Polypeptides comprising inteins for engineered evs

Abstract

The present invention relates to polypeptides comprising inteins (self-cleaving proteins), which are particularly suitable in the production and use of engineered extracellular vesicles (EVs). In particular, the present disclosure relates to fusion proteins comprising a newly developed intein variant and an EV polypeptide or EV loading moiety, which allow for more efficient loading of cargos into EVs. In addition, the present disclosure relates to polypeptides comprising a new intein variant alongside newly developed extein sequences that further improve EV loading and / or the bioactivity of the loaded EVs produced. The present invention also relates to polynucleotide constructs encoding these polypeptides and fusion proteins, as well as EVs comprising the polypeptides and fusion proteins in both their native and cleaved forms.

Description

[0001] Polypeptides comprising Inteins for Engineered EVs

[0002] Technical field

[0003] The present invention relates to polypeptides comprising inteins (self-cleaving proteins), which are particularly suitable in the production and use of engineered extracellular vesicles (EVs). In particular, the present disclosure relates to polypeptides comprising the AI-CM intein, alongside newly developed extein sequences that alter the properties of the intein mediated cleavage to allow for the production of cargo loaded EVs with improved bioactivity. The present disclosure also relates to fusion proteins comprising a newly developed intein variant and an EV polypeptide or EV loading moiety, which allow for more efficient loading of cargos into EVs. In addition, the present disclosure relates to polypeptides comprising the intein variant, alongside newly developed extein sequences that further improve EV loading and / or the bioactivity of the loaded EVs produced. The present invention also relates to polynucleotide constructs encoding these polypeptides and fusion proteins, as well as EVs comprising the polypeptides and fusion proteins in both their native and cleaved forms. The present invention also relates to methods of producing these EVs and the uses, including the medical use, of these EVs.

[0004] Sequence Listing

[0005] A sequence listing (P5291 PC00 sequence listing; Size: 17,474 bytes and Date of Creation: 20 September 2024) associated with this application is provided electronically in text format in lieu of a paper copy and is hereby incorporated by reference in its entirety.

[0006] Background

[0007] “Inteins”, or in-frame intervening sequences, are self-cleaving polypeptide sequences. “Exteins” are the external sequences to the C-terminal and / or the N-terminal of the intein.

[0008] A variety of inteins, derived from a from a range of different organisms and having different characteristics and activities, are known in the art. Some inteins have splicing activity and catalyse their own excision from a protein to yield the free intein and a mature protein consisting of the C-terminal extein fused to the N-terminal extein. Other inteins have cleaving activity, but display much reduced or no splicing activity, thus allow for the separation of the protein comprising the intein to release of the C-terminal extein and / or the N-terminal extein. One such intein, is the AI-CM mini-intein, which is derived from the Mycobacterium tuberculosis recA protein and characterised in D. W. Wood, et al., “A genetic system yields self-cleaving inteins for bioseparations". Nat. Biotechnol. 17, 889-892 (1999).

[0009] The bio-separation properties of inteins are useful in a variety of different fields, including the field of EV therapeutics. Therapeutic EVs include engineered EVs, which have been loaded with biotherapeutics and are used for the intracellular delivery of the loaded biotherapeutic. A variety of techniques for loading EVs with biotherapeutic cargos are known in the art, with some of the most effective techniques involving the fusion or conjugation of the cargo, or a protein that is capable of associating with the cargo, to a polypeptide or moiety that is naturally incorporated into an EV. However, when EVs are produced using this loading method, optimal bioactivity can only be achieved if the cargo is subsequently released from the EV polypeptide or EV localisation moiety. This release can be mediated by engineering an intein, or a polypeptide sequence comprising an intein, between the EV polypeptide or EV localisation moiety and the cargo (or the protein that associates with the cargo).

[0010] However, inclusion of an intein between the EV polypeptide or EV localisation moiety and the biotherapeutic can reduce the efficiency of cargo loading, due to premature intein cleavage that occurs prior to incorporation into the EV. Beyond reduced loading efficiency, such premature cleavage is associated with further detrimental effects in the EV producer cells, including increased cell stress, cell death and reduced EV production. Moreover, the premature release of the cargo can lead to difficulties in purifying the EV product from the non-EV associated biotherapeutic.

[0011] Therefore, there is a need to develop inteins and polypeptides comprising inteins that mediate cleavage when loaded into EVs, but which exhibit reduced cleavage prior to EV loading.

[0012] Summary of the invention

[0013] The present invention provides polypeptides comprising intein and extein sequences that exhibit reduced intein mediated cleavage in the cell cytoplasm and / or increased intein mediated cleavage in the lumen of an EV. The polypeptides of the present invention exhibit pH and / or temperature sensitive cleavage, thus allow for more controlled release of the cargo. Such polypeptides allow for improved EV loading efficiency and / or the production of cargo loaded EVs with improved bioactivity. The present invention provides a flexible platform tool for EV loading different cargoes that can be used in combination with any EV polypeptide or EV localisation moiety. In particular, the present invention provides for newly developed extein sequences for the Al- CM intein that increase intein mediated cleavage at a low pH. The present invention also provides fusion proteins comprising EV polypeptides fused to a newly developed intein variant (e.g. the 129 intein), which exhibits reduced intein mediated cleavage in the conditions of the cell cytoplasm (i.e. at 37°C and at pH 7.3). In addition, the present invention provides newly developed extein sequences for the 129 intein that further reduce intein mediated cleavage under these conditions.

[0014] In one aspect the present invention provides a fusion protein comprising:

[0015] (i) an intein and

[0016] (ii) an extracellular vesicle (EV) polypeptide; wherein the intein comprises or consists of a variant of SEQ ID NO. 1, wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41, a Valine residue at position 108, a Proline residue at position 111 and a Valine residue at position 152.

[0017] The present invention further provides a fusion protein comprising:

[0018] (i) a polypeptide comprising an intein and

[0019] (ii) an EV polypeptide; wherein the intein is capable of catalysing cleavage of the polypeptide; further wherein the intein comprises or consists of a variant of SEQ ID NO. 1 , wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41 ; a Valine residue at position 108; a Proline residue at position 111 and a Valine residue at position 152.

[0020] In a second aspect, the present invention provides a polypeptide comprising an intein, wherein the intein comprises or consists of a variant of SEQ ID NO. 1, wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41, a Valine residue at position 108, a Proline residue at position 111 and a Valine residue at position 152; wherein the polypeptide further comprises:

[0021] (i) a C-terminal extein sequence, wherein: a. the +1 residue of the C-terminal extein sequence is a Glutamate, Phenylalanine, Histidine, Leucine, Methionine, Glycine or Proline residue, preferably wherein the +1 residue of the C-terminal extein sequence is a Glycine residue, b. the +1 residue of the C-terminal extein sequence is a Glycine residue, the +2 residue of the C-terminal extein sequence is a Serine residue and the +3 residue of the C-terminal extein sequence is a Proline residue, c. the +1 residue of the C-terminal extein sequence is a Glycine residue, the +2 residue of the C-terminal extein sequence is a Glycine residue, the +3 residue of the C-terminal extein sequence is a Glycine residue, the +4 residue of the C-terminal extein sequence is a Glycine residue and the +5 residue of the C-terminal extein sequence is a Serine residue, or d. the +1 residue of the C-terminal extein sequence is a Threonine residue, the +2 residue of the C-terminal extein sequence is Arginine residue and the +3 residue of the C-terminal extein sequence is Histidine residue; and / or

[0022] (ii) an N-terminal extein sequence, wherein a. the -1 residue of the N-terminal extein sequence is a Glycine residue, the - 2 residue of the N-terminal extein sequence is a Serine residue, the -3 residue of the N-terminal extein sequence is a Glycine residue, the -4 residue of the N-terminal extein sequence is a Glycine residue and the -5 residue of the N-terminal extein sequence is a Glycine residue, b. the -1 residue of the N-terminal extein sequence is a Serine residue, the -2 residue of the N-terminal extein sequence is an Alanine residue and the -3 residue of the N-terminal extein sequence is a Phenylalanine residue, or c. the -1 residue of the N-terminal extein sequence is a Methionine residue, the -2 residue of the N-terminal extein sequence is an Arginine residue and the -3 residue of the N-terminal extein sequence is a Threonine residue.

[0023] The present invention further provides a polypeptide comprising an intein, wherein the intein is capable of catalysing cleavage of the polypeptide; further wherein the intein comprises or consists of a variant of SEQ ID NO. 1, wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41; a Valine residue at position 108; a Proline residue at position 111 and a Valine residue at position 152; wherein the polypeptide further comprises:

[0024] (i) a C-terminal extein sequence, wherein the C-terminal extein sequence wherein the +1 residue of the C-terminal extein sequence is Glu, Phe, His, Leu, Met, Gly or Pro; and / or

[0025] (ii) an N-terminal extein sequence, wherein the N-terminal extein sequence comprises Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and / or Gly as the -5 residue.

