, all specific nucleobase types can be modified. For example, all cytosines in mRNA can be 5-methylcytosine. In some embodiments, mRNA can include 5' cap structures, chain termination nucleotides, stem loops, polyA sequences and / or polyadenylation signals. Cap structures or cap types are compounds including two nucleoside moieties connected by a linker, which caps the mRNA at the 5' end of the mRNA, and can be selected from naturally occurring caps, non-naturally occurring caps or cap analogs, or anti-reverse cap analogs (ARCA). Cap types can include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap can include guanine nucleotides and 7 methylated guanine (G) nucleotides connected by triphosphate bonds at its 5' position, such as m7G(5')ppp(5')G, usually written as m7GpppG. Cap types can also be anti-reverse cap analogs. A non-limiting list of possible cap types includes m7GpppG, m7Gpppm7G, m73'dGpppG, iri27'03'GpppG, iri27'03'GppppG, iri27'02'GppppG, m7Gpppm7G, m73'dGpppG, iri27'03'GpppG, iri27'03'GppppG, and m27 02'GppppG. The mRNA may alternatively or additionally include chain terminating nucleosides. For example, chain terminating nucleosides may include those that are deoxygenated at the 2' and / or 3' positions of their sugar moieties. These species may include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytosine, 3'-deoxyguanosine, 3'-deoxythymidine and 2', 3'-dideoxynucleosides, such as 2', 3'-dideoxyadenosine, 2', 3'-dideoxyuridine, 2', 3'-dideoxycytidine, 2', 3'-dideoxyguanosine and 2', 3'-dideoxythymidine. The mRNA may alternatively or additionally include a stem loop, such as a histone stem loop. The stem loop may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or more nucleotide base pairs. For example, the stem loop may include 4, 5, 6, 7, 8, 9 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in, before or after the untranslated region (5' untranslated region or 3' untranslated region), the coding region or the polyA sequence or the tail. The mRNA may alternatively or additionally include a polyA sequence and / or a polyadenylation signal. The polyA sequence may consist entirely or mostly of adenine nucleotides or analogs or derivatives thereof. The polyA sequence may be a tail located near the 3' untranslated region of the mRNA.In addition to the region of the present invention (which encodes a fusion protein and can be codon-optimized), the modified mRNA of the present invention may also include one or more stem loops, chain termination nucleosides, miRNA binding sites, polyA sequences, polyadenylation signals, 3' and / or 5' untranslated regions (3'UTR and / or 5'UTR) and / or 5' cap structures. As described above, various nucleotide modifications are preferably incorporated into mRNA to modify it to increase translation, reduce immunogenicity and increase stability. Suitable modified nucleotides include, but are not limited to, N1-methyladenosine (m1A), N6-methyladenosine (m6A), 5-methylcytidine (m5C), 5-methyluridine (m5U), 2-thiouridine (s2U), 5-methoxyuridine (5moU), pseudouridine (ψ), N1-methylpseudouridine (m1ψ). Among these mRNA modifications, m5C and ψ are most preferred because they reduce the immunogenicity of mRNA and increase translation efficiency in vivo. In a preferred embodiment of the present invention, the composition herein comprises a modified mRNA as a polynucleotide cargo, wherein the mRNA is modified with at least 50% m5C and 50% ψ or m1ψ, preferably at least 75% m5C and 75% ψ or m1ψ, and even more preferably 90% m5C and 90% ψ or m1ψ, or even more preferably 100% modified with m5C and ψ or m1ψ.

[0161] In some embodiments, the POI can be a viral binding protein, for example, a protein that can bind to adeno-associated virus (AAV), such as an AAV receptor, or a protein that can bind to other types of viruses such as lentiviruses. Generally, any protein that can bind to a viral coat / envelope protein or can bind to a viral genome is within the scope of the present invention. Exemplary viral binding proteins include: AAVR GPR108, syndecan, and albumin.

[0162] In some embodiments, the POI can be a small molecule binding protein, such as any protein, polypeptide or peptide (i.e., any molecule comprising an amino acid sequence), to which a small molecule agent can be attached by non-covalent or covalent attachment, or by a combination of covalent and non-covalent interactions. Terms such as "binding protein-small molecule conjugate" or "binding protein-small molecule drug conjugate" or "binding protein-small molecule drug conjugate" or simply "conjugate" are used herein to describe the combination of a binding protein and a small molecule agent. A binding protein can play several different roles: for example, it can be (i) a carrier and / or delivery form, which essentially refers to transporting a small molecule agent attached thereto, (ii) a targeting agent that transports EVs carrying a binding agent protein-small molecule conjugate to a specific location, (iii) a therapeutically active protein that becomes therapeutically active or inactive by attaching a small molecule, which can have an agonistic or antagonistic effect, (iv) a signaling protein that, with or without its small molecule cargo, can exert or contribute to cellular and / or somatic changes and associated therapeutic and / or preventive effects, (v) a protein that only performs or catalyzes a specific reaction when in proximity with another protein, and so on. A binding protein can further contribute to somatic and / or cellular actions or activities and associated therapeutic effects by releasing the small molecule agent at the appropriate location, or it can contribute to such effects by retaining the small molecule agent bound thereto. An example of the first case is when the binding protein releases the small molecule drug within the target cell following EV-mediated delivery, while an example of the second case is the delivery of an antibody-small molecule drug conjugate into a tumor.

[0163] The terms "small molecule agent" or "small molecule" or "small molecule drug" or "small molecule therapeutic agent" are used interchangeably herein and should be understood to refer to any molecular agent that can be used to treat and / or diagnose a disease and / or condition, and is also used to modulate or alter, for example, the activity and / or binding and / or localization of a binding protein. Small molecule agents are typically synthesized by chemical synthesis methods, but can also be of natural origin, such as by purification from a natural source, or can be obtained by any other suitable method or combination of techniques. In short, the non-limiting definition of "small molecule" is any organic compound with a molecular weight of less than 900 g / mol (Daltons) that can modulate, act or affect a biological process in substantially any way. For the purposes of the present invention, a small molecule can be substantially greater than 900 g / mol, such as 1500 g / mol, 3000 g / mol, or occasionally even greater. In summary, molecular weight and / or molecular size are not limiting factors in constituting a small molecule agent. In fact, for the purposes of the present invention, any agent that can be bound by a binding protein displayed on an EV is considered a "small molecule agent."

[0164] When the POI is a viral binding protein, the EV of the present invention is loaded with a viral cargo. Exemplary viral cargoes include: a viral vector, which is an AAV vector or a lentiviral vector.

[0165] In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector comprises a capsid from human AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12. In some embodiments, the AAV vector comprises an AAV viral genome, and the AAV viral genome comprises an inverted terminal repeat (ITR) sequence from human AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, the AAV capsid and AAV ITR are from the same serotype or from different serotypes.

[0166] In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the lentiviral vector is derived from human immunodeficiency virus, simian immunodeficiency virus, or feline immunodeficiency virus. In some embodiments, the lentiviral vector is non-replicating. In some embodiments, the lentiviral vector is non-integrating.

[0167] In some embodiments, the viral vector comprises a viral capsid and a viral genome, the viral genome comprising one or more heterologous transgenes. In a preferred embodiment, the heterologous transgene encodes a polypeptide or protein. The protein encoded in the viral genome can be any of the protein cargoes according to the present invention that allow the viral cargo to be used as a gene replacement therapy.

[0168] In some embodiments, the cargo-loaded EV may additionally comprise one or more molecules that provide immune effector functions. Immune effector molecules are particularly useful in the case of EVs loaded with viral (e.g., AAV or lentiviral) cargo, but can also be used in the case of EVs loaded with any cargo according to the present invention. Immune effectors can reduce the immunogenicity of EVs. In some embodiments, immune effector functions stimulate immunosuppressants. In other embodiments, immune effector functions inhibit immunostimulatory molecules. In some embodiments, ABD-EVs comprise molecules that stimulate immunosuppressants and molecules that inhibit immunostimulatory molecules.

[0169] Exemplary immune effector molecules include, but are not limited to, one or more of CTLA4, B7-1, B7-2, PD-I, PD-L1, PD-L2, CD28, or VISTA. In some embodiments, the envelope comprises CTLA4 and PD-L1, CTLA and PD-L2, CTLA-4 and VISTA, PD-L1 and PD-L2, PD-L1 and VISTA, PD-L2 and VISTA, CTLA4 and PD-L1 and PD-L2, CTLA4 and PD-L1 and VISTA, CTLA4 and PD-L2 and VISTA, PD-L1 and PD-L2 and VISTA, or CTLA4 and PD-L1 and PD-L1 and VISTA.

[0170] The immune effector molecule may form part of a TSPAN2 fusion construct, or may form part of a separate fusion protein construct comprising the immune effector molecule fused to any EV protein according to the invention.

[0171] Examples of small molecules include anticancer agents such as doxorubicin, methotrexate, 5-fluorouracil or other nucleoside analogs such as cytosine arabinoside, proteasome inhibitors such as bortezomib, or kinase inhibitors such as imatinib or seliciclib, or NSAIDs such as naproxen, aspirin, or celecoxib, antibiotics such as heracillin, or antihypertensives such as ACE inhibitors such as enalapril, ARBs such as candesartan, cyclic dinucleotides, etc. It will be apparent to those skilled in the art that the present invention is naturally also applicable to other small molecules without departing from the spirit of the present invention.

[0172] In some embodiments, the POI may be a protein capable of binding to an Fc domain, also referred to as an Fc binding protein. The terms "Fc binding polypeptide" and "Fc binding protein" and "Fc binding agent" and "Fc-binding protein" and "binding agent" are used interchangeably herein and should be understood to refer to any protein, polypeptide or peptide (i.e., any molecule comprising an amino acid sequence) that is capable of binding to the Fc domain of any POI. Typically, the Fc binding polypeptides of the present invention are derived from various human or non-human sources (e.g., mammalian sources, bacteria, etc.), and they have high affinity for the Fc domains of various antibody isotypes, subtypes and species (e.g., IgG (as non-limiting examples in the case of IgG, IgG1, IgG2, IgG3, IgG4, IgG2a, IgG2d and / or IgG2c), IgA, IgM, IgM, IgD, etc.), and they can be fused to EV proteins. In addition to other Fc-binding polypeptides mentioned throughout the present application, non-limiting examples of Fc-binding polypeptides according to the present invention include protein A, protein G, protein A / G, Z domain, ZZ domain, human FCGRI, human FCGRIIA, human FCGRIIB (as a non-limiting example, accession number is 31994), human FCGRIIC (as a non-limiting example, accession number is 31995), human FCGRIIIA (as a non-limiting example, accession number is P08637), human FCGR3B (as a non-limiting example, accession number is O75015), human FCAMR, human FCERA, human FCAR, mouse FCGRI, mouse FCGRIIB, mouse FCGRIII, mouse, mouse FCGRn, and various combinations, derivatives or substitutes thereof.

[0173] The terms "Fc-containing protein" and "protein comprising an Fc domain" and "Fc domain-containing protein" and "Fc domain-containing protein" and "Fc domain protein" and similar terms are used interchangeably herein and should be understood to refer to any protein, polypeptide or peptide (i.e., any molecule comprising an amino acid sequence) comprising an Fc domain, either naturally or as a result of protein engineering to introduce an Fc domain. Fc stands for "fragment crystallizable" or "fragment constant," which is the name for the tail region of antibodies. However, Fc domains can also be produced and used on other proteins, not just antibodies. Non-limiting examples of such proteins containing an Fc domain include antibodies and antibody derivatives, Fc-modified decoy receptors (such as CD24-Fc or CD52-Fc) and / or signal transducers, such as decoy receptors for the following interleukins: IL1, IL2, IL3, IL4, IL5, IL6 (such as the signal transducer gp130 (as a non-limiting example, accession number P40189)), IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL17 (such as IL17R, as a non-limiting example, accession number Q96F46), IL23 (such as IL23R, as a non-limiting example, accession number Q5VWK5), etc., bispecific binders and multispecific binders containing Fc domains, any type of receptor or ligand containing an Fc domain, Fc domain-modified enzymes for use in, for example, enzyme replacement therapy or gene editing, nucleases such as Cas and Cas9 transplanted with Fc domains, tumor suppressors fused to Fc domains, etc. Suitable Fc domains that can be fused to a POI that natively lacks an Fc domain include the following non-limiting examples: human IGHM (as a non-limiting example, accession number P01871), human IGHA1 (as a non-limiting example, accession number P01876), human IGHA2 (as a non-limiting example, accession number P01877), human IGKC (as a non-limiting example, accession number P01834), human IGHG1 (as a non-limiting example, accession number P01857), human IGHG2 (as a non-limiting example, accession number P01859), human IGHG3 (as a non-limiting example, accession number P01860), human IGHG4 (as a non-limiting example, accession number P01861), human IGHD (as a non-limiting example, accession number P01880), human IGHE (as a non-limiting example, accession number P01854),

[0174] In some embodiments, the POI of the present invention is an endosomal escape domain or an endosomal escape moiety. In some embodiments, the endosomal escape domain may be a POI contained in a TSPAN2 fusion polypeptide. Alternatively, the endosomal escape domain may be present as a second POI expressed on a separate construct.

[0175] Endosomal escape domains according to the present invention include: HA2, VSVG, GALA, B18. Other exemplary endosomal escape peptides include: HIV TAT PDT (peptide / protein transduction domain), HIV Gp-120, KALA, GALA and INF-7 (derived from the N-terminal domain of the influenza virus hemagglutinin HA-2 subunit), endosomal escape moieties that act by causing membrane fusion, such as diphtheria toxin T domains, proton sponge endosomal escape moieties, such as peptides or lipids with histidine or imidazole moieties and CPPs, and other moieties that enable endosomal escape. CPPs are typically less than 50 amino acids, but can also be longer, are typically highly cationic and rich in arginine and / or lysine amino acids, and have the ability to access the interior of almost any cell type. Exemplary CPPs can be transferrin, transferrin 10, penetratin, MTS, VP22, CADY peptide, MAP, KALA, PpTG20, proline-rich peptides, MPG peptides, PepFect peptides, Pep-1, L-oligomers, calcitonin peptides, various arginine-rich CPPs such as poly-arg, tat, and combinations thereof).

[0176] The presence of the endosomal escape domain advantageously helps drive endosomal escape and thereby enhances the delivery of biological activity of the EV itself. The use of endosomal escape strategies is particularly important in the treatment of diseases where the cargo carried within the EV needs to be delivered to the cytosol of the recipient cell or to any other compartment outside the endolysosomal system.

[0177] In another embodiment, the POI can be a targeting moiety. The targeting moiety enables the EV to be engineered to allow targeted delivery of the EV to cells, tissues, organs and / or compartments of interest. In one embodiment, the targeting moiety can be a POI contained in a TSPAN2 fusion polypeptide. Alternatively, the targeting moiety can be present as a second POI expressed on a separate construct. In an advantageous embodiment, the targeting moiety is engineered to be displayed on the surface of the EV.

