Novel scaffold proteins for modified extracellular vesicles

Abstract

The present invention relates to genetically modified extracellular vesicles comprising an improved scaffold protein and a protein of interest. Also disclosed are related polypeptide compositions, polynucleotide compositions, cells, pharmaceutical compositions, and methods.

Description

Technical Field

[0001] [Reference to Electronic Sequence Listing] The content of the electronic sequence listing (EVOX_028_001WO_SeqList_ST26.xml; size: 8927 bytes; creation date: April 28, 2023) is hereby incorporated by reference in its entirety into this specification.

[0002] [Technical Field] The present invention relates to newly identified extracellular vesicle (EV) scaffold proteins, which are advantageously used for genetically modified extracellular vesicles. The present invention also relates to the use of said genetically modified EVs in therapy, said EVs, said populations of EVs, and methods for the production and purification of polypeptides and polynucleotide constructs encoding said new scaffold proteins, and cells comprising said polypeptide or polynucleotide constructs.

Background Art

[0003] EVs (such as exosomes) are typically nanometer-sized vesicles produced by most cell types and function as a natural transport system in the body that carries proteins, nucleic acids, peptides, lipids, and various other molecules between cells. EVs have many potential therapeutic applications and are promising natural delivery vehicles for proteins, nucleic acids, and small molecule therapeutics. Modifying EVs to contain a protein of interest (POI) is important for their utility as delivery vehicles for therapeutic cargos; however, it has proven difficult and unpredictable to identify suitable EV proteins that act as fusion partners for transporting therapeutic cargos into EVs.

[0004] Loading of therapeutic molecules of interest can be achieved by multiple means. In the simplest scenario, small EVs from producer cells with inherent therapeutic potential, such as immunomodulatory mesenchymal stem cells and chimeric antigen receptor T cells, have therapeutic molecules passively loaded and can thus be directly utilized for disease management. Alternatively, exogenous loading of therapeutic molecules using physical and chemical methods such as incubation, sonication, and electroporation has been widely applied. However, this strategy is limited to small RNAs (e.g., siRNA, miRNA, shRNA) and low molecular weight chemicals and has significant risks due to technical artifacts such as RNA precipitation. On the other hand, POIs are usually loaded into small EVs through genetic modification. In a typical workflow, producer cells are modified to overexpress the desired protein fused to the EV sorting / carrier domain, thereby promoting the endogenous sorting of the POI into the EVs during its biosynthesis. Additionally, potential RNA therapeutics can be indirectly loaded via the same strategy by using compatible RNA-binding proteins.

[0005] Depending on the topology and intracellular location of the EV protein (so-called "scaffold domain" or "scaffold protein") used to transport the POI into the modified EVs, and the termini used for fusion, the cargo molecule can be presented on the EV surface or loaded into the vesicles. In contrast to surface presentation, intracellular loading protects the cargo molecule from premature release and rapid degradation. EV scaffold domains reported so far for intracellular loading are classified into three major categories: cytoplasmic proteins (SDCB-1, ARRDC1, and BASP1), single-pass transmembrane proteins (LAMP2B and PTGFRN), and tetraspanin proteins (CD9, CD63, and CD81).

[0006] Conventionally, "common" or "classical" EV proteins that are originally highly expressed in EVs have been considered the most suitable EV proteins for use as a "scaffold" for transporting POIs into modified EVs. The generation of EV protein-POI fusion proteins has been shown to be an effective means of loading the selected POI into EVs. As described above, classical EV proteins such as CD63, CD9, and CD81, single-pass transmembrane EV proteins such as Lamp2, and membrane-associated EV proteins such as BASP have been previously demonstrated to be used as fusion partners for POIs.

[0007] However, the EV sorting efficiency of the above domains depends on the ability of the producing cells to constantly express a given scaffold domain and the specific type and size of the cargo molecules carried by the domain, which has previously caused highly variable results.

[0008] For example, the expression of the transgene CD63-POI is known to significantly decrease over time in producing cells, which impairs the yield of EV products. In other words, when using CD63 as an EV scaffold protein, even after generating a stable cell line, the stability of the cell line is very prone to variation and has been shown to decrease over time. As a result, not only does the number of CD63-POI fusion constructs on individual EVs decrease, but also the number of EVs containing the transgene fusion protein construct decreases.

[0009] In addition, when CD63 is modified to be fused to contain multiple POIs (e.g., different POIs are fused to the N-terminus, C-terminus, loop 1, and / or loop 2), the number of EVs expressing the CD63 fusion construct and the number of CD63 fusion constructs expressed per EV decrease.

[0010] As a result, there remains a need to improve the means of introducing POIs into EVs.

Summary of the Invention

Problems to be Solved by the Invention

[0011] In addition to the aforementioned known limitations, the inventors have also revealed that it is problematic to use these classical EV proteins when additional proteins or fusion protein constructs need to be added to therapeutic EVs. For example, it is impossible to create a dual-stable cell line that always expresses multiple POIs that would be expressed on the surface or inside the same EV population. The inventors have found that when a dual-stable cell line is produced using "classical EV proteins" such as CD63, CD9, or CD81, the expression of the second construct decreases very rapidly (for example, when CD63-POI is co-expressed with another POI such as VSVG, the expression of VSVG is rapidly suppressed as a result); as a result, the population of EVs produced will rapidly contain only the protein construct containing the "classical EV protein". When it is necessary to include multiple POIs in therapeutic EVs, it becomes impossible to create a consistent EV-producing cell line, which is of course a problem. The cause of this is unknown.

[0012] Therefore, while classical EV proteins such as CD63, CD81, and CD9 have been found to be highly expressed on native EVs / exosomes when modified to contain POIs, they have also been found to prevent the introduction of subsequent (different) proteins into the same EV, and thus prevent the co-localization of multiple protein constructs on the same EV. This greatly restricts the combination of proteins that can be introduced into EVs and hinders the multiplexing of functions. This also limits the development of therapeutic EVs as a platform technology and significantly reduces the effectiveness of modified EVs.

[0013] In addition, while classical tetraspanin EV proteins such as CD63 can theoretically be modified to contain multiple POIs (i.e., on the N-terminus, C-terminus, loop 1, and / or loop 2), when such classical tetraspanin EV proteins are modified to be fused to contain multiple POIs (e.g., different POIs are fused to the N-terminus, C-terminus, loop 1, and / or loop 2), it has been found that the number of EVs expressing these fusion constructs, and the number of these fusion constructs expressed per EV, decreases.

[0014] As a result, there remains a need for new means of introducing multiple POIs into EVs.

[0015] To address these limitations and meet the future challenges of genetic engineering, there is a need to identify new EV sorting domains. Thus, the present invention has arisen from studies designed to discover novel EV sorting domains and investigate the differences between multiple producer cells and cargo molecules.

[0016] An object of the present invention relates to overcoming the above problems by finding a novel EV carrier / scaffold protein that can be used as a fusion partner for efficiently and stably transporting a POI into an EV over a long period of time. The inventors sought to identify a novel EV carrier / scaffold protein that acts as a "universal EV carrier / scaffold protein", i.e., is effective across a variety of cell types and can carry a wide range of cargo.

[0017] Second, an object of the present invention is to identify an EV scaffold / carrier protein that maintains the expression of EV-protein-POI fusion constructs and does not prevent the co-localization of a second or subsequent POI on the same EV. The inventors sought to identify an alternative scaffold protein that can be modified to contain a POI while at the same time allowing a second or additional protein to be incorporated into the interior / surface of the same EV, i.e., allowing co-localization.

[0018] In addition, the inventors sought to identify an EV scaffold / carrier protein that can be modified to contain multiple POIs and that continues to be highly expressed in EVs even when so modified. Furthermore, the inventors have been constantly seeking to identify a scaffold protein that can increase the loading of RNA-containing cargo into EVs.

[0019] For these purposes, the inventors performed a large-scale screening of EV proteins and identified TSPAN2 as a novel and highly flexible scaffold protein, which, unlike classical EV proteins, is highly flexible and thus enables the development of a versatile platform technology. TSPAN2 exhibits a marked improvement in function as an EV scaffold protein compared to classical EV proteins (up to 10-fold improvement), and by enabling the co-localization of other protein components within the same EV, it significantly increases the modularity of this therapeutic exosome platform technology.

[0020] In addition, both TSPAN2 and TSPAN3 can increase mRNA loading compared to CD63, a classical EV tetraspanin. Furthermore, the expression of TSPAN2 fused to a POI increases the proportion of generated EVs that express the fusion construct, and also the number of these fusion constructs expressed per EV, compared to the expression of the classical EV tetraspanin CD63 fused to a POI. This also holds true when TSPAN2 is fused to multiple POIs.

Means for Solving the Problems

[0021] Accordingly, in a first aspect, the present invention provides an EV comprising a fusion protein, said fusion protein comprising TSPAN2 fused to a POI.

[0022] In second and third aspects, the present invention provides a polypeptide composition comprising TSPAN2 fused to a POI, and a polynucleotide composition encoding such a polypeptide composition.

[0023] In a fourth aspect, the present invention provides a cell comprising the polypeptide composition of the second aspect or the polynucleotide composition of the third aspect. Optionally, the cell may further comprise a second polypeptide composition or polynucleotide composition capable of expressing a POI.

[0024] 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.

[0025] In a sixth aspect, the present invention provides a method for producing the EV of the first aspect. The method for producing the EV comprises: (i) introducing a polynucleotide composition encoding a TSPAN2-POI fusion construct into an EV-producing cell; and (ii) expressing the construct in the EV-producing cell, thereby generating an EV containing the TSPAN2-POI fusion protein. Optionally, the method may further comprise an intermediate step of introducing a second polynucleotide composition encoding a second POI into the same EV-producing cell (wherein the second POI may be present in the form of a fusion protein with a classical EV protein), and then expressing both constructs in the EV-producing cell, thereby generating an EV containing the TSPAN2-POI fusion protein and the second POI.

[0026] In a seventh aspect, the present invention provides the EV of the first aspect for use in therapy. In an eighth aspect, the present invention also provides a method of treatment comprising administering an effective amount of the EV according to the first aspect of the invention, or the pharmaceutical composition of the fifth aspect, to a patient in need thereof.

[0027] As is apparent from this example, the discovery of TSPAN2 as a novel scaffold protein would not have been possible without the large-scale screening method described herein and the great inventive efforts of the inventors.

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18-19

Figure 20

Figure 21

BEST MODE FOR CARRYING OUT THE INVENTION

[0029] [BRIEF DESCRIPTION OF THE SEQUENCE LISTING] SEQ ID NO: 1 TSPAN2 protein SEQ ID NO: 2 TSPAN2 nucleic acid SEQ ID NO: 3 TSPAN3 protein SEQ ID NO: 4 TSPAN3 nucleic acid SEQ ID NO:5 TSPAN18 protein SEQ ID NO:6 TSPAN18 nucleic acid

[0030] [Detailed Description of the Invention] The present invention relates to EVs containing a fusion protein, said fusion protein comprising TSPAN2 fused to a POI. The present invention also relates to cells capable of producing these EVs, methods of making and purifying these EVs, and their use in therapy. The EVs of the present invention have a number of distinct advantages by using TSPAN2 in the fusion protein.

[0031] Primarily, the use of TSPAN2 improves the stability of the cell line producing the modified EVs and further enables high-level expression of the TSPAN2-POI construct on the surface or inside the EVs. Furthermore, the use of TSPAN2 enables co-localization of other protein constructs within the same EVs and solves the problem of generating cell lines that stably express multiple fusion constructs, thereby significantly increasing the modularity of this therapeutic exosome platform technology.

[0032] The inventors have shown that TSPAN2 is a superior scaffold protein compared to classical scaffold proteins. This is because it consistently shows high expression across a wide range of cell sources, consistently expresses a wide variety of POIs, shows up to 10-fold superior expression, and EVs expressing TSPAN2 as a scaffold protein also show superior biological availability compared to those expressing classical EV proteins. TSPAN2 was surprisingly found to be a very tractable scaffold protein that allows for large-scale modifications without any of the expression or co-localization problems observed with classical EV protein scaffolds. TSPAN2 consistently showed the highest performance in very large-scale scaffold screening. Importantly, out of 244 candidates, only 36 were found to be effective, indicating that EV sorting activity is highly heterogeneous even among closely related proteins. As is clear from the examples provided herein, the inventors used a novel functional screening approach and discovered that there is no correlation between the ranking of encapsulation and the total secreted reporter protein, and that the results obtained here are very difficult to predict.

[0033] In a first aspect, the present invention provides an EV comprising a fusion protein, said fusion protein comprising TSPAN2 fused to a POI.

[0034] In one embodiment, the POI is engineered into loop 1, loop 2, or both loops of TSPAN2. Advantageously, this allows the POI to be presented on the surface of the EV.

[0035] 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 lumenally loaded within the EV, and thus protected from degradation and / or the immunogenicity associated with the POI can be reduced.

[0036] In a further embodiment, TSPAN2 is fused to at least two POIs. In a preferred embodiment, one POI is incorporated within loop 1, loop 2, or both loops of TSPAN2, and a different POI is fused to the N-terminal domain (NTD) and / or C-terminal domain (CTD). In a preferred embodiment, TSPAN2 is fused to one POI incorporated within loop 2 and is fused to a second POI incorporated within the C-terminal domain (CTD).

[0037] In some embodiments, the fusion protein further comprises: (i) a release domain that is cleavable to release the POI; (ii) a linker or spacer; (iii) a multimerization domain; or (iv) at least one additional POI. TSPAN2 has been found by the inventors to be highly adaptable to large-scale modifications and, for example, any of the additional domains listed above can be added without loss of expression of the TSPAN2 scaffold. This clearly demonstrates that TSPAN2 is a very effective and reliable scaffold protein for the development of platform EV technology, where the ability to reliably add and exchange various protein domains within the scaffold protein is highly advantageous for the flexibility and modularity of the technology.

[0038] In some embodiments, the EV of the first aspect further comprises a second POI expressed on another construct. Optionally, the second POI may be present as a fusion protein with TSPAN2 or any classical exosomal polypeptide. Very advantageously, TSPAN2 has been found to not only tolerate large-scale modifications but also consistently enable stable co-localization with other protein constructs. This ability to express another construct well and thus enable co-localization of two constructs on the same EV population was not possible with classical EV proteins, making TSPAN2 highly versatile as a scaffold protein for EV platform modification. Also, the ability to consistently incorporate multiple constructs into EVs enables functional multiplexing, which is also understood to be another major advantage of the EVs of the present invention compared to EVs that utilize classical EV proteins as scaffold proteins. The inventors unexpectedly found that while TSPAN2 can be used as a very effective EV protein scaffold when modifying a POI, a second construct can be simultaneously localized on the same EV. This discovery is very unexpected because TSPAN2 is a paralog of CD9, and CD9 has been shown to prevent co-localization with a second construct when modified to include a POI, similar to other classical EV proteins such as CD63 and CD81.

[0039] In certain embodiments of the present invention, the POI can be the following: (i) a therapeutic protein, (ii) a binding protein for a therapeutic agent (e.g., an RNA-binding protein, a viral-binding protein, an Fc-binding protein, or a small molecule-binding protein), (iii) an endosomal escape site, (iv) a target site, (v) an albumin-binding domain, or (vi) a purification site. More specifically, the therapeutic protein can be selected from the following: 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 containing a Cas protein, a TALEN, a meganuclease. As can be seen from the examples, it has been very advantageously found that TSPAN2 is not only useful for loading a very wide variety of POIs, but also surprisingly up to 10-fold more effective than classical EV scaffold proteins. TSPAN2 has never been identified as a useful scaffold protein for loading POIs into exosomes, let alone thought to be more effective than currently preferred EV scaffold proteins. TSPAN2 has no known function and has therefore been little studied or understood. The fact that TSPAN2 (and TSPAN3, which was also identified as a useful scaffold protein in the examples) shows only a very weak interaction with CD63 or another EV protein further demonstrates the surprising nature of this discovery.

[0040] In another embodiment of the present invention, EVs containing TSPAN2 are further externally loaded with therapeutic cargo. The external loading methods can be by electroporation, use of transfection reagents, co-incubation, or contact with cell-penetrating peptides (CPPs), or any combination of the above. The cargo that can be externally loaded into EVs expressing TSPAN2 can be a protein, nucleic acid, virus, viral genome, antigen, or small molecule. When the cargo is a nucleic acid cargo, this can be: an RNA molecule, a DNA molecule, or a mixmer, messenger mRNA (mRNA), an antisense or splice-switching oligonucleotide, gRNA, siRNA, shRNA, miRNA, Doggybone® DNA (dbDNA®), plasmid DNA (pDNA), supercoiled or non-supercoiled plasmid, minicircle, etc. The external addition of therapeutic cargo has the additional advantage of further enhancing the versatility of EVs as a platform technology, i.e., EVs expressing multiple components that can act, for example, as brain target sites and endosomal escape sites can be utilized as versatile brain-targeting EVs into which any number of various therapeutic cargos can be added.

[0041] Another embodiment of the present invention relates to a population of EVs as described in the first aspect, wherein the TSPAN2-POI fusion protein and the second POI construct are expressed on the same EV. Such a population of EVs that reliably express two constructs co-localized on the same EV is advantageous for the same reasons as above, i.e., such co-localization is not possible with existing EV scaffold proteins, and is easily utilized as a platform tool that provides the ability to multiplex POIs.

[0042] In the second and third aspects, the present invention provides a polypeptide construct comprising TSPAN2 fused to a POI, and a polynucleotide construct encoding such a polypeptide construct.

[0043] In a fourth aspect, the 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. Such cells may be transiently modified to contain such polynucleotide or polypeptide constructs, or stably modified to contain at least one monocistronic, bicistronic, or polycistronic polynucleotide construct. One of the great advantages of using TSPAN2 as a scaffold protein is that it enables the development of cell lines that reliably express the modified fusion protein construct, whether in transiently transfected cell lines or stable cell lines, especially when the expression of multiple constructs is required. This demonstrates the unique utility of TSPAN2 in the development of clinical-grade cell lines for the production of therapeutic EVs / exosomes. This is because clinical-grade cell lines need to reliably express the desired POI(s) over a sustained period. This is also a requirement for clinical-grade cell lines based on quality control and batch release criteria to ensure consistency in EV products. In the specific case of establishing a stable cell line, this avoids transfection of the parental cells and thus significantly simplifies the EV production procedure. In addition, the expression level of the transgene in stable production cells is more easily controlled compared to transient transfection, reducing variability in EV products.

[0044] The term "stably modified" refers to a cell line that can pass on the introduced polynucleotide construct to its progeny (i.e., daughter cells), which can occur when the transfected DNA is integrated into the endogenous chromosome or through stable inheritance of an exogenous chromosome. A stable cell line can be defined as a homogeneous cell population that retains a volumetric productivity titer of 70% or more, 80% or more, and even 90% or more for at least 50 generations, optionally 60 generations, 70 generations, 85 generations, and even 100 generations. This can indicate that there are "no clinically meaningful differences" as determined by structure, function, purity, chemical identity, and / or biological activity over at least 50 generations, optionally 60 generations, 70 generations, 85 generations, and even 100 generations. Ideally, at the end of 100 doubling cycles, the cells can show a deviation of less than 15%, less than 10%, and even less than 5% (within the limits of measurement) in the productivity titer.

[0045] As shown in the examples, the inventors have also identified TSPAN3 and TSPAN18 as improved EV scaffold proteins. Accordingly, the present invention also relates to EVs comprising a fusion protein, said fusion protein comprising TSPAN3 or TSPAN18 fused to a POI. Optionally, the POI may be incorporated within loop 1, loop 2, or both loops of TSPAN3 or TSPAN18, facilitating surface presentation of the POI. Alternatively, the POI may be incorporated within the NTD and / or CTD of TSPAN3 or TSPAN18, or other luminal portions, facilitating luminal loading of the POI. In another embodiment, the TSPAN3 or TSPAN18 fusion protein further comprises: a release domain cleavable to release the POI; a linker or spacer; a multimerization domain; and / or at least one additional POI. In certain embodiments, the TSPAN3- or TSPAN18-containing EVs further comprise a second POI expressed on a separate construct, said second POI may be present as a fusion protein with an exosomal polypeptide. Similar to the above situation for TSPAN2, TSPAN3 or TSPAN18 EVs may be externally loaded with therapeutic cargo (e.g., nucleic acids, viruses, or small molecule cargo) by any known external loading mechanism comprising: electroporation, transfection reagents, co-incubation, or contact with a CPP, or any combination of these methods. The present invention also relates to pharmaceutical compositions comprising TSPAN3-POI or TSPAN18-POI EVs. Further, the present invention relates to a method of treatment comprising administering an effective amount of TSPAN3-POI or TSPAN18-POI EVs to a patient in need thereof, and similarly, to the use of TSPAN3-POI or TSPAN18-POI EVs for medical use.

[0046] The present invention also relates to a polypeptide composition comprising TSPAN3 or TSPAN19 fused to a POI, or a polynucleotide composition encoding such a polypeptide composition. The present invention also relates in the future to cells comprising such a polypeptide or polynucleotide composition of TSPAN3 or TSPAN18, said cells optionally comprising a second polypeptide or polynucleotide composition capable of expressing a second POI.