[0026] In a third aspect, the present invention provides a fusion protein comprising:

[0027] (i) a polypeptide of the present invention and

[0028] (ii) an EV polypeptide. In one embodiment of the polypeptide of the present invention or fusion protein of the present invention, the variant of SEQ ID NO. 1 exhibits a reduced rate of cleavage in the cytoplasm of a cell and / or at a pH of 7.0-7.5 and at a temperature of 34-40°C, as compared to SEQ ID NO 1.

[0029] In a preferred embodiment, the variant of SEQ ID NO. 1 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4, preferably over its entire length.

[0030] In a further preferred embodiment, the variant of SEQ ID NO. 1 further comprises a Glutamine residue at position 167. In one embodiment, variant of SEQ ID NO. 1 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 23, preferably over its entire length.

[0031] In one embodiment of the polypeptide of the present invention or fusion protein of the present invention, the C-terminal extein sequence reduces the rate of cleavage in the cytoplasm of a cell and / or at a pH of 7.0-7.5 and at a temperature of 34-40°C, as compared with where the C-terminal extein sequence is SEQ ID NO. 3. In a preferred embodiment, the +1 residue of the C-terminal extein sequence is Gly, the +2 residue of the C-terminal extein sequence is Ser and / or the +3 residue of the C-terminal extein sequence is Pro. In an alternative preferred embodiment, the +1 residue of the C-terminal extein sequence is Gly, the +2 residue of the C-terminal extein sequence is Gly, the +3 residue of the C-terminal extein sequence is Gly, the +4 residue of the C-terminal extein sequence is Gly and the +5 residue of the C-terminal extein sequence is Ser.

[0032] In one embodiment of the polypeptide of the present invention or fusion protein of the present invention, the N-terminal extein sequence reduces the rate of cleavage in the cytoplasm of a cell and / or at a pH of 7.0-7.5 and at a temperature of 34-40°C, as compared with where the N-terminal extein sequence is SEQ ID NO. 2 and / or SEQ ID NO. 5

[0033] In one embodiment of the fusion protein of the present invention, the EV polypeptide is fused to the N-terminus of the intein. In a preferred embodiment, the EV polypeptide comprises the N-terminal extein residues or the EV polypeptide is fused to the N-terminus of the N-terminal extein residues. In one embodiment of the fusion protein of the present invention, the intein is comprised in or fused to a domain or terminus of the EV polypeptide that is localised to and / or displayed in the lumen of the EV.

[0034] In one embodiment of the fusion protein of the present invention, the fusion protein further comprises a protein cargo, a protein capable of binding to a cargo or a ribonucleoprotein (RNP) complex, preferably wherein the RNP complex comprises a Cas9, a Cas12 such as Cas12a, a Cas3, a base editor or a prime editor. In a preferred embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the C-terminus of the intein. In a preferred embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex comprises the C-terminal extein residues or the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the C-terminus of the C-terminal extein residues. In a further preferred embodiment, the protein capable of binding to a cargo is associated with a cargo.

[0035] In a fourth aspect, the present invention provides a polynucleotide encoding the polypeptide of the present invention or fusion protein of the present invention.

[0036] In a fifth aspect, the present invention provides an EV comprising the polypeptide of the present invention, the fusion protein of the present invention or the polynucleotide of the present invention.

[0037] In a sixth aspect, the present invention provides an EV comprising the polypeptide of the present invention or the fusion protein of the present invention, wherein the intein has catalysed the cleavage of the polypeptide comprising the intein sequence or the fusion protein.

[0038] In a seventh aspect, the present invention provides an EV comprising the fusion protein of the present invention, wherein the protein cargo, protein capable of binding to a cargo or RNP complex has been released from the EV polypeptide.

[0039] In one embodiment of the EV of the present invention, the EV is an exosome, a microvesicle and / or a related extracellular vesicle.

[0040] In an eighth aspect, the present invention provides a method of preparing an EV of the present invention, comprising: (i) introducing into an EV-producing cell a polynucleotide of the present invention; and

[0041] (ii) expressing the polynucleotide in the EV-producing cell, thereby generating EVs comprising a polypeptide or fusion protein of the present invention. Optionally, the method comprises an additional step of collecting the EVs of step (ii) and incubating the EVs at a temperature that is sufficient to allow for the intein to cleave the polypeptide comprising the intein sequence or the fusion protein.

[0042] In a ninth aspect, the present invention provides a cell comprising the polypeptide of the present invention, the fusion protein of the present invention, the polynucleotide of the present invention or the EV of the present invention.

[0043] In a tenth aspect, the present invention provides a pharmaceutical composition comprising an EV of the present invention, and a pharmaceutically acceptable excipient and / or carrier.

[0044] In an eleventh aspect, the present invention provides the EV of the present invention or the pharmaceutical composition of the present invention for use as a medicament for the treatment or prevention of a disease in a subject, optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure or ALS.

[0045] In a twelfth aspect, the present invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the EV of the present invention or the pharmaceutical composition of the present invention to a subject suffering from or susceptible to the disease; optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure or ALS.

[0046] In a further aspect, the present invention provides the EV of the present invention or the pharmaceutical composition of the present invention for the preparation of a medicament for treatment or prevention of a disease in a subject; optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure or ALS. Description of Figures

[0047] Figure 1 shows C-terminal and N-terminal extein sequences can influence the rate of AI-CM intein mediated cleavage. (A) Schematic diagram of constructs comprising the AI-CM intein. “X” denotes the different +1 C-terminal extein residues (His, Met, Glu, Leu or Phe) assessed. (B) Bar chart showing the cleaving rates at room temperature and a pH of 6.2 of the AI-CM intein constructs shown in (A) with different +1 C-terminal extein residues (His, Met, Glu, Leu or Phe). Different C-terminal extein cargos (maltose binding protein (MBP), thioredoxin (TrxA), and p-galactosidase (p-gal)) were also tested. (C) Bar chart showing the cleaving rates at room temperature and a pH 6.2 (light grey) or at room temperature and a pH 8.5 (dark grey) of constructs comprising the AI-CM intein with different -1 N-terminal extein residues.

[0048] Figure 2 shows relatively low EV loading efficiency is achieved with the AI-CM intein. (A) Schematic diagram of constructs used to assess EV loading efficiency of the AI-CM intein (“Int”). (B) Bar chart showing the fluorescence signals (mScarlet single positive - “mScarlet SP”); eGFP single positive - “eGFP SP”; or double positive for both mScarlet and eGFP - “DP”) detected in exosomes isolated from cells expressing one of the three constructs shown (A).

[0049] Figure 3 shows the +1 C-terminal extein residue sequences can influence the rate of I29 intein mediated cleavage. (A) Schematic diagram of constructs comprising the I29 intein (“Intein”). “X” denotes the different +1 C-terminal extein residues (Glu, Phe, His, Leu, Met, Gly and Pro) tested. (B) Bar chart showing the estimated half-life of intein cleavage reactions in different conditions (pH 7.3 and pH 6.0 as well as 37°C and 20°C) of the I29 intein constructs shown in (A) with different +1 C-terminal extein residues Glu (bar 1, 8, 15 and 22), Phe (bar 2, 9, 16 and 23), His (bar 3, 10, 17 and 24), Leu (bar 4, 11 , 18 and 25), Met (bar 5, 12, 19 and 26), Gly (bar 6, 13, 20 and 27) and Pro (bar 7, 14, 21 and 28).

[0050] Figure 4 shows the I29 intein improves EV loading efficiency, compared with the AI-CM intein. (A) Schematic diagram of constructs used to assess relative EV loading efficiency: construct (1) comprises the I29 intein, construct (2) comprises the AI-CM intein and construct (3) comprises an N440A variant of the I29 intein that is non-cleaving. (B) Western blot image shows the presence of the non-cleaved protein (between 70 and 100kDa) and the cleaved cargo (between 25 and 35 kDa) in samples derived from conditioned media taken from cells expressing the constructs shown in (A). “In” is magnetically labelled conditioned media from which the exosome samples were then isolated; “FT” is the flow-through of magnetic columns of the exosome isolation kit; “WS” is the wash flow through of magnetic columns of the exosome isolation kit; “EL” is the exosome lysate elution. (C) The FACs plots show the fluorescent signals detected in exosomes derived from cells transfected with a CD63- RISEFAS-mScarlet-129-GSP-eGFP-His construct, a CD63-RISEFAS-mScarlet-AI- CM-GSP-eGFP-His construct or a CD63-RISEFAS-mScarlet-l29(N440A)-GSP-eGFP-His construct. The eGFP signal is plotted on the x-axis and the mScarlet signal is plotted on the y-axis. Each point on the graph represents a fluorescence measurement of an individual exosome. (D) Graph shows comparative cleavage rates at 37°C and pH 7.3 of polypeptides comprising the 129 intein with G-S-P C-terminal extein sequence and different C-terminal extein cargos (eGFP, mScarlet and AsCpfl).