[0178] The targeting molecule can be a protein, peptide, single chain fragment or any other derivative of an antibody and can be obtained from human or non-human animals, etc.

[0179] The presence of a targeting moiety combined with the presence of an ABD is particularly useful in organ targeting. For example, an EV may contain TSPAN2 fused to both a therapeutic protein / binding protein (luminescent-loaded POI) and a targeting moiety for a therapeutic cargo, and an ABD fused to a tetraspanin on a second construct. The PPOI-TSPAN2-targeting moiety and tetraspanin-ABD construct are expressed on the same EV, and once the EV is coated in albumin, it can remain in the circulation for a longer time, avoiding uptake by cells of the liver or immune system, and thus being able to reach the desired target organ.

[0180] Targeting moieties can be used to target EVs to cells, subcellular locations, tissues, organs, or other body compartments. Targetable organs and cell types include: brain, neuronal cells, blood-brain barrier, muscle tissue, eye, lung, liver, kidney, heart, stomach, intestine, pancreas, red blood cells, white blood cells including B cells and T cells, lymph nodes, bone marrow, spleen, and cancer cells.

[0181] Targeting can be achieved in many ways, for example using a targeting peptide. The length of this targeting peptide can vary from several amino acids to hundreds of amino acids, for example between 3-100 amino acids, 3-30 amino acids, 5-25 amino acids, etc., for example 7 amino acids, 12 amino acids, 20 amino acids. Targeting peptides of the present invention can also include full-length proteins, such as receptors, receptor ligands, etc. In addition, targeting peptides of the present invention can also include antibodies and antibody derivatives, for example monoclonal antibodies, single-chain variable fragments (scFv), nano antibodies, other antibody domains, etc.

[0182] Exemplary targeting moieties include brain targeting moieties such as rabies virus glycoprotein (RVG), nerve growth factor (NGF) that binds to the NGF receptor, melanotransferrin, and FC5 peptide, and muscle targeting moieties such as muscle specific peptide (MSP).

[0183] The term "albumin binding domain" (ABD) is understood to refer to any protein, peptide, antibody or nanobody capable of binding to albumin, or a fragment or domain thereof. The ABD may be derived from any species, preferably the ABD has a specific binding affinity for human serum albumin. Commonly known ABDs are antibodies or nanobodies produced against albumin, or the PAB protein from Peptostreptococcus magnus and the ABDs of protein G from group C and group G streptococci, both of which bind to albumin with high affinity. Alternatively, the albumin binding domain of the present invention may be an antibody, a scFv nanobody, a heavy chain antibody (hcAb), a single domain antibody (sdAb) such as VHH or VNAR, or a fragment thereof capable of binding to albumin. Particularly preferred are sdAbs and antibody fragments because their small size allows the introduction of other additional domains into fusion proteins and simple construct generation and expression.

[0184] In certain embodiments, the EV of the present invention comprises a second POI expressed on a second construct. Optionally, the second POI on the second construct may be present in the form of a fusion protein with TSPAN2, or in the form of a classic EV protein.

[0185] The classical EV protein according to the present invention may be selected from the group comprising the following non-limiting examples: CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, AAAT, AT1B3, AT2B4, ALIX, Annexin, BASI, BASP1, BSG, Syntenin-1, Syntenin-2, Lamp2, Lamp2a, Lamp2b, TNFR, TfR1, syndecan-1, syndecan-2, syndecan-3. , syndecan-4, CD37, CD82, CD151, CD224, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d / ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, CLIC1, CLIC4, interleukin receptor, immunoglobulin, MHC-I or MHC-II component, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD53, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD 274, CD362, COL6A1, AGRN, EGFR, FPRP, GAPDH, GLUR2, GLUR3, GP130, GPI-anchored protein, GTR1, HLAA, HLA-DM, HSPG2, ITA3, Lactadherin, L1CAM, LAMB1, LAMC1, LIMP2, MYOF, ARRDC1, ATP2B2, ATP2B3, ATP2B4, BSG, IGSF2, IGSF3, IGSF8, ITGB1, ITGA4, ATP1A2, ATP1A3, ATP1A4, ITGA4, SLC3A2, ATP transporter, ATP1A1, ATP1B3, ATP2B1, LFA-1, LGALS3BP, Mac-1alpha, Mac-1beta, MFGE8, members of the myristoylated alanine-rich protein kinase C substrate (MARCKS) protein family, such as MARCKSL1,Matrix metalloproteinase-14 (MMP14), PDGFR, PTGFRN, PRPH2, ROM1, SLIT2, SLC3A2, SSEA4, STX3, TCRA, TCRB, TCRD, TCRG, TFR1, UPK1A, UPK1B, VTI1A, VTI1B and any other EV protein, and any combination, derivative, domain, variant, mutant or region thereof. Mutations can be introduced into the wild-type sequence of the EV protein to alter its function, and a preferred mutant according to the present invention is CD63 (Y235A). The use of EV proteins has the effect of driving POI loading into EVs, so that POIs are actively loaded into EVs, and as a result EV proteins are sometimes referred to as carrier proteins. Particularly preferred classical EV proteins include transmembrane proteins such as tetraspanins, LAMP2B, LIMP2, ICAM, integrins, ARRDC1, syndecan, syntenin, TNFR, TfR1 and Alix. ,

[0186] As described above, the present invention relates to EVs comprising fusion proteins of TSPAN2-POI, TSPAN3-POI or TSPAN18-POI. In other aspects, the present invention also relates to polypeptide constructs of TSPAN2-POI, TSPAN3-POI or TSPAN18-POI and any polynucleotide constructs encoding the constructs. As described above, the TSPAN2 / 3 / 18-POI fusion protein may also comprise additional domains, including: (i) a release domain capable of being cleaved to release the POI; (ii) a linker or spacer and / or (iii) a multimerization domain.

[0187] In an advantageous embodiment, the TSPAN2 / 3 / 18-POI fusion protein further comprises a multimerization domain. The multimerization domain according to the present invention may be a homotypic multimerization domain or a heterotypic multimerization domain. The multimerization domain of the present invention may be a dimerization domain, a trimerization domain, a tetramerization domain or any higher-order multimerization domain. The multimerization domain enables dimerization, trimerization or any higher-order multimerization of the fusion polypeptide, which increases the sorting and transport of the fusion polypeptide into EVs, and may also help increase the yield of vesicles produced by EV-producing cells. Exemplary multimerization domains include: leucine zippers, folding domains, fragment X, collagen domains, 2G12 IgG homodimers, mitochondrial antiviral signaling protein CARD filaments, cardiac phospholamban transmembrane pentamers, parathyroid hormone dimerization domains, blood group glycoprotein A transmembrane, HIV Gp41 trimerization domains, HPV45 oncoprotein E7 C-terminal dimer domains and any combination thereof.

[0188] In another advantageous embodiment, the TSPAN2 / 3 / 18-POI fusion protein further comprises a linker and / or a spacer. The presence of linkers, spacers and / or scaffold sequences allows flexibility and enables the POI to be optimally positioned for display on the surface of the EV or luminescently positioned as desired. Linkers according to the present invention can be used to provide increased flexibility, improved pharmacokinetics (PK), increased expression and improved biological activity of fusion polypeptide constructs and corresponding polynucleotide constructs, and can also be used to ensure that steric hindrance is avoided and the functionality of the fusion polypeptide is maintained. Exemplary linkers according to the present invention include glycine or serine linkers that increase stability or flexibility, such as (GGGGS) n (n=1, 2, 4) or (Gly) 6、 (Gly)8 rigid linkers, such as (EAAAK)n (n=1-3) and A(EAAAK)4ALEA(EAAAK)4A, curved linkers (XP) n Or cleavable linkers, such as disulfides, protease-sensitive sequences.

[0189] Suitable release domains according to the present invention can be cis-cleavage or "self-cleavage" sequences, such as inteins, light-induced monomeric or dimeric release domains, such as Kaede, KikGR, EosFP, tdEosFP, mEos2, PSmOrange, GFP-like Dendra proteins Dendra and Dendra2, CRY2-CIBN, etc. Alternatively, a nuclear localization signal (NLS)-nuclear localization signal binding protein (NLSBP) (NLS-NLSBP) release system can be used. Depending on the desired function of the fusion polypeptide, a protease cleavage site can also be incorporated into the fusion protein for spontaneous release, etc. In the case of nucleic acid cargo, a specific nucleic acid cleavage domain can be included. Non-limiting examples of nucleic acid cleavage domains include endonucleases, such as Cas6, Cas13, engineered PUF nucleases, site-specific RNA nucleases, etc.

[0190] Including release domains is very advantageous because they can release specific parts or domains from the original fusion polypeptide. This is particularly advantageous when the release of parts of the fusion polypeptide will increase the delivery of the biological activity of the cargo and / or when a specific function of the fusion polypeptide works better when it is part of a smaller construct.

[0191] The term "self-cleaving protein" may refer to a naturally occurring protein that excises itself from a native host protein by self-cleavage. A suitable example of a self-cleaving protein is an intein. It will be appreciated that certain modifications are required to provide a protein that has only self-cleavage capability (i.e., no self-splicing). A suitable example of a protein that is only capable of self-cleavage (and no splicing) is ΔI-CM.

[0192] The term "self-splicing protein" may refer to a naturally occurring protein that excises itself from its native host protein by self-splicing and ligation of its flanking peptide bonds. A suitable example of a self-splicing protein is an intein.

[0193] The terms "small intein" or "delta intein" are used interchangeably herein and are understood to refer to a modified intein, preferably from the parent RecA, and lacking an endonuclease domain.

[0194] The term "rapidly cleaved intein" is understood to relate to an intein or miniintein modified at the +C-extein position and / or the -N-extein position such that the cleavage rate of the intein is faster / rapid than the original RecA, intein or miniintein.

[0195] The term "slow cleavage intein" is understood to refer to an intein or miniintein modified at the +C-extein position and / or the -N-extein position such that the cleavage rate of the intein is slower / slower than the original RecA, intein or miniintein. An example of a slow cleavage intein is ΔI-CM.

[0196] The cis cleavage sequence may be a self-cleaving protein, such as an intein. The intein may be a slow-cleaving or fast-cleaving intein. The intein may be a small intein, such as a small intein modified to optimize the cleavage rate. The intein may be a delta-intein-CM. Thus, the cis cleavage sequence (or self-cleavage) according to the present disclosure may include, but is not limited to, the following:

[0197] Inteins, mini-inteins, delta-inteins and certain variants, mutations and domains thereof having desired functions (including but not limited to self-cleavage rather than splicing), such as mini-inteins modified to optimize cleavage rate. For example, mini-inteins having C1A, D24G, V67L and / or D150G substitutions at the N-terminal portion (or appropriate -C-extein position). Such suitable substitutions can be found in SEQ ID No. 20. For example, splicing is achieved through the +1 position of the intein, where the +1 position is Cys. Substitution of Cys with Ala in ΔI-CM removes the splicing ability and only supports cleavage. It should be understood that other mutations, substitutions, etc. may also function similarly. Therefore, the substitutions mentioned above, while exemplary, are not limiting in any way and may also vary from intein to intein.

[0198] In some cases, it may be possible to choose to use a slow-cleaving intein. When more time is needed to ensure that the EV is efficiently loaded with the desired cargo, a slow separation system may be preferred. Slow-cleaving inteins according to the present disclosure may include, but are not limited to, the following: small inteins, delta inteins, delta intein-CM and small inteins and certain variants, mutations and domains thereof having desired functions (such as but not limited to cleavage rather than splicing and cleavage rates). In a preferred embodiment, the slow-cleaving cis-cleavage release system is based on an intein system, wherein the C-terminal portion of the intein may comprise the amino acid sequence Val-Val-Val-His-Asn, more preferably wherein the C-terminal portion of the intein is modified to comprise Val-Val-Val-His-Asn-Gly. It has been observed that certain modifications at the +1C-extein position slow the cleavage rate (i.e., slow cleavage).

[0199] In some cases, one may choose to use a fast-cleaving cis-cleaving release system (such as a fast-cleaving cis-cleaving intein). A fast-cleaving system may be preferred when rapid harvesting of EVs is desired. In a preferred embodiment, the fast-cleaving cis-cleaving release system is based on an intein system, wherein the C-terminal portion of the intein may comprise the amino acid sequence Val-Val-Val-His-Asn or Val-Val-Val-His-Asn-Cys. It has been observed that certain modifications at the +1C-extein position, such as the examples described above, accelerate the cleavage rate (i.e., rapid cleavage).

[0200] In a specific embodiment, the EV of the present invention comprises more than one POI. This may be the result of the presence of more than one POI in a single TSPAN2 fusion protein, or it may be the result of multiple fusion proteins being loaded into a single EV. The multiple fusion proteins may comprise the same or different scaffold proteins and the same or different POIs. The presence of more than one POI on a single EV is advantageous because it increases the therapeutic effect of the EV and, in the case of different POIs, allows multiplexing of cargo to improve delivery, therapeutic effect, targeting, etc.

[0201] In certain embodiments, the POI cargo carried by the EV may be present inside the EV, outside the EV, or in the membrane of the EV. The desired location of the therapeutic cargo will depend on the nature of the cargo and its mechanism of action, for example, a membrane protein will preferably be located in the membrane of the EV, a decoy receptor, an endosomal escape moiety, or a targeting moiety will preferably be present on the surface of the EV, but cargo designed to be delivered to the cytosol or nucleus of a recipient cell, such as a silencing RNA or AAV, will preferably be located in the lumen of the EV. The design of the fusion protein of the present invention allows expression on the surface or in the lumen of the EV.

[0202] Fusion of POIs to TSPAN2 or other canonical EV proteins as described above should be understood as endogenous loading of the EV. In alternative embodiments, therapeutic cargo can be passively loaded into EVs or actively loaded by exogenous loading methods as described below.

[0203] Therapeutic cargo can be passively loaded into EVs via the presence of the therapeutic cargo in the cytosol of the EV-producing cell. This passive loading is applied, for example, to nucleic acids, small molecules, viruses, soluble proteins, or membrane proteins that are naturally loaded into EVs.

[0204] In certain embodiments, therapeutic cargo is actively loaded into the EV of the present invention. One form of active loading of cargo involves exogenous active loading, which involves loading cargo using any known exogenous loading method, including: electroporation, transfection with a transfection reagent such as a cationic transfection agent lipofectamine (RTM), conjugation of cargo with a membrane anchoring moiety such as a lipid or cholesterol tail, or loading by CPP in the form of a CPP-cargo conjugate or in the form of a CPP-cargo non-covalent complex or any combination of these methods. Similarly, this type of active loading can result in the therapeutic cargo being located on the inside of the EV, on the outside of the EV, or within the membrane of the EV. Any cargo as defined above can be exogenously loaded. Particularly preferred embodiments include exogenously loading nucleic acids or viral cargoes by electroporation, CPP loading, or co-incubation with lipid-labeled cargoes.