[0047] Naturally, the present invention also relates to a method for producing EVs, said method comprising: (i) introducing into EV-producing cells a polynucleotide composition encoding a TSPAN3 / TSPAN18-POI fusion construct; and (ii) expressing said construct in said EV-producing cells, thereby generating EVs comprising a TSPAN3 / 18-POI fusion protein. The method described may also comprise an intermediate step of introducing into the same EV-producing cells a second polynucleotide composition encoding a second POI, wherein said second POI may be present in the form of a fusion protein with an EV protein; as a result, both constructs are expressed in said EV-producing cells, thereby generating EVs comprising a TSPAN3 / 18-POI fusion protein and said second POI.

[0048] [Definitions] For convenience and clarity, certain terms used herein are summarized 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.

[0049] When features, aspects, embodiments, or alternatives of the present invention are described with respect to a Markush group, one of ordinary skill in the art will recognize that the invention thereby describes the individual components of the Markush group or subgroups of components. One of ordinary skill in the art further recognizes that the invention thereby describes any combination of the individual components of the Markush group or subgroups of components. Further, note that embodiments and features described in connection with one aspect and / or embodiment of the present invention also apply mutatis mutandis to all other aspects and / or embodiments of the present invention. For example, the TSPAN2 fusion protein described herein should be understood as disclosed, relevant, and conforming for all other aspects, teachings, and embodiments herein, e.g., in connection with methods for producing or purifying EVs, or corresponding polynucleotide constructs described herein, or aspects and / or embodiments related to modified EV-producing cells from which EVs originate. Further, certain embodiments described in connection with one aspect (e.g., the route of administration of EVs comprising a therapeutic cargo molecule and optionally a fusion polypeptide) may also be relevant in connection with other aspects and / or embodiments, such as those related to pharmaceutical compositions comprising such EVs, when described in connection with aspects related to the treatment of certain medical indications. Further, all polypeptides and proteins identified herein can be freely combined within a fusion protein using conventional strategies for fusing polypeptides. By way of non-limiting example, the TSPAN2-POI fusion protein described herein may be freely combined in any combination with multiple target sites, endosomal escape sites, albumin binding domains, additional POIs, and all other polypeptide domains, regions, sequences, peptides, groups (e.g., any multimerization domain, linker sequence, release domain, etc.) described herein. Further, it should be understood that all such teachings herein that refer to EVs in the singular and / or refer to EVs as individual native nanoparticle-like vesicles are equally relevant and applicable to multiple EVs and populations of EVs.In general, the therapeutic POI, binding proteins to therapeutic agents, endosomal escape sites, albumin binding domains, or purification domains, and all other aspects, embodiments, and alternatives according to the present invention can be freely combined in any combination without departing from the scope and gist of the present invention. Furthermore, any polypeptide or polynucleotide of the present invention, or any polypeptide or polynucleotide sequence (amino acid sequence or nucleotide sequence, respectively), can deviate significantly from the original polypeptide, polynucleotide, and sequence as long as each molecule retains the ability to exert the desired technical effect associated therewith. As long as its biological properties are maintained, the polypeptide and / or polynucleotide sequence according to the present application can deviate up to 50% compared to the original sequence (e.g., calculated using BLAST or ClustalW), but it is preferred that the sequence identity or similarity is as high as possible (e.g., 60%, 70%, 80%, or e.g., 90% or more). Standard methods in the art can be used to determine homology. For example, the PILEUP and BLAST algorithms can be used to calculate homology or align sequences. For example, a combination (fusion) of multiple polypeptides means that specific segments of each polypeptide can be substituted and / or modified, and / or its sequence can be interrupted by the insertion of other amino acid contiguous regions, and as long as the major properties (e.g., maintaining its therapeutic effect, or the ability to bind to therapeutic cargo, or the ability to bind to albumin and extend the half-life, the ability to transport the fusion construct into EVs, targeting ability, etc.) are preserved, the deviation from the original sequence can be significant. Thus, similar reasoning naturally applies to the polynucleotide sequence encoding such a polypeptide. Any accession number or sequence number mentioned herein in relation to peptides, polypeptides, and proteins is for illustrative purposes only and should be regarded as information only, and all peptides, polypeptides, and proteins are given the ordinary meaning understood by those skilled in the art.Accordingly, as described above, one of ordinary skill in the art will also understand that the present invention encompasses not only the specific accession numbers and / or sequence numbers mentioned herein, but also their variants and derivatives. All accession numbers mentioned herein are UniProtKB accession numbers, and all proteins, polypeptides, peptides, nucleotides, and polynucleotides mentioned herein are to be construed according to their conventional meanings as understood by one of ordinary skill in the art.

[0050] [EV:] The terms "extracellular vesicle" or "EV" are used interchangeably herein and can be understood to relate to any type of vesicle that can be obtained from cells in any form. The size of EVs can vary significantly, but EVs typically contain a volume defined by a bilayer lipid membrane and have a nanosized hydrodynamic radius, i.e., a radius of less than 1000 nm. The volume can include the vesicular secretome. Different types of EVs are defined by their various forms, structures, cargo, and functions. EVs can be broadly classified into two categories: (1) exosomes, and (2) ectosomes. Examples of EVs include, for example, exosomes, apoptotic bodies, ARRDC1-mediated microvesicles (ARMMs), ectosomes (e.g., microparticles or microvesicles), or cardiosomes. In essence, the terms "extracellular vesicle" and / or "EV" can relate to any type of lipid-based structure (having a vesicular form or other suitable form) that can function as a delivery or transport vehicle or have an inherent therapeutic or pharmacological effect.

[0051] Furthermore, in some embodiments, the terms can be understood to also relate to cell membrane vesicles obtained through techniques such as extracellular vesicle mimetics, membrane extrusion, sonication, or other techniques.

[0052] Obviously, EVs can be derived from any type of cell, either in vivo, ex vivo, or in vitro (further details of suitable sources or producing cells are described below in this specification).

[0053] Exosomes, microvesicles, and ARMMs are merely some examples of several subtypes that fall within the broad description of EVs above, representing particularly preferred EVs, but it will be understood that other EVs may also be advantageous in certain situations. Advantageously, the EVs are exosomes.

[0054] The terms "apoptotic body" or "apoptotic bodies" are used synonymously herein and can be understood to relate to any type of vesicle obtained from or derived from apoptotic cells. Typically, apoptotic bodies have a diameter in the range of about 1 μm to about 5 μm.

[0055] The terms "cardiosome" or "cardiosomes" are used synonymously herein and can be understood to relate to any type of vesicle obtained from or derived from cardiac cells.

[0056] The terms "ectosome" or "ectosomes" are used synonymously herein and can be understood to relate to any type of heterogeneous vesicle obtained from or derived from the plasma membrane of a cell 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 large vesicles. Typically, ectosomes are in the size range of about 50 nm to about 1 μm.

[0057] The terms "microparticle" or "microparticles" are used interchangeably herein and can be understood to relate to any type of vesicle obtained from or derived from platelets.

[0058] The terms "microvesicle" and "microvesicles" are used interchangeably herein and can be understood to relate to any type of vesicle obtained from, derived from, or released from the plasma membrane or cell membrane of a cell.

[0059] The term "ARMM" can be understood to relate to any type of vesicle that buds directly from the plasma membrane or cell membrane of a cell or is derived therefrom. Such microvesicles are mediated by ARRDC1 and typically lack known late endosome markers. Therefore, ARMMs are distinguished from the exosomes described herein.

[0060] The terms "exosome" or "exosomes" are used interchangeably herein and can be understood to relate to any type of vesicle obtained from or derived from the endosomal pathway, lysosomal pathway, and / or endolysosomal pathway and / or from inward budding of the plasma membrane and / or cell membrane. Exosomes often have a size of about 30 to about 300 nm, typically in the range of about 40 to about 250 nm, and sometimes about 40 to about 160 nm, which is a very suitable size range.

[0061] The term "modified" can mean that the vesicle has been modified using genetic or chemical techniques, for example, via genetic modification of EV-producing cells, preferably exosome-producing cells, or, for example, via chemical conjugation, such as attaching a moiety to the EV, preferably to the exosome surface.

[0062] The terms "genetically modified" and "genetically engineered" are used interchangeably herein, and mean that an EV, preferably an exosome, is derived from a genetically modified / engineered cell or is otherwise genetically modified to effect the expression and / or modification of the expression of a protein presented within the lumen, on the outer vesicle membrane, and / or on the surface of the EV (e.g., exosome), which are typically incorporated into EVs (preferably exosomes) produced by these cells. Such genetically modified or engineered EVs do not occur naturally.

[0063] Furthermore, the terms should be understood to relate also to cell membrane vesicles obtained through, for example, EV mimetics, membrane extrusion, sonication, or other techniques in some embodiments.

[0064] As will be apparent to those skilled in the art, when describing the medical and scientific uses and applications of EVs, the present invention typically relates to a population of EVs, which may include multiple EVs, i.e., thousands, millions, billions, or even trillions of EVs. As can be seen from the following experimental section, EVs are present at 10 5 、10 8 、10 10 、10 11 、10 12 、10 13 、10 14 、10 15 、10 18 、10 25 、10 30They may be present in concentrations such as EVs (often referred to as "particles"), or in some other number that is larger, smaller, or intermediate. Similarly, the term "population" may be associated with, for example, EVs that contain a certain POI, but should be understood to encompass the multiple entities that together make up such a population. In other words, when there are multiple individual EVs, they constitute an EV population. Thus, of course, the present invention relates to both individual EVs and populations containing EVs, as will be apparent to those skilled in the art. The dosage of EVs when applied in vivo can, of course, vary widely depending on the disease being treated, the route of administration, the activity and effect of the albumin-binding domain (ABD), the therapeutic cargo, any target sites present on the EVs, the pharmaceutical formulation, and so on.

[0065] [TSPAN2:] TSPAN2 is to be understood to mean tetraspanin-2 (also called TSN2 or tetraspan NET-3), and its derivatives, domains, variants, mutants, or regions. (Database references for TSPAN2 include the following: HGNC:20659, NCBI Entrez Gene:10100, Ensembl:ENSG00000134198, OMIM®:613133, UniProtKB / Swiss-Prot:O60636). The TSPAN2 protein is a multi-pass transmembrane protein having four transmembrane regions. TSPAN2 is a paralog of CD9 and is thought to play a role in signal transduction at an early stage in the final differentiation of oligodendrocytes into myelinating glia and may also function in the stabilization of mature myelin sheaths. The sequences of TSPAN2 are shown in SEQ ID NOs:1 and 2. Derivatives, domains, variants, mutants, or regions of TSPAN2 are assumed to form part of the present invention and may be proteins having at least 10% sequence identity with SEQ ID NOs:1 and / or 2. For example, the derivative, domain, variant, mutant, or region may have at least 20%, or even at least 30% sequence identity with SEQ ID NOs:1 and / or 2. The derivative, domain, variant, mutant, or region may “substantially” have the nucleotide and / or amino acid sequences of SEQ ID NOs:1 and / or 2, which may mean a sequence having at least 40% sequence identity with the sequences of SEQ ID NOs: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 with SEQ ID NOs: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.

[0066] [TSPAN3:] TSPAN3 is to be understood to mean tetraspanin 3 (TSN3, also known as 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 the following: HGNC:17752, NCBI Entrez Gene:10099, Ensembl:ENSG00000140391, OMIM®:613134, UniProtKB / Swiss-Prot:O60637). TSPAN3 is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. TSPAN3 is a paralog of CD82 and is thought to control the proliferation and migration of oligodendrocytes, a process essential for normal myelin formation and repair. The sequences of TSPAN3 are shown in SEQ ID NOs: 3 and 4. Derivatives, domains, variants, mutants, or regions of TSPAN3 are assumed to form part of the present invention and can be proteins having at least 10% sequence identity with SEQ ID NO: 3 and / or 4. For example, the derivative, domain, variant, mutant, or region can have at least 20%, or even at least 30% sequence identity with SEQ ID NO: 3 and / or 4. The derivative, domain, variant, mutant, or region may "substantially" have the "nucleotide and / or amino acid sequence" of SEQ ID NO: 3 and / or 4, which can mean 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 with SEQ ID NO: 3 and / or 4.

[0067] [TSPAN18:] TSPAN18 should be understood to mean tetraspanin-18, and its derivatives, domains, variants, mutants, or regions. (Database references for TSPAN18 include the following: HGNC:20660, NCBI Entrez Gene:90139, Ensembl:ENSG00000157570, OMIM®:619399, UniProtKB / Swiss-Prot:Q96SJ8). Little is known about the function of TSPAN18, but it is predicted to be an integral component of the plasma membrane and is a paralog of TSPAN1. The sequences of TSPAN18 are shown in SEQ ID NOs: 5 and 6. Derivatives, domains, variants, mutants, or regions of TSPAN18 are assumed to form part of the present invention and can be proteins having at least 10% sequence identity with SEQ ID NO: 5 and / or 6. For example, the derivative, domain, variant, mutant, or region can have at least 20%, or even at least 30% sequence identity with SEQ ID NO: 5 and / or 6. The derivative, domain, variant, mutant, or region may “substantially” have the “nucleotide and / or amino acid sequence” of SEQ ID NO: 5 and / or 6, which can mean a sequence having at least 40% sequence identity with the sequences 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 with SEQ ID NO: 5 and / or 6.

[0068] [POI:] A POI can be any protein or polypeptide that is desirably loaded inside or on the surface of an EV. Exemplary POIs can be (i) a therapeutic protein, (ii) a binding protein for a therapeutic agent (e.g., an RNA-binding protein, a virus-binding protein, a small molecule-binding protein, an fc-binding protein), (iii) an endosomal escape site, (iv) a target site, (v) an albumin-binding domain, or (vi) a purification site. In the detailed embodiments described below, any of the specific POIs can be included within a TSPAN2 fusion polypeptide. Alternatively or additionally, the POI can be included within the EV as a second POI expressed on another construct.

[0069] As described above, the advantage of using TSPAN2 as a scaffold protein is that it enables the development of cell lines that have undergone large-scale modifications while maintaining robust and consistent expression of single or multiple protein constructs. As noted above, this is particularly advantageous when incorporated into the loops / NTD / CTD of TSPAN2 and is also very advantageous when expressing multiple constructs within the same EV, as TSPAN2 enables co-localization of the constructs. Shown below are some non-limiting and exemplary advantageous constructs and combinations of constructs that can be more reliably and stably incorporated into EVs when using TSPAN2:

[0070] [A single POI fused to TSPAN2] - TSPAN2-surface displayed therapeutic protein - TSPAN2-lumen loaded therapeutic protein - TSPAN2-binding protein for a therapeutic agent - TSPAN2-surface displayed target site - TSPAN2-surface displayed ABD site - TSPAN2-endosomal escape site

[0071] Furthermore, any of the fusion constructs listed above can be expressed in combination with a second construct containing any POI within the EV / cell of the present invention such that the EV can contain at least two different protein constructs. The second construct may be a fusion protein of an EV protein - POI.

[0072] [Two POIs fused to TSPAN2] - Therapeutic proteins within TSPAN2 - loop1 and loop2 - Therapeutic proteins within TSPAN2 - loop1 / 2 and therapeutic proteins on NTD / CTD - Therapeutic proteins within TSPAN2 - loop1 / 2 and target sites within loop1 / 2 - Therapeutic proteins within TSPAN2 - loop1 / 2 and endosomal escape sites within loop1 / 2 - Therapeutic proteins within TSPAN2 - loop1 / 2 and ABD within loop1 / 2 - Therapeutic proteins on TSPAN2 - NTD and CTD - Therapeutic proteins on TSPAN2 - NTD / CTD (lumen) and target sites within loop1 / 2 - Therapeutic proteins on TSPAN2 - NTD / CTD (lumen) and ABD within loop1 / 2 - Therapeutic proteins on TSPAN2 - NTD / CTD (lumen) and endosomal escape sites within loop1 / 2 - Binding proteins within TSPAN2 - loop1 and loop2 - Binding proteins within TSPAN2 - loop1 / 2 and target sites within loop1 / 2 - Binding proteins within TSPAN2 - loop1 / 2 and endosomal escape sites within loop1 / 2 - Binding proteins within TSPAN2 - loop1 / 2 and ABD within loop1 / 2 - Binding proteins on TSPAN2 - NTD and CTD - Binding proteins on TSPAN2 - NTD / CTD (lumen) and target sites within loop1 / 2 - Binding proteins on TSPAN2-NTD / CTD (lumen) and ABDs within loops 1 / 2 - Binding proteins on TSPAN2-NTD / CTD (lumen) and endosomal escape sites within loops 1 / 2 - Target sites within TSPAN2-loops 1 / 2 and endosomal escape sites within loops 1 / 2 - Target sites within TSPAN2-loops 1 / 2 and ABDs within loops 1 / 2 - Target sites within TSPAN2-loops 1 / 2 and purification sites within loops 1 / 2 - Endosomal escape sites within TSPAN2-loops 1 / 2 and ABDs within loops 1 / 2 - Endosomal escape sites within TSPAN2-loops 1 / 2 and purification sites within loops 1 / 2 - Purification sites within TSPAN2-loops 1 / 2 and ABDs within loops 1 / 2

[0073] Furthermore, any of the fusion constructs listed above can be expressed in combination with a second construct containing any POI within the EV / cell of the present invention such that the EV contains at least two different protein constructs. The second construct may be a fusion protein of an EV protein-POI.

[0074] Particularly preferred combinations include a second construct comprising a binding protein for a therapeutic protein or therapeutic agent fused to TSPAN2, and the endosomal escape site VSVG, where VSVG may be fused to TSPAN2 or a classical EV protein.

[0075] In other embodiments, the TSPAN2 component of the constructs listed above can be replaced with TSPAN3 or TSPAN18 having similar effects. The present invention includes polynucleotides encoding any of the polypeptide fusion constructs listed above, including TSPAN3 or TSPAN18 instead of TSPAN2.

[0076] In certain embodiments, the POI, or at least one of the POIs, can be a therapeutic protein. In one embodiment, the therapeutic POI is included within a TSPAN2 fusion polypeptide. Alternatively, the POI fused to TSPAN2 can be any of the other exemplary POIs, and the therapeutic POI can instead be expressed on another construct.

[0077] The therapeutic protein can be selected from the following: enzymes, receptors such as 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 such as CRISPR effector proteins including Cas proteins, transcription activator-like effector nucleases (TALENs), meganucleases.

[0078] More specifically, the therapeutic protein cargo (POI) according to the present invention includes the following: antibodies, intrabodies, nanobodies, single-chain variable fragments (scFv), affibodies, bispecific and multispecific antibodies or binders including bispecific T cell engager antibodies (BiTE), receptors, ligands, transporters, such as ERT or enzymes for gene editing, tumor suppressors, viral or bacterial inhibitors, cell constituent proteins, DNA repair inhibitors, nucleases, proteases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins (e.g., Pseudomonas exotoxin), structural proteins, neurotrophic factors (e.g., NT3 / 4, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF), and their individual subunits (e.g., 2.5S β subunit)), ion channels, membrane transporters, proteostasis factors, proteins involved in cell signaling, translation and transcription-related proteins, nucleotide-binding proteins, protein-binding proteins, lipid-binding proteins, glycosaminoglycans (GAG) and GAG-binding proteins, metabolic proteins, cell stress regulatory proteins, inflammation and immune system regulatory proteins (e.g., cytokines and inhibitors of such cytokines) (cytokines may include the following: 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 INF-α / 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.

[0079] In a preferred embodiment, the encoded protein is a CRISPR-associated (Cas) polypeptide having full nuclease activity (e.g., Cas9), which is bound to an RNA strand (i.e., carried with the RNA strand) and, when delivered by a peptide, allows the Cas polypeptide to exert its nuclease activity in a target cell. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) components according to the present invention include CRISPR components from any bacterial source. The CRISPR components may be derived from class 1 or class 2, and specifically, the Cas type may be Cas type I, II, III, IV, V, or VI. Specific Cas proteins 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 and enable target gene modification. Yet another alternative includes another type of CRISPR effector, such as the single RNA-guided endonuclease Cpf1. Inclusion of Cpf1 is a particularly preferred embodiment of the present invention because it cleaves target DNA via staggered double-strand breaks. Cpf1 can be obtained from species such as Acidaminococcus or Lachnospiraceae. In yet another exemplary embodiment, the Cas polypeptide may also be fused to a transcriptional activator (e.g., the P3330 core protein) to specifically induce gene expression.