[0051] Figure 5 shows that the 129 cleavage activity at pH 6.2 over time. (A) Western blot image shows the presence of the non-cleaved protein (between 70 and 100kDa) and the cleaved cargo (between 25 and 35 kDa) in samples derived from conditioned media taken from cells expressing a CD63-RISEFAS-l29-GSP-eGFP-His construct. “In” is magnetically labelled conditioned media from which the exosome samples were then isolated; “FT” is the flow- through of magnetic columns of the exosome isolation kit; “WS” is the wash flow through of magnetic columns of the exosome isolation kit; “Oh” is the exosome lysate elution stored at 20°C, pH 6.2 for 0 hours; “5h” is the exosome lysate elution stored at 20°C, pH 6.2 for 5 hours; “24h” is the exosome lysate elution stored at 20°C, pH 6.2 for 24 hours.

[0052] Figure 6 shows the improved EV loading efficiency achieved with the 129 intein is not dependent on the EV polypeptide. (A) Schematic diagram of constructs used to assess relative EV loading efficiency comprising either the 129 intein (129), the AI-CM intein (dICM) or a non-cleavable control amino acid sequence (AA) and either CD63 or TSN2 as the EV polypeptide. (B) JESS image shows the cleaved GFP cargo and non-cleaved protein present in conditioned media taken from cells transfected with the fusion protein constructs shown in (A), comprising the non-cleavable AA sequence (1), the AI-CM intein sequence (2) or the I29 intein sequence (3). (C) Bar chart shows the proportion of GFP positive particles in conditioned media taken from cells transfected with each of the constructs shown in (A). (D) Bar chart shows the percentage of genome editing as the mTtr211 locus induced by different dosages of EVs purified from cells expressing one of the following constructs: Gag-GGGSG- I29-GGGGS-Cas9 (bar 1 and bar 9), Gag-GGGSG-l29-GSP-Cas9 (bar 2 and bar 10), Myristilation-GGGSG-I29-GGGGS-Cas9 (bar 3 and bar 11), Myristilation-GGGSG-l29-GSP- Cas9 (bar 4 and bar 12), Single-pass EV polypeptide-GGGSG-l29-GGGGS-Cas9 (bar 5 and bar 13), Single-pass EV polypeptide-GGGSG-l29-GSP-Cas9 (bar 6 and bar 14), TSN2- GGGSG-I29-GGGGS-Cas9 (bar 7 and bar 15), TSN2-GGGGG-l29-GSP-Cas9 (bar 8 and bar 16).

[0053] Figure 7 shows the N-terminal and C-terminal extein sequences can influence the rate of I29 intein mediated cleavage. (A) Schematic diagram of constructs comprising the I29 intein and different N-terminal and C-terminal extein sequences. (B) Bar chart shows the number of GFP positive particles in conditioned media taken from cells transfected with each of the constructs shown (A) (bars 3 to 12) or with a construct comprising a non-cleaving amino acid sequence in the place of the I29 intein (bar 1) or with a construct comprising the AI-CM intein in the place of the I29 intein (bar 2). (C) JESS image shows the amount of the cleaved GFP cargo and non-cleaved protein present in conditioned media taken from cells transfected with the fusion protein constructs comprising the non-cleavable AA sequence (lane 1), the dICM intein sequence (lane 2) or the constructs shown in (A) (lanes 3 to 12).

[0054] Figure 8 shows EVs comprising the I29 intein have increased bioactivity in target cells, as compared with EVs comprising the AI-CM intein. (A) and (B) Bar chart shows the percentage of tdTomato positive cells, which is a read-out for the bioactive delivery of the Cre cargo to target cells. In (A) the target cells (HSCs) are dosed with 1 x 108EVs and in (B) the target cells (HSCs) are dosed with 1 x 109EVs.

[0055] Figure 9 is further evidence that EVs comprising the I29 intein have increased bioactivity in target cells, as compared with EVs comprising the AI-CM intein, with Cas9 as a cargo. (A) Bar Chart shows the percentage of GFP positive cells, which is a read-out for the bioactive delivery of the Cas9 RNP complex cargo to target cells. (B) Graph shows the percentage of genome editing at the endogenous gene target Ttr. (C) Bar charts show the percentage of genome editing at the endogenous gene target Ttr. (D) Bar chart shows the number of gRNA copies loaded into EVs when the following constructs are expressed in EV producer cells alongside guide RNA: TSN2-GGGSG-I29-GGGGS-Cas (bar 1 , labelled “I29”), TSN2- GGGSG-I29aa-GGGGS-Cas where “I29aa” is a non-cleaving I29 mutant (bar 2), TSN2- FAS-l29-GSP-Cas9 (bar 3), TSN2-TRM-I29-TRH-Cas9 (bar 4) and TSN2-GGGSG-AI-SM- GGGGS where “DISM” (AI-SM) is a slow cleaving variant of the AI-CM intein (bar 5).

[0056] Figure 10 is further evidence that EVs comprising the I29 intein have increased bioactivity in target cells, as compared with EVs comprising the AI-CM intein, with Cas12a as a cargo. (A) to (D) Bar Charts show the percentage of GFP positive cells, which is a read-out for the bioactive delivery of the Cas12a RNP complex cargo to target cells. In (A) and (B) the target cells are dosed with 1 x 1O10EVs and in (C) and (D) the target cells are dosed with 1 x 109EVs.

[0057] Figure 11 shows that that EVs comprising an I29 intein that includes an additional H167Q mutation (referred to as Intein29 H439Q in the figure) also have increased bioactivity in target cells, as compared with EVs comprising the AI-CM intein. This intein also offers a further improvement in bioactivity as compared with EVs comprising the I29 intein. (A) to (D) Bar Charts show the percentage of GFP positive cells, which is a read-out for the bioactive delivery of the Cas9 RNP complex cargo to target cells, 96 hours after different doses of EVs were added as shown on the X-axis. In (A) and (B) the target cells are HEK-SL cells and in (C) and (D) the target cells are B16F10-SL cells.

[0058] Detailed Description of the Invention

[0059] The present invention relates to polypeptides comprising inteins that are particularly suitable in the production and use of engineered extracellular vesicles. Provided in the present disclosure are polypeptides comprising the AI-CM intein, alongside newly developed extein sequences. The newly developed extein sequences increase AI-CM intein self-cleavage at pH 6.2 and / or at room temperature and therefore allow for the production of cargo loaded EVs where a higher proportion of the cargo is released from the EV polypeptide or EV localisation moiety used for loading. Thus, polypeptides comprising the AI-CM intein alongside these newly developed extein sequences allow for the production of EVs with improved bioactivity.

[0060] The present disclosure also relates to polypeptides comprising a newly developed intein variant (e.g. the I29 intein) fused to an EV polypeptide or conjugated to an EV localisation moiety. The I29 intein is described in the patent application entitled “Modified Inteins and Methods of Use” filed at the United States Patent and Trademark Office by Ohio State University on 29 September 2023. The newly developed I29 intein is a variant of the AI-CM intein that exhibits reduced self-cleavage, as compared to the AI-CM intein, at pH 7.2-7.3 at 37°C. This newly developed intein therefore exhibits a reduction in premature cleavage, as compared with the AI-CM intein, and thus mediates improved loading of cargos and other proteins of interest into EVs. Importantly, like the AI-CM intein, the I29 intein still allows for the cargos to have bioactivity in target cells, perhaps due to the I29 intein maintaining a more similar ability to self-cleave at an acidic pH (e.g. pH 6.0 to 6.2) at both room temperature and at 37°C, as compared to the AI-CM intein. Thus, the I29 intein also mediates the production of cargo-loaded EVs that display increased bioactivity. The newly developed H429Q 129 intein, which includes all the same mutations present in the 129 intein (along with an additional H167Q) relative to the AI-CM intein, also allows for the production of cargo-loaded EVs that display increased bioactivity, as compared with when the AI-CM intein is used.

[0061] In addition, the present disclosure relates to polypeptides comprising the new intein variant (e.g. I29 intein), alongside newly developed extein sequences. The newly developed extein sequences further reduce the new intein variant (e.g. I29 intein) self-cleavage at pH 7.2-7.3 at 37°C and therefore display a further reduction in premature cleavage. Thus, polypeptides comprising the new intein variant (e.g. I29 intein) alongside newly developed extein sequences, allow for further improvements in loading cargos and other proteins of interest into EVs.

[0062] For convenience and clarity, certain terms employed herein are collected and described below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0063] Where features, aspects, embodiments, or alternatives of the present invention are described in terms of Markush groups, a person skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. The person skilled in the art will further recognize that the invention is also thereby described in terms of any combination of individual members or subgroups of members of Markush groups.

[0064] Additionally, it should be noted that embodiments and features described in connection with one of the aspects and / or embodiments of the present invention also apply mutatis mutandis to all the other aspects and / or embodiments of the invention. For example, the inteins, extein, fusion proteins and conjugated proteins described herein in connection with the EVs are to be understood to be disclosed, relevant, and compatible with all other aspects, teachings and embodiments herein, for instance aspects and / or embodiments relating to the methods for producing or the EVs, or relating to the corresponding polynucleotide constructs described herein or the engineered EV-producing cells from which the EVs derive.