[0205] The present invention also relates to a population of EVs comprising at least one TSPAN2-POI fusion protein. In addition, the present invention also relates to a population of EVs comprising at least one TSPAN2-POI fusion protein and a second POI construct expressed on the same EV. As detailed above, the main advantage of using TSPAN2 as a scaffold protein for loading POIs into engineered EVs is the ability to reliably and consistently co-localize additional constructs onto the same EV.

[0206] In certain embodiments, the average number of POIs per EV in the EV population according to the present invention is higher than one POI per EV, but may also be lower than one POI per EV. In addition, in certain embodiments, in the EV population according to the present invention, the average number of POIs per EV is higher or lower than one POI per EV. In another embodiment, in the EV population according to the present invention, at least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and / or at least 95% of all EVs comprise at least one TSPAN2-POI construct, and optionally also comprise at least one additional POI.

[0207] Generally, EV can be derived from substantially any cell source, whether it is a primary cell source or an immortalized cell line. EV source cells can be any embryonic, fetal and adult stem cell types, including induced pluripotent stem cells (iPSC) and other stem cells derived by any method, as well as any adult cell source. The source cells of the present invention can be selected from a variety of cells and cell lines, such as mesenchymal stem cells or stromal cells (available from, for example, bone marrow, adipose tissue, Wharton's jelly, perinatal tissue, chorion, placenta, tooth germ, umbilical cord blood, skin tissue, etc.), fibroblasts, amniotic cells, more specifically amniotic epithelial cells, optionally expressing various early markers, bone marrow suppressor cells, M2 polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc. Cell lines of particular interest include human umbilical cord endothelial cells (HUVEC), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular or lymphatic endothelial cells, erythrocytes, erythroid progenitor cells, chondrocytes, mesenchymal stromal cells (MSC) of various origins, amniotic cells, amniotic epithelial (AE) cells, CEVEC Cells, any cells obtained by amniocentesis or obtained from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, etc. In addition, immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DC) also belong to the scope of the present invention, and basically any type of cell as long as it can produce EVs is also included in the present invention. The source cells can be allogeneic, autologous, or even xenogeneic, that is, the cells can come from the patient himself or from an unrelated, matched or unmatched donor for the patient to be treated.

[0208] In particular, the present invention relates to cells of a monocistronic, bicistronic or multicistronic polynucleotide construct (as defined above) according to the present invention that has been stably modified to contain at least one fusion protein encoding TSPAN2-POI. These cells can be stably or transiently transfected with the polynucleotides of the present invention, making them engineered EV production cells. Such cells can also be stably or transiently modified to include a second construct encoding a second POI, which optionally can form part of a fusion protein with a classical EV protein. The cells of the present invention can be monoclonal cells or polyclonal cell lines.

[0209] Preferred production cells according to the present invention may be: HEK cells, HEK293 cells, HEK293T cells, mesenchymal stem cells (MSC), in particular WJ-MSC or BM-MSC, fibroblasts, amniotic cells, amniotic epithelial cells, CEVEC Cells, placenta-derived cells, umbilical cord blood cells, immune system cells, endothelial cells, epithelial cells or any other type of cells, wherein the cells can be adherent cells, suspension cells and / or suspension adaptor cells. In a more preferred embodiment, the production cells are HEK293 cells. In another more preferred embodiment, the production cells are CAP cells.

[0210] Therefore, in a preferred embodiment, the producer cell is a HEK293 cell or a CAP cell, and the polynucleotide construct encodes a fusion protein of TSPAN2-POI.

[0211] The present invention also relates to a pharmaceutical composition comprising at least one EV according to the present invention and a pharmaceutically acceptable excipient or carrier.

[0212] The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The term encompasses any agent approved by a regulatory agency such as the FDA or EMEA or listed in the U.S. Pharmacopoeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to the subject and does not abrogate the biological activity and properties of the therapeutic cargo. Excipients and carriers that are useful in preparing pharmaceutical compositions and are generally safe and non-toxic are included.

[0213] Exemplary excipients include stabilizer excipients that degrade or lose activity, such as proteins, such as human serum albumin; polyols, such as glycerol, sorbitol, and erythritol; amino acids, such as arginine, aspartic acid, glutamic acid, lysine, proline, glycine, histidine, and methionine; polymers, such as polyvinylpyrrolidone and hydroxypropylcellulose; surfactants, such as polysorbate 80, polysorbate 20, and pluronic F68; antioxidants, such as ascorbic acid and α-tocopherol (vitamin E); buffers, such as acetate, succinate, citrate, phosphate, histidine, tris(hydroxymethyl)aminomethane (TRIS); metal ion / chelating agents, such as Ca 2+ 、Zn 2+ and EDTA; based on cyclodextrins, such as hydroxypropyl β-cyclodextrin and others such as polyanions and salts; stabilizers or fillers, such as lactose, trehalose, dextrose, sucrose, sorbitol, glycerol, albumin, gelatin, mannitol and dextran; or preservatives, such as benzyl alcohol, m-cresol, phenol, 2-phenoxyethanol.

[0214] The pharmaceutical composition according to the present invention can be formulated by any known formulation method, including but not limited to:

[0215] Oral preparations - tablets, capsules, sustained-release preparations, liquid preparations

[0216] Intravenous preparations

[0217] Parenteral preparations

[0218] Topicals - Skin application: creams, ointments, gels, pastes, powders

[0219] Modified release preparations-sustained release preparations

[0220] Liquid or lyophilized preparation

[0221] EVs according to the present invention can be administered to a human or animal subject via a variety of different routes of administration, such as auricle (ear), cheek, conjunctiva, skin, teeth, electroosmosis, intracervical, intranasal sinus, intratracheal, enteral, epidural, extra-amniotic, in vitro, hemodialysis, infiltration, interstitial, intraperitoneal, intraamniotic, intraarterial, intra-articular, intra-biliary, intra-bronchial, intracapsular, intra-cardiac, intra-cartilaginous, intra-caudal, intra-cavernous, intra-cavity, intra-cerebral, intra-ventricular, intra-cistern, intra-corneal, intra-coronary (tooth), intra-coronary, intra-cavernous, intra-dermal, intra-discal, intra-ductal, intra-duodenal, intra-dural, intra-epidermal, intra-esophageal, intra-gastric, intra-gingival, intra-ileal, intra-lesional, intra-luminal, intra-lymphatic, intra-medullary, intra-meningeal, intra-muscular, intra-ocular, intra-ovarian, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, Intrapulmonary, intrasinus, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous push, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing, ocular, oral, oropharyngeal, other, parenteral, transdermal, periarticular, epidural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral and / or vaginal administration, and / or any combination of the foregoing routes of administration, the route of administration generally depending on the disease to be treated and / or the characteristics of the EV, the cargo molecule in question, or the EV population itself.

[0222] It will be clear to those skilled in the art that when describing the medical and scientific uses and applications of EVs, the present invention generally relates to a plurality of EVs, i.e. a population of EVs that may contain thousands, millions, billions or even trillions of EVs. EVs may be present in, for example, 10 5 , 10 8 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 ,10 30EVs (often referred to as "particles") are present in a concentration per unit volume or per unit weight (e.g. per ml or per L or per kg of body weight), or any other greater, lesser or intermediate figure. Similarly, the term "population", which may, for example, relate to EVs containing a specific cargo, should be understood to cover a plurality of entities constituting such a population. In other words, a single EV when present in plurality constitutes an EV population. Therefore, naturally, the present invention relates to individual EVs and populations comprising EVs, as will be clear to a person skilled in the art. When administered in vivo, the dosage of EVs may naturally vary significantly depending on the disease to be treated, the route of administration, the activity and effect of the cargo of interest, any targeting moieties present on the EVs, the pharmaceutical formulation, etc.

[0223] It is contemplated that any dosage regimen is suitable for use with the engineered EVs of the present invention. The dosage regimen selected will depend on the cargo delivered by the EV and the disease to be treated as well as any additional therapy applied which will be determined by a skilled physician.

[0224] It is envisioned that the EVs of the invention will be administered multiple times, i.e., more than 1 time but typically more than 2 times or potentially for chronic, long-term treatment (i.e., administered tens to hundreds to thousands of times). Preferably, if the cargo is an antigen administered as a vaccine, the immunization regimen will involve two or more administrations of the polypeptide, spread out over several weeks. Similarly, if the cargo is, for example, an RNA agent such as siRNA or mRNA or a protein such as an antibody or enzyme or transporter, the EVs containing the cargo will likely be administered more than once, typically multiple times as part of a chronic treatment regimen.

[0225] The present invention relates to a method for producing an EV according to the present invention. A method for producing an EV, comprising: (i) introducing a polynucleotide construct encoding a TSPAN2-POI fusion construct into a cell producing an EV; and (ii) expressing the construct in the cell producing the EV, thereby producing an EV comprising the TSPAN2-POI fusion protein.

[0226] The method for producing EVs according to the invention may further comprise a second step of introducing a second polynucleotide construct encoding a second POI into the same EV-producing cell, optionally wherein the second POI is present in the form of a fusion protein with TSPAN2 or a classical EV protein. The POI may of course be any POI as defined above.

[0227] The above-mentioned methods for producing fusion proteins and EVs containing fusion proteins can be achieved by protein and cell engineering techniques well known in the art, such as molecular cloning and transfecting cells with vectors encoding fusion constructs.

[0228] The method of producing EVs may further comprise the step of exogenously loading EVs with cargo molecules. As described above, the exogenous loading step may comprise loading cargo by any exogenous loading method, including electroporation, microfluidics, transfection with a transfection agent such as the cationic transfection agent lipofectamine (RTM), conjugation of cargo to a membrane anchoring moiety such as a lipid or cholesterol tail, or loading by CPP in the form of a CPP-cargo conjugate or a CPP-cargo non-covalent complex. Particularly preferred embodiments relate to methods of exogenously loading nucleic acid or viral cargo by electroporation, CPP loading, or co-incubation with a lipid-tagged cargo.

[0229] The method for producing engineered EVs according to the present invention may further include a purification step of EVs. The purification of EVs is achieved by any method, including but not limited to: the following techniques, including liquid chromatography (LC), high performance liquid chromatography (HPLC), bead elution chromatography, ion exchange chromatography, spin filtration, tangential flow filtration (TFF), hollow fiber filtration, centrifugation, immunoprecipitation, flow field fractionation, dialysis, microfluidic-based separation, etc., or any combination thereof. In an advantageous embodiment, the purification of EVs is performed using a sequential combination of filtration (preferably ultrafiltration (UF) tangential flow filtration (TFF) or hollow fiber filtration) and affinity chromatography, optionally also including size exclusion LC or bead elution LC. The combined purification steps generally increase the purity of the resulting sample and thereby lead to excellent therapeutic activity. In addition, compared to the UC conventionally used to purify exosomes, sequential filtration-chromatography is significantly faster and may be scaled up to a larger manufacturing volume, which is a major disadvantage of the UC method currently dominating the prior art. Another advantageous purification method is TFF, which provides scalability and purity, and it can be combined with any other type of purification technology.

[0230] Alternatively, the present invention also relates to a method for producing EVs in a patient (i.e., patient-derived engineered EVs), the method comprising administering to the patient a delivery vector comprising a polynucleotide according to the present invention, wherein the target cells in the patient produce EVs comprising a TSPAN2-POI or TSPAN3-POI or TSPAN18-POI fusion protein, optionally, the delivery vector may further comprise a polynucleotide encoding a second construct comprising a POI, which is also co-expressed on the same patient-derived EV as the TSPAN2-POI construct. In a preferred embodiment, the polynucleotide is an mRNA, a circular mRNA, Linear DNA, circular DNA, plastid DNA, linear RNA, circular RNA, self-amplifying RNA or DNA, "naked" or viral genome in a capsid, or any modified form of the above. In a preferred embodiment, the delivery vector is a viral vector or non-viral vector selected from the group consisting of lipid nanoparticles (LNP), virus-like particles (VLP), CPP, polymers or pharmaceutically acceptable carriers.

[0231] Thus, in one embodiment, the present invention also relates to a delivery vector comprising a polynucleotide according to the present invention, wherein the polynucleotide cargo encodes a fusion protein comprising a POI and is arranged to be translated into a fusion protein by a cell producing an EV, said translation resulting in the production of at least one EV comprising a TSPAN2-POI, TSPAN3-POI or TSPAN18-POI fusion protein, optionally wherein the delivery vector further comprises a polynucleotide encoding a second construct comprising a POI, which is also co-expressed on the same patient-derived EV as the TSPAN2-POI construct. Such TSPAN2 / 3 / 18-POI EVs derived from patients will have the same benefits as the above-mentioned bioreactor-produced EVs, such as reliability of construct expression and the ability to co-express multiple constructs.

[0232] The present invention also relates to an EV according to the present invention for use in medicine.

[0233] The present invention also relates to a method of treatment, comprising administering an effective amount of EV according to the present invention or a pharmaceutical composition according to the present invention to a patient in need thereof.

[0234] The use in medicine or treatment methods (collectively referred to as "treatment") can be to deliver any kind of goods according to the present invention. For example, treatment can be as a protein replacement therapy by delivering functional proteins, and delivery of mRNA encoding functional proteins is also used as a protein replacement therapy. This protein replacement therapy can be, for example, enzyme replacement therapy (ERT), which is used for diseases caused by inborn errors of metabolism, such as phenylketonuria, urea cycle disorders, or lysosomal storage disorders. It can be treated by delivering gene silencing RNA, RNA conversion or CRISPR-Cas9 for gene editing. Treatment can be gene therapy by delivering plasmid DNA, small circles, such as viral gene therapy of AAV or lentivirus. Treatment can be carried out by presenting antigens or new antigens for immunotherapy, effectively acting as a vaccine for inducing an immune response. For example, EV can act by delivering and / or presenting tumor antigens for cancer immunotherapy or for viral, bacterial or fungal antigens for immunity to pathogens. Treatment can be carried out by delivering small molecules, antibodies and antibody-drug conjugates, which can mediate therapeutic effects once delivered to cells or extracellular matrix. In one embodiment, treatment or therapy can be achieved by EVs containing more than one type of therapeutic cargo, i.e., the therapeutic cargo can be a mixture of proteins, nucleic acids, viruses, viral genomes, antigens and / or small molecules.

[0235] Importantly, the present invention relates to the use of the EV compositions described herein in the prevention and / or treatment and / or alleviation of various diseases, generally by delivering essentially any type of drug cargo, such as, for example: nucleic acids, such as RNA molecules, DNA molecules or mixers, mRNA, antisense or splice-switching oligonucleotides, siRNA, shRNA, miRNA, plasmid DNA (pDNA), supercoiled or non-supercoiled plasmids, minicircles, peptides or proteins, including: transporters, enzymes, receptors, such as decoy receptors, membrane proteins, cytokines, antigens and neoantigens, ribonucleoproteins, nucleic acid binding proteins, antibodies, nanobodies, antibody fragments, antibody-drug conjugates, small molecule drugs, gene editing technologies, such as CRISPR-Cas9, TALEN, homing endonucleases (meganuclease), or vesicle-based cargo, such as viruses (e.g., AAV, lentivirus, etc.). In one embodiment, the cargo can be a mixture of proteins, nucleic acids, viruses, viral genomes, antigens and / or small molecules.