[0080] Additional preferred embodiments include therapeutic protein cargos selected from the group comprising enzymes or transporters for lysosomal storage disorders, such as glucocerebrosidase, e.g., imiglucerase, α-galactosidase, α-L-iduronidase, iduronate-2-sulfatase and idursulfase, arylsulfatase, galactosulfase, acid α-glucosidase (GAA), sphingomyelinase, galactocerebrosidase, galactosylceramidase, ceramidase, α-N-acetylgalactosaminidase, β-galactosidase, lysosomal acid lipase, acid sphingomyelinase, NPC1, NPC2, heparan sulfamidase, N-acetylglucosaminidase, heparan-α-glucosaminide-N-acetyltransferase, N-acetylglucosamine 6-sulfatase, galactose-6-sulfatase, galactose-6-sulfatase, hyaluronidase, αN-acetylneuraminidase, GlcNAc phosphotransferase, mucolipin 1, palmitoyl protein thioesterase, tripeptidyl peptidase I, palmitoyl protein thioesterase 1, tripeptidyl peptidase 1, battenin, linclin, α-D-mannosidase, β-mannosidase, aspartylglucosaminidase, α-L-fucosidase, cystinosin, cathepsin K, sialin, LAMP2, and hexoaminidase.

[0081] Additional preferred embodiments include therapeutic protein cargos selected from the group comprising enzymes associated with urea cycle disorders, including: N-acetylglutamate synthase, carbamoyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, arginase, mitochondrial ornithine transporter, citrulline, y+L amino acid transporter 1, uridine monophosphate synthase UMPS.

[0082] In other preferred embodiments, the POI may be, for example, an intracellular protein that modifies an inflammatory response, such as an epigenetic protein such as a methylase and a bromodomain, or may be an intracellular protein that modifies muscle function, such as a transcription factor (e.g., MyoD or Myf5), a protein that regulates muscle contraction (e.g., a calcium / binding protein such as myosin, actin, troponin, etc.), or a structural protein (e.g., dystrophin, minidystrophin, microdystrophin, utrophin, titin, nebulin, dystrophin-related proteins (e.g., dystrobrevin, syntrophin, syncoilin, desmin, sarcoglycan, dystroglycan, sarcospan, agrin, and / or fukutin)). The POI is typically a protein or peptide of human origin, unless otherwise indicated by its name, other nomenclature, or methods known to those skilled in the art, and these can be verified in various public databases such as Uniprot, RCSB, etc.

[0083] In another preferred embodiment, the therapeutic cargo is an antigen / novel antigen, where the antigen / novel antigen may be suitable for use in cancer immunotherapy.

[0084] Any antigen / novel antigen can be incorporated into the EVs of the present invention. The antigen may be suitable for enhancing the immune response against pathogens such as bacteria, viruses, fungi, etc., or the antigen may be a tumor antigen useful for inducing an immune response against tumors for cancer immunotherapy. Any EV according to the present invention may contain one or more antigens / novel antigens. The one or more antigens / novel antigens may be endogenous / self (derived from the subject itself) or exogenous / allogeneic (derived from another subject), or when multiple antigens / novel antigens are incorporated inside / on the surface of the EV, the antigens / novel antigens may be any mixture of self / allogeneic antigens. Preferably, the antigen is self. Also, the one or more antigens / novel antigens may have any origin, such as viral or bacterial, etc., or may be tumor antigens, and may further be immunostimulatory or immunosuppressive, or a combination thereof. The antigen / novel antigen may be useful in the treatment of any disease by immunotherapy. The treatment of cancer by immunotherapy is a particularly preferred embodiment. When the antigen is a novel antigen, it can be identified by tumor sequencing to identify the novel antigen.

[0085] Exemplary tumor antigens can be the following: 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, peptides derived from chromosomal translocations such as CTAG1B, 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).

[0086] When the therapeutic cargo is an antigen or neoantigen, the EV or 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 can be the following: inorganic compounds such as aluminum hydroxide, aluminum phosphate, calcium phosphate hydroxide, mineral oils such as paraffin oil, bacterial products (e.g., killed bacteria of Bordetella pertussis, Mycobacterium bovis), toxoids, non-bacterial organic substances such as squalene, detergents such as Quil A, plant saponins, cytokines (e.g., IL-1, IL-2, IL-12), or ISCOM (immunostimulating complex) such as RIBI (muramyl dipeptide) or a STING (stimulator of interferon genes) agonist containing cyclic dinucleotides. Such adjuvants may protect the therapeutic EV from rapid dispersion by sequestering it in local deposits, or may include substances that stimulate the host to secrete chemotactic factors for macrophages and other components of the immune system. Adjuvants that can be incorporated into vaccines are well known to those skilled in the art and are selected such that they do not adversely affect the immunological activity of the EV. When the adjuvant is a protein (e.g., cytokine), in a specific embodiment, the adjuvant can be incorporated into the EV itself as part of a TSPAN2 fusion protein or a second POI.

[0087] In certain embodiments, the POI or at least one of the POIs can be a binding protein for a therapeutic agent. Such binding proteins can be, for example, RNA or DNA binding proteins, viral binding proteins, small molecule binding proteins, or Fc binding proteins. In some embodiments, the binding protein capable of binding to the therapeutic agent can be the POI contained in a TSPAN2 fusion polypeptide. Alternatively, the binding protein can exist as a second POI expressed on another construct.

[0088] 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.

[0089] Non-limiting examples of NA binding proteins include Ago2, Dicer, Drosha, DGCR8, hnRNPA1, hnRNPA2B1, DDX4, ADAD1, DAZL, ELAVL4, IGF2BP3, SAMD4A, TDP43, FUS, FMR1, FXR1, FXR2, EIF4A13, MS2 coat protein, and any domains, portions, or derivatives thereof. More broadly, specific subclasses of RNA binding proteins and domains, such as messenger RNA binding proteins (mRBPs), precursor 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). Further, various domains and derivatives can also be used as NA binding domains to transport NA cargo into EVs. Non-limiting examples of RNA binding domains include those containing 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, etc.). Such RNA binding domains can be present singly or in multiple combinations with others, and as long as their major function (i.e., the ability to transport the NA cargo of interest, such as mRNA or short-chain RNA) is maintained, they can remain as part of a larger RNA binding protein construct or form a larger construct.

[0090] In preferred embodiments, the present invention relates to two groups of NA-binding domains, namely PUF proteins and CRISPR-related polypeptides (Cas), specifically Cas9, Cas6, and Cas13, and various NA-binding aptamers. In the present invention, the term PUF protein is used to encompass all related proteins and domains of such proteins (which may also be called PUM proteins), for example, human Pumilio homolog 1 (PUM1), PUMx2 or PUFx2 (which are dimers of PUM1), or any NA-binding domain obtained from any PUF (PUM) protein. PUF proteins are typically characterized by the presence of eight consecutive PUF repeats, each approximately 40 amino acids, often flanked by two related sequences, Csp1 and Csp2. Each repeat has a "core consensus" that includes aromatic and basic residues. The entire cluster of PUF repeats is required for RNA binding. The PUF proteins according to the present invention may be natural or modified to bind at any location within an RNA molecule, or alternatively, PUF proteins with various binding affinities for various sequences may be selected to modify the RNA molecule to include said sequences. Furthermore, there are also PUF domains that have been modified and / or duplicated to bind 16 nucleotides sequence-specifically, which can also be used to further increase the specificity for NA cargo molecules. Thus, the PUF domain may be modified to bind to any sequence with various affinities and sequence lengths, whereby the system is highly modular and adaptable to any RNA cargo molecule according to the present invention.PUF proteins, their regions and derivatives that can be used as NA-binding domains according to 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 Drosophila melanogaster; Puf5p / Mpt5p / Uth4p, Puf4p / Ygl014wp / Ygl023p, Puf5p / Mpt5p / Uth4p, Puf5p / Mpt5p / Uth4p, Puf3p (all from Saccharomyces cerevisiae); PufA from Dictyostelium; human PUM1 (also known as Pumilio1, PUF-8R) and any of its domains, polypeptides containing NA-binding domains from at least two PUM1s, any truncated, or modified, or altered PUF proteins, such as PUF-6R, PUF-9R, PUF-10R, PUF-12R, and PUF-16R, or their derivatives, etc.; and X-Puf1 from Xenopus. Particularly suitable NA-binding PUFs according to the present invention include: PUF531, PUF mRNA loc (also called PUF modified or PUFeng), and / or PUFx2 (whose sequence is available in PCT / EP2018 / 080681), and any of their derivatives, domains, and / or regions. PUF / PUM proteins are very advantageous because those of human origin can be selected. Furthermore, similar to the case of PUF proteins, Cas proteins such as Cas6, Cas9, and Cas13 are very preferred examples of releasable NA-binding domains that bind to NA cargo molecules with appropriate affinity, thereby enabling the releasable and reversible attachment of Cas proteins to NA cargo. Similar to the PUF-based NA-binding domains, Cas proteins represent releasable and irreversible NA-binding domains with programmable and modifiable sequence specificity for target NA cargo molecules, enabling higher specificity with lower overall affinity, thereby enabling both loading of NA cargo into EVs and release of NA cargo at the target location.

[0091] "Nucleic acid" refers to polynucleotides and includes polyribonucleotides and polydeoxyribonucleotides. The nucleic acids according to the present invention include any polymer or oligomer of pyrimidine and purine bases (for example, cytosine, thymine, and uracil, and adenine and guanine, respectively) (see Albert L. Lehninger, Principles of Biochemistry, 793 - 800 (Worth Pub. 1982) and G. Michael Blackburn, Michael J. Gait, David Loakes and David M. Williams, Nucleic Acids in Chemistry and Biology 3rd edition (RSC publishing 2006). These are hereby incorporated by reference in their entirety for all purposes). In fact, the present invention contemplates any deoxyribonucleotide or ribonucleotide component, and any chemical variants thereof. The polymer or oligomer may have a heterogeneous or homogeneous composition, may be isolated from a naturally occurring source, or may be produced artificially or synthetically. Further, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or temporarily in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

[0092] "Oligonucleotide" or "polynucleotide" refers to a nucleic acid having a length in the range of at least 2, at least 8, at least 15, or at least 25 nucleotides, which may reach up to 50, 100, 1000, 5000, 10000, 15000, or 20000 nucleotides in length, or is a compound that specifically hybridizes to a polynucleotide. Polynucleotides include DNA or RNA sequences, or mimetics thereof, which may be isolated from natural sources, produced recombinantly, or synthesized artificially. Further examples of polynucleotides used in the present invention may include peptide nucleic acids (PNA; see U.S. Patent No. 6,156,501, which is hereby incorporated by reference in its entirety). The present invention also encompasses situations where there is non-traditional base pairing, such as Hoogsteen-type base pairing, which has been identified in certain tRNA molecules and is hypothesized to exist in triple helices. "Polynucleotide" and "oligonucleotide" are used synonymously herein. When a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C), it is understood to also include the corresponding RNA sequence (e.g., A, U, G, C) where "U" replaces "T".

[0093] As used herein, "polynucleotide" includes, for example, cDNA, RNA, DNA / RNA hybrids, antisense RNA, siRNA, mRNA, ribozymes, genomic DNA, synthetic forms, and hybrid polymers (both sense and antisense strands), and may be chemically or biochemically modified to include unnatural or derivatized, synthetic, or semi-synthetic nucleotide bases. Also contemplated are modifications of wild-type or synthetic genes, including, but not limited to, deletions, insertions, substitutions, or fusions with other polynucleotide sequences of one or more nucleotides.

[0094] The present invention is specifically related to ABD-EV further loaded with nucleic acids such as siRNA targeting cancer genes known to be involved in the occurrence of cancer. Genes targeted by the nucleic acids according to the present invention may 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.

[0095] More specifically, the nucleic acid cargo molecules that bind to the RNA / DNA-binding protein (POI) of the present invention or are externally loaded into the exosome (further described herein) can be selected from the group consisting of shRNA, siRNA, saRNA, miRNA, anti-miRNA, mRNA, modified mRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA, lncRNA, ribozyme, minicircle DNA, plasmid DNA, RNA / DNA vector, trans-splicing oligonucleotide, splice-switching oligonucleotide, CRISPR guide strand, morpholino (PMO) antisense oligonucleotide (ASO), peptide nucleic acid (PNA), viral genome and viral genetic material (e.g., naked AAV genome), but essentially any kind 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 may be naturally occurring (e.g., RNA or DNA) or chemically synthesized RNA and / or DNA molecules, which may include 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, tricyclo-DNA, thionucleotide, phosphoramidate, PNA, PMO, etc.

[0096] When the cargo is mRNA, the mRNA can be a naturally or non-naturally occurring mRNA. The mRNA can include one or more modified nucleobases, nucleosides, or nucleotides. The nucleobases of the 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 but not limited to one or more substitutions or modifications such as: alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitution; one or more fused or open rings; oxidation; and / or reduction. Thus, the nucleobases can be selected from the non-limiting group consisting of: adenine, guanine, uracil, cytosine, 7-methylguanine, 5-methylcytosine, 5-hydroxymethylcytosine, thymine, pseudouridine, dihydrouridine, hypoxanthine, and xanthine. The nucleosides of the mRNA are compounds that include a sugar molecule (e.g., a 5- or 6-carbon sugar such as pentose, ribose, arabinose, xylose, glucose, galactose, or their deoxy derivatives) combined with a nucleobase. The nucleosides can be standard nucleosides (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine, and thymidine) or analogs thereof, and can include one or more substitutions or modifications of the nucleobase and / or sugar moiety, including but not limited to: alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitution; one or more fused or open rings; oxidation; and / or reduction. The nucleotides of the mRNA are compounds that include a nucleoside and a phosphate group or alternative group (e.g., boranophosphate, thiophosphate, selenophosphate, phosphonate, alkyl group, amidate, and glycerol).The nucleotide may be a standard nucleotide (e.g., adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine monophosphate) or an analog thereof, and may include one or more substitutions or modifications of a nucleobase, sugar, and / or phosphate or alternative components including, but not limited to: alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitution; one or more fused or open rings; oxidation; and / or reduction. The nucleotide may include one or more phosphates or alternative groups. For example, the nucleotide may include a nucleoside and a triphosphate group. "Nucleoside triphosphate" (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) may refer to a standard nucleoside triphosphate, or an analog or derivative thereof, and may include one or more substitutions or modifications described herein. For example, "guanosine triphosphate" should be understood to include standard guanosine triphosphate, 7-methylguanosine triphosphate, or other definitions encompassed herein. The mRNA may include a 5' untranslated region, a 3' untranslated region, and / or a coding or translated sequence, which is translated to create the fusion protein of the present invention. The mRNA may include any number of base pairs, including dozens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) of nucleobases, nucleosides, or nucleotides may be analogs, substituted, modified, or otherwise non-natural of standard species. In certain embodiments, all of a particular type of nucleobase may be modified. For example, all cytosines in the mRNA may be 5-methylcytosine. In some embodiments, the mRNA may include a 5' cap structure, a chain-terminating nucleotide, a stem-loop, a polyA sequence, and / or a polyadenylation signal.A cap structure or cap species is a compound that includes two nucleoside sites linked by a linker, which caps mRNA at its 5′ end and can be selected from a naturally occurring cap, a non-natural cap or cap analog, or an anti-reverse cap analog (ARCA). The cap species can include one or more modified nucleosides and / or linker sites. For example, a natural mRNA cap can include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position and linked by a triphosphate bond at their 5′ positions (e.g., m7G(5′)ppp(5′)G, commonly denoted as m7GpppG). The cap species can also be an anti-reverse cap analog. A non-limiting list of possible cap species includes the following: 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 can alternatively or additionally include a chain-terminating nucleoside. For example, chain-terminating nucleosides can include those nucleosides that are deoxygenated at the 2′ and / or 3′ positions of their sugar group. Such species can include the following: 3′-deoxyadenosine (cordycepin), 3′-deoxythymidine, 3′-deoxycytidine, 3′-deoxyguanosine, 3′-deoxythymidine, and 2′,3′-dideoxynucleosides (e.g., 2′,3′-dideoxyadenosine, 2′,3′-dideoxythymidine, 2′,3′-dideoxycytidine, 2′,3′-dideoxyguanosine, and 2′,3′-dideoxythymidine). The mRNA can alternatively or additionally include a stem-loop such as a histone stem-loop. The stem-loop can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or more nucleotide base pairs. For example, the stem-loop can include 4, 5, 6, 7, 8, 9 nucleotide base pairs. The stem-loop can be located in any region of the mRNA.For example, a stem loop can be located within, before, or after a non-translated region (5' untranslated region or 3' untranslated region), coding region, or polyA sequence or tail. The mRNA may alternatively or additionally include a polyA sequence and / or polyadenylation signal. The polyA sequence can be composed entirely or mostly of adenine nucleotides or analogs or derivatives thereof. The polyA sequence can be a tail located adjacent to the 3' untranslated region of the mRNA. The modified mRNA of the present invention can include, in addition to the coding regions (which can encode a fusion protein and can be codon-optimized), one or more stem loops, chain-terminating 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 the mRNA to modify it, improve translation efficiency, reduce immunogenicity, and improve 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 for reducing the immunogenicity of the mRNA and improving translation efficiency in vivo. In a preferred embodiment of the present invention, the compositions herein include mRNA modified as a polynucleotide cargo, where the mRNA is modified using at least 50% m5C and 50% ψ or m1ψ, preferably at least 75% m5C and 75% ψ or m1ψ, more preferably 90% m5C and 90% ψ or m1ψ, or even more preferably 100% modified using m5C and ψ or m1ψ.

[0097] In some embodiments, the POI may be a viral binding protein, such as the following: a protein capable of binding to adeno-associated virus (AAV) (e.g., AAV receptor), or a protein capable of binding to other types of viruses such as lentivirus. Generally, any protein capable of binding to the coat / envelope protein of a virus or capable of binding to the viral genome is within the scope of the present invention. Exemplary viral binding proteins include the following: AAVR GPR108, syndecan, and albumin.

[0098] In some embodiments, the POI may be a small molecule binding protein, for example, any protein, polypeptide, or peptide (i.e., any molecule containing an amino acid sequence), and the small molecule agent can be attached thereto via non-covalent or covalent bonds, or via a combination of both covalent and non-covalent interactions. The combination of the binding protein and the small molecule agent is described herein using terms such as "binding protein-small molecule conjugate" or "binding protein-small molecule drug conjugate" or "binding protein-small molecule agent conjugate", or simply "conjugate". The binding protein can play multiple different roles as follows: for example, (i) a carrier and / or delivery means mainly for attaching and transporting the small molecule drug, (ii) a targeting agent that directly transports the EV carrying the binder protein-small molecule conjugate to a specific location, (iii) a therapeutically active protein that becomes therapeutically active or inactive through the attachment of the small molecule and may have an agonist or antagonist effect, (iv) a signaling protein that can exert or contribute to changes in cells and / or the body and related therapeutic and / or prophylactic effects with or without the small molecule cargo, (v) a protein that can perform or catalyze a specific reaction only when placed in the vicinity of another protein, and so on. The binding protein may further contribute to the action or activity in the body and / or cells and the related therapeutic effect by releasing the small molecule agent at an appropriate location, or may contribute to such an effect by binding and retaining the small molecule agent. As a first example, the binding protein may release the small molecule drug within the target cell after EV-mediated delivery, while as a second example, there is the delivery of an antibody-small molecule drug conjugate into a tumor.

[0099] The terms "small molecule agent", "small molecule", "small molecule drug", or "small molecule therapeutic" are used interchangeably herein and are to be understood as relating to any molecular agent that can be used for the treatment and / or diagnosis of diseases and / or disorders and, for example, to modulate or alter the activity and / or binding and / or location of a binding protein. Small molecule agents are typically synthesized via chemical synthetic means, but may also be of natural origin, such as via purification from a natural source, or may be obtained through other suitable means or combinations of techniques. A concise and non-limiting definition of "small molecule" is any organic compound having a molecular weight of less than 900 g / mol (Daltons) that can, in some way, control, affect, or influence a biological process. For the purposes of the present invention, a small molecule may be substantially greater than 900 g / mol, for example 1500 g / mol, 3000 g / mol, or in some cases even greater. Overall, molecular weight and / or molecular size are not decisive factors in constituting a small molecule agent. Indeed, for the purposes of the present invention, any agent that can be bound by a binding protein presented on an EV is considered to be a "small molecule agent".

[0100] When the POI is a viral binding protein, the EVs of the present invention are loaded with viral cargo. Exemplary viral cargo includes: viral vectors that are AAV vectors or lentiviral vectors.

[0101] In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector comprises a capsid derived 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 comprising inverted terminal repeat (ITR) sequences derived from human AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In some embodiments, the AAV capsid and the AAV ITR are derived from the same serotype or from different serotypes.

[0102] 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-proliferative. In some embodiments, the lentiviral vector is non-integrating.

[0103] In some embodiments, the viral vector comprises a viral capsid and a viral genome, and the viral genome comprises one or more heterologous transgenes. In a preferred embodiment, the heterologous transgene encodes a polypeptide or a protein. The protein encoded within the viral genome may be any one of the protein cargos according to the present invention, whereby the viral cargo can act as a gene replacement therapy.