[0065] Furthermore, certain embodiments described in connection with certain aspects, for instance the administration routes of the EVs comprising the inteins, extein, fusion proteins and conjugated proteins and optionally further proteins of interest, including cargos, as described in relation to aspects pertaining to treating certain medical indications, may naturally also be relevant in connection with other aspects and / or embodiment such as those pertaining to the pharmaceutical compositions comprising such EVs. Furthermore, all polypeptides and proteins identified herein can be freely combined in fusion proteins using conventional strategies for fusing polypeptides. As a non-limiting example, the polypeptides comprising an intein and an extein sequence described herein, the fusion proteins described herein and the conjugated proteins described herein may be freely combined in any combination with one or more exosomal polypeptides, optionally combined with all other polypeptide domains, regions, sequences, peptides, groups herein, e.g. any multimerization domains, release domains, fusogens, and / or targeting peptides. Also, exosomal polypeptides and / or NA- binding domains, including NA-binding proteins such as nucleases and other proteins having related functions, e.g. Cas9, Cas12, Cas12a, base editors, prime editors, may be combined with each other to generate constructs. Moreover, any and all features (for instance any and all members of a Markush group) can be freely combined with any and all other features (for instance any and all members of any other Markush group), e.g. any polypeptides comprising an intein and an extein sequence described herein, the fusion proteins described herein and the conjugated proteins described herein may be combined with any exosomal polypeptide. Furthermore, when teachings herein refer to EVs in singular and / or to EVs as discrete natural nanoparticle-like vesicles it should be understood that all such teachings are equally relevant for and applicable to a plurality of EVs and populations of EVs. As a general remark, polypeptides comprising an intein and an extein sequence described herein, the fusion proteins described herein and the conjugated proteins described herein, the exosomal polypeptides, the EV-producing cell sources, the additional domains and peptides, the cargo molecule, and all other aspects, embodiments, and alternatives in accordance with the present invention may be freely combined in any and all possible combinations without deviating from the scope and the gist of the invention.

[0066] Furthermore, any polypeptide or polynucleotide or any polypeptide or polynucleotide sequences (amino acid sequences or nucleotide sequences, respectively) of the present invention may deviate considerably from the original polypeptides, polynucleotides and sequences as long as any given molecule retains the ability to carry out the desired technical effect associated therewith. As long as their biological properties are maintained the polypeptide and / or polynucleotide sequences according to the present application may deviate with as much as 50% (calculated using for instance BLAST or ClustalW) as compared to the native sequence, although a sequence identity or similarity that is as high as possible is preferable (for instance 60%, 70%, 80%, or e.g. 90% or higher). Standard methods in the art may be used to determine homology. For example, the LIWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et al (1990) J Mol Biol 215:403-10. The combination (fusion) of e.g. several polypeptides implies that certain segments of the respective polypeptides may be replaced and / or modified and / or that the sequences may be interrupted by insertion of other amino acid stretches, meaning that the deviation from the native sequence may be considerable as long as the key properties (e.g. catalysing cleavage of an intein-extein bond / NA- binding / trafficking into EVs / targeting capabilities, etc.) are conserved. Similar reasoning thus naturally applies to the polynucleotide sequences encoding for such polypeptides. Any accession numbers or SEQ ID NOs mentioned herein in connection with peptides, polypeptides and proteins shall only be seen as examples and for information only, and all peptides, polypeptides and proteins shall be given their ordinary meaning as the skilled person would understand them. Thus, as above-mentioned, the skilled person will also understand that the present invention encompasses not merely the specific SEQ ID NOs and / or accession numbers referred to herein but also variants and derivatives thereof. All accession numbers referred to herein are UniProtKB accession numbers, and all proteins, polypeptides, peptides, nucleotides and polynucleotides mentioned herein are to be construed according to their conventional meaning as understood by a skilled person.

[0067] As used herein, the term “intein” refers to an in-frame intervening sequence in a protein as described by Perler (Perler, Davis et al. 1994). The term intein, as used herein, encompasses modified or mutated inteins and mini-inteins. As used herein, the term “extein” refers to an external sequence to the C-terminal and / or the N-terminal of the intein.

[0068] The term intein, as used herein, includes split inteins and contiguous inteins. As used herein, the term “contiguous intein” refers to an intein expressed from a single gene sequence. As used herein, the term “split intein” refers to an intein that consists of two separate fragments: an N-terminal intein connected to N-extein and a C-terminal intein connected to C-extein. Split inteins only display activity when the two fragments are fused together to form a complete intein. In a preferred embodiment, the intein of the present disclosure is a contiguous intein as defined herein.

[0069] The term intein, as used herein, includes inteins that have splicing activity and inteins that have reduced, limited or no splicing activity. Such inteins having limited or preferably no splicing activity are referred to herein as “cleaving inteins”. Cleaving inteins are capable of mediating the separation of a polypeptide in which they are comprised and releasing the C- terminal extein and / or the N-terminal extein. Cleaving inteins are particularly advantageous in the context of engineered EVs, as they allow for loaded cargoes to have improved bioactivity, as they are be released and can move to the subcellular location that is optimal for their activity. In a preferred embodiment, the intein of the present disclosure is a cleaving intein as defined herein. In a more preferred embodiment, the intein of the present invention has, or is capable of, C-terminal cleavage activity. In a most preferred embodiment, the intein of the present invention has, or is capable of, C-terminal cleavage activity, but has reduced, or preferably no, N-terminal cleavage and extein ligation activity.

[0070] In one embodiment, where the intein has, or is capable of, C-terminal cleavage activity, the intein is derived from Mycobacterium tuberculosis recA (such as where the intein comprises or consists of SEQ ID NO: 1 , SEQ ID NO: 4, SEQ ID NO: 23 or a variant of SEQ ID NO: 1 , SEQ ID NO: 4 or SEQ ID NO: 23 as described herein) and the last (most C-terminal) amino acid (i.e. position 168 of SEQ ID NO: 1 , SEQ ID NO: 4, SEQ ID NO: 23 or a variant of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 23 as described herein) is (i) not an alanine residue or preferably (ii) is an asparagine residue. In one embodiment, where the intein has reduced, or preferably no, N-terminal cleavage and extein ligation activity, the intein is derived from Mycobacterium tuberculosis recA (such as where the intein comprises or consists of SEQ ID NO: 1 , SEQ ID NO: 4, SEQ ID NO: 23 or a variant of SEQ ID NO: 1 , SEQ ID NO: 4 or SEQ ID NO: 23 as described herein) and the first (most N-terminal) amino acid (i.e. position 1 of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 23 or a variant of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 23 as described herein) is an alanine residue.

[0071] As used herein, the term “rate of cleavage” or “cleavage rate” refers to the intein’s cleavage activity (i.e. ability to self-cleave). Thus, an increased rate of cleavage results in an intein, or a polypeptide comprising an intein, that has a reduced half-life. A reduced rate of cleavage results in an intein, or a polypeptide comprising an intein, that has an increased half-life. In a preferred embodiment, the rate of cleavage refers to the rate of C-terminal cleavage (i.e. mediating separation between the C-terminal extein and the intein).

[0072] As used herein in relation to an intein, or a polypeptide comprising an intein, the term “halflife” refers to the amount of time it takes for half of the intein, or polypeptide comprising an intein, molecules present to be cleaved, preferably self-cleaved due to the activity of the intein. As used herein, the term “AI-CM intein”, “AI-CM”, “AI-CM mini-intein”, “delta l-CM intein”, “Al- CM mini-intein”, “dICM Intein” or “dICM” refers to an intein derived from the Mycobacterium tuberculosis recA protein and characterised in D. W. Wood, et al., “A genetic system yields self-cleaving inteins for bioseparations’’. Nat. Biotechnol. 17, 889-892 (1999). The AI-CM intein has 100% sequence identity to SEQ ID NO. 1 over its entire length.

[0073] As used herein, the term “I29 intein”, “AI-29” or “Intein29” refers to an intein having 100% sequence identity to SEQ ID NO. 4 over its entire length. The I29 intein comprises the following specific mutations relative to the AI-CM intein: H41 R, 1108V, R111 P and E152V.

[0074] As used herein, the term “H439Q I29 intein”, “H439Q Intein29” or “Intein29 H439Q” refers to an intein having 100% sequence identity to SEQ ID NO. 23 over its entire length. The H439Q I29 intein has the following specific mutations relative to the AI-CM intein: H41R, 1108V, R111P, E152V and H167Q. The H439Q I29 intein comprises the H167Q relative to the I29 intein.

[0075] In one aspect, the present disclosure provides a polypeptide sequence comprising or consisting of an intein and at least one extein sequence.

[0076] In one embodiment, the polypeptide sequence of the present disclosure comprises an extein sequence to the N-terminus of the intein. In one embodiment, the polypeptide sequence of the present disclosure comprises an extein sequence to the C-terminus of the intein. In a preferred embodiment, the polypeptide sequence of the present disclosure comprises an extein sequence to the N-terminus of the intein and an extein sequence to the C-terminus of the intein.

[0077] The N-terminal and / or C-terminal extein may be contiguous with the intein, and so there are no intervening sequences.