[0236] Non-limiting examples of diseases and conditions that are suitable targets for treatment using the peptide delivery systems described herein include the following non-limiting examples: autoimmune diseases (e.g., celiac disease, Crohn's disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus), ulcerative colitis, ankylosing spondylitis, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), interleukin-1 receptor antagonist deficiency (DIRA), endometriosis, autoimmune hepatitis, scleroderma, myositis, stroke, Acute spinal cord injury, vasculitis, Guillain-Barré syndrome, acute myocardial infarction, ARDS, sepsis, meningitis, encephalitis, liver failure, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), renal failure, heart failure or any acute or chronic organ failure and associated underlying etiology, graft-versus-host disease; Duchenne muscular dystrophy and other muscular dystrophies; inborn errors of metabolism, including disorders of carbohydrate metabolism, such as G6PD-deficient galactosemia, hereditary fructose intolerance, fructose 1,6-bisphosphatase deficiency, and glycogen storage diseases; organic acid metabolism disorders (organic aciduria), such as melanocortin Uric aciduria, 2-hydroxyglutaric aciduria, methylmalonic acidemia or propionic acidemia, polycarboxylase deficiency; amino acid metabolism disorders such as phenylketonuria, maple syrup urine disease, glutaric acidemia type 1; amino acid metabolism diseases such as hereditary tyrosinemia, nonketotic hyperglycinemia and homocystinuria; hereditary tyrosinemia; Fanconi syndrome; primary lactic acidemias such as pyruvate dehydrogenase, pyruvate carboxylase and cytochrome oxidase deficiencies; fatty acid oxidation and mitochondrial metabolism disorders such as short, medium and long chain acyl-CoA dehydrogenase deficiencies, also known as beta oxidation deficiencies; Reye syndrome; medium chain acyl-CoA dehydrogenase deficiency (MCADD); MELAS, MERFF, pyruvate dehydrogenase deficiency; porphyrin metabolism disorders such as acute intermittent porphyria; purine or pyrimidine metabolism disorders such as Lesch–Nyhan syndrome; steroid metabolism disorders such as lipid congenital adrenal hyperplasia, congenital adrenal hyperplasia; mitochondrial dysfunction such as Kearns-Sayre syndrome; peroxisomal dysfunction such as Zellweger syndrome and neonatal adrenoleukodystrophy, congenital adrenal hyperplasia or Smith-Lemli-Opitz, Menkes syndrome, neonatal hemochromatosis; urea cycle disorders,Such as N-acetylglutamate synthase deficiency, carbamoyl phosphate synthase deficiency, ornithine transaminase deficiency, citrullinemia (arginine succinate synthase deficiency), argininosuccinic aciduria (arginyl succinate lyase deficiency), argininemia (arginase deficiency), hyperornithinemia, hyperammonemia, hyperornithinemia hyperammonemia hypercitrullinuria (HHH) syndrome (mitochondrial ornithine transporter deficiency), citrullinemia II (aspartate-glutamate transporter citrate deficiency), lysinuria protein intolerance (y+L amino acid transporter 1 mutation), orotic aciduria (uridine monophosphate synthase (UMPS) deficiency), all lysosomal storage diseases, such as alpha-mannosidosis, beta-mannosidosis , aspartylglucosaminuria, cholesterol ester storage disease, cystinosis, Danon disease, Fabry disease, Farber disease, fucosidosis, galactosialidosis, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, GM1 gangliosidosis type I, GM1-gangliosidosis type II, GM1-gangliosidosis type III, GM2-Sandhoff disease, GM2-Tay-Sachs disease, GM2-gangliosidosis, AB variant, mucolipidosis type II, Krabbe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, MPS I-Hurler syndrome, MPS I-Scheie syndrome, MPS I-Hurler-Schie syndrome, MPS II-Hunter syndrome,MPS IIIA-Sanfilippo syndrome type A, MPS IIIB-Sanfilippo-syndrome type B, MPS IIIB-Sanfilippo syndrome type C, MPS-IIIB-Sanfilippo syndrome type D, MPS IV Morquio type A, MPS IV-Morquio type B, MPS IX-Hyaluronidase deficiency, MPS VI-Maroteaux-Lamy, MPS VII-Sly syndrome, Mucolipidosis I-Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV, Mucopolysaccharidosis, Multiple sulfatase deficiency, Neuronal ceroid lipofuscinosis T1, Neuronal ceroid lipofuscinosis T2, Neuronal ceroid lipofuscinosis T3, Neuronal ceroid lipofuscinosis T4, Neuronal ceroid lipofuscinosis T5, Neuronal ceroid lipofuscinosis T6, Neuronal ceroid lipofuscinosis T7, Neuronal ceroid lipofuscinosis T8, Neuronal ceroid lipofuscinosis T9, Neuronal ceroid lipofuscinosis T10, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C, Pompe disease type e) disease, pycnodysostosis, cystic fibrosis including Salla, Schindler and Wolman diseases, primary ciliary dyskinesia, pulmonary alveolar proteinosis, ARC syndrome, Ret syndrome, neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, GBA-associated Parkinson's disease, Huntington's disease and other trinucleotide repeat-related diseases, prion diseases, dementia including frontotemporal dementia, ALS, motor neuron disease, multiple sclerosis, cancer-induced cachexia, anorexia, type 2 diabetes and various cancers.

[0237] Specifically, it is envisioned that the present invention can be used to treat cancer by cancer immunotherapy, i.e., presenting cancer antigens on the surface of EVs so that these antigens generate an immune response against the cancer antigens. Virtually all types of cancer are relevant disease targets of the present invention, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendix cancer, astrocytoma, cerebellar or brain cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumors, brain stem gliomas, brain cancer, brain tumors (cerebellar astrocytoma, brain astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma, carcinoid tumors (children, gastrointestinal tract), tumors of unknown primary, central nervous system Lymphoma, cerebellar astrocytoma / glioblastoma, cervical cancer, chronic lymphocytic leukemia, chronic myeloproliferative disease, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (intraocular melanoma, retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (extracranial, extragonadal or ovarian), gestational trophoblastic tumor, glioma (brain stem glioma, brain astrocytoma, visual pathway hypothalamic glioma), gastric carcinoid, hairy cell leukemia, head and neck cancer, heart Cancer, Hepatocellular Carcinoma, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi's Sarcoma, Kidney Cancer (Renal Cell Carcinoma), Laryngeal Cancer, Leukemia (Acute Lymphoblastic (also known as Acute Lymphocytic Leukemia), Acute Myeloid (also known as Acute Myeloid Leukemia), Chronic Lymphocytic (also known as SLL), Chronic Myeloid (also known as Chronic Myeloid Leukemia), Hairy Cell Leukemia), Lip and Oral Cancer, Liposarcoma, Liver Cancer (Primary), Lung Cancer (Non-Small Cell, Small Cell), Lymphoma, AIDS-Related Lymphoma, Burkitt's Lymphoma, Cutaneous T-Cell Lymphoma, Hodgkin ) lymphoma, non-Hodgkin's lymphoma, medulloblastoma, Merkel cell carcinoma, mesothelioma, squamous neck cancer with occult metastatic primary, oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic / myeloproliferative diseases, myeloid leukemia, chronic myeloid leukemia (acute, chronic), myeloma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, islet cell carcinoma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma,Pineal astrocytoma, pineal germ cell tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (Ewing family tumor sarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma), Sézary syndrome, skin cancer (non-malignant tumors, melanoma), small intestine cancer, squamous cell carcinoma, squamous neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Macroglobulinemia and / or Wilm's tumor.

[0238] The present invention is also particularly advantageous for treating brain and central nervous system diseases as it enables targeting of TSPAN2-expressing EVs to the brain while continuing to express another POI, such as a therapeutic protein or an endosomal escape moiety. The present invention specifically relates to EVs having an ABD present on the surface of the EV, which are loaded with any type of cargo according to the present invention, but preferably a nucleic acid cargo, such as a silencing RNA, such as a siRNA targeting a known neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar ataxia, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, motor neuron disease, multiple sclerosis, cystic wall degeneration and bullous retinoschisis, Goldberg-Shprintzen syndrome, Kuru disease, autoimmune GFAP astrocytopathy, MECP2 duplication syndrome, AQP4-astrocytopathy, familial pain syndromes such as erythromelalgia, paroxysmal extreme pain disorder and congenital insensitivity to pain, Pelizaeus–Merzbacher disease, prion diseases including Creutzfeldt-Jakob disease (CJD), Gerstmann- - Scheinker syndrome (GSS) and fatal familial insomnia (FFI), leukodystrophies including demyelinating, adult-onset, autosomal dominant and leukodystrophies.

[0239] The present invention specifically relates to EVs loaded with therapeutic cargo, which can be protein and / or nucleic acid cargo, for the treatment of Duchenne muscular dystrophy (DMD), diseases caused by inborn errors of metabolism, including lysosomal storage disorders, including Niemann-Pick type C (NPC) and Pompe disease, urea cycle disorders, such as argininosuccinic aciduria (ASA) and citrullinemia and ornithine transcarbamylase (OTC) deficiency, metachromatic leukodystrophy and phenylketourea (PKU).

[0240] Wherein the POI is a binding protein, such as an albumin binding protein, an Fc binding agent, a nucleic acid binding protein, a virus binding protein or a small molecule binding protein, the present invention also relates to a nanoparticle complex comprising any TSPAN2 EV according to the present invention bound to a corresponding binding partner (such as albumin, an Fc-containing protein, a nucleic acid, a virus or a small molecule). In this embodiment, some or all EVs are bound to their corresponding binding partners. The present invention also relates to a pharmaceutical composition comprising the nanoparticle complex of the present invention in combination with a pharmaceutically acceptable excipient or carrier. Example

[0241] Example 1 - Establishment of a high-throughput screening protocol

[0242] To date, only a few scaffold domains have been used to endogenously load protein therapeutics into EVs. To expand the range of available scaffold domains, the inventors compiled a list of potential EV scaffold domains based on literature review and proteomics databases ( Figure 1 A). Candidate proteins were selected based on, but not limited to, the following criteria: (1) common EV proteins from the NCI-60 database; (2) proteins enriched in EVs derived from human embryonic kidney (HEK-)293T cells; (3) reported EV scaffold proteins used as references, and (4) proteins from the tetraspanin family that have no known function / association with EVs. The fourth group of proteins was included because it was hypothesized that other proteins in the tetraspanin family might also exhibit high loading efficiency, as CD9, CD63, and CD81 are widely used for genetic engineering ( Figure 1 A). It is noteworthy that all proteins with a molecular weight greater than 130 kDa were excluded. Finally, a total of 244 candidate proteins with a medium size of 38 kDa were included, of which 115 were transmembrane proteins ( Figure 1 A).

[0243] In order to be able to screen all these domains in a high-throughput manner, the inventors chose a reporter system based on bioluminescence using ThermoLuc because it is a sensitive and stable reporter for marking engineered EVs. A proof-of-concept study was conducted to ensure that Tluc-based assays can distinguish cargo from the rest (surface and soluble fractions) in vesicles. HEK-293T cells were transfected with plasmids encoding Tluc alone or fused with CD63, and their conditioned medium was fractionated using size exclusion chromatography (SEC). Tluc activity in each fraction was quantified in the presence or absence of the detergent Triton. As expected, Tluc activity was mainly detected in the soluble fraction of the conditioned medium of Tluc-transfected cells, but when fused with CD63, it was transferred to the EV fraction. In addition, Tluc activity was detected only after the membrane was destroyed with Triton, indicating that its intravesicular location ( Figure 1 B), thus showing the feasibility of our method. In order to compare and analyze the EV sorting ability, Tluc was fused to the C-terminus of 244 EV scaffold domains, and the resulting fusion box was cloned into a mammalian expression plasmid. These plasmids were then introduced into HEK-293T cells in the form of microplates. A small amount of plasmid encoding another highly sensitive luciferase (i.e., NanoLuc (Nluc)) was incorporated into the transfection mixture as an internal control to adjust confounding variables, such as transfection efficiency. For downstream analysis, candidate proteins were compared mainly in terms of total secretion Tluc, intravesicular Tluc (total secretion Tluc minus surface / soluble Tluc) and encapsulation index (total secretion Tluc to surface / soluble Tluc ratio).

[0244] Figure 1 High throughput screening protocol is shown. (1A) Selection and overview of potential domains for EV-sorting. (1B) SEC elution profiles of Tluc and CD63-Tluc media. HEK-293T cells were transfected with plasmids encoding independent Tluc or CD63-Tluc fusion proteins, and conditioned medium was fractionated. Tluc activity in each fraction was quantified using Triton with or without membrane lysis. (1C) Experimental and data analysis protocol. HEK-293T cells were grown in 96-well plates and co-transfected with 0.75 μg / mL fusion plasmid and 7.5 ng / mL Nluc plasmid for 48 h. The samples were then centrifuged and Tluc activity was measured in cell pellets and conditioned medium. Nluc activity was quantified only in conditioned medium.

[0245] Example 2-Validation of Tluc high-throughput screening platform

[0246] To determine the robustness of our high-throughput screening platform, all candidate proteins were ranked according to total secreted Tluc as well as the ratio of total secreted Tluc / Nluc.

[0247] Figure 2 Results of screening Tluc in the secretome of HEK-293T cells are shown. (2A) Correlation between the ranking of total secreted Tluc and the ranking of total secreted Tluc / Nluc. (2B) Correlation between the ranking of total secreted Tluc and the ranking of the encapsulation index. (2C) Overview of the encapsulation index. Candidate proteins with an encapsulation index higher than 1 (dashed line) are considered EV-sorting domains. N=5. (2D) Total secreted and intravesicular Tluc of 36 EV-sorting domains. The figure shows the ratio of intravesicular to total secreted Tluc. N=5. (2E) Correlation of the ranking of the encapsulation index obtained from two independent batches. Each point in the scatter plot refers to a candidate protein, and the red dot marks the reference EV-sorting domain CD63.

[0248] The results demonstrated that normalization to secreted Nluc did not affect the relative secretion of Tluc ( Figure 2 A), thus transfection efficiency was not considered a major confounding variable. The presence of three well-studied EV-sorting tetraspanins (CD9, CD63, and CD81) and two reported domains (gag and PTGFRN) in this subset suggests the robust utility of our screening protocol ( Figure 2 D). The inventors found that the candidate proteins exhibited heterogeneous EV-sorting abilities, as seen from the poor correlation between the ranking of encapsulation index and the ranking of total secreted Tluc ( Figure 2 B). In fact, only 36 domains had a packaging index higher than 1, which were therefore considered promising EV-sorting domains in HEK-293T cells ( Figure 2 C). The poor correlation observed and the fact that only 36 of 244 promising candidate proteins were effective suggest that it is difficult to predict which carrier / scaffold proteins will be useful for loading POIs into EVs without extensive screening and creative efforts.