[0104] In some embodiments, the cargo-loaded EV may further comprise one or more molecules that provide an immune effector function. Immune effector molecules are particularly useful in the case of EVs loaded with viral (e.g., AAV or lentivirus) cargo, but may be used equally well when the EVs are loaded with any cargo according to the present invention. The immune effector may act to reduce the immunogenicity of the EV. In some embodiments, the immune effector function stimulates an immune inhibitory factor. In other embodiments, the immune effector function inhibits an immune stimulatory molecule. In some embodiments, the ABD-EV comprises a molecule that stimulates an immune inhibitory factor and a molecule that inhibits an immune stimulatory molecule. Exemplary immune effector molecules include, but are not limited to, one or more of the following: CTLA4, B7-1, B7-2, PD-1, 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-L and VISTA. The immune effector molecule may form part of a TSPAN2 fusion construct or may form part of another fusion protein construct comprising an immune effector molecule fused to any EV protein according to the present invention.

[0105] Examples of small molecules include the following: anticancer agents (e.g., other nucleoside analogs such as doxorubicin, methotrexate, 5-fluorouracil, or cytarabine), proteasome inhibitors (e.g., bortezomib), or kinase inhibitors (e.g., imatinib or seliciclib), or NSAIDs (e.g., naproxen, aspirin, or celecoxib), antibiotics (e.g., heracillin), or antihypertensive agents (e.g., ACE inhibitors such as enalapril), ARBs (e.g., candesartan), cyclic dinucleotides, and the like. As will be apparent to those skilled in the art, the present invention is of course also applicable to other small molecules without departing from the gist of the invention.

[0106] In some embodiments, the POI may be a protein capable of binding to the Fc domain (also known as an Fc-binding protein). The terms "Fc binding polypeptide", "Fc binding protein", "Fc binder", "Fc-binding protein", and "binder" are used interchangeably herein and are to be understood as relating to any protein, polypeptide, or peptide (i.e., any molecule containing an amino acid sequence) capable of binding to the Fc domain of any POI. Typically, the Fc-binding polypeptides of the present invention are derived from a variety of sources, human or non-human (e.g., mammalian origin, bacteria, etc.), and they have high affinity for the Fc domains of various antibody isotypes, subtypes, and species (e.g., IgG (in the case of IgG, IgG1, IgG2, IgG3, IgG4, IgG2a, IgG2d, and / or IgG2c as non-limiting examples), IgA, IgM, IgD, etc.), and they can be fused to EV proteins. Non-limiting examples of Fc-binding polypeptides according to the present invention include, in addition to other Fc-binding polypeptides referred to throughout this application, the following: Protein A, Protein G, Protein A / G, Z domain, ZZ domain, human FCGRI, human FCGRIIA, human FCGRIIB (accession number 31994 as a non-limiting example), human FCGRIIC (accession number 31995 as a non-limiting example), human FCGRIIIA (accession number P08637 as a non-limiting example), human FCGR3B (accession number O75015 as a non-limiting example), human FCAMR, human FCERA, human FCAR, mouse FCGRI, mouse FCGRIIB, mouse FCGRIII, mouse, mouse FCGRn, and various combinations, derivatives, or alternatives thereof.

[0107] The terms "Fc-containing protein", "protein comprising an Fc domain", "Fc domain-containing protein", "Fc domain containing protein", "Fc domain protein", and similar terms are used synonymously herein and are to be understood as relating to any protein, polypeptide, or peptide (i.e., any molecule containing an amino acid sequence) that contains an Fc domain either naturally or as a result of a modification that introduces an Fc domain into the protein. Fc means "fragment crystallizable" or "fragment constant", which is the name for the tail region of an antibody. However, Fc domains can also be created and used in proteins other than antibodies. Non-limiting examples of such Fc domain-containing proteins include: antibodies and antibody derivatives, Fc-modified decoy receptors (e.g., CD24-Fc or CD52-Fc), and / or signaling factors (e.g., interleukin decoy receptors for: IL1, IL2, IL3, IL4, IL5, IL6 (e.g., the signaling factor gp130 (non-limiting example, accession number P40189)), IL7, IL8, IL9, IL10, IL11, IL12, IL13, IL14, IL15, IL17 (e.g., IL17R, non-limiting example having accession number Q96F46), IL23 (e.g., IL23R, non-limiting example having accession number Q5VWK5), etc.), Fc domain-containing bispecific and multispecific binders, any type of Fc domain-containing receptor or ligand, e.g., Fc domain-modified enzymes for enzyme replacement therapy or gene editing, nucleases such as Cas and Cas9 with an Fc domain grafted thereon, tumor suppressors fused to an Fc domain, etc.Suitable Fc domains that can be fused to a POI that originally lacks an Fc domain include the following non-limiting examples: human IGHM (non-limiting example, accession number P01871), human IGHA1 (non-limiting example, accession number P01876), human IGHA2 (non-limiting example, accession number P01877), human IGKC (non-limiting example, accession number P01834), human IGHG1 (non-limiting example, accession number P01857), human IGHG2 (non-limiting example, accession number P01859), human IGHG3 (non-limiting example, accession number P01860), human IGHG4 (non-limiting example, accession number P01861), human IGHD (non-limiting example, accession number P01880), human IGHE (non-limiting example, accession number P01854).

[0108] In some embodiments, the POI in the present invention is an endosomal escape domain or an endosomal escape site. In some embodiments, the endosomal escape domain can be the POI included in the TSPAN2 fusion polypeptide. Alternatively, the endosomal escape domain can exist as a second POI expressed on another construct.

[0109] The endosomal escape domains according to the present invention include the following: HA2, VSVG, GALA, B18. Other exemplary endosomal escape peptides include the following: HIV TAT PDT (peptide / protein transduction domain), HIV Gp-120, KALA, GALA and INF-7 (derived from the N-terminal domain of the hemagglutinin HA-2 subunit of influenza virus), endosomal escape sites that act by causing membrane fusion (e.g., diphtheria toxin T domain), proton sponge-type endosomal escape sites (e.g., peptides or lipids having histidine or imidazole sites), and CPPs and other sites that enable endosomal escape. CPPs are typically less than 50 amino acids in length, but may be longer, are typically highly cationic, rich in arginine and / or lysine amino acids, have the ability to access the interior of virtually any type of cell, and exemplary CPPs can be the following: transportan, transportan 10, penetratin, MTS, VP22, CADY peptide, MAP, KALA, PpTG20, proline-rich peptide, MPG peptide, PepFect peptide, Pep-1, L-oligomer, calcitonin peptide, various arginine-rich CPPs (e.g., polyarginine), tat, and combinations thereof).

[0110] The presence of endosomal escape domains is advantageous for promoting endosomal escape, thereby enhancing the bioactive delivery of EVs per se. The use of endosomal escape strategies is particularly important in the treatment of diseases where the cargo carried within EVs needs to be delivered into the cytoplasm of recipient cells or into other compartments outside of the endo-lysosomal system.

[0111] In a further embodiment, the POI can be a target site. The target site can modify the EV and enable targeted delivery of the EV to a target cell, tissue, organ, and / or compartment. In one embodiment, the target site can be the POI included in the TSPAN2 fusion polypeptide. Alternatively, the target site can exist as a second POI expressed on another construct. In an advantageous embodiment, the target site is modified to be presented on the surface of the EV.

[0112] The target site can be a protein, peptide, single-stranded fragment, or other derivative of an antibody obtained from humans or non-human animals such as.

[0113] Combining the presence of the target site with the presence of the ABD is particularly useful in organ targeting. For example, the EV can contain TSPAN2 fused to both a therapeutic protein / binding protein for a therapeutic cargo (POI loaded in the lumen) and the target site, and an ABD fused to tetraspanin on a second construct. The POI-TSPAN2-target site and tetraspanin-ABD constructs are expressed on the same EV, and when that EV is coated with albumin, it remains in circulation much longer, avoiding uptake by liver or immune system cells and thus reaching the desired target organ.

[0114] The target site can be used to target the EV to a cell, intracellular location, tissue, organ, or other body compartment. Organs and cell types that can be targeted include: brain, nerve 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.

[0115] Targeting can be achieved by various means, such as the use of targeting peptides. Such targeting peptides can be of any length from a few amino acids to hundreds of amino acids, for example, in the range of 3 to 100 amino acids, 3 to 30 amino acids, 5 to 25 amino acids, such as 7 amino acids, 12 amino acids, 20 amino acids, etc. The targeting peptides of the present invention may also include full-length proteins such as receptors, receptor ligands, etc. Furthermore, the targeting peptides according to the present invention may also include antibodies and antibody derivatives, such as monoclonal antibodies, single-chain variable fragments (scFv), nanobodies, and other antibody domains.

[0116] Exemplary target sites include the following: brain target sites (e.g., rabies virus glycoprotein (RVG), nerve growth factor (NGF) that binds to the NGF receptor, melanotransferrin, and FC5 peptide), and muscle target sites (e.g., muscle-specific peptide (MSP)).

[0117] The term "albumin binding domain" (ABD) should be understood to relate to any protein, peptide, antibody or nanobody, or fragment or domain thereof that can bind to albumin. 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 ABDs derived from the PAB protein from Peptostreptococcus magnus and protein G from group C and G streptococci, both of which bind to albumin with high affinity. Alternatively, the albumin binding domain of the present invention can be a single-domain antibody (sdAb) such as an antibody, scFv nanobody, heavy-chain antibody (hcAb), VHH or VNAR, or a fragment thereof that can bind to albumin. sdAbs and antibody fragments are particularly preferred because their small size allows other additional domains to be introduced into the fusion protein, facilitating the production and expression of the construct.

[0118] In certain embodiments, the EVs of the invention comprise a second POI that is 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 alternatively a classical EV protein.

[0119] Classical EV proteins according to the present invention can 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, CD274, 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-1 alpha, Mac-1 beta, MFGE8, myristoylated alanine-rich protein kinase C substrate (MARCKS) protein family member (e.g., 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 may be introduced into the wild-type sequences of the EV proteins to alter their function, and a preferred mutant according to the invention is CD63(Y235A). The use of the EV protein has the effect of promoting the loading of the POI into the EV, as a result of which the POI is actively loaded into the EV, and as a result the EV protein may be referred to as a carrier protein. Particularly preferred classical EV proteins include transmembrane proteins such as tetraspanins, LAMP2B, LIMP2, ICAM, integrins, ARRDC1, syndecan, syntenin, TNFR, TfR1, and Alix.,

[0120] As described above, the present invention relates to EVs comprising a fusion protein of TSPAN2-POI, TSPAN3-POI, or TSPAN18-POI. In other aspects, the present invention also relates to a polypeptide composition of TSPAN2-POI, TSPAN3-POI, or TSPAN18-POI, and any polynucleotide composition encoding said composition. As described above, the TSPAN2 / 3 / 18-POI fusion protein may also comprise additional domains including: (i) a release domain that is cleavable to release the POI; (ii) a linker or spacer; and / or (iii) a multimerization domain.,

[0121] In an advantageous embodiment, the TSPAN2 / 3 / 18-POI fusion protein further comprises a multimerization domain. The multimerization domain according to the invention can be a homomultimerization domain or a heteromultimerization domain. The multimerization domain of the present invention can be a dimerization domain, a trimerization domain, a tetramerization domain, or a higher-order multimerization domain. The multimerization domain enables dimerization, trimerization, or higher-order multimerization of the fusion polypeptide, thereby increasing the sorting and transport of the fusion polypeptide into EVs and also contributing to an increase in the yield of vesicles produced by EV-producing cells. Exemplary multimerization domains include the following: leucine zipper, fold-on domain, fragment X, collagen domain, 2G12 IgG homodimer, mitochondrial antiviral signaling protein CARD filament, cardiac phospholamban transmembrane pentamer, parathyroid hormone dimerization domain, glycophorin A transmembrane, HIV Gp41 trimerization domain, HPV45 oncoprotein E7 C-terminal dimer domain, and any combination thereof.

[0122] In another advantageous embodiment, the TSPAN2 / 3 / 18-POI fusion protein further comprises a linker and / or a spacer. The presence of the linker, spacer, and / or scaffold sequence creates flexibility, enabling the POI to be positioned optimally on the surface of the EV or, if necessary, in the lumen. The linker according to the invention is useful for improving the flexibility, pharmacokinetics (PK), expression, and biological activity of the fusion polypeptide construct and the corresponding polynucleotide construct, and also for avoiding steric hindrance and ensuring the maintenance of the functionality of the fusion polypeptide. Exemplary linkers according to the invention include glycine or serine linkers that improve stability or flexibility (e.g., (GGGGS) n (n = 1, 2, 4) or (Gly) 6 , (Gly) 8 ), rigid linkers (e.g., (EAAAK)n (n = 1 - 3), and A(EAAAK)4ALEA(EAAAK)4A), bend linkers (XP) nor a cleavable linker (e.g., disulfide, protease-sensitive sequence).

[0123] Suitable release domains according to the present invention can be cis-cleaving or "self-cleaving" sequences (e.g., intein), photoinducible monomeric or dimeric release domains (e.g., 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. Protease cleavage sites can also be incorporated into the fusion protein to allow for spontaneous release, etc., depending on the desired functionality of the fusion polypeptide. In the case of nucleic acid cargo, specific nucleic acid cleavage domains can be included. Non-limiting examples of nucleic acid cleavage domains include endonucleases such as, for example, Cas6, Cas13, modified PUF nucleases, site-specific RNA nucleases, etc.

[0124] The incorporation of a release domain is highly advantageous as it allows for the release of specific portions or domains from the original fusion polypeptide. This is particularly advantageous when the physiological delivery of the cargo is improved by releasing a portion of the fusion polypeptide and / or when a specific function of the fusion polypeptide works better as part of a smaller construct.

[0125] The term "self-cleaving protein" can mean a naturally occurring protein that cleaves itself through self-cleavage from the original host protein. Suitable examples of self-cleaving proteins include intein. It is understood that certain modifications are desirable to provide a protein that only has the ability to self-cleave (i.e., does not self-splice). Suitable examples of proteins that can only self-cleave (do not splice) include ΔI-CM.

[0126] The term "self-splicing protein" can mean a naturally occurring protein that cleaves itself from the original host protein through self-splicing and ligation of its adjacent peptide bond. A suitable example of a self-splicing protein is an intein.

[0127] The terms "mini-intein" or "delta-intein" are used synonymously herein and can be understood to relate to a modified intein, preferably derived from the parental RecA, that lacks an endonuclease domain.

[0128] The term "Fast-cleaving intein" is modified at the +C-extein position and / or the -N-extein position such that the cleavage rate of said intein is faster than that of the original RecA, intein, or mini-intein, and can be understood to relate to an intein or mini-intein.

[0129] The term "Slow-cleaving intein" is modified at the +C-extein position and / or the -N-extein position such that the cleavage rate of said intein is slower than that of the original RecA, intein, or mini-intein, and can be understood to relate to an intein or mini-intein. An example of a slow-cleaving intein is ΔI-CM.

[0130] A cis-cleavage sequence can be a self-cleaving protein, such as an intein. The intein can be a slow-cleaving or fast-cleaving intein. The intein can be a mini-intein, such as a mini-intein modified to optimize the cleavage rate. The intein can be a delta-intein-CM. Thus, a cis-cleavage sequence (or self-cleavage) according to the present disclosure can include, but is not limited to:

[0131] Inteins, mini-inteins, delta-inteins, and certain variants, mutants, and domains thereof that have a desired functionality (including, but not limited to, self-cleavage instead of splicing), such as a mini-intein modified to optimize cleavage rate. For example, a mini-intein having C1A, D24G, V67L, and / or D150G substitutions at the N-terminal portion (or appropriate -C-extein position). Said appropriate substitutions can be found in SEQ ID NO: 20. For example, splicing is enabled by the +1 position of the intein, where the +1 position is Cys. Substituting Cys with Ala in ΔI-CM removes the splicing function and allows only cleavage. Other mutations, substitutions, etc. will be understood to work similarly. Thus, the substitutions described hereinabove are exemplary and in no way limiting and may vary for each intein.

[0132] In some cases, the use of a slow-cleaving intein may be selected. A slow-cleaving system may be preferred when more time is required to efficiently load a desired cargo into an EV. Slow-cleaving inteins according to the present disclosure may include, but are not limited to: mini-inteins, delta-inteins, delta-intein-CM, and mini-inteins, and certain variants, mutants, and domains thereof that have a desired functionality (such as, but not limited to, cleavage action and cleavage rate instead of splicing). In a preferred embodiment, the slow-cleaving cis-cleavage release system is based on an intein system, where the C-terminal portion of the intein may include the amino acid sequence Val-Val-Val-His-Asn, and more preferably, the C-terminal portion of the intein is modified to include Val-Val-Val-His-Asn-Gly. Certain modifications at the +1 C-extein position have been observed to slow down the cleavage rate (i.e., result in slow cleavage).

[0133] In some cases, it may be selected to utilize a high-speed cleavage system (e.g., high-speed cleavage intain), such as a high-speed cleavage release system. When it is necessary for the EV to be rapidly taken up, the high-speed cleavage system may be preferred. In a preferred embodiment, the high-speed cleavage release system is based on an intain system, where the C-terminal portion of the intain may include the amino acid sequence Val-Val-Val-His-Asn or Val-Val-Val-His-Asn-Cys. Certain modifications at the +1C-extein position (e.g., the examples described above) have been observed to increase the cleavage rate (i.e., result in high-speed cleavage).

[0134] In a specific embodiment, the EVs of the present invention contain a plurality of POIs. This may be as a result of the plurality of POIs being present within a single TSPAN2 fusion protein, or alternatively as a result of a plurality of fusion proteins being loaded within a single EV. The plurality of fusion proteins may include the same or different scaffold proteins, and the same or different POIs. The presence of a plurality of POIs on a single EV is advantageous when improving the therapeutic effect of the EV. When the POIs are different, multiplexing of the cargo is possible, improving delivery, therapeutic effect, targeting, etc.

[0135] In certain embodiments, the POI cargo carried by the EV can be present on the inside of the EV, on the outside of the EV, or within the membrane of the EV. The desired location of the therapeutic cargo depends on the nature of the cargo and its mechanism of action. For example, membrane proteins are preferably located within the membrane of the EV, while decoy receptors, endosomal escape sites, or target sites are preferably present on the surface of the EV. However, cargo designed to be delivered into the cytoplasm or nucleus of the recipient cell, such as silencing RNA or AAV, is preferably located within the lumen of the EV. The design of the fusion proteins of the present invention enables expression on the surface or within the lumen of the EV.

[0136] As described above, fusing a POI to TSPAN2 or other classical EV proteins should be understood as an endogenous loading of said EVs. In alternative embodiments, therapeutic cargo may be passively or actively loaded into EVs by the exogenous loading methods described below.

[0137] Therapeutic cargo may be passively loaded into EVs by virtue of the therapeutic cargo being present in the cytoplasm of the EV-producing cells. Such passive loading applies, for example, to nucleic acids, small molecules, viruses, soluble proteins, or membrane proteins that are naturally loaded into EVs.

[0138] In certain embodiments, therapeutic cargo is actively loaded into the EVs of the present invention. One form of active cargo loading is exogenous active loading, which involves loading the cargo using any known exogenous loading method including: electroporation, transfection with transfection reagents such as cationic transfection agents and lipofectamine®, conjugation of the cargo to membrane anchor sites such as lipids or cholesterol tails, or loading by means of CPPs (in the form of CPP-cargo conjugates, or in the form of CPP-cargo non-covalent complexes, or any combination of these methods). Also, as a result of this type of active loading, the therapeutic cargo can be located on the inside, outside, or within the membrane of the EVs. Any of the cargoes defined above can be exogenously loaded. Particularly preferred embodiments include exogenous loading of nucleic acid or viral cargo by electroporation, CPP loading, or co-incubation with lipid-tagged cargo.

[0139] The present invention also relates to a population of EVs comprising at least one TSPAN2-POI fusion protein. Further, 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 noted above, the main advantage of using TSPAN2 as a scaffold protein for loading POIs into modified EVs is that it enables reliable and persistent co-localization of additional constructs on the same EV.

[0140] In certain embodiments, the average number of POIs per EV in a population of EVs according to the present invention may be greater than 1 POI per EV but less than 1 per EV. Further, in certain embodiments, the average number of POIs per EV in a population of EVs according to the present invention is greater than or less than 1 POI per EV. In another embodiment, in a population of EVs 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 may optionally comprise at least one additional POI.

[0141] Generally, EVs may essentially be derived from any cell source, which may be a primary cell source or an immortalized cell line. The EV-supplying cells may be of any of embryonic, fetal, and adult somatic stem cell types, including induced pluripotent stem cells (iPS cells) and other stem cells obtained by any method, as well as any adult cell source. The supplying cells according to the present invention may be selected from a wide range of cells and cell lines, for example, mesenchymal stem cells or stromal cells (e.g., obtainable from bone marrow, adipose tissue, Wharton's jelly, peripartum tissue, chorion, placenta, dental pulp, umbilical cord blood, skin tissue, etc.), fibroblasts, amniotic cells, more specifically amniotic epithelial cells that optionally express various early markers, myeloid-derived suppressor cells, M2-polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc. Particularly notable cell lines include the following: human umbilical vein endothelial cells (HUVEC), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular or lymphatic endothelial cells, erythrocytes, erythroblast progenitor cells, chondrocytes, mesenchymal stromal cells (MSC) of different origins, amniotic cells, amniotic epithelium (AE) cells, CAP® cells of CEVEC, any cells obtained through amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, etc. Also, immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DC), etc. are also within the scope of the present invention, and essentially any type of cell capable of producing EVs is also encompassed herein. The source cells may be essentially allogeneic, autologous, or xenogeneic to the patient to be treated, i.e., the cells may be derived from the patient himself or from unrelated, compatible, or incompatible donors.