[0078] In one embodiment, the intein of the present disclosure is capable of catalysing the cleavage of an intein-extein bond. In a preferred embodiment, the intein of the present disclosure catalyses cleavage of the C-terminal intein-extein bond. In a most preferred embodiment, the intein of the present disclosure mediates the release of the C-terminal extein.

[0079] In a preferred embodiment, the intein of the present disclosure is capable of catalysing cleavage of an intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. In a more preferred embodiment, the intein of the present disclosure is capable of catalysing cleavage of an intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at 34-40°C, preferably at 37°C. In a more preferred embodiment, the intein of the present disclosure is capable of catalysing cleavage of an intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at room temperature.

[0080] As used herein, the term “room temperature” refers to a temperature falling within the range of 16-25°C, 17-23°C or preferably 19-21°C or most preferably a temperature of 20°C. Cleavage within an EV lumen at room temperature is advantageous, as it allows for easier and more efficient production of an EV product with improved bioactivity (i.e. due to the release of loaded cargo(s)). Production is considered easier and more efficient since there is no need to include further incubation steps in bioreactors at higher temperatures.

[0081] The terms “extracellular vesicle” or “EV” are used interchangeably herein and can be understood to relate to any type of vesicle that is obtainable from a cell in any form. In one embodiment, the EV is a microvesicle (e.g. any vesicle shed from the plasma membrane of a cell), an exosome (e.g. any vesicle derived from the endo-lysosomal pathway or from any other cellular pathway producing exosomes), ARRCD1 Mediated Microvesicles (ARMM), an apoptotic body (e.g. obtainable from apoptotic cells), a microparticle (which may be derived from e.g. platelets), an ectosome (derivable from e.g. neutrophils and monocytes in serum), prostatosome (e.g. obtainable from prostate cancer cells), or a cardiosome (e.g. derivable from cardiac cells), etc. Furthermore, the said terms shall be understood to also relate to in some embodiments to extracellular vesicle mimics, cellular membrane vesicles obtained through membrane extrusion, sonication or other techniques, etc. Essentially, the present invention may relate to any type of lipid-based structure (with vesicular morphology or with any other type of suitable morphology) that can act as a delivery or transport vehicle. Exosomes, microvesicles and ARRDCI-mediated microvesicles (ARMMs) represent particularly preferable EVs, but other EVs may also be advantageous in various circumstances. In preferred embodiments of the present invention the EV is an exosome or microvesicle or any other type of extracellular vesicle related to such exosomes and / or microvesicles.

[0082] The sizes of EVs may vary considerably but an EV typically has a nano-sized hydrodynamic radius, i.e. a radius below 1000 nm. Exosomes often have a sized of between 30 and 300 nm, typically in the range between 50 and 250 nm, which is a highly suitable size range. EVs may be derived from any cell type, both in vivo, ex vivo, and in vitro. The term “EV producer cells” as defined herein are the cells from which the EVs are derived. The terms “EV cell source”, “EV source cell”, “source cell” “EV producing cell” and “producer cell” are used interchangeably with the term “EV producer cells”. Generally, EVs may be derived from essentially any cell source, be it a primary cell source or an immortalized cell line. The EV source cells may be any embryonic, fetal, and adult somatic stem cell types, including induced pluripotent stem cells (iPSCs) and other stem cells derived by any method, as well as any adult cell source. In one aspect, the source cells may be selected from a wide range of cells and cell lines, for instance mesenchymal stem or stromal cells (obtainable from e.g. bone marrow, adipose tissue, Wharton’s jelly, perinatal tissue, chorion, placenta, tooth buds, umbilical cord blood, skin tissue, etc.), fibroblasts, amnion cells and more specifically amnion epithelial cells optionally expressing various early markers, myeloid suppressor cells, M2 polarized macrophages, adipocytes, endothelial cells, fibroblasts, etc. Cell lines of particular interest include human umbilical cord endothelial cells (HLIVECs), human embryonic kidney (HEK) cells, endothelial cell lines such as microvascular or lymphatic endothelial cells, erythrocytes, erythroid progenitors, chondrocytes, mesenchymal stromal cells (MSCs) of different origin, amnion cells, amnion epithelial (AE) cells, CEVEC’s CAP® cells any cells obtained through amniocentesis or from the placenta, airway or alveolar epithelial cells, fibroblasts, endothelial cells, etc. In a further aspect, the source cells are immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DCs). Essentially any type of cell which is capable of producing EVs is also encompassed herein. In a preferred embodiment, the EV producer cells are a cultured cell line. Preferred producer cell-lines according to the present invention are HEK cells, in particular a HEK293 cells, most preferably HEKVPC2 cells (also referred to as Gibco™ Viral Production Cell 2.0, Viral Production Cells 2.0, VPC 2.0 cells and 2.0-HEK293F cells) or HEKVPC1 cells (Gibco™ Viral Production Cell 1.0, Viral Production Cells 1.0, VPC 1.0 cells and 1.0-HEK293F cells). The cell lines from which EVs are derived may be adherent or suspension cells and may be generated as stable cell lines or single clones.

[0083] In one embodiment, the EV producer cell is allogeneic, autologous, or even xenogeneic in nature to the patient to be treated, i.e. the cells may be from the patient themself or from an unrelated, matched or unmatched donor. In certain contexts, allogeneic cells may be preferable from a medical standpoint, as they could provide immuno-modulatory effects that may not be obtainable from autologous cells of a patient suffering from a certain indication. For instance, in the context of treating systemic, peripheral and / or neurological inflammation, EVs may enable immuno-modulation via e.g. macrophage and / or neutrophil phenotypic switching (from pro-inflammatory M1 or N1 phenotypes to anti-inflammatory M2 or N2 phenotypes, respectively).

[0084] In particular, the present invention relates to EV producer cells that have been stably modified to comprise at least one monocistronic, bicistronic or multicistronic polynucleotide construct of the present invention. Such cells may be stably or transiently transfected with the polynucleotides of the present invention to render them engineered EV producing cells. Such cells may also be stably or transiently modified so as to include a second construct encoding for a (or an additional) protein cargo, protein capable of binding a cargo, purification moiety, targeting moiety, endosomal escape moiety, pharmokinetic moiety and / or pharmoeffector moiety, which optionally may form part of a fusion protein with a classical EV protein. The cells of the present invention may be a monoclonal cell or a polyclonal cell line.

[0085] The term “engineered EV” is used interchangeably with “modified EV” and “genetically engineered EV” herein. As used herein, the term “engineered EV” refers to an EV, preferably an exosome or a microvesicle or similar vesicle, that is derived from a genetically modified / engineered cell or is otherwise genetically engineered to express the proteins in the lumen, in the extravesicular membrane and / or on the surface of the EV (e.g., exosome), which are not typically incorporated into the EVs, preferably exosomes, microvesicles and / or related extracellular vesicles, produced by those cells, or to modify the expression of the EV proteins that are typically incorporated into the EVs, preferably exosomes, microvesicles and / or a related extracellular vesicles, produced by those cells.

[0086] It will be clear to the skilled artisan that when describing medical and scientific uses and applications of the EVs, the present invention normally relates to a plurality of EVs, i.e. a population of EVs which may comprise thousands, millions, billions or even trillions of EVs. As can be seen from the experimental section below, EVs may be present in concentrations such as 105, 108, 1O10, 1011, 1012, 1013, 1014, 1015, 1018, 1025,103° EVs (often termed “particles”) per unit of volume (for instance per ml or per litre), or any other number larger, smaller or anywhere in-between, or within any range in-between these values. In the same vein, the term “population”, which may e.g. relate to an EV comprising a certain protein of interest shall be understood to encompass a plurality of entities which together constitute such a population. In other words, individual EVs when present in a plurality constitute an EV population. Thus, naturally, the present invention pertains both to individual EVs and populations comprising EVs, as will be clear to the skilled person. In a preferred embodiment, the intein of the present disclosure is capable of catalysing the cleavage of an intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at a pH 6.2. In a more preferred embodiment, the intein of the present disclosure is capable of catalysing cleavage of an intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2, at 34-40°C, preferably at 37°C. In a more preferred embodiment, the intein of the present disclosure is capable of catalysing cleavage of an intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2, at room temperature.

[0087] The ability of an intein to catalyse the cleavage of an intein-extein bond in the lumen of an EV and / or under particular conditions (i.e. at a particular pH and / or temperature) may be verified by the methods outlined in the examples.

[0088] In one embodiment, the intein of the present disclosure comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1. SEQ ID NO. 1 is the sequence of the AI-CM mini intein described in US Patent 6,933,362 (herein incorporated by reference in its entirety for its teaching concerning AI-CM and its uses). In a preferred embodiment, the intein sequence comprises the following specific residues, relative to SEQ ID NO. 1: a Leucine at position 67 and / or a Glycine at position 150. Optionally, the intein sequence may also comprise a Glycine at position 24.