[0249] Notably, for TSPAN2 and TSPAN3, more than 90% of total secreted Tluc was inside EVs, with TSPAN2 loading even more intravesicular Tluc than CD63. The reproducibility of the screen was assessed by comparing the ranking of candidate proteins with respect to total secreted Tluc or secretion efficiency (ratio of total secreted Tluc to cellular Tluc). The high reproducibility was well exemplified by the linear correlation between two independent batches and different doses. Importantly, regarding the ranking of encapsulation efficiency, the overall correlation between two independent batches was rather poor. However, only in the most promising 36 EV-sorting domains ( Figure 2 E), the correlation increases dramatically, which emphasizes the reliability of these domains.

[0250] Example 3 - Verification of the sorting capability of small EVs

[0251] Based on the broad EV sorting efficiency obtained from the microplate-based screening, some representative and / or promising candidate proteins were further examined using SEC. Unlike the screening protocol, the conditioned medium was additionally filtered through a 200 nm membrane and concentrated prior to SEC fractionation to enrich for small EVs ( Figure 3 A). In addition to using Triton to distinguish intravesicular Tluc from other fractions, proteinase K (ProK) was also incorporated into each fraction to discriminate resistant protein aggregates ( Figure 3B). Consistent with the exceptional EV sorting capabilities found in the screen, TSPAN2 and TSPAN3 ranked highest in terms of percentage of intravesicular Tluc to total secreted Tluc, showing improved activity over CD63 (>80%; Figure 3 C).

[0252] Figure 3 Results of screening for the ability to sort into small EVs are shown. (3A) Validation protocol using size exclusion chromatography (SEC). HEK-293T cells were grown in 15 mm petri dishes and transfected with 1.5 μg / mL plasmid for 6 h. The culture medium was changed to Opti-MEM and collected after 48 hours. After centrifugation, the samples were filtered through a 0.2 μm membrane and then concentrated, and the Tluc activity in each fraction was quantified in the presence / absence of Triton or proteinase K. (3B) Relative Tluc activity in SEC fractions of conditioned medium. (3C) Percentage of Tluc in vesicles to total secreted Tluc. Validation data are from two independent experiments.

[0253] TSPAN2 and TSPAN3 ranked highest (80%) and fourth (45%) in terms of the percentage of Tluc within vesicles, respectively, while CD63 sorted only 28% of Tluc into EVs. Consistent with the results from HEK-293T cells, SDCBP, CALM1, YWHAG or BASP1 showed poor EV sorting efficiency.

[0254] Example 4 - Quantification of engineered small EVs at the single vesicle level

[0255] To analyze EVs in more detail and obtain information about the concentration and relative abundance (by mean fluorescence intensity, MFI) of certain transgenic molecules per EV, the method of choice was single vesicle flow cytometry. Luciferase is a useful reporter for quantifying bulk engineered EVs. Tluc ( Figure 4 A). HiBiT can complement its partner subunit LgBiT and form a fully functional luciferase complex (Nluc), which enables the amount of engineered EVs to be compared with Tluc-based screening results. A total of 18 domains were studied, including the top 5 domains in terms of encapsulation index, all secreted Tluc or secretion efficiency from screening readout, three EV markers (CD9, CD81, SDCBP), PTGFRN and CALM1.

[0256] Figure 4The results of this validation using a dual-function bioluminescent and fluorescent reporter are shown. (4A) Validation scheme using a hybrid HiBiT-mNG reporter. HEK-293T cells were grown in 6-well plates and transfected with 1.5 μg / mL plasmid for 6 hours. The medium was then changed to Opti-MEM and collected after 48 hours. After centrifugation and filtration through a 0.2 μm membrane, the samples were quantified based on HiBiT activity and mNG fluorescence within the vesicles. (4B) Comparison of HiBiT and Tluc within the vesicles. (4C) Single vesicle flow cytometric analysis of mNG-labeled small extracellular vesicles. (4D) Quantification of concentration and mean fluorescence intensity of mNG-labeled small extracellular vesicles.

[0257] TSPAN3, TSPAN2, and CD63 consistently exhibited high EV sorting capacity, as demonstrated by high levels of intravesicular HiBiT and encapsulation index ( Figure 4 B). In addition, results from flow cytometry showed that TSPAN3 and TSPAN2 produced the highest number of engineered EVs, approximately 3.5 times more than CD63 ( Figure 4 C; Figure 4 D). Interestingly, using the above dual-function screen TSPAN18 also emerged as a potent EV-sorting domain, second only to TSPAN3.

[0258] Removal of large particles resulted in negligible concentrations of engineered EVs targeting the domain CALM1. Calmodulins appear to be ideal domains for sorting cargo molecules into large EVs. This finding is useful for applications using large EVs for drug / gene delivery of larger cargoes.

[0259] Example 5 - Tluc screening results in different cell lines

[0260] The inventors attempted to identify EV scaffold proteins that are highly effective not only in a single production cell line, but also in a variety of different cell lines, so that they can be used as a platform technology in a universal manner, rather than having narrow functionality. To test EV sorting capabilities, four additional cell lines were tested, including: Freestyle 293-F, MSC (mesenchymal stem cells), Huh-7 (human hepatocytes), and TCMK-1 (mouse kidney epithelial cells) were studied. Based on the screening in HEK-293T cells, the inventors tested the top 95 domains, measuring encapsulation index, total secreted Tluc, intravesicular Tluc, or secretion efficiency. In parallel with the discovery of HEK-293T cells, transfection efficiency did not seem to confound the performance of the domains.

[0261] Figure 5Results of evaluating Tluc in the secretome of other EV producing cell lines are shown. (5A) Top ten scaffold domains implicated in intravesicular Tluc in different producing cell lines. N=3. (5B) Number of EV-sorting domains in each producing cell line and across several cell lines. (5C) Relative ranking of 24 conserved EV-sorting domains implicated in intravesicular Tluc in each cell line and their averages. (5D) Subcellular locations of 24 conserved EV-sorting domains. (5E) Interaction network of 24 conserved EV-sorting domains. Line thickness indicates the strength of data support.

[0262] TSPAN2 sorted the greatest amount of Tluc into EVs in Freestyle 293-F and TCMK-1 cells, respectively, whereas CALM2 was the optimal domain in MSC and Huh-7 cells ( Figure 5 A). Overall, the number of EV-sorting domains (packaging index above one) ranged from 30 to 37 for the five cell lines studied. Figure 5 B). A total of 24 domains are conserved, indicating robust EV-sorting capabilities across different cellular contexts. The relative performance of each domain in the conserved subset of each cell line was determined based on intravesicular Tluc activity ( Figure 5 C) On average, TSPAN2, CD63, and TSPAN3 showed the highest EV sorting capacity in a reproducible manner.

[0263] The inventors then consulted online databases to understand the potential mechanism of the superior EV sorting ability of this conserved subset. According to the annotations available on UniProtKB, in addition to the three calmodulin proteins and ANXA11 ( Figure 5 D), most of the domains are characterized by four-pass transmembrane structures. A molecular interaction map derived from the STRING database was also drawn ( Figure 5 E), showing a central role for CD9, CD63, and CD81, which may reflect their widespread use in EV engineering. Interestingly, the calmodulin protein appears to function independently of the remaining proteins, while there is weak evidence that TSPAN2 and TSPAN3 interact with CD63, which is due to the fact that TSPAN2 and TSPAN3 have never been previously identified as EV proteins and therefore have never been used or thought to function as scaffold proteins for loading POIs into EVs.

[0264] Example 6 - TSPAN2 and TSPAN3 as potential EV-sorting domains - comparison with CD63

[0265] TSPAN2 and TSPAN3 were considered promising candidates for sorting proteins of interest into EVs from the screening and validation results. Further characterization of both types of engineered EVs revealed a narrow size distribution with a median hydrodynamic diameter of approximately 120 nm ( Figure 6 A). Common EV markers such as CD81, syntenin-1, and TSG101 were found in these EV preparations ( Figure 6 B). The morphological appearance of TSPAN2- and TSPAN3-engineered EVs is typical for EV features such as membrane structure and size, as determined by cryo-electron microscopy ( Figure 6 C).

[0266] To explore whether TSPAN2 and TSPAN3 confer unique biological properties to engineered EVs compared to CD63, the present inventors used mNG and Tluc reporters to examine cellular uptake and in vivo distribution, respectively. As shown in confocal microscopy images, all types of engineered EVs were efficiently internalized by Huh-7 cells and transported to lysosomes in large quantities ( Figure 6 D). Similar trends in cellular uptake were observed by titrating EV dose ( Figure 6 E; Figure S6). Due to the rapid distribution and catabolism of EVs in vivo, luciferin substrate was intraperitoneally administered 5 min before intravenous administration of engineered EVs, and Tluc activity in mice was continuously monitored by the IVIS Spectrum in vivo imaging system ( Figure 6 F). For all three types of EVs, we observed rapid distribution to the liver and spleen and a steady decrease in systemic activity within 30 min ( Figure 6 G). Interestingly, TSPAN2 and TSPAN3 engineered EVs showed high retention over the time frame obtained ( Figure 6 H). Tluc activity was also measured in major organs ex vivo and in plasma, with results supporting its predominant liver and spleen accumulation and, most importantly, improved exposure compared to CD63 ( Figure 6 I; Figure 6 J).

[0267] In detail, Figure 6Characterization results of TSPAN2 and TSPAN3 engineered EVs are shown. (6A) Size distribution of engineered EVs measured by NTA. (6B) Western blot of engineered EVs. (6C) Cryo-EM images of engineered EVs. (6D) Confocal micrographs of Huh-7 cells treated with mNG-labeled EVs for 4 h. (6E) Mean fluorescence intensity of Huh-7 cells treated with mNG-labeled EVs for 8 h. N=3. (6F) Scheme for examining the in vivo distribution of Tluc-labeled EVs. Mice were sacrificed immediately after the IVIS period, and major organs and plasma were collected. N=3. (6G) Representative IVIS images of mice injected with Tluc-labeled-EVs. (6H) Quantification of whole-body irradiation of mice injected with Tluc-labeled-EVs. (6I) Quantification of Tluc-labeled EVs in isolated organs. (6J) Quantification of Tluc-labeled EVs in plasma.

[0268] In summary, the inventors surprisingly found for the first time that two mammalian proteins, TSPAN2 and TSPAN3, outperformed benchmark domains such as CD63, CD9, and CD81. Certain domains are known to have excellent EV sorting capabilities in specific cell lines but not in other cell lines (e.g. Figure 5 B). For example, the viral glycoprotein gag operates significantly in HEK-293T, Freestyle 293-F, Huh-7, operates to a lower degree in TCMK-1 cells, but operates quite poorly in MSC. The inventors have identified up to 24 domains that maintain EV sorting capabilities in a wide range of cell lines tested. In addition, the overall weak internal molecular interactions of the conservative subsets indicate heterogeneous mechanisms of biogenesis of engineered EVs, and subsequently indicate different molecular fingerprints and biological properties, which also emphasizes that the discovery of these specific scaffold proteins is unpredictable and only as a result of highly creative and large-scale screening efforts. The results of the screening and validation experiments highlight the abnormal EV-sorting capabilities of TSPAN2 and TSPAN3. Their splicing isoforms were also studied, but hardly any EV-sorting capabilities were shown (data not shown). In addition, TSPAN2- or TSPAN3-engineered EVs are effectively absorbed by cells in vitro and are even more bioavailable than benchmark CD63 over time after intravenous administration in mice.

[0269] Example 7: Initial results of a large-scale functional screen to identify novel EV protein candidates that allow co-expression of multiple constructs

[0270] Following the experiments detailed in Examples 1-6, the delivery of functional cargo was further investigated. A total of approximately 280 potential EV proteins were screened. Importantly, not only were the scaffold / carrier EV proteins screened for presence / prevalence on exosomes, but they were also tested for functional delivery of reporter proteins by two different systems (co-culture system and method of adding purified EVs directly to cells). These tests were also performed in a large number of different cell lines to ensure that the findings were robust and not cell line specific.

[0271] Figure 7 A shows a schematic diagram of the Cre reporter protein used to identify promising EV protein candidates. Figure 7 The co-culture method is depicted in B. In detail, the method involves mixing EV-producing cells with reporter cells, allowing the EVs to be taken up by the cells and measuring by FACS on day 4 to analyze GFP-positive cells. Figure 7 C depicts the EV addition system. In more detail, the method involves the isolation and purification of EVs, followed by their addition to reporter cells, which are then analyzed by FACS to detect GFP-positive cells. These two different systems were used in combination because the co-culture system is very economical, rapid, easy to operate, and suitable for primary screening, while the addition system provides a second, more accurate but time-consuming screen.

[0272] HEK-293T cells were used as EV producer cells. HEK293T cells were co-transfected with EV-sortin-intein-Cre plasmid and VSV-G plasmid. In the co-culture and EV addition system, the following reporter cell lines were tested: Hela TL; T47D TL and B16F10 TL. These reporter cells respond to the recombination of the Cre cargo delivered by EV using the traffic light reporter system. Therefore, the delivery of functional Cre by EV can be detected by the observed color change. In the co-culture system ( Figure 7 In B), EV-producer and reporter cells were mixed at ratios of 1:1 and 1:5 (producer cells:reporter cells). In the EV addition system, EVs were added directly to reporter cells at doses of 1E10, 1E9, and 1E8.

[0273] The results of this subsequent functional screening are given in Examples 7-12 below. The materials and methods of Examples 7-12 are as follows:

[0274] Construct generation

[0275] Codon-optimized DNA sequences encoding scaffold proteins including tetraspanins (2-33), late endosomal proteins (STR3N, LAP4B, STAR3, SPP2A, SNX14, NTRK1), cytoplasmic proteins (syntenin1, ANXA4, CALM2, ANX11, CALM3, CALM1, GDIB), single-pass transmembrane proteins (CD316, ICAM1, PTGFRN, Lamp2B), membrane-associated proteins (ARRDC1, Basp1, Myr) and other proteins (GAG, CLD1, AAAT) were ordered from TB (Twist Bioscience, US). Tetraspanin-1, intein-cre and VSV-G were purchased from IDT (Integrated DNA Technologies, USA). The scaffold proteins were first cloned into the pLEX vector, and then the intein-cre was inserted into the plex-scaffold plasmid using the corresponding restriction endonuclease sites.