[0142] Specifically, the present invention relates to cells stably modified to contain a polynucleotide construct according to the invention (as defined above), which encodes a TSPAN2-POI fusion protein, which is at least monocistronic, bicistronic, or polycistronic. Such cells may be stably or transiently transfected with the polynucleotide according to the invention and become modified EV-producing cells. Such cells may also be stably or transiently modified to contain a second construct encoding a second POI, which may optionally form part of a fusion protein with a classical EV protein. The cells of the present invention may be monoclonal or polyclonal cell lines.

[0143] Preferred producer cells according to the invention can be: HEK cells, HEK293 cells, HEK293T cells, MSCs, specifically WJ-MSC cells or BM-MSC cells, fibroblasts, amniotic cells, amniotic epithelial cells, CEVEC's CAP® cells, placenta-derived cells, umbilical cord blood cells, immune system cells, endothelial cells, epithelial cells, or any other cell type, where said cells may be, for example, adherent cells, suspension cells, and / or suspension-adapted cells. In a more preferred embodiment, the producer cells are HEK293 cells. In another more preferred embodiment, the producer cells are CAP cells.

[0144] Thus, in a preferred embodiment, the producer cells are HEK293 cells or CAP cells, and the polynucleotide construct encodes a TSPAN2-POI fusion protein.

[0145] The present invention also relates to a pharmaceutical composition comprising at least one EV according to the invention and a pharmaceutically acceptable excipient or carrier.

[0146] The terms "excipient" or "carrier" refer to inert substances that are added to a pharmaceutical composition to further facilitate the administration of a compound. This term encompasses any agent approved by a regulatory authority such as the FDA or EMEA, or described in the United States Pharmacopeia for use in animals (including humans), and any carrier or diluent that does not cause significant irritation to the subject and does not impair the biological activity and properties of the therapeutic cargo. Excipients and carriers are included that are useful in the preparation of pharmaceutical compositions and are generally safe and non-toxic.

[0147] Exemplary excipients include the following: stabilizing excipients that prevent degradation or loss of activity, such as proteins (e.g., human serum albumin), polyols (e.g., glycerol, sorbitol, and erythritol), amino acids (e.g., arginine, aspartic acid, glutamic acid, lysine, proline, glycine, histidine, and methionine), polymers (e.g., polyvinylpyrrolidone and hydroxypropylcellulose), surfactants (e.g., polysorbate 80, polysorbate 20, and pluronic F68), antioxidants (e.g., ascorbic acid and α-tocopherol (vitamin E)), buffers (e.g., acetic acid, succinic acid, citric acid, phosphoric acid, histidine, tris(hydroxymethyl)aminomethane (TRIS)), metal ions / chelating agents (e.g., Ca 2+ , Zn 2+ , and EDTA), cyclodextrin-based (e.g., hydroxypropyl β-cyclodextrin), and others (e.g., polyanions and salts), stabilizers or fillers (e.g., lactose, trehalose, dextrose, sucrose, sorbitol, glycerol, albumin, gelatin, mannitol, and dextran), or preservatives (e.g., benzyl alcohol, m-cresol, phenol, 2-phenoxyethanol).

[0148] The pharmaceutical compositions according to the present invention can be formulated by any known formulation method, including but not limited to the following: · Oral formulations - tablets, capsules, sustained release formulations, solutions · Intravenous formulations · Parenteral formulations · Topical formulations - dermal administration: creams, ointments, gels, pastes, powders · Modified release formulations - sustained release formulations · Liquid or lyophilized formulations

[0149] The EVs according to the present invention may be administered to human or animal subjects via various routes of administration, for example, auricle (ear), cheek, conjunctiva, skin, dental, iontophoresis, intracervical, intranasal, intratracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraperitoneal, intra-amniotic, intra-arterial, intra-articular, intra-biliary, intra-bronchial, intra-synovial, intra-cardiac, intra-cartilaginous, intra-caudal, intra-cavernous, intracavitary, intracerebral, intraventricular, intracisternal, intra-corneal, intra-coronal (dental), intra-coronary, intra-cavernous, intradermal, intra-laminar, intra-ductal, intra-duodenal, intradural, intra-epidermal, intra-esophageal, intra-gastric, intra-gingival, intra-ileal, intra-lesional, intra-luminal, intra-lymphatic, intramedullary, intrameningeal, intramuscular, intra-ocular, intra-ovarian, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intranasal, intraspinal, intra-synovial, intra-tendinous, intra-testicular, sub-arachnoid, intra-thoracic, intra-tubular, intra-tumoral, intra-tympanic, intra-uterine, intra-vascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intra-bladder, intra-vitreous, iontophoresis, perfusion, intra-laryngeal, intranasal, nasogastric, occlusive dressing, ophthalmic, oral, oropharyngeal, other, parenteral, transdermal, perijoint, peridural, perineural, periodontal, rectal, respiratory (inhalation), retro-orbital, soft tissue, sub-arachnoid, sub-conjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, trans-tympanic, ureteral, urethral, and / or intravaginal administration, and / or any combination of the above routes of administration, which usually depends on the disease to be treated and / or the characteristics of the EVs, the cargo molecules, or the EV population itself.

[0150] As will be apparent to those skilled in the art, when describing the medical and scientific uses and applications of EVs, the present invention is generally related to a population of EVs, which may typically include multiple EVs, i.e., thousands, millions, billions, or even trillions of EVs. EVs are present at a concentration of 10 per unit volume or per unit weight (e.g., per ml, or per L, or per kg of body weight).5 , 10 8 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 , 10 30 EVs (often referred to as "particles") may be present at concentrations, or at some other number that is greater than, less than, or intermediate to, such concentrations. Similarly, the term "population" is to be understood to relate, for example, to EVs containing a certain cargo, and to encompass the plurality of entities that make up such a population. In other words, when multiple individual EVs are present, they constitute an EV population. Thus, naturally, the present invention relates to both individual EVs and populations containing EVs, as will be apparent to those skilled in the art. The dosage of EVs when applied in vivo will, of course, vary widely depending on the disease being treated, the route of administration, the activity and effect of the cargo of interest, any target sites present on the EVs, the pharmaceutical formulation, and the like.

[0151] Any dosing schedule is contemplated to be applicable to the modified EVs of the present invention. The dosing schedule selected will depend on the cargo being delivered by the EVs, the disease being treated, and the administration of additional therapies determined by a physician skilled in the art.

[0152] The EVs of the present invention are contemplated to be administered multiple times, i.e., more than once, but usually more than twice, or possibly for chronic long-term treatment (i.e., administered dozens to hundreds, thousands of times). Preferably, when the cargo is an antigen administered as a vaccine, the immunization schedule includes more than two administrations of the polypeptide given over a period of several weeks. Similarly, when the cargo is an RNA agent such as, for example, 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 and usually multiple times as part of a chronic treatment regimen.

[0153] The present invention relates to a method for producing EVs according to the present invention. The method for producing EVs includes the following: (i) introducing a polynucleotide construct encoding a TSPAN2-POI fusion construct into EV-producing cells; and (ii) expressing the construct in the EV-producing cells, thereby generating EVs containing the TSPAN2-POI fusion protein.

[0154] The method for producing EVs according to the present invention may further include a second step of introducing a second polynucleotide construct encoding a second POI into the same EV-producing cells, wherein the second POI may be present in the form of a fusion protein with TSPAN2 or a classical EV protein. The POI may of course be any POI defined above.

[0155] The above methods for generating fusion proteins and EVs containing fusion proteins can be achieved by protein and cell modification techniques well known in the art, such as molecular cloning and transfection of cells using vectors encoding fusion constructs.

[0156] The method for producing EVs may further include the step of externally loading cargo molecules onto the EVs. As described above, the external loading step may include loading of cargo by any external loading method including the following: electroporation, microfluidic techniques, transfection using transfection reagents such as cationic transfection agents and lipofectamine®, conjugation of cargo to membrane anchor sites such as lipids or cholesterol tails, or loading by means of CPPs (in the form of CPP-cargo conjugates or CPP-cargo non-covalent complexes). Particularly preferred embodiments include methods for external loading of nucleic acid or viral cargo by electroporation, CPP loading, or co-incubation with lipid-tagged cargo.

[0157] The method for producing modified EVs according to the present invention may further include a step of purifying the EVs. Purification of the EVs is achieved by any method including but not limited to: 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, microfluidics-based separation, etc., or any combination thereof. In an advantageous embodiment, purification of the EVs is performed using a sequential combination of filtration (preferably ultrafiltration (UF), tangential flow filtration (TFF) or hollow fiber filtration) and affinity chromatography, optionally including size exclusion LC or bead elution LC. By combining the purification steps, the purity of the sample usually obtained is improved, resulting in excellent therapeutic activity. Furthermore, sequential filtration-chromatography is very rapid and scalable to larger production volumes compared to UC, which is routinely used to purify exosomes, and this is a major drawback of the current UC methods that dominate the prior art. Another advantageous purification method is TFF, which offers scalability and purity and can be combined with any other type of purification technique.

[0158] Alternatively, the present invention also relates to a method of producing EVs in a patient (i.e., patient-derived modified EVs), said method comprising administering to the patient a delivery vector comprising a polynucleotide according to the present invention, wherein the target cells within said patient produce EVs comprising a TSPAN2-POI, or TSPAN3-POI, or TSPAN18-POI fusion protein, and optionally said delivery vector also comprises a polynucleotide encoding a second construct comprising a POI that is also co-expressed on the same patient-derived EVs as the TSPAN2-POI construct. In a preferred embodiment, the polynucleotide is mRNA, circular mRNA, dbDNA®, linear DNA, circular DNA, plasmid DNA, linear RNA, circular RNA, self-amplifying RNA or DNA, "naked" or viral genome within a capsid, or a modified version of any of the foregoing. In a preferred embodiment, the delivery vector is a viral vector or a non-viral vector selected from the group consisting of lipid nanoparticles (LNPs), virus-like particles (VLPs), CPPs, polymers, or pharmaceutically acceptable carriers.

[0159] In one embodiment, the present invention thus also relates to a delivery vector comprising a polynucleotide according to the present invention, said polynucleotide cargo encoding a fusion protein comprising a POI and being configured to be translated into said fusion protein by EV-producing cells, said translation resulting in the production of at least 1 EV comprising a TSPAN2-POI, TSPAN3-POI, or TSPAN18-POI fusion protein, and optionally said delivery vector also comprises a polynucleotide encoding a second construct comprising a POI that is also co-expressed on the same patient-derived EVs as the TSPAN2-POI construct. Such patient-derived TSPAN2 / 3 / 18-POI EVs have all the same advantages as the above-described bioreactor-produced EVs, such as reliability of construct expression and the ability to co-express multiple constructs.

[0160] The present invention also relates to EVs according to the present invention for use in medicine.

[0161] The present invention also relates to a method of treatment comprising the step of administering to a patient in need thereof an effective amount of an EV according to the present invention or a pharmaceutical composition of the present invention.

[0162] Use in medicine or methods of treatment (collectively "treatment") can be effected by delivery of any kind of cargo according to the present invention. For example, treatment can be effected by delivery of a functional protein as protein replacement therapy, delivery of an mRNA encoding a functional protein that also acts as protein replacement therapy. Such protein replacement therapy can be, for example, enzyme replacement therapy (ERT) for diseases caused by inborn errors of metabolism such as phenylketonuria, urea cycle disorders, or lysosomal diseases. Treatment can be effected by delivery of: gene silencing RNA, splice-switching RNA, or CRISPR-Cas9 for gene editing. Treatment can be gene therapy by delivery of plasmid DNA, mini-circles, or viral gene therapy such as AAV or lentivirus. Treatment may be effected by presentation of an antigen or neoantigen for immunotherapy, acting as a vaccine that substantially induces an immune response. For example, EVs can act by delivery and / or presentation of tumor antigens for cancer immunotherapy, or viral, bacterial, or fungal antigens for immunity against pathogens. Treatment can be effected by delivery of small molecules, antibodies, and antibody-drug conjugates capable of mediating a therapeutic effect after delivery into the intracellular or extracellular matrix. In one embodiment, the treatment or therapy may be effected by EVs comprising multiple types of therapeutic cargo, i.e., the therapeutic cargo may be a mixture of proteins, nucleic acids, viruses, viral genomes, antigens, and / or small molecules.

[0163] Importantly, the present invention relates to the use of the EV compositions described herein for the prevention and / or treatment and / or alleviation of various diseases and is typically effected via the delivery of essentially any kind of pharmaceutical cargo such as, for example: nucleic acids (e.g., RNA molecules, DNA molecules, or mixmers, mRNA, antisense or splicing-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 (e.g., CRISPR-Cas9, TALEN, meganucleases), or vesicle-based cargo such as viruses (e.g., AAV, lentivirus, etc.). In one embodiment, the cargo may be a mixture of proteins, nucleic acids, viruses, viral genomes, antigens, and / or small molecules.

[0164] 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 molecule 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, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), kidney failure, heart failure, or any acute or chronic organ failure and related underlying etiologies, graft-versus-host disease, Duchenne muscular dystrophy and other muscular dystrophies, inborn errors of metabolism including: carbohydrate metabolism disorders (e.g., G6PD deficiency, galactosemia, hereditary fructose intolerance, fructose 1,6-diphosphatase deficiency, and glycogenosis), organic acid metabolism disorders (organic acidurias) (e.g., alkaptonuria, 2-hydroxyglutaric aciduria, methylmalonic acidemia, or propionic acidemia, multiple carboxylase deficiency), amino acid metabolism disorders (e.g., phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, amino acid metabolism disorders (e.g., hereditary tyrosinemia, non-ketotic hyperglycinemia, and homocystinuria), hereditary tyrosinemia, Fanconi syndrome, primary lactic acidosis (e.g., pyruvate dehydrogenase, pyruvate carboxylase, and cytochrome oxidase deficiency)), fatty acid oxidation and mitochondrial metabolism disorders (e.g., short-chain, medium-chain, and long-chain acyl-CoA dehydrogenase deficiency (alias beta-oxidation deficiency), Leigh syndrome, medium-chain acyl-CoA dehydrogenase deficiency (MCADD), MELAS, MERFF, pyruvate dehydrogenase deficiency), porphyrin metabolism disorders (e.g., acute intermittent porphyria), purine or pyrimidine metabolism disorders (e.g., Lesch - Nyhan syndrome), steroid metabolism disorders (e.g., congenital lipoid adrenal hyperplasia, congenital adrenal hyperplasia), mitochondrial dysfunction (e.g.,Kearns-Sayre syndrome), peroxisomal dysfunction (e.g., Zellweger syndrome and neonatal adrenoleukodystrophy, congenital adrenal hyperplasia or Smith-Lemli-Opitz syndrome, Menkes disease, neonatal hemochromatosis), urea cycle disorders (e.g., N-acetylglutamate synthase deficiency, carbamoyl phosphate synthase deficiency, ornithine transcarbamylase deficiency, citrullinemia (argininosuccinate synthase deficiency), argininosuccinic aciduria (argininosuccinate lyase deficiency), argininemia (arginase deficiency), hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome (mitochondrial ornithine transporter deficiency), type II citrullinemia (deficiency of citrin, an aspartate-glutamate transporter), lysinuric protein intolerance (mutation of y+L amino acid transporter 1), orotic aciduria (deficiency of the enzyme uridine monophosphate synthase UMPS)), all lysosomal diseases, e.g., α-mannosidosis, β-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, mucopolysaccharidosis (MPS) type I (Hurler syndrome), MPS I type (Scheie syndrome), MPS I type (Hurler-Scheie syndrome), MPS II type (Hunter syndrome), MPS IIIA type (Sanfilippo syndrome type A), MPS IIIB type (Sanfilippo syndrome type B), MPS IIIB type (Sanfilippo syndrome type C), MPS IIIB type (Sanfilippo syndrome type D), MPS IV type (Morquio type A), MPS IV type (Morquio type B), MPS IX type (hyaluronidase deficiency), MPS VI type (Maroteaux-Lamy syndrome), MPS VII type (Sly syndrome), mucolipidosis type I (sialidosis), mucolipidosis IIIC type,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, pycnodysostosis, Schindler disease, and Wolman disease, such as cystic fibrosis, primary ciliary dyskinesia, alveolar proteinosis, ARC syndrome, Rett syndrome, neurodegenerative diseases (including Alzheimer's disease, Parkinson's disease, GBA-related Parkinson's disease, Huntington's disease, and other triplet repeat-related diseases), prion diseases, dementia (including frontotemporal dementia), ALS, motor neuron diseases, multiple sclerosis, cancer cachexia, anorexia, type 2 diabetes, and various cancers.,

[0165] Specifically, the present invention is useful for the treatment of cancer by cancer immunotherapy, i.e., presenting cancer antigens on the surface of EVs, and as a result, these antigens elicit an immune response against the cancer antigens. Virtually all types of cancer are disease targets related to the present invention, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma (cerebellar or cerebral), basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone tumor, brainstem glioma, brain cancer, brain tumor (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic pathway and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor (pediatric, gastrointestinal), cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colorectal cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, 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 (brainstem glioma, cerebral astrocytoma, optic pathway and hypothalamic glioma), gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver cancer), Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, kidney cancer (renal cell carcinoma), laryngeal cancer, leukemia (acute lymphoblastic (also called acute lymphocytic leukemia), acute myeloid (also called acute myelogenous leukemia), chronic lymphocytic (also called chronic lymphocytic leukemia), chronic myelogenous (also called chronic myeloid leukemia), hairy cell leukemia), lip and oral cavity cancer, liposarcoma, liver cancer (primary), lung cancer (non-small cell, small cell), lymphoma, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, medulloblastoma, Merkel cell carcinoma, mesothelioma, metastatic cervical squamous cell carcinoma of unknown primary origin, mouth cancerCancer, multiple endocrine neoplasia, multiple myeloma / plasma cell tumor, fungating polyposis, myelodysplastic / myeloproliferative diseases, myeloid leukemia, chronic myeloid leukemia (acute phase, chronic phase), myeloma, nasal and paranasal cavity 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, low malignant potential ovarian tumor, pancreatic cancer, pancreatic islet cell cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pinealoblastoma 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 sarcoma family of tumors, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma), Sézary syndrome, skin cancer (non-melanoma, melanoma), small intestine cancer, squamous cell carcinoma, cervical squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and / or Wilms tumor.

[0166] The present invention is also particularly advantageous for the treatment of brain and central nervous system (CNS) disorders, as TSPAN2-expressing EVs are targeted to the brain while also being able to consistently express another POI such as a therapeutic protein or an endosomal escape site. Specifically, the present invention relates to EVs having an ABD on their surface and loaded with any kind of cargo according to the present invention, preferably a nucleic acid cargo, such as a silencing RNA, e.g., siRNA, targeting an RNA known to be involved in neurodegenerative diseases, such diseases being for example the following: Alzheimer's disease, Parkinson's disease, Huntington's disease, spinocerebellar degeneration, amyotrophic lateral sclerosis (ALS), frontotemporal dementia, motor neuron disease, multiple sclerosis, Wallerian degeneration and vitreoretinal detachment, Goldberg-Shprintzen Syndrome, Kuru disease, autoimmune GFAP astrocytopathy, MECP2 duplication syndrome, AQP4 astrocytopathy, familial pain syndromes (e.g., acroparaesthesia, paroxysmal extreme pain disorder, and congenital insensitivity to pain), Pelizaeus-Merzbacher disease, prion diseases (including Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI)), white matter dystrophies (including demyelinating, adult-onset, autosomal dominant, and white matter dystrophies).

[0167] The present invention particularly relates to EVs loaded with a therapeutic cargo (which can be a protein and / or a nucleic acid cargo) for the following treatments: Duchenne muscular dystrophy (DMD), diseases caused by inborn errors of metabolism (including lysosomal diseases (including Niemann-Pick disease type C (NPC) and Pompe disease), urea cycle disorders (e.g., argininosuccinic aciduria (ASA), and citrullinemia, and ornithine transcarbamylase (OTC) deficiency), metachromatic leukodystrophy, and phenylketonuria (PKU)).

[0168] When the POI is a binding protein such as an albumin-binding protein, an Fc binder, a nucleic acid-binding protein, a virus-binding protein, or a small molecule-binding protein, the invention also targets nanoparticle complexes comprising any TSPAN2 EV according to the invention bound to a corresponding binding partner (e.g., albumin, an Fc-containing protein, a nucleic acid, a virus, or a small molecule). In this embodiment, some or all of the EVs are bound to their corresponding binding partners. The invention is also related to pharmaceutical compositions comprising the nanoparticle complexes of the invention in combination with a pharmaceutically acceptable excipient or carrier.