[0089] "Percent (%) sequence identity" as used herein refers to the percentage of nucleotides or amino acids in a candidate sequence that are identical with a reference sequence after aligning the respective sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the skill in the art. Standard methods in the include the use of PILEUP and BLAST algorithms to calculate homology or line up sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. “100% sequence identity” or “sequence identity” as used herein refers to two or more referenced entities that are the same when they are "aligned" sequences. For example, when two polynucleotide sequences are identical, they have the same nucleic acid base sequence. Similarly, when two amino acid sequences are identical, they have the same amino acid sequence. An "aligned" sequence refers to multiple polynucleotide or amino acid sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence. Sequence identity is interchangeable with the term homology or sequence homology. Preferably, the percent (%) sequence identity is over the entire length of the sequence.

[0090] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 1 , the C-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3 in the lumen of an extracellular vesicle, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In a preferred embodiment, the C-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3 in the lumen of an extracellular vesicle, preferably an exosome, a microvesicle and / or a related extracellular vesicle, at room temperature. In a preferred embodiment, the C-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3 in the lumen of an extracellular vesicle, preferably an exosome, a microvesicle and / or a related extracellular vesicle, at 34-40°C, preferably at 37°C.

[0091] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the C-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3, at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2. In a preferred embodiment, the C- terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3, at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2, at room temperature. In a preferred embodiment, the C-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3, at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2, at 34-40°C, preferably at 37°C.

[0092] The ability of an extein to increase the rate of cleavage of an intein-extein bond in the lumen of an EV and / or under particular conditions (i.e. at a particular pH and / or temperature) may be verified by the methods outlined in the examples.

[0093] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the C-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3, in the cytoplasm of a cell, preferably a cultured mammalian cell. In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the C-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with the SEQ ID NO. 3, at pH 7.2 or pH 8.5. In one embodiment, the C-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with the SEQ ID NO. 3, at pH 7.2 or pH 8.5 at room temperature. In one embodiment, the C- terminal extein sequence does not significantly increase the rate of cleavage of the intein- extein bond, as compared with the SEQ ID NO. 3, at pH 7.2 or pH 8.5 at 34-40°C, preferably at 37°C.

[0094] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the C-terminal extein sequence comprises a +1 amino acid residue that has a side chain of a relatively large volume and / or high molecular weight. In one embodiment, the +1 amino acid residue has a molecular weight of 131 Da or more, 149 Da or more or 155 Da or more. In a preferred embodiment, the C-terminal extein sequence comprises His, Met, Glu, Leu or Phe as the +1 amino acid residue. In a more preferred embodiment, the C-terminal extein sequence comprises His or Met as the +1 amino acid residue. In a most preferred embodiment, the C-terminal extein sequence comprises His as the +1 amino acid residue.

[0095] As used herein, the term “+1 amino acid residue” or “+1 residue” refers to the amino acid of the extein closest to the C-terminal of the intein. Preferably, the intein of the present disclosure is capable of catalysing cleavage of the intein-extein bond at the intein C- terminus, thus the “+1 residue” is the amino acid immediately downstream, or C-terminally, of this cleavage site.

[0096] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the C-terminal extein sequence comprises Ser as the +2 amino acid residue, Pro as the +3 amino acid residue, Pro as the +4 amino acid residue and / or Phe as the +5 amino acid residue.

[0097] The term “+2 amino acid residue” or “+2 residue” refers to the amino acid of the extein immediately downstream, or C-terminally, of +1 amino acid residue. The term “+3 amino acid residue” or “+3 residue” refers to the amino acid of the extein immediately downstream, or C- terminally, of +2 amino acid residue. The term “+4 amino acid residue” or “+4 residue” refers to the amino acid of the extein immediately downstream, or C-terminally, of +3 amino acid residue. The term “+5 amino acid residue” or “+5 residue” refers to the amino acid of the extein immediately downstream, or C-terminally, of +4 amino acid residue etc.

[0098] In an embodiment, wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, the C-terminal extein sequence comprises His as the +1 amino acid residue, Ser as the +2 amino acid residue, Pro as the +3 amino acid residue, Pro as the +4 amino acid residue and Phe as the +5 amino acid residue.

[0099] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the polypeptide of the present disclosure further comprises a C-terminal extein sequence as described herein, and an N- terminal extein sequence comprising or consisting of SEQ ID NO. 2.

[0100] In an alternative embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, the N-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2 in the lumen of an extracellular vesicle, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In a preferred embodiment, the N-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2 in the lumen of an extracellular vesicle, preferably an exosome, a microvesicle and / or a related extracellular vesicle, at room temperature. In a preferred embodiment, the N-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2 in the lumen of an extracellular vesicle, preferably an exosome, a microvesicle and / or a related extracellular vesicle, at 34-40°C, preferably at 37°C.

[0101] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the N-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2, at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2. In a preferred embodiment, the N- terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2, at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2, at room temperature. In a preferred embodiment, the N-terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2, at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably at pH 6.2, at 34-40°C, preferably at 37°C.

[0102] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the N-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3 or preferably SEQ ID NO. 2, in the cytoplasm of a cell, preferably a cultured mammalian cell.

[0103] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the N-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with the SEQ ID NO. 2, at pH 7.2 or pH 8.5. In one embodiment, the N-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with the SEQ ID NO. 2, at pH 7.2 or pH 8.5 at room temperature. In one embodiment, the N- terminal extein sequence does not significantly increase the rate of cleavage of the intein- extein bond, as compared with the SEQ ID NO. 2, at pH 7.2 or pH 8.5 at 34-40°C, preferably at 37°C.

[0104] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the N-terminal extein sequence comprises a -1 amino acid residue that has a hydrophilic side chain. In a preferred embodiment, the N-terminal extein sequence comprises Ala, Asp, Gly, Gin, His, Glu, Lys, Pro, Thr, Arg, Cys, Trp or Met as the -1 amino acid residue, preferably Ala, Asp, Gly, Gin or His as the -1 amino acid residue.

[0105] As used herein, the term “-1 amino acid residue” or “-1 residue” refers to the amino acid of the extein closest to the N-terminal of the intein.

[0106] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the N-terminal extein sequence comprises Glu as the -2 amino acid residue, Ser as the -3 amino acid residue, lie as the -4 amino acid residue and / or Arg as the -5 amino acid residue.

[0107] The term “-2 amino acid residue” or “-2 residue” refers to the amino acid of the extein immediately downstream, or N-terminally, of -1 amino acid residue. The term “-3 amino acid residue” or “-3 residue” refers to the amino acid of the extein immediately downstream, or N- terminally, of -2 amino acid residue. The term “-4 amino acid residue” or “-4 residue” refers to the amino acid of the extein immediately downstream, or N-terminally, of -3 amino acid residue. The term “-5 amino acid residue” or “-5 residue” refers to the amino acid of the extein immediately downstream, or N-terminally, of -4 amino acid residue etc.

[0108] In an embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, the N-terminal extein sequence comprises Ala, Asp, Gly, Gin or His as the -1 amino acid residue, Glu as the -2 amino acid residue, Ser as the -3 amino acid residue, lie as the -4 amino acid residue and Arg as the -5 amino acid residue.

[0109] In one embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 , the polypeptide of the present disclosure further comprises an N-terminal extein sequence as described herein and a C- terminal extein sequence comprising or consisting of SEQ ID NO 3.

[0110] In a preferred embodiment wherein the intein comprises or consists of a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1, the polypeptide of the present disclosure further comprises an N-terminal extein sequence as described herein and a C- terminal extein sequence as described herein.

[0111] In an alternative embodiment, the intein of the present disclosure comprises or consists of a variant of SEQ ID NO. 1.

[0112] As used herein, “variant” may refer to a molecule having a structure sufficiently similar to the structure of a parent molecule (e.g., a protein or peptide disclosed herein) that one skilled in the art would expect the variant to exhibit the same or similar activities and utilities compared to the parent molecule. For example, substituting specific amino acids in a given peptide can yield a variant peptide with similar activity to the parent.

[0113] In one embodiment, the variant of SEQ ID NO. 1 exhibits a reduced rate of cleavage of the intein-extein bond, as compared with the SEQ ID NO. 1, in the cytoplasm of a cell, preferably a cultured mammalian cell, most preferably an EV producer cell.

[0114] In one embodiment, the variant of SEQ ID NO. 1 exhibits a reduced rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 1 , at pH 7.0-7.6, more preferably at pH 7.3, preferably at 34-40°C, most preferably at 37°C.

[0115] The ability of an intein to reduce the rate of cleavage of an intein-extein bond in the cytoplasm of a cell and / or under particular conditions (i.e. at a particular pH and / or temperature) may be verified by the methods outlined in the examples.

[0116] In a one embodiment, the variant of SEQ ID NO. 1 is capable of cleaving the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. In a preferred embodiment, the variant of SEQ ID NO. 1 is capable of cleaving the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at 34-40°C, preferably at 37°C. In a preferred embodiment, the variant of SEQ ID NO. 1 is capable of cleaving the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at room temperature.

[0117] In one embodiment, the variant of SEQ ID NO. 1 is capable of cleaving the intein-extein bond at pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2. In a preferred embodiment, the variant of SEQ ID NO. 1 is capable of cleaving the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, at 34-40°C, preferably at 37°C. In a preferred embodiment, the variant of SEQ ID NO. 1 is capable of cleaving the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, at room temperature.