[0276] Cell culture

[0277] In this study, HEK-293T cells were used to produce functional EVs and cultured in DMEM medium (high glucose) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% anti-antibodies (Gibco, USA). Cells were cultured at 37 ° C in a humidified air atmosphere with 5% CO2. Reporter cell lines (Hela-TL, T47D-TL, MSC-TL, and B16F10-TL) were cultured in the same culture medium and conditions as HEK-293T cells. THP1-TL, RAW246.7-TL, K562-TL were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% anti-antibodies (Gibco, USA).

[0278] EV Production

[0279] EVs were produced by transient transfection of transgenes using polyethyleneimine (PEI). Specifically, HEK-293T cells were seeded into 15 cm dishes with complete DMEM medium at 10 million cells / dish. The next day, cells were transfected with transgenes, and 6 hours after transfection, the medium was replaced with Opti-MEM medium (Gibco, USA) containing 1% anti-anti-. After transfection for 48 hours, conditioned medium (CM) was collected and centrifuged (700 g, 5 min, then 2000 g, 10 min). The supernatant was then filtered through a 0.22 μm filtration system.

[0280] EV isolation

[0281] Tangential flow filtration (TFF, MicroKross, 20 cm 2 , Spectrum labs) to separate EVs from filtered CM. The cutoff value of TFF was 300 kDa, and particles larger than 300 kDa were retained in the system and concentrated. The concentrated particles were then further concentrated by Amicon Ultra-15 100 kDa (Millipore) spin filter, centrifuged at 4000 × g for 30 min to several hours at 4 ° C, depending on the amount of EVs in the sample. Finally, the concentrated EVs were collected in maxirecovery1.5 ml Eppendorf tubes (Axygene, USA) and the concentration was detected by nanoparticle tracking analysis (NTA).

[0282] Nanoparticle tracking analysis (NTA).

[0283] EV samples were diluted with fresh 0.22 μm filtered PBS before checking the particle size and concentration of samples with NanoSight Ns500 instrument. Five videos of more than 30 seconds were taken at a camera level of 15 in light scattering mode. The acquired data were analyzed by the equipped NTA 2.3 software and all samples were analyzed with the same constant settings.

[0284] MACSQuant Flow Cytometry

[0285] After adding different traffic light reporter cells to EV at different time points, or after co-culturing the reporter cells with EV-producing cells for 24 hours, their GFP expression was examined by MACSQuant flow cytometer (Miltenyi biotec, Germany). Briefly, after discarding the supernatant, the cells in the 96-well plate were washed once with PBS, and then the cells were centrifuged for 5 minutes at 37°C. The cells were then resuspended in cell culture medium supplemented with 10% FBS. After adding DAPI to check cell viability, the cells were sampled by MACSQuant, and the settings were the same for all measurements of a specific reporter cell line. Finally, the data were analyzed by flowjo to calculate the percentage of GFP-positive cells.

[0286] EV uptake in reporter cells

[0287] Reporter cells were seeded into 96-well plates one day before the addition of engineered EVs. After 48 h, the percentage of GFP-positive cells was determined by MACSQuant.

[0288] Co-culture of EV-producing cells with reporter cells

[0289] HEK-293T cells were seeded into 6-well plates at 0.5 million cells / well. The next day, when the cells reached 60-70% confluence, lipofectamine2000 (Invitrogen, USA) was used to transfect the corresponding constructs into the wells according to the protocol provided by the manufacturer. 6h after transfection, the culture medium was replaced with fresh culture medium (DMEM+10% FBS+1% anti-anti) to reduce the toxicity of the lipofectamine2000 used. 24h after plasmid transfection, the cells were trypsinized and counted and mixed with the corresponding reporter cells in a ratio of 1:1 or 1:5 (ratio = EV-producing cells: reporter cells) in a 96-well plate. After 24 hours of co-culture, the cells were trypsinized and measured by MACSQuant to check the percentage of GFP-positive cells.

[0290] Different classes of possible EV scaffold / carrier proteins were tested, including cytoplasmic, late endosome, monospanins, and tetraspanins. Of the 280 proteins screened, tetraspanins showed the most promise. Thirty-three tetraspanins were tested (see Fig. 9 ), and then the first 13 of them were taken forward for further analysis after the initial screening (see Fig. 9 and 10).

[0291] from Fig. 8A -F As can be seen, by far the most effective tetraspanin EV protein for co-delivery of VSVG and Cre is TSPAN2 (results for the classic EV protein CD63 are highlighted for comparison). This is true in every cell line and in both systems tested. This further confirms the evidence from Examples 1-6, which identified TSPAN2 as a new and highly effective EV scaffold protein that is able to transport cargo into EVs from different EV cell sources for cargo transport.

[0292] Fig. 9 The same data are shown, but depicted as a heat map of % GFP-positive cells. Fig. 9 Also shown are the cutoff points for the top 13 tetraspanin candidates selected for further validation. TSPAN2 performed best in all cell lines tested.

[0293] Example 8 - Further validation of the top 13 tetraspanin candidates using other difficult-to-transfect cell lines

[0294] Then further testing in different cell lines that are more difficult to transfect Fig. 9 The top 13 candidate proteins identified in: MSC, THP1, Raw264.7 and K562 cells. Figure 7 The EV addition system shown in C was used for this experiment.

[0295] As can be seen in Figure 10, TSPAN2 is also the best candidate among the top 13 scaffold protein candidates tested in difficult-to-transfect cell lines. This demonstrates the broad utility of TSPAN2 as a scaffold protein.

[0296] Example 9 - Comparison of TSPAN2 and classical EV scaffold proteins (CD9 / 63 / 81)

[0297] The inventors then wanted to compare the efficacy of the newly identified EV protein candidate TSPAN2 with the classic EV proteins. The above co-culture method was used to test the ability of TSPAN2 to allow multiple constructs to be co-expressed on the same vesicle relative to classic EV proteins. The experiment was performed using three cell lines, Hela, T47D, and B16F10, and the ability of TSPAN2 to act as a scaffold protein was compared with three classic EV proteins: CD9, CD63, and CD81. In this experiment, the cargo protein was an albumin binding domain (ABD) plus NanoLuc. As a reporter (Figure 14 shows the ABD construct design).

[0298] Fig.11 It was shown that the albumin binding domain (ABD) in the second loop of traditional tetraspanins (CD9, CD63 and CD81) counteracts the VSV-G delivery effect, while the ABD in the second loop of TSPAN2 enhances the VSV-G delivery effect. This surprisingly suggests that TSPAN2 is superior to its symbiotic homologs CD9 and other classical EV proteins CD63 and CD81 when it reaches a level that allows genetic manipulation to carry POIs and co-localize multiple POIs on the same EV. The inventors have found that, unlike the "classical" EV-associated tetraspanins (CD9, CD81 and CD63), TSPAN2 peptides can be engineered while maintaining co-localization with another construct (in this case VSV-G-intein-Cre). This enables TSPAN2 to function as a more flexible tool for EV engineering, allowing for simpler development of "platform technologies" compared to previous scaffold proteins.

[0299] Example 10 - Demonstration that Lack of Colocalization Leads to Reduced POI Delivery

[0300] The inventors subsequently confirmed by FACS that in Examples 9 and Fig.11 The reduced POI delivery seen in is due to the lack of co-localization of the two essential constructs. Details of the FACS protocol are as above.

[0301] Fig.12Shown are the results of FACS analysis comparing two fusion protein constructs (i) VSVG-mNG+TSPAN2-mCardinal and (ii) VSVG-mNG+CD63-mCardinal. Fig.12 It is shown that although the mCardinal values ​​are fairly similar between TSPAN2 and CD63, it can be seen that when CD63 is overexpressed, the mNG values ​​(corresponding to the level of co-localized VSVG) are significantly lower than when TSPAN2 is the scaffold used. The inventors believe that the variation in results between EVs expressing CD63 and TSPAN2 is due to these proteins being localized to different populations of EVs, which allows TSPAN2 to co-localize with the second construct more easily than CD63. This suggests that it is difficult to introduce multiple constructs into exosomes when engineering POIs using the CD63 scaffold. Fig.11 As shown, engineering CD63 resulted in lower expression of the other constructs, and thus TSPAN2 is much better than CD63 at loading cargo into EVs. It should also be noted that this data also indicates that TSPAN2 can deliver both luminal and surface displayed POIs simultaneously.

[0302] Example 11 - In vivo data showing functional delivery of Cre protein

[0303] The inventors then went on to test the ability of TSPAN2 to deliver a functional POI in vivo.To test the in vivo delivery, an intratumoral mouse model was used.

[0304] C57BL / 6 mice were ordered at approximately 5 weeks of age and 20 g body weight. Animals were acclimated to their new environment for at least one week before the experiment. B16F10-TL cells were harvested and resuspended in PBS, and then subcutaneously inoculated at a number of 0.5 million cells per mouse. Ten days after inoculation, when palpable tumors were formed, engineered EVs were injected directly into the tumors. The injection volume was 50 μL / mouse, 7.5×10 10 EV. Four days after intratumoral injection of EV, mice were sacrificed and tumors were harvested. Half of the tumor tissue was fixed with PFA and sent for slides, and the other half was immersed in lysis buffer (0.1% TritonX-100) for DNA isolation. When the slides with tumor tissue were ready, IHC staining for GFP expression was performed and the tissue in lysis buffer was homogenized using a tissue lyser. 50 μL tissue lysate was extracted for DNA isolation using RSC tissue DNA kit (Promega, USA).

[0305] Fig.13 A shows a schematic diagram of intratumoral injection and primer design of the melanoma model. Fig.13 B shows PCR results demonstrating efficient Cre recombination in vivo. Fig.13 The band intensity in B shows that TSN2-engineered EVs exhibit better in vivo reconstitution than CD63-engineered EVs. Fig.13 C shows a microscopic photograph, again demonstrating that TSPAN2-engineered EVs functionally deliver Cre protein in a melanoma model. Fig.13 TSPAN2 was demonstrated to be more efficient than CD63 in delivering functional cargo in vivo.

[0306] Example 12 - Using TSN2 to display ABD as an alternative POI

[0307] Next, the inventors tested the ability of TSPAN2 to express an albumin binding domain (ABD) on EVs. The ABD is used to attract albumin to EVs, thereby extending the half-life of EVs in circulation. Figure 14A shows a schematic diagram of the TSPAN2 construct used in this experiment. The ABD is displayed on the surface of EVs.

[0308] Binding of ABD-engineered EVs was evaluated with commercially available FITC-labeled human serum albumin (HSA-FITC). Isolated EVs were incubated with HSA-FITC and then size excluded by QEV. 48 fractions of 300 μl were collected from each sample. Therefore, fractions 1 to 9 were considered to be the fractions containing only EVs (EV fractions), while fractions 10 to 48 contained mainly soluble proteins. The results are shown in Fig. 14B , it is shown that the ABD expressed in the second loop of TSPAN2 obtains the best HSA binding.

[0309] Figure 14 shows in vivo data demonstrating that EVs modified with albumin by displaying ABD on the surface of EVs fused to TSPAN2 have extended in vivo circulation time. It has previously been observed that using CD63 as a scaffold protein, half-life extension of approximately 10-fold has been observed. Here using TSPAN2, the ability to extend half-life beyond CD63 was increased to 12-fold.

[0310] This demonstrates that TSPAN2 is an effective scaffold as a binding protein for the POI and furthermore demonstrates once again that the delivery of the POI, in this case the albumin binding domain, via TSPAN2 is superior to that achieved using conventional EV proteins. This would not have been anticipated without the extensive screening work of the inventors. Prior to the development of the above complex screening method, TSPAN2 had never been considered a viable scaffold protein because its native form is not highly enriched on EVs.

[0311] Example 13 - TSPAN2 delivery of Ago2 RNA complexes

[0312] The inventors next tested the ability of TSPAN2 to deliver ribonucleoprotein complexes as POIs. To this end, an EV-mediated silencing model of Gapdh at the mRNA and protein levels was used. By fusing the RNA-binding protein Argonaute 2 (AGO2) with the EV-sorting domain MYR and tetraspanin 2 (TSN2), shRNA (shGapdh) for Gapdh was indirectly loaded into EVs. In addition, VSVG was used as a fusion protein to ensure that the cargo was delivered to the cytosol of the recipient cell. The constructs were transiently co-transfected into parental HEK293T cells to produce engineered EVs, which were then isolated and purified. After addition to the recipient cells, EVs achieved dose-dependent target knockdown at the mRNA and protein levels 48 hours later. When shGapdh was enriched in EVs by AGO2 fused to the EV-sorting domain, an enhanced silencing effect was observed.

[0313] Cell culture

[0314] Unless otherwise stated, cells were cultured in complete growth medium consisting of Dulbecco's modified Eagle's medium with high glucose (DMEM, Gibco Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 1% antibiotic-antimycotic (AA, Gibco, ThermoFisher Scientific) and maintained at 37°C in a 5% CO2 atmosphere.

[0315] For EV production, 1×10 7 Human embryonic kidney cells (HEK293T) were seeded in 15 cm dishes and 24 h later, transfected with the plasmid of interest complexed with branched polyethyleneimine (Sigma-408727; 30 μg DNA: 45 μg PEI) in OptiMEM (Gibco, Thermo Fisher Scientific). 4 h after transfection, the medium was changed to OptiMEM reduced serum medium supplemented with 1% AA. Cells were cultured for 48 h before EV isolation.

[0316] For EV uptake studies, mouse neuroblasts (Neuro-2a) were seeded in 96-well plates (1 × 10 4 cells / well) or 24-well plates (5×10 4 cells / well) for RNA and protein extraction. After 24 h, EVs (5×10 7 -5×10 9 EV / well, 24-well plate; 1×10 7 -1×109 Ev / well, 96-well plate), the cells were incubated for another 48 h, and then RNA or protein was extracted.

[0317] Extracellular vesicle isolation

[0318] Conditioned medium (CM) from previously transfected HEK293T cells was collected and spun at 700 × g for 5 min, followed by 2000 × g for 10 min to remove cell debris and larger particles. The supernatant was passed through a 0.22 μm vacuum filter and concentrated to a final volume of 500 μL by ultrafiltration (UF) with an Amicon Ultra-15 100 kDa molecular weight cutoff spin filter (Millipore) and then loaded onto a qEVoriginal / 70 nm Legacy size exclusion column (Izon Science). The vesicle fraction was collected and further concentrated to a final volume of 100 μl with an Amicon Ultra-2 10 kDa molecular weight cutoff spin filter (Millipore) according to the manufacturer's instructions and stored at -80 ° C for further analysis.

[0319] Nanoparticle tracking analysis

[0320] The particle size and concentration of the samples were determined by nanoparticle tracking analysis (NTA) using a NanoSight NS500 equipped with NTA2.3 analysis software and a 488 nm laser. Briefly, samples were diluted in 0.22 μm filtered PBS and for each sample, five 30 s videos were recorded and analyzed.