Example

[0169] [Example 1 - Establishment of a high-throughput screening protocol] 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 created a list of potential EV scaffold domains based on a literature review and proteomics databases (Figure 1A). Candidates 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 from human embryonic kidney (HEK)-293T cells; (3) reported EV scaffold proteins used as references; and (4) proteins from the tetraspanin family with no known function / association with EVs to date. This fourth set of proteins was included because the hypothesis was made that other proteins within the tetraspanin family may also exhibit high loading efficiency, given that CD9, CD63, and CD81 are widely used in gene modification (Figure 1A). It should be noted that all proteins with a molecular weight exceeding 130 kDa were excluded. Finally, a total of 244 candidates with a median molecular weight of 38 kDa were included, 115 of which were transmembrane proteins (Figure 1A).

[0170] To enable screening of all these domains in a high-throughput manner, the inventors selected a reporter system based on bioluminescence using ThermoLuc, as it is a sensitive and stable reporter for labeling modified EVs. Proof-of-concept experiments were performed to confirm that Tluc-based assays can distinguish intravesicular cargo from the rest (surface and soluble fractions). HEK-293T cells were transfected with plasmids encoding Tluc alone or fused to CD63, and their conditioned media were fractionated using size-exclusion chromatography (SEC). Tluc activity in each fraction was quantified in the presence or absence of the surfactant Triton. As expected, Tluc activity was detected mainly in the soluble fraction in the conditioned media of Tluc-transfected cells, but shifted to the EV fraction when fused to CD63. Furthermore, Tluc activity was detected only after membrane disruption by Triton, indicating its location within vesicles (Figure 1B), demonstrating the feasibility of our approach. For comparative analysis of EV sorting capabilities, Tluc was fused to the C-terminus of 244 EV docking domains, and the resulting fusion cassettes were cloned into mammalian expression plasmids. These plasmids were then introduced into HEK-293T cells in a microplate format. A small amount of plasmid encoding another sensitive luciferase, namely NanoLuc (Nluc), was added to the transfection mixture as an internal control to adjust confounding factors such as transfection efficiency. In downstream analysis, candidates were compared mainly in the following aspects: total secreted Tluc, intravesicular Tluc (total secreted Tluc minus surface / soluble Tluc), and encapsulation index (ratio of surface / soluble Tluc to total secreted Tluc).

[0171] Figure 1 shows a high-throughput screening scheme. (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 either Tluc alone or the CD63-Tluc fusion protein, and their conditioned media were fractionated. Tluc activity in each fraction was quantified with or without membrane lysis using Triton. (1C) Experiment and data analysis scheme. HEK-293T cells were cultured in 96-well plates and co-transfected with 0.75 μg / mL of the fusion plasmid and 7.5 ng / mL of the Nluc plasmid for 48 hours. Samples were then centrifuged, and Tluc activity in the cell pellet and conditioned media was measured. Nluc activity was quantified only in the conditioned media.

[0172] [Example 2 - Validation of the Tluc High-Throughput Screening Platform] To confirm the robustness of our high-throughput screening platform, all candidates were ranked according to total secreted Tluc and the ratio of total secreted Tluc / Nluc.

[0173] Figure 2 shows the screening results for Tluc in the secretome of HEK-293T cells. (2A) Correlation between the rank of total secreted Tluc and the rank of total secreted Tluc / Nluc. (2B) Correlation between the rank of total secreted Tluc and the rank of the encapsulation index. (2C) Overview of the encapsulation index. Candidates with an encapsulation index greater than 1 (dashed line) were considered EV sorting domains. N = 5. (2D) Total secreted and intracellular Tluc for 36 EV sorting domains. The figure shows the ratio of intracellular Tluc to total secreted Tluc. N = 5. (2E) Correlation of the rank of the encapsulation index obtained from two independent batches. Each point in the scatter plot refers to one candidate, and the red points indicate the reference EV sorting domain CD63.

[0174] The results showed that normalization against secreted Nluc did not affect the relative secretion of Tluc (Figure 2A), and thus transfection efficiency was not considered a major confounding factor. The presence of three well-studied EV sorting tetraspanins (CD9, CD63, and CD81) and two reported domains (gag and PTGFRN) within this subset suggested the robust utility of our screening protocol (Figure 2D). The inventors found that candidates exhibited heterogeneous EV sorting capabilities, as evidenced by the low correlation between the ranking of encapsulation index and the ranking of total secreted Tluc (Figure 2B). Indeed, only 36 domains had an encapsulation index greater than 1, and these were consequently considered promising EV sorting domains in HEK-293T cells (Figure 2C). The observed low correlation and the fact that only 36 out of 244 promising candidates were effective indicate the difficulty of predicting which carrier / scaffold protein is useful in loading the POI into EVs without extensive screening and inventive efforts.

[0175] Notably, for both TSPAN2 and TSPAN3, over 90% of the total secreted Tluc was present within EVs, and TSPAN2 loaded more intracellular Tluc than CD63. The reproducibility of the screening was evaluated by comparing the rankings of candidates in terms of total secreted Tluc or secretion efficiency (the ratio of total secreted Tluc to cellular Tluc). The linear correlation between two independent batches and different doses showed good reproducibility. Importantly, the overall correlation between two independent batches was rather low with respect to the ranking of encapsulation efficiency. However, focusing only on the most promising 36 EV sorting domains (Figure 2E), the correlation improved dramatically, which corroborates the reliability of these domains.

[0176] [Example 3 - Verification of Sorting Ability into Small EVs] Based on the extensive EV sorting efficiency obtained from microplate-based screening, several representative and / or promising candidates were further investigated using SEC. Different from the screening protocol, the conditioned medium was further filtered through a 200 nm membrane prior to SEC fractionation and concentrated to enrich for small EVs (Figure 3A). In addition to using Triton to distinguish intracellular Tluc from others, proteinase K (ProK) was also added to each fraction to identify resistant protein aggregates (Figure 3B). Consistent with the excellent EV sorting ability found in the screening, TSPAN2 and TSPAN3 were ranked top in terms of the ratio of intracellular Tluc to total secreted Tluc and showed activity (>80%) superior to CD63 (Figure 3C).

[0177] Figure 3 shows the screening results regarding the sorting ability into small EVs. (3A) Validation scheme using size exclusion chromatography (SEC). HEK-293T cells were cultured in 15 mm Petri dishes and transfected with 1.5 μg / mL plasmid for 6 hours. The medium was changed to Opti-MEM and collected after 48 hours. After centrifugation, filtration through a 0.2 μm membrane, and subsequent concentration, the sample was fractionated and the Tluc activity in each fraction was quantified in the presence / absence of Triton or proteinase K. (3B) Relative Tluc activity in the SEC fractions of the conditioned medium. (3C) Ratio of intracellular Tluc to total secreted Tluc. The validation data were obtained from two independent experiments.

[0178] TSPAN2 and TSPAN3 were ranked top (80%) and fourth (45%) respectively in terms of the ratio of intracellular Tluc, while CD63 sorted only 28% of the Tluc into EVs. Consistent with the results from HEK-293T cells, SDCBP, CALM1, YWHAG, or BASP1 showed low EV sorting efficiency.

[0179] [Example 4 - Quantification of Engineered Small EVs at the Single Vesicle Level] To analyze EVs in more detail and obtain information about the concentration and relative abundance (through mean fluorescence intensity, MFI) of a certain transgene molecule per EV, single-vesicle flow cytometry was the method of choice. Luciferase is a useful reporter for quantifying modified EVs collectively. Tluc is a hybrid reporter consisting of the 11-mer peptide HiBiT (1.3 kDa) and the fluorescent protein mNeonGreen (mNG; 26.6 kDa), which was replaced (Figure 4A). HiBiT can complement its chaperone subunit LgBiT to form a fully functional luciferase complex (Nluc), allowing the amount of modified EVs to be compared with the results from Tluc-based screening. A total of 18 domains were investigated, including the top 5 domains in terms of encapsulation index, total secreted Tluc, or secretion efficiency obtained from screening reads, three EV markers (CD9, CD81, SDCBP), PTGFRN, and CALM1.

[0180] Figure 4 shows the results of this validation using a bifunctional bioluminescent and fluorescent reporter. (4A) Validation scheme using the hybrid HiBiT-mNG reporter. HEK-293T cells were cultured in 6-well plates and transfected with 1.5 μg / mL of plasmid for 6 hours. The medium was then changed to Opti-MEM and harvested after 48 hours. After centrifugation and filtration through a 0.2-μm membrane, the samples were quantified in terms of intracellular HiBiT activity and mNG fluorescence. (4B) Comparison of intracellular HiBiT to Tluc. (4C) Single-vesicle flow cytometry analysis of mNG-tagged small extracellular vesicles. (4D) Quantification of the concentration and mean fluorescence intensity of mNG-tagged small extracellular vesicles.

[0181] TSPAN3, TSPAN2, and CD63 consistently showed high EV sorting ability as demonstrated by high levels of intracellular HiBiT and encapsulation index (Figure 4B). Furthermore, flow cytometry results showed that TSPAN3 and TSPAN2 generated the largest number of modified EVs, approximately 3.5-fold more than CD63 (Figure 4C; Figure 4D). Interestingly, using the above two functional screenings, TSPAN18 also emerged as an efficient EV sorting domain, second only to TSPAN3.

[0182] As a result of removing large particles, the concentration of modified EVs in domain CALM1 was almost negligible. Calmodulin is considered an ideal domain for the sorting of cargo molecules into large EVs. This discovery is useful in applications that use large EVs for drug / gene delivery of larger cargo.

[0183] [Example 5 - Tluc Screening Results in a Range of Different Cell Lines] The inventors sought to identify EV scaffold proteins that are not only highly effective in a single producer cell line but also effective across multiple different cell lines and thus can be used multifunctionally as a platform technology rather than a narrow functionality. To test the EV sorting ability, four additional cell lines were tested: Freestyle 293-F, MSC (mesenchymal stem cells), Huh-7 (human hepatocytes), and TCMK-1 (mouse kidney epithelial cells). Based on the screening in HEK-293T cells, the inventors tested the top 95 domains to measure the encapsulation index, total secreted Tluc, intracellular Tluc, or secretion efficiency. Consistent with the findings in HEK-293T cells, transfection efficiency did not apparently confound the performance of the domains.

[0184] Figure 5 shows the results of evaluating Tluc in the secretomes of other EV-producing cell lines. (5A) Top 10 scaffold domains for intracellular Tluc in different producing cell lines. N = 3. (5B) Number of EV-sorting domains in each producing cell line and across multiple cell lines. (5C) Relative rank and its average value of 24 conserved EV-sorting domains for intracellular Tluc in each cell line. (5D) Intracellular location of 24 conserved EV-sorting domains. (5E) Interaction network of 24 conserved EV-sorting domains. The thickness of the line indicates the strength of data support.

[0185] TSPAN2 sorted the most Tluc into EVs in Freestyle 293-F and TCMK-1 cells, respectively, while CALM2 was the best domain in MSC and Huh-7 cells (Figure 5A). Overall, in the five cell lines tested, the number of EV-sorting domains (encapsulation index > 1) ranged from 30 to 37 (Figure 5B). A total of 24 domains were conserved, showing robust EV-sorting ability across different cellular contexts. The relative performance of each domain within the conserved subset of each cell line was determined based on intracellular Tluc activity (Figure 5C). On average, TSPAN2, CD63, and TSPAN3 showed the highest EV-sorting ability in a reproducible manner.

[0186] Subsequently, the inventors referred to online databases to gain insights into the underlying mechanisms responsible for the exceptional EV sorting ability of this stored subset. According to the annotations available on UniProtKB, most domains were characterized by a four-transmembrane structure, except for three calmodulin proteins and ANXA11 (Figure 5D). The intermolecular interactions obtained from the STRING database were also graphed (Figure 5E), revealing the central roles of CD9, CD63, and CD81, which is thought to reflect their previously widespread use in EV modification. Interestingly, while the calmodulin proteins appear to act independently of the other parts, the evidence for the interaction of TSPAN2 and TSPAN3 with CD63 is weak, which is due to the fact that TSPAN2 and TSPAN3 have never been recognized as EV proteins and have therefore not been used or considered for use as scaffold proteins for loading POIs into EVs.

[0187] [Example 6 - Comparison of TSPAN2 and TSPAN3 with CD63 as potential EV sorting domains] Both TSPAN2 and TSPAN3 stood out as promising candidates for sorting the protein of interest into small EVs, based on both screening and validation results. Further characterization of both modified EVs revealed a narrow size distribution with a median hydrodynamic diameter of approximately 120 nm (Figure 6A). Common EV markers such as CD81, syntenin-1, and TSG101 were found in these EV preparations (Figure 6B). As revealed by cryo-electron microscopy, the morphological appearance of TSPAN2- and TSPAN3-modified EVs was typical of EVs in terms of features such as membrane structure and size (Figure 6C).

[0188] To explore whether TSPAN2 and TSPAN3 confer distinct biological properties to modified EVs compared to CD63, the inventors utilized mNG and Tluc reporters to investigate intracellular uptake and in vivo distribution, respectively. As depicted in confocal microscopy images, all types of modified EVs were efficiently taken up by Huh-7 cells, and the majority of them were transported to lysosomes (Figure 6D). A similar trend in intracellular uptake was observed by gradually increasing the EV dose (Figure 6E; Figure S6). Since EVs are rapidly distributed and cleared in vivo, luciferin substrate was intraperitoneally administered 5 minutes prior to the intravenous injection of modified EVs, and Tluc activity in mice was continuously monitored through an IVIS spectrum in vivo imaging system (Figure 6F). For all three types of EVs, we observed rapid distribution to the liver and spleen and a stable decrease in systemic activity over 30 minutes (Figure 6G). Interestingly, TSPAN2- and TSPAN3-modified EVs showed a higher retention rate within the acquired time frame (Figure 6H). Tluc activity in major organs (ex vivo) and plasma was also measured, and the results supported that accumulation in the liver and spleen was dominant and, most importantly, improved the exposure compared to CD63 (Figure 6I; Figure 6J).

[0189] Specifically, Figure 6 shows the results of the characterization of TSPAN2- and TSPAN3-modified EVs. (6A) Size distribution of modified EVs measured by NTA. (6B) Western blot of modified EVs. (6C) Cryo-electron microscopy images of modified EVs. (6D) Confocal microscopy images of Huh-7 cells treated with mNG-labeled EVs for 4 hours. (6E) Mean fluorescence intensity of Huh-7 cells treated with mNG-labeled EVs for 8 hours. N = 3. (6F) Scheme for investigating the in vivo distribution of Tluc-labeled EVs. After the IVIS session, the mice were sacrificed immediately, and major organs and plasma were collected. N = 3. (6G) Representative IVIS images of mice injected with Tluc-labeled EVs. (6H) Quantification of total body luminescence in mice injected with Tluc-labeled EVs. (6I) Quantification of Tluc-labeled EVs in ex vivo organs. (6J) Quantification of Tluc-labeled EVs in plasma.

[0190] In summary, it was surprisingly discovered for the first time by the inventors that two mammalian proteins, TSPAN2 and TSPAN3, are superior to benchmark domains such as CD63, CD9, and CD81. It is known that a certain domain has excellent EV sorting ability in a specific cell line(s), but not in others (shown in Figure 5B). For example, the viral glycoprotein gag acted significantly in HEK-293T, Freestyle 293-F, and Huh-7 cells, acted to a lesser extent in TCMK-1 cells, but rather had little effect in MSCs. The inventors identified 24 domains that maintain EV sorting ability in a wide range of cell lines tested. Furthermore, overall weak intermolecular interactions in the conserved subset indicate a heterogeneous mechanism of modified EV biosynthesis and the resulting distinct molecular fingerprints and biological properties, which also emphasizes that the discovery of these specific scaffold proteins was unpredictable and possible only as a result of highly inventive and large-scale screening efforts. The results of the screening and validation experiments highlighted the excellent EV sorting ability of TSPAN2 and TSPAN3. Their splice isoforms were also investigated but showed little EV sorting ability (data not shown). In addition, TSPAN2- or TSPAN3-modified EVs were more efficiently taken up by cells in vitro and had even higher bioavailability over time after intravenous administration to mice than the benchmark CD63.

[0191] [Example 7: Initial results of large-scale functional screening to identify novel EV protein candidates enabling co-expression of multiple components] Following the experiments detailed in Examples 1-6, further investigations were conducted regarding the delivery of functional cargo. A total of approximately 280 potential EV proteins were screened. Importantly, the scaffold / carrier EV proteins were screened not only for their presence / distribution rate on exosomes, but also for the functional delivery of reporter proteins via two different systems: a co-culture system and a method of directly adding purified EVs to cells. These tests were also performed in a wide variety of different cell lines to ensure that the findings were robust and not cell-line specific.

[0192] Figure 7A shows a schematic diagram of the Cre reporter protein used to identify promising EV protein candidates. Figure 7B illustrates the co-culture method. Specifically, this method includes the steps of mixing EV-producing cells with reporter cells, enabling the EVs to be taken up by the cells, and analyzing GFP-positive cells by FACS measurement on day 4. Figure 7C illustrates the EV addition system. More specifically, this method includes the steps of isolating and purifying the EVs before they are added to the reporter cells, and then detecting GFP-positive cells by FACS analysis. These two different systems were used in combination. This is because the co-culture system is very economical, rapid, and easy to operate, and is suitable for preliminary screening, while the addition system provides a more accurate but time-consuming second screening.

[0193] HEK-293T cells were used as EV-producing cells. HEK-293T cells were co-transfected with an EV sorting protein-intein-Cre plasmid and a VSV-G plasmid. In both the co-culture and EV addition systems, the following reporter cell lines were tested: HeLa TL; T47D TL, and B16F10 TL. These reporter cells respond to recombination by Cre cargo delivered by EVs using a traffic light reporter system. Thus, it is possible to detect the delivery of functional Cre by EVs by the observed color change. In the co-culture system (Figure 7B), EV-producing cells and reporter cells were mixed at ratios of 1:1 and 1:5 (producing cells:reporter cells). In the EV addition system, EVs were added directly to reporter cells at doses of 1E10, 1E9, and 1E8.

[0194] The results of this tracking function screening are shown in Examples 7-12 below. The materials and methods for Examples 7-12 are as follows:

[0195] [Construct Generation] A codon-optimized DNA sequence encoding a scaffold protein comprising: tetraspanin (2 - 33), late endosomal proteins (STR3N, LAP4B, STAR3, SPP2A, SNX14, NTRK1), cytoplasmic proteins (syntenin 1, 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) was ordered from Twist Bioscience (USA). Tetraspanin-1, intein-cre, and VSV-G were ordered from Integrated DNA Technologies (USA). The scaffold protein was first cloned into the pLEX vector, and then intein-cre was inserted into the plex-scaffold plasmid by using the corresponding restriction endonuclease sites.

[0196] [Cell culture] In this study, HEK-293T cells were used to generate functional EVs and were cultured in DMEM medium (high glucose) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% anti-antibody (Gibco, USA). Cells were cultured at 37 °C in a humidified air atmosphere containing 5% CO2. Reporter cell lines (HeLa-TL, T47D-TL, MSC-TL, and B16F10-TL) were cultured in the same medium and conditions as HEK-293T cells. THP1-TL, RAW246.7-TL, and K562-TL were cultured using RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% Anti-anti (Gibco, USA).

[0197] [EV production] EVs were produced by transient transfection of the transgene using polyethyleneimine (PEI). Specifically, HEK-293T cells were seeded at 10 million cells / dish in 15-cm dishes using complete DMEM medium. The next day, the cells were transfected with the transgene, and 6 hours after transfection, the medium was changed to Opti-MEM medium (Gibco, USA) supplemented with 1% Anti-anti. At 48 hours after transfection, the conditioned medium (CM) was collected and centrifuged (700 g, 5 minutes, then 2000 g, 10 minutes). The supernatant was then filtered through a 0.22-μm filter system.

[0198] [EV Isolation] Tangential flow filtration (TFF, MicroKross, 20 cm 2 , Spectrum labs) was used to isolate EVs from the filtered CM. The cut-off of the TFF was 300 kDa, and particles larger than 300 kDa remained in the system and were concentrated. The subsequently concentrated particles were further concentrated by an Amicon Ultra-15 100-kDa (Millipore) spin filter and centrifuged at 4000 × g for 30 minutes to several hours at 4°C depending on the amount of EVs in the sample. Finally, the concentrated EVs were collected in an Eppendorf tube (Axygen, USA) with a maximum recovery of 1.5 ml, and the concentration was detected by nanoparticle tracking analysis (NTA).

[0199] [Nanoparticle Tracking Analysis (NTA)] The EV samples were diluted with freshly 0.22-μm filtered PBS, and then the particle size and concentration of the samples were confirmed using a NanoSight NS500 instrument. Five videos were taken at a camera level of 15 and in light scattering mode for longer than 30 seconds. The obtained data were analyzed using the attached NTA 2.3 software, and all samples were analyzed using the same constant settings.