[0118] The ability of an intein to cleave an intein-extein bond in the lumen of an EV and / or under particular conditions (i.e. at a particular pH and / or temperature) may be verified by the methods outlined in the examples.

[0119] In a preferred embodiment, the variant of SEQ ID NO. 1 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4 or SEQ ID NO: 23. In a preferred embodiment, the intein sequence retains the following specific residues, relative to SEQ ID NO. 4 or SEQ ID NO 23: a Leucine at position 67 and / or a Glycine at position 150. Optionally, the intein sequence may also retain a Glycine at position 24. In a preferred embodiment, the variant of SEQ ID NO. 1 comprises the following specific mutations relative to SEQ ID NO. 1 : H41 R, 1108V, R111 P and / or E152V. In an alternative preferred embodiment, the variant of SEQ ID NO. 1 further comprises a H167Q mutation. In a preferred embodiment, the variant of SEQ ID NO. 1 comprises the following specific mutations relative to SEQ ID NO. 1 : H41 R, 1108V, RW P and E152V. In an alternative preferred embodiment, the variant of SEQ ID NO. 1 comprises the following specific mutations relative to SEQ ID NO. 1 : H41 R, 1108V, R111 P, E152V and H167Q. In a most preferred embodiment, the variant of SEQ ID NO. 1 comprises the following specific residues, relative to SEQ ID NO. 4: a Leucine at position 67 and a Glycine at position 150, and further comprises the following specific mutations relative to SEQ ID NO. 1 : H41 R, 1108V, R111 P and E152V. In an alternative most preferred embodiment, the variant of SEQ ID NO. 1 comprises the following specific residues, relative to SEQ ID NO. 23: a Leucine at position 67 and a Glycine at position 150, and further comprises the following specific mutations relative to SEQ ID NO. 1 : H41 R, 1108V, R111 P, E152V and H167Q.

[0120] The intein of SEQ ID NO 4 was identified following random mutagenesis of the AI-CM intein (having SEQ ID NO. 1). Hence it is likely that not all of the mutations present in SEQ ID NO 4, as compared to SEQ ID NO 1 , are required in order to reduce the rate of cleavage, as compared with SEQ ID NO. 1 at pH 7.0-7.6, more preferably at pH 7.3, at 34-40°C, most preferably at 37°C. Thus, the variant of SEQ ID NO. 1 may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.1 over its entire length, wherein as compared with SEQ ID NO. 1 the variant comprises specific mutations that may be selected from the following: (i) H41 R, (ii) H41 R and 1108V, (iii) H41 R and R111 P, (iv) H41 R and E152V, (v) H41 R, 1108V and R111 P, (vi) H41 R, 1108V and E152V, (vii) H41 R, RW P and E152V, (viii) 1108V (ix) 1108V and R111 P, (x) 1108V and E152V, (xi) 1108V, RW P and E152V, (xii) R111 P (xiii) RW P and E152V, (xiv) E152V. In a preferred embodiment the variant further comprises a Leucine at position 67 and / or a Glycine at position 150. In a more preferred embodiment, the variant further comprises a H167Q mutation.

[0121] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence reduces the rate of cleavage of the 1 intein-extein bond, as compared with SEQ ID NO. 3, in the cytoplasm of a cell, preferably a cultured mammalian cell, most preferably an EV producer cell.

[0122] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence reduces the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 3, at pH 7.0-7.6, more preferably at pH 7.3, preferably at 37°C.

[0123] The ability of an extein to reduce the rate of cleavage of an intein-extein bond in the cytoplasm of a cell and / or under particular conditions (i.e. at a particular pH and / or temperature) may be verified by the methods outlined in the examples.

[0124] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence does not prevent the cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. In a preferred embodiment, the C-terminal extein sequence does not prevent the cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at 34-40°C, preferably at 37°C. In a preferred embodiment, the C-terminal extein sequence does not prevent the cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at room temperature.

[0125] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence does not prevent the cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2. In a preferred embodiment, the C-terminal extein sequence does not prevent the cleavage of the intein- extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, at a temperature of 34 -40°C, preferably 37°C. In a preferred embodiment, the C-terminal extein sequence does not prevent the cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, at room temperature.

[0126] The ability of an extein to affect the cleavage of an intein-extein bond in the lumen of an EV and / or under particular conditions (i.e. at a particular pH and / or temperature) may be verified by the methods outlined in the examples. In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence comprises Glu, Phe, His, Leu, Met, Gly or Pro as the +1 amino acid residue. In a preferred embodiment, the C-terminal extein sequence of the present disclosure comprises Phe or Gly as the +1 amino acid residue. In a more preferred embodiment, the C-terminal extein sequence of the present disclosure comprises Gly as the +1 amino acid residue.

[0127] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence comprises Ser as the +2 amino acid residue and / or Pro as the +3 amino acid residue.

[0128] In a preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence comprises Gly as the +1 amino acid residue, Ser as the +2 amino acid residue and / or Pro as the +3 amino acid residue.

[0129] In an alternative preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence comprises Gly as the +1 amino acid residue, Gly as the +2 amino acid residue, Gly as the +3 amino acid residue, Gly as the +4 amino acid residue and / or Ser as the +5 amino acid residue.

[0130] In an alternative preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the C-terminal extein sequence comprises Thr as the +1 amino acid residue, Arg as the +2 amino acid residue and / or His as the +3 amino acid residue.

[0131] In one embodiment wherein the intein comprises or consists of a variant of SEQ ID NO 1 , the polypeptide of the present disclosure comprises a C-terminal extein sequence as described herein and an N-terminal extein sequence, comprising or consisting of SEQ ID NO. 5.

[0132] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence reduces the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2 and / or SEQ ID NO. 5, in the cytoplasm of a cell, preferably a cultured mammalian cell, most preferably an EV producer cell. In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence reduces the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2 and / or SEQ ID NO. 5, at pH 7.0-7.6, more preferably at pH 7.3, preferably at 37°C.

[0133] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence does not prevent the cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. In a preferred embodiment, the N-terminal extein sequence does not prevent the cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at 34-40°C, preferably at 37°C. In a preferred embodiment, the N-terminal extein sequence does not prevent the cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, at room temperature.

[0134] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence does not prevent the cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2. In a preferred embodiment, the N-terminal extein sequence does not prevent the cleavage of the intein- extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, at a temperature of 34-40°C, preferably 37°C. In a preferred embodiment, the N-terminal extein sequence does not prevent the cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, at room temperature.

[0135] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the - 4 amino acid residue and / or Gly as the -5 amino acid residue. In a preferred embodiment, wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the -4 amino acid residue, Gly as the -5 amino acid residue, Ser as the -6 amino acid residue, Gly as the -7 amino acid residue and / or Gly as the -8 amino acid residue. In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Ser as the -1 amino acid residue, Ala as the -2 amino acid residue and / or Phe as the -3 amino acid residue.

[0136] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Met as the -1 amino acid residue, Arg as the -2 amino acid residue and / or Thr as the -3 amino acid residue.

[0137] In a particularly preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the -4 amino acid residue and / or Gly as the -5 amino acid residue and the C- terminal extein sequence comprises Gly as the +1 amino acid residue. In a further particularly preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the -4 amino acid residue, Gly as the -5 amino acid residue, Ser as the -6 amino acid residue, Gly as the -7 amino acid residue and / or Gly as the -8 amino acid residue and the C-terminal extein sequence comprises Gly as the +1 amino acid residue.

[0138] In a more preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the -4 amino acid residue and / or Gly as the -5 amino acid residue and the C- terminal extein sequence comprises Gly as the +1 amino acid residue, Ser as the +2 amino acid residue and / or Pro as the +3 amino acid residue. In a more preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the -4 amino acid residue, Gly as the -5 amino acid residue Ser as the -6 amino acid residue, Gly as the -7 amino acid residue and / or Gly as the -8 amino acid residue and the C-terminal extein sequence comprises Gly as the +1 amino acid residue, Ser as the +2 amino acid residue and / or Pro as the +3 amino acid residue.

[0139] In a more preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the -4 amino acid residue and / or Gly as the -5 amino acid residue and the C- terminal extein sequence comprises Gly as the +1 amino acid residue, Gly as the +2 amino acid residue, Gly as the +3 amino acid residue, Gly as the +4 amino acid residue and / or Ser as the +5 amino acid residue. In a more preferred embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Gly as the -1 amino acid residue, Ser as the -2 amino acid residue, Gly as the -3 amino acid residue, Gly as the -4 amino acid residue, Gly as the -5 amino acid residue Ser as the -6 amino acid residue, Gly as the -7 amino acid residue and / or Gly as the -8 amino acid residue and the C-terminal extein sequence comprises Gly as the +1 amino acid residue, Gly as the +2 amino acid residue, Gly as the +3 amino acid residue, Gly as the +4 amino acid residue and / or Ser as the +5 amino acid residue.