[0321] RNA isolation and RT-qPCR

[0322] Neuro-2a cells previously incubated with EVs in 96-well plates were detached with trypsin-EDTA (0.25%, Gibco, Thermo Fisher Scientific), transferred to a v-bottom 96-well plate and spun at 900 × g for 5 min. Optionally, cell pellets were stored at -80 °C until further processing. RNA was extracted with the Maxwell RSC simplyRNA Cell Kit (Promega) according to the manufacturer's instructions on the Maxwell RSC Instrument (Promega). The resulting RNA concentration was determined on a Qubit3 fluorometer (Invitrogen, Thermo Fisher Scientific) using the Qubit RNA High Sensitivity Assay (Invitrogen, Thermo Fisher Scientific). 100 ng of RNA was then reverse transcribed according to the instructions of the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific). The following primers and probes (Gapdh: forward GCC TTC CGTGTT CCT ACC, reverse CCT CAG TGT AGC CCAAGA TG, probe / 5HEX / CGC CTG GAG / ZEN / AAA CCTGCC AAG TA / 3IABkFQ / ; Hprt: forward GCC CTC TGT GTG CTC AAG, reverse CCC CGT TGA CTG ATCATT ACA, probe / 56-FAM / AGC AGG TCA / ZEN / GCA AAG AAC TTATAG CCC / 3IABkFQ / ) were used on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad), using the equivalent of 5 ng RNA per qPCR reaction with TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific).

[0323] Protein extraction and western blotting

[0324] Neuro-2a cells previously incubated with EVs in 24-well plates were detached with trypsin-EDTA (0.25%, Gibco, Thermo Fisher Scientific), transferred to tubes and spun at 900 × g for 5 min. Optionally, cell pellets were stored at -80 °C until further processing or lysed in RIPA buffer. Cell fragments were pelleted at 13,000 × g for 12 min, and the supernatant was transferred to a new tube. NuPAGE (Invitrogen, Thermo Fisher Scientific) was used according to the technical guidelines. 4–12% Bis-Tris protein gel (Invitrogen, Thermo Fisher Scientific) was used The subsequent steps were performed in MESSDS running buffer (Invitrogen, Thermo Fisher Scientific) at 120 V for 2 hours. The proteins on the gel were transferred to a nitrocellulose membrane (iBlot2 Transfer Stacks, Invitrogen, Thermo Fisher Scientific) using an iBlot 2 gel transfer apparatus (Invitrogen, Thermo Fisher Scientific) for 7 min. The membrane was blocked with Odyssey blocking buffer (LI-COR) for 60 min at room temperature with gentle shaking. After blocking, the membrane was incubated with primary antibody solution (1:10,000 dilution of anti-β-actin [A5441, Sigma] and anti-Gapdh [PA1-16777, Invitrogen, Thermo Fisher Scientific]]) at 4 ° C overnight or at RT for 1 h. The membrane was washed 5 times with PBS supplemented with 0.1% Tween 20 (PBS-T) for 5 min and incubated with the corresponding secondary antibodies (LI-COR) for 1 h at room temperature (1:15,000 anti-mouse 680LT to detect β-actin; 1:15,000 dilution of anti-rabbit 800 CW to detect Gapdh). The membrane was washed 5 times with PBS-T within 25 min, washed once with PBS, and developed on an Odyssey infrared imaging system (LI-COR).

[0325] Fig.15 The results of this experiment are presented, showing that TSPAN2 can be used to deliver RNA cargo, again demonstrating the broad utility of TSPAN2 as a scaffold protein for the delivery of a variety of different types of cargo POIs.

[0326] Example 14 - TSPAN2 delivery of mRNA

[0327] Based on the highly promising results detailed above, the inventors also wished to test whether TSPAN2 could be used to load mRNA via nucleic acid binding proteins.

[0328] PUFeng was selected as the nucleic acid binding protein, and erythropoietin (EPO) containing PUFeng binding site mRNA was designed as the cargo nucleic acid. The following fusion constructs were compared to test their ability to load EPO mRNA into EVs:

[0329] TSPAN2-PUFeng

[0330] TSPAN2-Intein-PUFeng

[0331] CD63-PUFeng

[0332] CD63-Intein-PUFeng

[0333] TSPAN2-MS2 (no binding control)

[0334] TSPAN2-intein-MS2 (no binding control)

[0335] CD63-MS2 (no binding control)

[0336] CD63-intein-MS2 (no binding control)

[0337] EV-producing cells were cultured and transfected in a total volume of 2 ml or 15 ml according to the manufacturer's protocol, and the EV material was harvested after 72 h. To purify the harvested material, the cell culture was centrifuged and then passed through a 0.2 μm conical filter, and the culture medium was added to a Proteus X-spinner spin filter and centrifuged at 2000 × g for 30 min. Using Maxwell RSC, 'from plasma or serum RNA was extracted from EVs using the RSC miRNA' kit according to the manufacturer's instructions and quantified by nanodrop. RT-PCR was then performed using a high capacity cDNA reverse transcription kit with oligo dT primers.

[0338] Fig.16 TSPAN2 was shown to have more than 10-fold superior loading capacity compared to the classic EV scaffold protein CD63. When TSPAN2 was used as the scaffold protein, 44,572 copies of mRNA were loaded, while only 3,667 copies were loaded when CD63 was used.

[0339] In detail, Fig.16It was shown that a clear enrichment of EPO mRNA loading into EVs was observed in PUFeng samples compared to MS2 (no binding) controls, with mRNA copy numbers in producer cells remaining similar (data not shown), indicating that loading is an active process. Again, this was a very surprising finding, as TSPAN2 was not previously known to be highly expressed on EVs, and thus had never previously been considered a promising scaffold protein, especially not in the context of nucleic acid cargo.

[0340] Example 15 - TSPAN2 and TSPAN3 delivering NanoLuc and msOx40L mRNA

[0341] The inventors went on to investigate whether TSPAN2 and TSPAN3 could be used to load other mRNAs via PUF nucleic acid binding proteins. In these experiments, a construct containing MS2 without the RNA binding domain was used as a control.

[0342] NanoLuc mRNA and codon-optimized murine Ox40L mRNA designed to contain the PUFeng binding site mRNA were used as cargo nucleic acids. The following fusion constructs were compared to test their ability to load NanoLuc or msOx40L mRNA into EVs:

[0343] TSPAN2-PUFeng

[0344] TSPAN2-MS2 (no binding control)

[0345] TSPAN3-PUFeng

[0346] TSPAN3-MS2 (no binding control)

[0347] CD63-PUFeng

[0348] CD63-MS2 (no binding control)

[0349] HEK293T cells were cultured and transfected with the construct by standard methods, and conditioned medium was collected from the cells 48 hours after transfection. The Ev material was purified by centrifugation at 700 × g for 5 min to remove residual cells, and then centrifuged at 2000 × g for 10 minutes to remove cell debris. The centrifuged medium was then filtered through a polyethersulfone (PES) membrane filter (Techno Plastic Products) with a pore size of 0.22 μm. The filtered conditioned medium was ultracentrifuged at 100,000 × g for 90 min at 4 ° C, and the pellet was resuspended in 200 μl sterile PBS for immediate use.

[0350] High-capacity cDNA was then reverse transcribed using oligo dT primers RT-PCR was performed using the RNAi kit (Thermo Fisher Scientific).

[0351] Fig.17a and 17b It was demonstrated that both TSPAN2 and TSPAN3 have the ability to load more NanoLuc mRNA than the classic EV scaffold protein CD63 ( Fig.17a ) and msOx40L mRNA( Fig.17b ). When TSPAN2 was used as a scaffold protein, 8715 copies of NanoLuc mRNA and 41042 copies of Ox40L mRNA were loaded, when TSPAN3 was used as a scaffold protein, 4473 copies of NanoLuc mRNA and 19231 copies of Ox40L mRNA were loaded, and when CD63 was used as a scaffold protein, 3165 and 7902 copies of NanoLuc mRNA and Ox40L mRNA were loaded, respectively. Interestingly, the data also showed that passive loading of mRNA (which occurs independently of the PUF mRNA binding domain) was also increased when TSPAN2 or TSPAN3 was expressed compared to when CD63 was expressed.

[0352] Example 16 - Improved Engineering with TSN2

[0353] The inventors wanted to investigate how EV expression of engineered TSPAN2 (also known as TSN2) constructs compares to EV expression of constructs containing classical EV scaffold proteins.

[0354] HEK-CTS cells (Thermo Fisher Scientific) were transfected with the following constructs:

[0355] -CD63-Flag-eGFP (Flag at the C-terminus of CD63, eGFP at the C-terminus of Flag)

[0356] -CD63-Z(L2)-Flag-eGFP (Flag at the C-terminus of CD63, eGFP at the C-terminus of Flag; Z domain is shown on the second loop of CD63)

[0357] -TSN2-Flag-eGFP (Flag at the C-terminus of TSN2, eGFP at the C-terminus of Flag)

[0358] -TSN2-Z(L2)-Flag-eGFP (Flag at the C-terminus of TSN2, eGFP at the C-terminus of Flag; Z domain is shown on the second loop of TSN2)

[0359] Three days after transfection, the conditioned medium (CM) was collected and centrifuged at 4000×g for 10 minutes to remove cells and cell debris. The supernatant was then concentrated (up to 10 times) to approximately 200 μL using a spin filter (Proteus X-spinner 2.5 Pack, 300 kDa MWCO, PES membrane, Cat. No. PAL-X-300-96; Generon).

[0360] Filtered CM was analyzed for eGFP content by EV flow cytometry performed on a NanoAnalyser N30E (NanoFCM).

[0361] Fig.18a It is shown that EVs derived from cells transfected with the TSN2 construct are more eGFP positive than EVs derived from cells transfected with the CD63 construct (compare columns 1 and 3), thus the TSN2 construct is more efficiently engineered. Insertion of the domain sequence on the second loop reduced the engineering efficiency of both scaffolds (compare columns 1 and 2 and columns 3 and 4), however TSN2 was less affected by this phenomenon.

[0362] Fig.18b The mean fluorescence intensity (MFI) of EVs derived from cells transfected with the TSN2 construct was shown to be almost doubled compared to EVs derived from cells transfected with the CD63 construct (compare columns 1 and 3), thus the TSN2 construct resulted in more scaffold molecules localized per engineered EV. While insertion of a domain sequence on the second loop of CD63 reduced the MFI (compare columns 1 and 2), this was not the case when the domain sequence was engineered on the second loop of TSN2 (compare columns 3 and 4). Fig.18a The data in a and b show that TSN2 undergoes significant engineering while still maintaining remarkably high levels of expression at the POI on EVs.

[0363] Example 17 - Further Evidence of Improved Engineering with TSN2

[0364] The inventors wanted to investigate whether the results in Example 16 above occurred in different cell lines and with different modifications to the second loop of the scaffold protein.

[0365] EK was transfected with the following fusion constructs VPC.2 cells (Thermo Fisher Scientific):

[0366] - Lamp2b, engineered with eGFP at the C-terminus and VHH to transferrin receptor (TfR) at the N-terminus.

[0367] -TSN2, engineered with eGFP at the terminus and VHH to TfR at the loop.

[0368] -CD63, engineered with eGFP at the terminus and VHH to TfR at the loop.

[0369] According to the manufacturer's instructions, use (PolyPlus Transfection) was used as a transfection agent and plasmid DNA was used to transfect cells. After 72 h, the conditioned medium was harvested by differential centrifugation steps, starting at 300 × g and 700 × g for 5 minutes each to remove cells and larger debris. Then at 4000 × g for 20 minutes to remove smaller debris. The conditioned medium was then ultracentrifuged at 100,000 × g for 2 hours to precipitate EVs. The formed pellet was then resuspended in storage buffer and centrifuged at 4000 × g to remove any debris that may have formed.

[0370] EV flow cytometry was performed on a Nanoanalyzer N30E (NanoFCM).

[0371] Fig.19a It was shown that a larger proportion (more than 10%) of EVs derived from cells transfected with the TSN2 construct were positive for eGFP compared to EVs from cells transfected with the Lamp2b construct or the CD63 construct.

[0372] Fig.19b It was shown that the fluorescence intensity of EVs derived from cells transfected with the TSN2 construct increased by about 3-fold compared to EVs derived from cells transfected with the Lamp2b construct. The fluorescence intensity of EVs derived from cells transfected with the TSN2 construct increased by about 4-fold compared to EVs derived from cells transfected with the CD63 construct. This suggests that EVs derived from cells transfected with the TSN2 construct have a much higher ligand density than EVs derived from cells transfected with the Lamp2b construct or the CD63 construct.

[0373] Example 18 - Long-term stability of cell lines

[0374] To test the long-term stability of TSPAN2EVs (both the stability of expression of the TSPAN2-POI construct alone and the stability of simultaneous expression of TSPAN2-POI when co-expressed with a second construct encoding POI), EV-producing cells were prepared by transduction with a lentiviral vector to express the desired fusion protein. These producer cells and the produced EVs were then longitudinally examined by Western blotting, flow cytometry, bioluminescence, etc. to observe the stability of cargo protein expression.

[0375] Example 19 - Stability of TSN2 constructs in EV producer cells over time.

[0376] A known problem in the field of engineered EV production is that expression of constructs of typical tetraspanin EV scaffold proteins is continually lost from EV producer cells and secreted EVs over time. The inventors continued to investigate the stability of constructs containing TSPAN2 in EV producer cells compared to constructs containing the classic tetraspanin EV scaffold CD63.

[0377] EV production cells (HEK293VPC1.0CTS (Thermo Fisher Scientific) or CEVEC CAP (CEVEC Pharmaceuticals) cells) were transfected with CD63-G4S-eGFP construct or TSN2-G4S-eGFP construct. The G4S linker consists of four glycines and one serine (GlyGlyGlyGlySer), which forms a flexible peptide connecting eGFP and the scaffold protein. This allows the presence of the scaffold to be inferred by the presence of eGFP.

[0378] Cells were sorted for high eGFP expression using FACS before cryopreservation. After recovery from resuscitation, antibiotic selection was reintroduced into the culture. Cells were maintained in continuous culture in the presence and absence of antibiotic selection during the study. Only cells expressing the CD63-G4S-eGFP construct or TSN2-G4S-eGFP survived antibiotic selection. The G4S linker consists of four glycines and one serine (GlyGlyGlyGlySer), which forms a flexible peptide connecting eGFP and the scaffold protein. This allows the presence of the scaffold to be inferred by the presence of eGFP.

[0379] At two time points, RNA from cell pellets was extracted according to the manufacturer's instructions ( RSCsimply RNA extraction kit; Promega). Time point 1 was performed when the cells were at passage 2, and time point 2 was performed when the cells were at passage 13. For HEK293 cells, the two time points were approximately 7 weeks apart, and for CAP cells, the two time points were approximately 6 weeks apart.

[0380] 30 ng RNA was used for cDNA synthesis using a high capacity cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer's instructions, and the random primer was replaced by 15mer oligoDT. The cDNA was evaluated in triplicate using PowerUp Sybergreen PCR. The final cycle threshold (CT) values ​​for GAPDH and eGFP were used to calculate ΔΔCT to determine the absolute change in mRNA expression between eGFP and GAPDH during the study period.