[0200] [MACSQuant Flow Cytometry] After different traffic light reporter cells were added to EVs at different time points, or after reporter cells were co-cultured with EV-producing cells for 24 hours, their GFP expression was confirmed by using MACSQuant flow cytometry (Miltenyi biotec, Germany). Briefly, the cells in 96-well plates were washed once with PBS after discarding the supernatant, and then the cells were trypsinized at 37°C for 5 minutes. Subsequently, the cells were resuspended in cell culture medium supplemented with 10% FBS. After adding DAPI to confirm cell viability, the cells were sampled by MACSQuant using the same settings for all measurements of a specific reporter cell line. Finally, the data were analyzed by flowjo to calculate the percentage of GFP-positive cells.

[0201] [EV Uptake in Reporter Cells] Reporter cells were seeded in 96-well plates one day before adding modified EVs. After 48 hours, the percentage of GFP-positive cells was measured by MACSQuant.

[0202] [Co-Culture of Reporter Cells with EV-Producing Cells] HEK-293T cells were seeded in 6-well plates at 0.5 million cells per well. The next day, when the cells reached 60 - 70% confluence, the corresponding constructs were transfected into the wells by using lipofectamine2000 (Invitrogen, USA) according to the protocol provided by the manufacturer. Six hours after transfection, the medium was changed to fresh medium (DMEM + 10% FBS + 1% Anti-anti) to reduce the toxicity of the lipofectamine2000 used. Twenty-four hours after plasmid transfection, the cells were trypsinized and counted, and then mixed with the corresponding reporter cells at a ratio of 1:1 or 1:5 (ratio = EV-producing cells: reporter cells) in 96-well plates. After 24 hours of co-culture, the cells were trypsinized and measured by MACSQuant to confirm the percentage of GFP-positive cells.

[0203] Potential EV scaffold / carrier proteins in different categories, including cytoplasm, late endosomes, single-pass transmembrane proteins, and tetraspanins, were tested. Among the 280 proteins screened, tetraspanins were the most promising. 33 tetraspanins were tested (see Figure 9), and the top 13 of them advanced to further analysis after the initial screening (see Figures 9 and 10).

[0204] As can be seen from Figures 8A - F, the most efficient tetraspanin EV protein, excluding the co-delivery of VSVG and Cre, was TSPAN2 (the results of the classical EV protein CD63 are highlighted for comparison). This was true for each cell line and both test systems. This further supported the evidence from Examples 1 - 6 that identified TSPAN2 as a novel and highly effective EV scaffold protein capable of transporting cargo into EVs across a wide range of different EV cell sources.

[0205] Figure 9 shows the same data, presented as a heatmap of the percentage of GFP-positive cells. Figure 9 also shows the cut-off points for the top 13 tetraspanin candidates selected for further validation. TSPAN2 functioned best in all cell lines tested.

[0206] [Example 8 - Further validation of the top 13 tetraspanin candidates using other cell lines difficult to transfect] The top 13 candidates identified in Figure 9 were then further tested in different cell lines difficult to transfect: MSC, THP1, Raw264.7, and K562 cells. The EV addition system illustrated in Figure 7C above was used for this experiment.

[0207] As can be seen from Figure 10, among the top 13 scaffold protein candidates tested in cell lines difficult to transfect, TSPAN2 again remained the best candidate. This indicates the broad utility of TSPAN2 as a scaffold protein.

[0208] [Example 9 - Comparison between TSPAN2 and classical EV scaffold proteins (CD9 / 63 / 81)] The inventors then desired to compare the effectiveness of the newly identified EV protein candidate, TSPAN2, against classical EV proteins. The co-culture method described above was used to test the ability to co-express multiple constructs on the same vesicles when comparing TSPAN2 with classical EV proteins. This experiment was performed using three cell lines, HeLa, T47D, and B16F10, and the ability of TSPAN2 acting as a scaffold protein was compared with three classical EV proteins: CD9, CD63, and CD81. In this experiment, the cargo protein was the albumin-binding domain (ABD) and NanoLuc as a reporter (Figure 14 shows the design of the ABD construct).

[0209] Figure 11 shows that the albumin-binding domain (ABD) in the second loop of conventional tetraspanins (CD9, CD63, and CD81) cancels out the VSV-G delivery effect, while the ABD in the second loop of TSPAN2 enhances the VSV-G delivery effect. This surprisingly indicates that TSPAN2 is superior to its paralog CD9, and the other classical EV proteins CD63 and CD81, with respect to enabling both genetic manipulation for the purpose of transporting a POI and genetic manipulation for co-localizing multiple POIs on the same EV. The inventors discovered that, unlike the "classical" EV-related tetraspanins (CD9, CD81, and CD63), TSPAN2 can be modified while maintaining co-localization with another construct (in this case, VSV-G-intein-Cre). Thereby, TSPAN2 can function as a tool for much more flexible EV modification, enabling the development of a much simpler "platform technology" compared to conventional scaffold proteins.

[0210] [Example 10 - Confirmation that lack of co-localization causes a decrease in POI delivery] Subsequently, the inventors further confirmed by FACS that the decrease in POI delivery observed in Example 9 and Figure 11 was due to the lack of co-localization of the two required components. The details of the FACS protocol are as described above.

[0211] Figure 12 shows the results of FACS analysis comparing two fusion protein constructs: (i) VSVG-mNG + TSPAN2-mCardinal and (ii) VSVG-mNG + CD63-mCardinal. Figure 12 shows that while the mCardinal values are approximately the same between TSPAN2 and CD63, the mNG values (corresponding to the co-localized VSVG levels) are significantly lower when CD63 is overexpressed compared to when TSPAN2 is used as the scaffold. The inventors believe that the difference in results between CD63-expressing EVs and TSPAN2-expressing EVs is due to the fact that these proteins localize to different EV populations, and it is much easier for TSPAN2 to co-localize with the second construct than for CD63 to do so. This indicates the difficulty in introducing multiple constructs into exosomes when using the CD63 scaffold to modify the POI. As shown in Figure 11, modifying CD63 decreases the expression of other constructs, and thus TSPAN2 is much better at loading cargo into EVs than CD63. It should also be noted that this data shows that TSPAN2 can simultaneously deliver luminal and surface-presented POIs.

[0212] [Example 11 - In vivo data showing functional delivery of Cre protein] Subsequently, the inventors further tested the ability of TSPAN2 to deliver functional POI in vivo. A tumor-bearing mouse model was used to test in vivo delivery.

[0213] C57BL / 6 mice were ordered at approximately 5 weeks of age with a body weight of 20 g. The animals were acclimated to the new environment for at least 1 week before the experiment. B16F10-TL cells were harvested, resuspended in PBS, and then subcutaneously inoculated with 0.5 million cells per mouse. Ten days after inoculation, when obvious tumors were formed, the modified EV was directly injected into the tumor. The injection volume was 50 μL per mouse and contained 7.5×10 10 EV. Four days after intratumoral injection of EV, the mice were sacrificed and the tumors were harvested. Half of the tumor tissue was fixed with PFA and sent for slide preparation, and the other half was immersed in lysis buffer (0.1% TritonX-100) for DNA isolation. When slides with tumor tissue were prepared, IHC staining for GFP expression was performed, and the tissue in the lysis buffer was homogenized using a tissue lyser. 50 μL of tissue lysate was taken out for DNA isolation by using the Maxwell® RSC tissue DNA Kit (Promega, USA).

[0214] Figure 13A shows a schematic diagram of intratumoral injection and primer design in the melanoma model. Figure 13B shows the PCR results, indicating effective Cre recombination in vivo. The band intensity in Figure 13B shows that the TSN2-modified EV is superior in in vivo recombination to the CD63-modified EV. Figure 13C shows microscopic observations, again indicating the functional delivery of Cre protein by the TSPAN2-modified EV in the melanoma model. Figure 13 confirms that TSPAN2 is more effective in the delivery of functional cargo in vivo than CD63.

[0215] [Example 12 - Use of TSN2 for presenting ABD as an alternative POI] Next, the inventors tested the ability of TSPAN2 to express the albumin-binding domain (ABD) on EVs. ABD is used to attract albumin to EVs, thereby extending the half-life of circulating EVs. Figure 14A shows a schematic diagram of the TSPAN2 construct used in this experiment. ABD is presented on the surface of the EV.

[0216] The binding of ABD-modified EVs was evaluated using commercially available FITC-labeled human serum albumin (HSA-FITC). The isolated EVs were incubated with HSA-FITC and then size-excluded by QEV. 300 μL per fraction, and 48 fractions were collected from each sample. As a result, fractions 1-9 were assumed to contain only EVs (EV fraction), while fractions 10-48 were assumed to mainly contain soluble proteins. The results are shown in Figure 14B, indicating that the ABD expressed in the second loop of TSPAN2 provides the best HSA binding.

[0217] Figure 14 shows in vivo data, indicating that EVs decorated with albumin by presenting ABD on the surface of EVs fused to TSPAN2 extended the circulation time in vivo. When CD63 was conventionally used as a scaffold protein, an approximately 10-fold increase in half-life was observed. Here, when using TSPAN2, the increase in half-life was up to 12-fold more than what was possible with CD63.

[0218] This shows that TSPAN2 is an effective scaffold for the binding protein as a POI, and again shows that the delivery of the POI by TSPAN2, in this case the albumin-binding domain, achieves better results than those seen using classical EV proteins. This would not have been expected without the extensive screening efforts by the inventors. Before the development of the complex screening methods described above, TSPAN2 was not considered a practical scaffold protein because it was not highly enriched in its native form on EVs.

[0219] [Example 13 - Delivery of Ago2 ribonucleic acid complex by TSPAN2] Next, the inventors tested the ability of TSPAN2 to deliver ribonucleoprotein complexes as POIs. To do this, a model of EV-mediated silencing of Gapdh was used for both mRNA and protein levels. ShRNA against Gapdh (shGapdh) was indirectly loaded into EVs by fusing the RNA-binding protein Argonaute 2 (AGO2) to the EV sorting domain MYR and tetraspanin 2 (TSN2). Furthermore, VSVG was used as a fusogenic protein to ensure delivery of the cargo into the cytoplasm of recipient cells. The constructs were transiently co-transfected into parental HEK293T cells to produce modified EVs, which were then isolated and purified. When added to recipient cells, the EVs achieved dose-dependent target knockdown for both mRNA and protein levels after 48 hours. An enhanced silencing effect was observed when shGapdh was enriched by AGO2 fused to the EV sorting domain within the EVs.

[0220] [Cell culture] Cells were cultured in complete culture medium consisting of Dulbecco's Modified Eagle Medium high glucose (DMEM, Gibco Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific) and 1% antibiotic-antifungal (AA, Gibco, Thermo Fisher Scientific), and maintained at 37 °C in a 5% CO2 atmosphere unless otherwise specified.

[0221] For EV production, 1×10 7 human embryonic kidney cells (HEK293T) were seeded in 15 cm culture dishes and transfected in OptiMEM (Gibco, Thermo Fisher Scientific) with the plasmid of interest complexed with branched polyethyleneimine (Sigma-408727; 30 μg DNA: 45 μg PEI) 24 hours later. Four hours after transfection, the medium was changed to OptiMEM low serum medium supplemented with 1% AA. Cells were incubated for 48 hours before EV isolation.

[0222] For the EV uptake assay, mouse neuroblastoma (Neuro-2a) cells 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, respectively. After 24 hours, EVs were added (5×10 7 ~5×10 9 EVs / well for 24-well plates; 1×10 7 ~1×10 9 EVs / well for 96-well plates), and the cells were incubated for an additional 48 hours before RNA or protein extraction.

[0223] [Isolation of extracellular vesicles] Conditioned medium (CM) from pre-transfected HEK293T cells was collected and centrifuged at 700×g for 5 minutes, followed by centrifugation at 2000×g for 10 minutes to remove cell debris and large particles. The supernatant was passed through a 0.22 μm vacuum filter and concentrated to a final volume of 500 μL by ultrafiltration (UF) using an Amicon Ultra-15 100 kDa molecular weight cut-off spin filter (Millipore), and then loaded onto a qEV original / 70 nm Legacy size exclusion column (Izon Science). According to the manufacturer's instructions, the vesicle fraction was collected and further concentrated to a final volume of 100 μL using an Amicon Ultra-2 10 kDa molecular weight cut-off spin filter (Millipore), and stored at -80 °C for subsequent analysis.

[0224] [Nanoparticle tracking analysis] The particle size and concentration of the samples were determined via nanoparticle tracking analysis (NTA) using NTA 2.3 analysis software and a NanoSight NS500 equipped with a 488 nm laser. Briefly, the samples were diluted in 0.22 μm filtered PBS, and five 30-second videos were recorded and analyzed for each sample.

[0225] [RNA Isolation and RT-qPCR] Neuro-2a cells pre-incubated with EVs in 96-well plates were detached using trypsin-EDTA (0.25%, Gibco, Thermo Fisher Scientific), transferred to a V-bottom 96-well plate, and centrifuged at 900×g for 5 minutes. Optionally, the cell pellet was stored at -80°C until further processing. RNA was extracted on a Maxwell RSC Instrument (Promega) using the Maxwell RSC simplyRNA Cells Kit (Promega) according to the manufacturer's instructions. The resulting RNA concentration was determined on a Qubit 3 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). 5 ng equivalent of RNA was used per qPCR reaction on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using TaqMan Fast Advanced Master Mix (Applied Biosystems, Thermo Fisher Scientific) with the following primers and probes (Gapdh: forward GCC TTC CGT GTT CCT ACC, reverse CCT CAG TGT AGC CCA AGA TG, probe / 5HEX / CGC CTG GAG / ZEN / AAA CCT GCC AAG TA / 3IABkFQ / ; Hprt: forward GCC CTC TGT GTG CTC AAG, reverse CCC CGT TGA CTG ATC ATT ACA, probe / 56-FAM / AGC AGG TCA / ZEN / GCA AAG AAC TTA TAG CCC / 3IABkFQ / ).

[0226] [Protein Extraction and Western Blot] Neuro-2a cells pre-incubated with EV in a 24-well plate were detached using trypsin-EDTA (0.25%, Gibco, Thermo Fisher Scientific), transferred to tubes, and centrifuged at 900×g for 5 minutes. Optionally, the cell pellet was stored at -80°C until the next treatment or lysed in RIPA buffer. The cell debris was pelleted at 13000×g for 12 minutes, and the supernatant was transferred to a new tube. The subsequent steps were performed according to the technical guide of NuPAGE (Invitrogen, Thermo Fisher Scientific), using a NuPAGE® Novex® 4-12% Bis-Tris protein gel (Invitrogen, Thermo Fisher Scientific) and running in NuPAGE® MES SDS running buffer (Invitrogen, Thermo Fisher Scientific) at 120V for 2 hours. Proteins on the gel were transferred to a nitrocellulose membrane (iBlot 2 Transfer Stacks, Invitrogen, Thermo Fisher Scientific) using an iBlot 2 Gel Transfer Device (Invitrogen, Thermo Fisher Scientific) for 7 minutes. The membrane was blocked with Odyssey blocking buffer (LI-COR) for 60 minutes at room temperature with gentle shaking. After blocking, the membrane was incubated with a primary antibody solution (anti-β-actin [A5441, Sigma] and anti-Gapdh [PA1-16777, Invitrogen, Thermo Fisher Scientific], diluted 1:10000) overnight at 4°C or for 1 hour at room temperature. The membrane was washed 5 times for 5 minutes each with PBS containing 0.1% Tween 20 (PBS-T) and incubated with the corresponding secondary antibody (LI-COR) for 1 hour at room temperature (anti-mouse 680LT diluted 1:15000 for detecting β-actin; anti-rabbit IRDye® 800CW diluted 1:15000 for detecting Gapdh).The membrane was washed 5 times with PBS-T within 25 minutes and 1 time with PBS, and visualized on an Odyssey infrared imaging system (LI-COR).

[0227] Figure 15 shows the results of this experiment, indicating that TSPAN2 can be used to deliver ribonucleic acid cargo, and demonstrating once again the broad utility of TSPAN2 as a scaffold protein for delivering a wide variety of different types of cargo POIs.

[0228] [Example 14 - Delivery of mRNA by TSPAN2] In view of the very promising results described above, the inventors also wished to test whether TSPAN2 could be used to load mRNA via a nucleic acid-binding protein.

[0229] PUFeng was selected as the nucleic acid-binding protein, and mRNA of erythropoietin (EPO) designed to contain the PUFeng binding site was utilized as the cargo nucleic acid. To test the ability of EPO mRNA to be loaded into EVs, the following fusion constructs were compared:

[0230] TSPAN2-PUFeng TSPAN2-intein-PUFeng CD63-PUFeng CD63-intein-PUFeng TSPAN2-MS2 (non-binding control) TSPAN2-intein-MS2 (non-binding control) CD63-MS2 (non-binding control) CD63-intein-MS2 (non-binding control)

[0231] EV-producing cells were cultured and transfected according to the manufacturer's protocol in a total volume of 2 ml or 15 ml, and EV material was harvested after 72 hours. To purify the harvested material, the cell culture supernatant was centrifuged to pellet and then passed through a 0.2 um conical filter, after which the medium was added to a Proteus X-spinner spin filter and centrifuged at 2000×g for 30 minutes. RNA was extracted from EVs using Maxwell RSC with the "Maxwell (registered trademark) RSC miRNA from Plasma or Serum" kit according to the manufacturer's instructions and quantified by nanodrop. RT-PCR was then performed using the High-Capacity cDNA Reverse Transcription Kit with oligo dT primers.

[0232] Figure 16 shows that the loading capacity of TSPAN2 is more than 10-fold superior compared to the classical EV scaffold protein CD63. When using TSPAN2 as the scaffold protein, 44,572 copies of mRNA were loaded, whereas only 3,667 copies were loaded when using CD63.

[0233] Specifically, Figure 16 shows that loading of EPO mRNA into EVs with clear enrichment was observed in the PUFeng sample compared to the MS2 (unbound) control, and the mRNA copy number in the producing cells remained approximately the same (data not shown), indicating that loading is an active process. Once again, this is a very surprising discovery because it has not been previously known that TSPAN2 is highly expressed on EVs and thus has not been previously considered as a promising scaffold protein, especially in the context of nucleic acid cargo.

[0234] [Example 15 - Delivery of NanoLuc and msOx40L mRNA by TSPAN2 and TSPAN3] The inventors further investigated whether TSPAN2 and TSPAN3 could be used to load other mRNAs via PUF nucleic acid-binding proteins. In these experiments, constructs containing MS2 (which is not an RNA-binding domain) were used as controls.

[0235] NanoLuc mRNA and codon-optimized mouse Ox40L mRNA designed to contain the PUFeng binding site were used as cargo nucleic acids. To test the ability of NanoLuc or msOx40L mRNA to be loaded into EVs, the following fusion constructs were compared:

[0236] TSPAN2-PUFeng TSPAN2-MS2 (non-binding control) TSPAN3-PUFeng TSPAN3-MS2 (non-binding control) CD63-PUFeng CD63-MS2 (non-binding control)

[0237] HEK293T cells were cultured and transfected with the constructs by standard methods, and the conditioned medium was collected from the cells 48 hours after transfection. The EV material was purified by centrifugation at 700×g for 5 minutes to remove residual cells and then at 2000×g for 10 minutes to remove cell debris. The centrifuged medium was then filtered through a polyethersulfone (PES) membrane filter with a pore size of 0.22 μm (Techno Plastic Products). The filtered conditioned medium was ultracentrifuged at 100000×g for 90 minutes at 4°C, and the pellet was resuspended in 200 μL of sterile PBS for immediate use.

[0238] RT-PCR was then performed using the High-Capacity cDNA Reverse Transcription Kit® (Thermo Fisher Scientific) with oligo dT primers.

[0239] Figures 17a and 17b show that both TSPAN2 and TSPAN3 have the ability to load more NanoLuc mRNA (Figure 17a) and msOx40L mRNA (Figure 17b) than the classical EV scaffold protein CD63. When TSPAN2 was used as the scaffold protein, 8715 copies of NanoLuc mRNA and 41042 copies of Ox40L mRNA were loaded. When TSPAN3 was used as the scaffold protein, 4473 copies of NanoLuc mRNA and 19231 copies of Ox40L mRNA were loaded, compared to 3165 and 7902 copies of NanoLuc mRNA and Ox40L mRNA, respectively, when CD63 was used as the scaffold protein. Interestingly, the data also show that passive loading of mRNA (occurring independently of the PUF mRNA binding domain) was also increased when TSPAN2 or TSPAN3 was expressed compared to when CD63 was expressed.

[0240] [Example 16 - Improvement of Modification Using TSN2] The inventors wished to investigate how the EV expression of a modified TSPAN2 (also known as TSN2) construct compares to that of constructs containing classical EV scaffold proteins.