[0381] Figure 20a shows that expression of the CD63 construct is lost from HEK293T cells over time. Surprisingly, despite also being a tetraspanin, Figure 20b demonstrates that HEK293T cells continue to express the TSN2 construct at comparable levels over time.

[0382] Figures 20c and 20d show the results observed with CAP cells. In this case, the expression of the CD63 construct was lost over time (Figure 20c), but the expression of the TSN2 construct increased over time (Figure 20d).

[0383] Thus, in both HEK and CAP cells, a clear improvement in the stability of eGFP mRNA expression was observed when eGFP was expressed in a construct fused to TSN2 rather than CD63. Thus, the use of TSN2 as a scaffold protein overcomes the problem of decreased expression over time observed when CD63 is used as a scaffold.

[0384] Example 20 - Stability of engineered EV production over time by EV producing cells expressing TSN2 constructs.

[0385] In view of the positive results in Example 19, the inventors continued to investigate the expression of eGFP in EVs secreted from cells transfected with the TSPAN2 construct compared to cells transfected with the CD63 construct.

[0386] In these experiments, conditioned media was collected from EV-producing cells (HEK293 VPC1.0 CTS or CEVEC CAP cells, as described previously) at two time points. Time point 1 was performed when the cells were at passage 2, and time point 2 was performed when the cells were at passage 13. For HEK293 cells, the two time points were approximately 7 weeks apart, and for CAP cells, the two time points were approximately 6 weeks apart.

[0387] To isolate EVs, 20 mL of conditioned medium was centrifuged at 300 × g for 5 min, and the resulting supernatant was centrifuged at 4000 × g and concentrated to 1 mL using an Amicon MWCO column with a 100,000 kDa molecular weight limit.

[0388] Pierce Micro BCA assay (Thermo Fisher Scientific) was performed according to the manufacturer's instructions, and 20 μg of protein was evaluated using Western blotting under reducing conditions. Reduced protein separation was performed for 35 minutes in 1x MES electrophoresis buffer at 200V using Bolt 4-12% Bis-Tris Gels (ThermoFisher Scientific). Proteins were transferred to nitrocellulose membranes using iBlot2 membranes and then blocked with Intercept TBS blocking buffer at room temperature for 1 h. eGFP (Chromotek clone 3H9) and Syntenin (Abcam EPR8102) were detected with the blocked membranes. Densitometry analysis was performed for the relevant eGFP bands of 50 kDa by standardizing the corresponding syntenin bands.

[0389] Fig.21a Shown is the loss of eGFP expression over time in EVs secreted by HEK293T cells expressing the CD63 construct. Figure 21b It is shown that the expression of eGFP in EVs secreted by HEK293T cells expressing the TSN2 construct is maintained in the cells under antibiotic selection. Thus, in a cell population in which all cells express the TSN2-G4S-eGFP construct, the amount of engineered EVs produced remains constant over time. Even in the absence of antibiotic selection, in cases where the cell population comprises a mixed population in which only a portion of the cells express the TSN2-G4S-eGFP construct, the relative expression of eGFP in secreted EVs remains above 0.5 at later time points, thus greatly improving the relative expression of eGFP compared to that observed when the CD63-G4S-eGFP construct was expressed.

[0390] Fig.21c and 21d The results observed with CAP cells are shown. In this case, the expression of eGFP in EVs from CAP cells expressing the CD63 construct was also lost over time ( Fig.21c ), but eGFP expression in EVs from CAP cells expressing the TSN2 construct increased over time ( Fig.21d ).

[0391] Thus, in EVs derived from HEK and CAP cells, a clear improvement in the stability of eGFP expression was observed when eGFP was expressed in constructs fused to TSN2 rather than to CD63. Thus, the use of TSN2 as a scaffold protein helps to overcome the problem of decreased production of engineered EVs over time, which was observed when CD63 was used as a scaffold.

[0392] sequence

[0393] SEQ ID NO:1(TSPAN2 protein)

[0394] MGRFRGGLRCIKYLLLGFNLLFWLAGSAVIAFGLWFRFGGAIKELSSEDKSPEYFYVGLYVLVGAGALMMAVGFFGCCGAMRESQCVLGSFTCLLVIFAAEVTTGVFAFIGKGVAIRHVQTMYEEAYNDYLKDRGKGNGTLITFHSTFQCCGKESSEQVQPTCPKELLGHKNCIDEIETIISVKLQLIGIVGIGIAGLTIFGMIFSMVLCCAIRNSRDVI

[0395] SEQ ID NO:2 (TSPAN2 nucleic acid)

[0396] ATGGGCCGGTTTAGGGGCGGTCTGAGATGTATCAAGTACCTCCTCCTTGGTTTCAACCTCCTGTTTTGGTTGGCCGGAAGCGCTGTGATCGCCTTCGGATTGTGGTTTAGATTCGGAGGCGCAATCAAGGAACTGTCATCTGAGGACAAGTCACCAGAGTACTTTTACGTGGGGCTCTATGTACTCGTGGGAGCCGGGGCCCTGATGATGGCCGTGGGTTTCTTCGGGTGTTGCGGAGCAATGAGGGAAAGCCAGTGCGTGCTTGGGTCTTTCTTTACTTGTCTGCTGGTCATCTTTGCCGCCGAGGTGACTACTGGTGTCTTTGCATTCATCGGAAAGGGCGTCGCTATCAGGCACGTGCAGACCATGTATGAAGAAGCCTATAACGACTATCTCAAGGACCGGGGAAAGGGTAACGGCACCCTTATCACATTTCACTCTACTTTTCAGTGCTGTGGCAAAGAATCAAGTGAGCAGGTCCAACCCACTTGTCCCAAAGAACTGCTCGGCCATAAGAACTGCATCGACGAAATCGAGACTATCATCTCCGTGAAGTTGCAGCTCATAGGGATCGTAGGCATCGGTATTGCTGGTTTAACTATCTTCGGTATGATCTTCAGCATGGTACTGTGTTGTGCTATTAGAAATAGCCGAGATGTTATA

[0397] SEQ ID NO:3 (TSPAN3 protein)

[0398] MGQCGITSSKTVLVFLNLIFWGAAGILCYVGAYVFITYDDYDHFFEDVYTLIPAVVIIAVGALLFIIGLIGCCATIRESRCGLATFVIILLLVFVTEVVVVVLGYVYRAKVENEVDRSIQKVYKTYNGTNPDAASRAIDYVQRQLHCCGIHNYSDWENTDWFKETKNQSVPLSCCRETASNCNGSLAHPSDLYAEGCEALVVKKLQEIMMHVIWAALAFAAIQLLGMLCACIVLCRRSRDPAYELLITGGTYA

[0399] SEQ ID NO:4 (TSPAN3 nucleic acid)

[0400] ATGGGGCAGTGTGGGATCACTAGTAGCAAAACCGTGCTGGTCTTTCTGAATCTTATCTTTTGGGGAGCCGCGGGGATCCTGTGCTATGTGGGGGCATACGTGTTCATTACCTACGACGACTATGATCATTTCTTTGAAGATGTGTACACTCTCATACCCGCAGTTGTCATTATTGCAGTTGGTGCGCTGCTGTTCATAATAGGACTCATCGGCTGTTGCGCAACTATTCGAGAGAGCAGGTGTGGCCTGGCTACCTTCGTAATTATCCTGCTTCTGGTATTCGTGACAGAAGTAGTGGTAGTGGTCCTGGGTTACGTTTACAGGGCGAAGGTGGAGAATGAAGTTGACCGGTCTATTCAAAAAGTCTACAAGACCTATAATGGGACGAATCCAGATGCCGCGTCTAGAGCTATTGATTATGTGCAGCGACAGCTGCACTGCTGCGGAATCCACAATTATTCCGATTGGGAGAACACAGATTGGTTCAAAGAAACAAAGAACCAATCCGTGCCACTGTCCTGCTGCAGGGAGACAGCATCCAACTGCAATGGATCACTCGCCCATCCCAGTGACCTCTATGCCGAGGGCTGTGAGGCCTTAGTGGTCAAGAAACTCCAAGAAATTATGATGCACGTGATCTGGGCTGCCTTGGCCTTCGCAGCTATCCAACTGCTGGGGATGTTATGTGCGTGTATTGTTCTGTGCCGCCGCAGCAGGGACCCTGCTTACGAGCTCCTGATTACGGGGGGCACCTATGCA

[0401] SEQ ID NO:5 (TSPAN18 protein)

[0402] MEGDCLSCMKYLMFVFNFFIFLGGACLLAIGIWVMVDPTGFREIVAANPLLLTGAYILLAMGGLLFLLGFLGCCGAVRENKCLLLFFFLFILIIFLAELSAAILAFIFRENLTREFFTKELTKHYQGNNDTDVFSATWNSVMITFGCCGVNGPEDFKFASVFRLLTLDSEEVPEACCRREPQSRDGVLLSREECLLGRSLFLNKQGCYTVILNTFETYVYLAGALAIGVLAIELFAMIFAMCLFRGIQ

[0403] SEQ ID NO:6 (TSPAN18 nucleic acid)

[0404] ATGGAAGGCGATTGTCTCAGTTGCATGAAGTACCTGATGTTCGTTTTCAATTTTTTTATTTTCCTCGGGGGGGCGTGTCTGCTGGCCATTGGGATTTGGGTGATGGTAGACCCTACAGGCTTTAGGGAAATAGTGGCTGCCAACCCTCTGCTCCTTACTGGAGCCTACATTCTTCTGGCCATGGGCGGGCTGCTGTTTCTCTTGGGCTTCTTGGGGTGTTGCGGAGCCGTCAGAGAAAATAAGTGTTTGCTGCTTTTCTTCTTTTTGTTCATACTTATAATATTTCTGGCAGAGCTCAGCGCTGCTATTTTGGCATTTATTTTTAGGGAAAATCTCACCCGCGAATTTTTCACCAAAGAACTGACCAAACATTATCAGGGCAACAATGACACAGATGTATTTTCTGCAACGTGGAACTCTGTTATGATTACTTTTGGCTGCTGCGGGGTAAATGGCCCTGAGGACTTCAAATTCGCTTCCGTCTTCCGCCTGCTGACCTTGGACTCAGAGGAGGTACCCGAAGCCTGTTGTCGCAGGGAACCCCAATCAAGAGACGGCGTGCTGCTGTCTAGGGAAGAGTGCCTGCTGGGGCGTAGTTTATTCCTGAACAAACAGGGTTGTTATACCGTAATCCTGAATACGTTCGAAACATACGTTTATCTTGCTGGAGCCCTGGCTATTGGGGTGCTCGCTATTGAGCTGTTCGCAATGATATTTGCAATGTGTCTTTTCCGGGGCATCCAG

Claims

1. An extracellular vesicle (EV) comprising a fusion protein, characterized in that The fusion protein comprises TSPAN2 fused to a protein of interest (POI).

2. The EV according to claim 1, wherein: The POIs are engineered into loop 1, loop 2, or both loops of TSPAN2.

3. The EV according to claim 1 or 2, characterized in that: The POI is fused to the N-terminal domain (NTD) or C-terminal domain (CTD) or other luminal parts of TPSAN2.

4. An EV according to any preceding claim, characterized in that The fusion protein further comprises: (i) a release domain that can be cleaved to release the POI; (ii) a linker or spacer; (iii) a multimerization domain; and / or (iv) at least one additional POI.

5. An EV according to any preceding claim, characterized in that The EV further comprises a second POI expressed on a separate construct.

6. The EV according to claim 5, characterized in that The second POI exists as a fusion protein with the exosomal polypeptide.

7. An EV according to any preceding claim, characterized in that The POIs are: (i) Therapeutic proteins; (ii) a binding protein of a therapeutic agent (optionally an RNA binding protein, a virus binding protein, an Fc binding protein or a small molecule binding protein); (iii) endosomal escape fraction; (iv) a targeting moiety; (v) an albumin binding domain; or (vi) Purification part.

8. The EV of claim 7, wherein the therapeutic protein is selected from the group consisting of: enzymes, receptors (optionally decoy receptors), membrane proteins, transporters, cytokines, antigens, neoantigens, immune effector molecules, ribonucleoproteins, nucleic acid binding proteins, antibodies, nanobodies, antibody fragments, antibody-drug conjugates, gene editing proteins (optionally CRISPR-Cas), TALENs, and large-range nucleases.

9. An EV according to any preceding claim, characterized in that The EVs can be obtained by a method comprising further exogenously loading the EVs with a therapeutic cargo.

10. The EV according to claim 9, wherein: The further step of exogenously loading the EVs with therapeutic cargo is by electroporation, transfection reagents, co-incubation or contact with a cell penetrating peptide (CPP) or any combination thereof.

11. The EV according to claim 9 or 10, characterized in that: The therapeutic cargo is a protein, a nucleic acid, a virus, a viral genome, an antigen or a small molecule.

12. The EV of claim 11, wherein the nucleic acid cargo is: an RNA molecule, a DNA molecule or a mixture, optionally mRNA, an antisense or splice-switching oligonucleotide, gRNA, siRNA, shRNA, miRNA, dbDNA, plasmid DNA (pDNA), a supercoiled or non-supercoiled plasmid or a minicircle.

13. The population of EVs according to claim 5, characterized in that The TSPAN2-POI fusion protein and the second POI construct were expressed on the same EV.

14. A polypeptide construct comprising TSPAN2 fused to a POI.

15. A polynucleotide construct encoding the polypeptide construct of claim 14.

16. A cell comprising the polypeptide construct of claim 14 or the polynucleotide construct of claim 15.

17. The cell of claim 16, further comprising a second polypeptide construct or polynucleotide construct capable of expressing the POI.

18. A pharmaceutical composition comprising the EV according to any one of claims 1 to 12, the population of EVs according to claim 13, or the cells according to claim 16, and a pharmaceutically acceptable excipient or carrier.

19. A method for producing an EV according to any one of claims 1 to 12, the method comprising: (i) introducing a polynucleotide construct encoding a TSPAN2-POI fusion construct into an EV producer cell; and (ii) expressing the construct in EV producer cells, thereby producing EVs containing the TSPAN2-POI fusion protein.

20. A method of producing the EV of claim 5, the method comprising: (i) introducing a polynucleotide construct encoding a TSPAN2-POI fusion construct into an EV producer cell; (ii) introducing a second polynucleotide construct encoding a second POI into the same EV producer cell (optionally, wherein the second POI is present as a fusion protein with an EV protein); and (iii) expressing both constructs in EV producer cells, thereby generating EVs containing the TSPAN2-POI fusion protein and a second POI.

21. The EV according to any one of claims 1-12 for use in medicine.

22. A method of treatment comprising administering an effective amount of the EV of claims 1-12 or the pharmaceutical composition of claim 18 to a patient in need thereof.

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