[0241] HEK-CTS cells (Thermo Fisher Scientific) were transfected with the following constructs: - CD63-Flag-eGFP (Flag at the C-terminus of CD63 and eGFP at the C-terminus of Flag) - CD63-Z(L2)-Flag-eGFP (Flag at the C-terminus of CD63 and eGFP at the C-terminus of Flag; the Z domain was presented on the second loop of CD63) - TSN2-Flag-eGFP (Flag at the C-terminus of TSN2 and eGFP at the C-terminus of Flag) - TSN2-Z(L2)-Flag-eGFP (Flag at the C-terminus of TSN2 and eGFP at the C-terminus of Flag; the Z domain was presented on the second loop of TSN2).

[0242] Three days after transfection, the conditioned medium (CM) was collected and centrifuged at 4000 × g for 10 minutes to pellet cells and cell debris. The supernatant was then concentrated (up to 10-fold) using a spin filter (Proteus X-spinner 2.5 Pack, 300 kDa MWCO, PES membrane, catalog number PAL-X-300-96; Generon) and reduced to approximately 200 μL.

[0243] The filtered CM was analyzed for eGFP content by EV flow cytometry performed on a NanoAnalyser N30E (NanoFCM).

[0244] Figure 18a shows that EVs derived from cells transfected with the TSN2 construct are more eGFP-positive (compare columns 1 and 3) than EVs derived from cells transfected with the CD63 construct, indicating that the TSN2 construct results in more efficient modification. Insertion of the domain sequence into the second loop decreases the modification efficiency of both scaffolds (compare columns 1 and 2, and columns 3 and 4), but TSN2 is less affected by this phenomenon.

[0245] Figure 18b shows that for EVs derived from cells transfected with the TSN2 construct, the mean fluorescence intensity (MFI) is almost twice that of EVs derived from cells transfected with the CD63 construct (compare columns 1 and 3), indicating that the TSN2 construct results in more scaffold molecules localized to each modified EV. Inserting the domain sequence into the second loop of CD63 decreases the MFI (compare columns 1 and 2), while this is not the case when the domain sequence is incorporated into the second loop of TSN2 (compare columns 3 and 4). The data in Figures 18a and b show that TSN2 is highly adaptable to large-scale modifications while maintaining a significantly higher expression level of the POI on EVs.

[0246] [Example 17 - Further Evidence for Improved Modification Using TSN2] The inventors wished to investigate whether the above results in Example 16 would occur in different cell lines and when different modifications were made to the second loop of the scaffold protein.

[0247] EK CTS® VPC.2 cells (Thermo Fisher Scientific) were transfected with the following fusion constructs: - Lamp2b, in which eGFP was incorporated at the C-terminus and a VHH against the transferrin receptor (TfR) was incorporated at the N-terminus. - TSN2, in which eGFP was incorporated at the terminus and a VHH against TfR was incorporated into the loop. - CD63, in which eGFP was incorporated at the terminus and a VHH against TfR was incorporated into the loop.

[0248] The cells were transfected using plasmid DNA according to the manufacturer's instructions, using FectoPro® (PolyPlus Transfection) as the transfection reagent. After 72 hours, the conditioned medium was harvested through differential centrifugation steps at 300×g and 700×g for 5 minutes each to remove cells and larger debris. Then, it was centrifuged at 4000×g for 20 minutes to remove smaller debris. The conditioned medium was then ultracentrifuged at 100000×g for 2 hours to precipitate EVs. The formed pellet was then resuspended in storage buffer and centrifuged at 4000×g to remove any possible debris.

[0249] EV flow cytometry was performed on a NanoAnalyser N30E (NanoFCM).

[0250] Figure 19a shows that EVs derived from cells transfected with the TSN2 construct have a higher percentage (more than 10% higher) of eGFP positivity compared to EVs derived from cells transfected with the Lamp2b construct or the CD63 construct.

[0251] Figure 19b shows that the fluorescence intensity was improved by about 3-fold in EVs derived from cells transfected with the TSN2 construct compared to EVs derived from cells transfected with the Lamp2b construct. In EVs derived from cells transfected with the TSN2 construct, the fluorescence intensity was improved by about 4-fold compared to EVs derived from cells transfected with the CD63 construct. This indicates 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.

[0252] [Example 18 - Long-Term Stability of Cell Lines] To test the long-term stability of TSPAN2 EVs (both the stability of expression of the TSPAN2-POI construct alone and the co-expression stability of TSPAN2-POI when co-expressed with a second construct encoding the POI), EV-producing cells were generated by transduction using a lentiviral vector and expressed the desired fusion protein(s). Both these producer cells and the produced EVs were then investigated longitudinally, and the stability of cargo protein expression was observed by Western blotting, flow cytometry, bioluminescence, etc.

[0253] [Example 19 - Stability of the TSN2 Construct over Time in EV-Producing Cells] In the field of modified EV production, it is a known problem that the expression of constructs containing classical tetraspanin EV scaffold proteins is lost over time from EV-producing cells and secreted EVs. The inventors further investigated the stability of constructs containing TSPAN2 in EV-producing cells compared to constructs containing the classical tetraspanin EV scaffold CD63.

[0254] EV-producing cells (HEK293 VPC1.0 CTS (Thermo Fisher Scientific) or CEVEC CAP (CEVEC Pharmaceuticals) cells) were transfected with the CD63-G4S-eGFP construct or the TSN2-G4S-eGFP construct. The G4S linker consists of four glycines and one serine (GlyGlyGlyGlySer), which makes a flexible peptide that connects eGFP to the scaffold protein. This allows the presence of the scaffold to be inferred by the presence of eGFP.

[0255] Cells with high eGFP expression were sorted using FACS before cryopreservation. Following recovery from thawing, antibiotic selection was reintroduced into the culture. Cells were maintained in continuous culture during the course of the study, with or without antibiotic selection. Only cells expressing the CD63-G4S-eGFP construct or TSN2-G4S-eGFP should survive antibiotic selection. The G4S linker consists of four glycines and one serine (GlyGlyGlyGlySer), which makes a flexible peptide that connects eGFP to the scaffold protein. This allows the presence of the scaffold to be inferred by the presence of eGFP.

[0256] At two time points, RNA was extracted from cell pellets according to the manufacturer's instructions (Maxwell® RSC simplyRNA Tissue Kit; Promega). Time point 1 was taken when the cells were at passage 2, and time point 2 was taken when the cells were at passage 13. In HEK293 cells, the two time points were approximately 7 weeks apart, and in CAP cells, the two time points were approximately 6 weeks apart.

[0257] 30 ng of RNA was used for cDNA synthesis using the High-Capacity cDNA Synthesis Kit (Thermo Fisher Scientific), and the random primer was replaced with a 15-mer oligo DT according to the manufacturer's instructions. 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 and determine the absolute change in mRNA expression between eGFP and GAPDH over the test period.

[0258] Figure 20a shows that the expression of the CD63 construct was lost over time from HEK293T cells. Surprisingly, despite being the same tetraspanin, Figure 20b shows that the TSN2 construct continued to be expressed at comparable levels over time by HEK293T cells.

[0259] Figures 20c and 20d show the results observed using CAP cells. In this case, the expression of the CD63 construct was lost over time (Figure 20c), while the expression of the TSN2 construct increased over time (Figure 20d).

[0260] Therefore, in both HEK and CAP cells, when eGFP is expressed in a construct fused to TSN2 rather than CD63, it is observed that the stability of eGFP mRNA expression is clearly improved. Thus, using TSN2 as a scaffold protein overcomes the problem of decreasing expression over time observed when using CD63 as a scaffold.

[0261] [Example 20 - Temporal Stability of Modified EV Production by EV-Producing Cells Expressing the TSN2 Construct] Based on the positive results in Example 19, the inventors further investigated the expression of eGFP within EVs secreted by cells transfected with the TSPAN2 construct compared to cells transfected with the CD63 construct.

[0262] In these experiments, conditioned media were collected from EV-producing cells (HEK293 VPC1.0 CTS or CEVEC CAP cells, as before) at two time points. Time point 1 was taken when the cells were at passage 2, and time point 2 was taken 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.

[0263] To isolate EVs, 20 mL of conditioned media were centrifuged at 300×g for 5 minutes, and the resulting supernatant was centrifuged at 4000×g and concentrated to 1 mL using an Amicon MWCO column with a molecular weight cut-off of 100000 kDa.

[0264] The Pierce Micro BCA assay (Thermo Fisher Scientific) was performed according to the manufacturer's instructions, and 20 μg of protein was evaluated under reducing conditions using Western blot. The reduced proteins were separated in 1×MES running buffer at 200 V for 35 minutes using a Bolt 4-12% Bis-Tris gel (Thermo Fisher Scientific). The proteins were transferred onto a nitrocellulose membrane using an iBlot 2 Membrane and then blocked with Intercept TBS blocking buffer for 1 hour at room temperature. The blocked membrane was probed for eGFP (Chromotek clone 3H9) and syntenin (Abcam EPR8102). Concentration measurement analysis was performed by normalizing the syntenin band corresponding to the eGFP band at 50 kDa.

[0265] Figure 21a shows that the expression of eGFP in EVs secreted by HEK293T cells expressing the CD63 construct was lost over time. Figure 21b shows that the expression of eGFP in EVs secreted by HEK293T cells expressing the TSN2 construct was maintained in cells under antibiotic selection. Thus, in a cell population where all cells express the TSN2-G4S-eGFP construct, the amount of modified EVs produced remains constant over time. Even in the absence of antibiotic selection (where the cell population consists of a mixed population and only some cells express the TSN2-G4S-eGFP construct), the relative expression of eGFP in the secreted EVs remained above 0.5 at later time points and was thus significantly improved compared to the results observed when the CD63-G4S-eGFP construct was expressed.

[0266] Figures 21c and 21d show the results observed using CAP cells. In this case, the expression of eGFP in EVs from CAP cells expressing the CD63 construct was also lost over time (Figure 21c), while the expression of eGFP in EVs from CAP cells expressing the TSN2 construct increased over time (Figure 21d).

[0267] Thus, it is observed that in EVs derived from both HEK and CAP cells, the stability of eGFP expression is clearly improved when eGFP is expressed in a construct fused to TSN2 rather than CD63. Thus, using TSN2 as a scaffold protein helps to overcome the problem of decreased production of modified EVs over time, which is observed when CD63 is used as a scaffold.

[0268] [Sequence Listing] SEQ ID NO: 1 (TSPAN2 protein) MGRFRGGLRCIKYLLLGFNLLFWLAGSAVIAFGLWFRFGGAIKELSSEDKSPEYFYVGLYVLVGAGALMMAVGFFGCCGAMRESQCVLGSFFTCLLVIFAAEVTTGVFAFIGKGVAIRHVQTMYEEAYNDYLKDRGKGNGTLITFHSTFQCCGKESSEQVQPTCPKELLGHKNCIDEIETIISVKLQLIGIVGIGIAGLTIFGMIFSMVLCCAIRNSRDVI

[0269] Sequence number 2 (TSPAN2 nucleic acid) ATGGGCCGGTTTAGGGGCGGTCTGAGATGTATCAAGTACCTCCTCCTTGGTTTCAACCTCCTGTTTTGGTTGGCCGGAAGCGCTGTGATCGCCTTCGGATTGTGGTTTAGATTCGGAGGCGCAATCAAGGAACTGTCATCTGAGGACAAGTCACCAGAGTACTTTTACGTGGGGCTCTATGTACTCGTGGGAGCCGGGGCCCTGATGATGGCCGTGGGTTTCTTCGGGTGTTGCGGAGCAATGAGGGAAAGCCAGTGCGTGCTTGGGTCTTTCTTTACTTGTCTGCTGGTCATCTTTGCCGCCGAGGTGACTACTGGTGTCTTTGCATTCATCGGAAAGGGCGTCGCTATCAGGCACGTGCAGACCATGTATGAAGAAGCCTATAACGACTATCTCAAGGACCGGGGAAAGGGTAACGGCACCCTTATCACATTTCACTCTACTTTTCAGTGCTGTGGCAAAGAATCAAGTGAGCAGGTCCAACCCACTTGTCCCAAAGAACTGCTCGGCCATAAGAACTGCATCGACGAAATCGAGACTATCATCTCCGTGAAGTTGCAGCTCATAGGGATCGTAGGCATCGGTATTGCTGGTTTAACTATCTTCGGTATGATCTTCAGCATGGTACTGTGTTGTGCTATTAGAAATAGCCGAGATGTTATA

[0270] Accession number 3 (TSPAN3 protein) MGQCGITSSKTVLVFLNLIFWGAAGILCYVGAYVFITYDDYDHFFEDVYTLIPAVVIIAVGALLFIIGLIGCCATIRESRCGLATFVIILLLVFVTEVVVVVLGYVYRAKVENEVDRSIQKVYKTYNGTNPDAASRAIDYVQRQLHCCGIHNYSDWENTDWFKETKNQSVPLSCCRETASNCNGSLAHPSDLYAEGCEALVVKKLQEIMMHVIWAALAFAAIQLLGMLCACIVLCRRSRDPAYELLITGGTYA

[0271] Accession number 4 (TSPAN3 nucleic acid) ATGGGGCAGTGTGGGATCACTAGTAGCAAAACCGTGCTGGTCTTTCTGAATCTTATCTTTTGGGGAGCCGCGGGGATCCTGTGCTATGTGGGGGCATACGTGTTCATTACCTACGACGACTATGATCATTTCTTTGAAGATGTGTACACTCTCATACCCGCAGTTGTCATTATTGCAGTTGGTGCGCTGCTGTTCATAATAGGACTCATCGGCTGTTGCGCAACTATTCGAGAGAGCAGGTGTGGCCTGGCTACCTTCGTAATTATCCTGCTTCTGGTATTCGTGACAGAAGTAGTGGTAGTGGTCCTGGGTTACGTTTACAGGGCGAAGGTGGAGAATGAAGTTGACCGGTCTATTCAAAAAGTCTACAAGACCTATAATGGGACGAATCCAGATGCCGCGTCTAGAGCTATTGATTATGTGCAGCGACAGCTGCACTGCTGCGGAATCCACAATTATTCCGATTGGGAGAACACAGATTGGTTCAAAGAAACAAAGAACCAATCCGTGCCACTGTCCTGCTGCAGGGAGACAGCATCCAACTGCAATGGATCACTCGCCCATCCCAGTGACCTCTATGCCGAGGGCTGTGAGGCCTTAGTGGTCAAGAAACTCCAAGAAATTATGATGCACGTGATCTGGGCTGCCTTGGCCTTCGCAGCTATCCAACTGCTGGGGATGTTATGTGCGTGTATTGTTCTGTGCCGCCGCAGCAGGGACCCTGCTTACGAGCTCCTGATTACGGGGGGCACCTATGCA

[0272] Sequence number 5 (TSPAN18 protein) MEGDCLSCMKYLMFVFNFFIFLGGACLLAIGIWVMVDPTGFREIVAANPLLLTGAYILLAMGGLLFLLGFLGCCGAVRENKCLLLFFFLFILIIFLAELSAAILAFIFRENLTREFFTKELTKHYQGNNDTDVFSATWNSVMITFGCCGVNGPEDFKFASVFRLLTLDSEEVPEACCRREPQSRDGVLLSREECLLGRSLFLNKQGCYTVILNTFETYVYLAGALAIGVLAIELFAMIFAMCLFRGIQ

[0273] Sequence number 6 (TSPAN18 nucleic acid) ATGGAAGGCGATTGTCTCAGTTGCATGAAGTACCTGATGTTCGTTTTCAATTTTTTTATTTTCCTCGGGGGGGCGTGTCTGCTGGCCATTGGGATTTGGGTGATGGTAGACCCTACAGGCTTTAGGGAAATAGTGGCTGCCAACCCTCTGCTCCTTACTGGAGCCTACATTCTTCTGGCCATGGGCGGGCTGCTGTTTCTCTTGGGCTTCTTGGGGTGTTGCGGAGCCGTCAGAGAAAATAAGTGTTTGCTGCTTTTCTTCTTTTTGTTCATACTTATAATATTTCTGGCAGAGCTCAGCGCTGCTATTTTGGCATTTATTTTTAGGGAAAATCTCACCCGCGAATTTTTCACCAAAGAACTGACCAAACATTATCAGGGCAACAATGACACAGATGTATTTTCTGCAACGTGGAACTCTGTTATGATTACTTTTGGCTGCTGCGGGGTAAATGGCCCTGAGGACTTCAAATTCGCTTCCGTCTTCCGCCTGCTGACCTTGGACTCAGAGGAGGTACCCGAAGCCTGTTGTCGCAGGGAACCCCAATCAAGAGACGGCGTGCTGCTGTCTAGGGAAGAGTGCCTGCTGGGGCGTAGTTTATTCCTGAACAAACAGGGTTGTTATACCGTAATCCTGAATACGTTCGAAACATACGTTTATCTTGCTGGAGCCCTGGCTATTGGGGTGCTCGCTATTGAGCTGTTCGCAATGATATTTGCAATGTGTCTTTTCCGGGGCATCCAG

Claims

1. Extracellular vesicles (EVs) containing fusion proteins, The aforementioned fusion protein includes TSPAN2 fused to the target protein (POI). Extracellular vesicles.

2. An EV as described in claim 1, The aforementioned POI is integrated into loop 1, loop 2, or both loops of TSPAN2. EV.

3. An EV as described in claim 1, The POI is fused to the N-terminal domain (NTD), C-terminal domain (CTD), or other luminal portion of TSPAN2. EV.

4. An EV as described in claim 1, The aforementioned fusion tomato is: (i) an emission domain that can be cleaved to release the POI; (ii) Linker or spacer; (iii) Multimerization domain; and / or (iv) at least one further POI; Further including, EV.

5. An EV as described in claim 1, The EV further comprises a second POI expressed on another construct. EV.

6. An EV according to claim 5, The second POI exists as a fusion protein with an exosome polypeptide. EV.

7. An EV as described in claim 1, The aforementioned POI is: (i) Therapeutic proteins; (ii) A binding protein to the therapeutic agent (optionally, an RNA-binding protein, a virus-binding protein, an Fc-binding protein, or a small molecule-binding protein); (iii) Endosome escape site; (iv) target site; (v) Albumin-binding domain; or (vi) purification site; That is, EV.

8. An EV according to claim 7, The aforementioned therapeutic protein is: Enzymes, receptors (optionally, decoy receptors), membrane proteins, transporters, cytokines, antigens, nascent antigens, immune effector molecules, ribonucleoproteins, nucleic acid-binding proteins, antibodies, nanobodies, antibody fragments, antibody-drug conjugates, gene-editing proteins (optionally, CRISPR-Cas), TALEN, and meganucleases. Selected from, EV.

9. An EV as described in claim 1, The EV can be obtained by a process that includes the step of further loading therapeutic cargo onto the EV. EV.

10. An EV according to claim 9, The step of further loading therapeutic cargo onto the EV is carried out by electroporation, transfection reagents, co-incubation, or contact with cell membrane permeable peptides (CPPs), or any combination thereof. EV.

11. An EV according to claim 9, The aforementioned therapeutic cargo is a protein, nucleic acid, virus, viral genome, antigen, or small molecule. EV.

12. An EV according to claim 11, The aforementioned nucleic acid cargo is: RNA molecules, DNA molecules, or mixmers, mRNA, antisense or splice-switching oligonucleotides, gRNA, siRNA, shRNA, miRNA, dbDNA, plasmid DNA (pDNA), supercoiled or non-supercoiled plasmids, or minicircles may be used. EV.

13. A group of EVs as described in claim 5, Here, the TSPAN2-POI fusion protein and the second POI component are expressed on the same EV. A group of electric vehicles.

14. A polypeptide component containing TSPAN2 fused to POI.

15. A polynucleotide component encoding the polypeptide component described in claim 14.

16. A cell comprising the polypeptide component described in claim 14.

17. A cell comprising the polynucleotide component described in Claim 15.

18. A cell according to claim 16 or 17, Further comprising a second polypeptide or polynucleotide component capable of expressing POI, cell.

19. A pharmaceutical composition comprising an EV according to any one of claims 1 to 12, a population of EVs according to claim 13, or cells according to claim 16 or 17, and a pharmaceutically acceptable excipient or carrier.

20. A method for producing an EV according to any one of claims 1 to 12, The aforementioned method is: (i) A step of introducing a polynucleotide component encoding a TSPAN2-POI fusion component into EV-producing cells; and (ii) Expressing the above components in the EV-producing cells to generate an EV containing the TSPAN2-POI fusion protein; including, method.

21. A method for producing the EV described in claim 5, The aforementioned method is: (i) A step of introducing a polynucleotide component encoding a TSPAN2-POI fusion component into EV-producing cells; (ii) Introducing a second polynucleotide component encoding a second POI into the same EV-producing cell (wherein the second POI may exist in the form of a fusion protein with the EV protein); and (iii) Expressing both components in the EV-producing cells to generate an EV comprising the TSPAN2-POI fusion protein and the second POI; including, method.

22. A pharmaceutical composition for medical use, comprising an EV as described in any one of claims 1 to 12.

23. A therapeutic method comprising the step of administering an effective amount of EV described in claims 1 to 12 to a non-human subject in need thereof.

24. A therapeutic method comprising the step of administering an effective amount of the pharmaceutical composition described in claim 19 to a non-human subject in need thereof.

Images