[0140] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Ser as the -1 amino acid residue, Ala as the -2 amino acid residue and / or Phe as the -3 amino acid residue and the C-terminal extein sequence comprises Gly as the +1 amino acid residue, Ser as the +2 amino acid residue, Pro as the +3 amino acid residue.

[0141] In one embodiment wherein the intein comprises or consists of the variant of SEQ ID NO. 1 of the present disclosure, the N-terminal extein sequence comprises Met as the -1 amino acid residue, Arg as the -2 amino acid residue and / or Thr as the -3 amino acid residue and the C-terminal extein sequence comprises Thr as the +1 amino acid residue, Arg as the +2 amino acid residue, His as the +3 amino acid residue.

[0142] In other embodiments, the intein may be flanked by any combination of N-terminal and C- terminal sequences illustrated in Fig. 7.

[0143] In a particular aspect, there is provided a polypeptide comprising an intein, wherein the intein is capable of catalysing cleavage of the polypeptide and comprises or consists of a variant of SEQ ID NO. 1, wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41; a Valine residue at position 108; a Proline residue at position 111 ; a Valine residue at position 152 and optionally a Glutamine residue at position 167; wherein the polypeptide further comprises a C-terminal extein sequence, wherein the +1 residue of the C-terminal extein sequence is Glu, Phe, His, Leu, Met, Gly or Pro.

[0144] The intein may have any properties or features disclosed herein. For instance, the intein may comprise SEQ ID NO: 4 or be according to SEQ ID NO: 4. The intein may be a variant of SEQ ID NO: 4 with a lower degree of sequence identity, as disclosed herein. For instance, the intein may be of a sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4, wherein positions 41 , 108, 111 , and 152 are invariant ( / .e. are as recited in SEQ ID NO: 4). The intein may be according to SEQ ID NO: 4 but comprise one or more substitutions, deletions, or additions compared to SEQ ID NO: 4. The intein may be according to SEQ ID NO: 4 but comprise zero to five, zero to four, zero to three, zero to two, zero to one, or no substitutions, deletions, or additions compared to SEQ ID NO: 4. In some embodiments, positions 24, 41 , 67, 108, 111 , 150, and / or 152 are invariant.

[0145] Alternatively, the intein may comprise SEQ ID NO: 23 or be according to SEQ ID NO: 23. The intein may be a variant of SEQ ID NO: 23 with a lower degree of sequence identity, as disclosed herein. For instance, the intein may be of a sequence with at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 23, wherein positions 41 , 108, 111 , 152 and 167 are invariant ( / .e. are as recited in SEQ ID NO: 23). The intein may be according to SEQ ID NO: 23 but comprise one or more substitutions, deletions, or additions compared to SEQ ID NO: 23. The intein may be according to SEQ ID NO: 23 but comprise zero to five, zero to four, zero to three, zero to two, zero to one, or no substitutions, deletions, or additions compared to SEQ ID NO: 23. In some embodiments, positions 24, 41, 67, 108, 111 , 150, 152 and / or 167, are invariant.

[0146] The polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and the +1 residue of the C-terminal extein sequence is Gly or Phe. Hence, Gly or Phe may be the next residue following the end of SEQ ID NO: 4 or SEQ ID NO: 23 ( / .e. conjugated to the final Asn of SEQ ID NO: 4 or SEQ ID NO: 23). Experimental data relating to these residues is presented in Fig. 3B. In a particular embodiment, the +1 residue of the C-terminal extein sequence is Gly.

[0147] The polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and the +2 residue of the C-terminal extein sequence is Ser. The polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and the +3 residue of the C-terminal extein sequence is Pro.

[0148] In a particular embodiment, the polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and a sequence according to Gly- Ser-Pro (SEQ ID NO: 6) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23. In an alternative embodiment, the polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and a sequence according to Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7) positioned +1 to +5 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23. In an alternative embodiment, the polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and a sequence according to Thr-Arg-His (SEQ ID NO: 8) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0149] The polypeptide of this aspect may comprise an N-terminal extein. For instance, the polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and a sequence according to Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 10) positioned -5 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23. In an alternative embodiment, the polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and a sequence according to Gly-Gly-Ser- Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 22) positioned -8 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23. In an alternative embodiment, the polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and a sequence according to Thr-Arg-Met (SEQ ID NO: 9) positioned -3 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23. In an alternative embodiment, the polypeptide may comprise an intein according to SEQ ID NO: 4 or SEQ ID NO: 23, or variant as disclosed herein, and a sequence according to Phe-Ala-Ser (SEQ ID NO: 11) positioned -

[0150] 3 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23. Other suitable N- terminal extein sequences are illustrated in Fig. 7.

[0151] In a particular example, the polypeptide may comprise a sequence according to SEQ ID NO:

[0152] 4 or SEQ ID NO: 23 or variant as disclosed herein, and a sequence according to Gly-Gly- Gly-Ser-Gly (SEQ ID NO: 10) positioned -5 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23, and a sequence according to Gly-Ser-Pro (SEQ ID NO: 6) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23 or a sequence according to Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7) positioned +1 to +5 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0153] In a further example, the polypeptide may comprise a sequence according to SEQ ID NO: 4 or SEQ ID NO: 23 or variant as disclosed herein, a sequence according to Gly-Gly-Ser-Gly- Gly-Gly-Ser-Gly (SEQ ID NO: 22) positioned -8 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23, and a sequence according to Gly-Ser-Pro (SEQ ID NO: 6) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23 or a sequence according to Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 7) positioned +1 to +5 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0154] In a further example, the polypeptide may comprise a sequence according to SEQ ID NO: 4 or SEQ ID NO: 23 or variant as disclosed herein, a sequence according to Thr-Arg-Met (SEQ ID NO: 9) positioned -3 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23, and a sequence according to The-Arg-His (SEQ ID NO: 8) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0155] In a further example, the polypeptide may comprise a sequence according to SEQ ID NO: 4 or SEQ ID NO: 23 or variant as disclosed herein, a sequence according to Phe-Ala-Ser (SEQ ID NO: 11) positioned -3 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23 or variant as disclosed herein, and a sequence according to Gly-Ser-Pro (SEQ ID NO: 6) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23 or variant as disclosed herein.

[0156] In another particular aspect, there is provided a polypeptide comprising an intein, wherein the intein is capable of catalysing cleavage of the polypeptide and comprises or consists of a variant of SEQ ID NO. 1, wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41; a Valine residue at position 108; a Proline residue at position 111, a Valine residue at position 152 and optionally a Glutamine at position 167; wherein the polypeptide further comprises an N-terminal extein sequence, wherein the N-terminal extein sequence comprises Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue. For instance, the polypeptide may comprise an intein according to SEQ ID NO: 4, SEQ ID NO: 23 or variant as disclosed herein, and a sequence according to Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 10) positioned -5 to -1 relative to the N- terminal end of SEQ ID NO: 4 or SEQ ID NO: 23. Other suitable N-terminal extein sequences are illustrated in Fig. 7.

[0157] In one aspect, the present disclosure provides a fusion protein comprising or consisting of an intein or a polypeptide comprising or consisting of an intein and optionally an extein sequence of the present disclosure, fused to an EV polypeptide. The intein may be any as disclosed herein. The extein may be any as disclosed herein. The fusion protein may comprise an extein N-terminal to the intein and an extein C-terminal to the intein, and the combination of exteins and intein may be any disclosed herein. In an example, there is provided a fusion protein comprising an intein according to SEQ ID NO: 4, SEQ ID NO: 23 or variant as disclosed herein, and an EV polypeptide. The fusion protein may comprise a sequence according to Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 10) positioned -5 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23, and a sequence according to Gly-Ser-Pro (SEQ ID NO: 6) positioned +1 to +3 relative to the C- terminal end of SEQ ID NO: 4 or SEQ ID NO: 23 or a sequence according to Gly-Gly-Gly- Gly-Ser (SEQ ID NO: 7) positioned +1 to +5 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0158] In a further example, the fusion protein comprises an intein according to SEQ ID NO: 4, SEQ ID NO: 23 or variant as disclosed herein, and an EV polypeptide. The fusion protein may further comprise a sequence according to Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 22) positioned -8 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23, and a sequence according to Gly-Ser-Pro (SEQ ID NO: 6) positioned +1 to +3 relative to the C- terminal end of SEQ ID NO: 4 or SEQ ID NO: 23 or a sequence according to Gly-Gly-Gly- Gly-Ser (SEQ ID NO: 7) positioned +1 to +5 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0159] In a further example, the fusion protein comprises an intein according to SEQ ID NO: 4, SEQ ID NO: 23, or variant as disclosed herein, and an EV polypeptide. The fusion protein may further comprise a sequence according to Thr-Arg-Met (SEQ ID NO: 9) positioned -3 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23, and a sequence according to The-Arg-His (SEQ ID NO: 8) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0160] In a further example, the fusion protein comprises an intein according to SEQ ID NO: 4, SEQ ID NO: 23, or variant as disclosed herein, and an EV polypeptide. The fusion protein may comprise a sequence according to Phe-Ala-Ser (SEQ ID NO: 11) positioned -3 to -1 relative to the N-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23, and a sequence according to Gly- Ser-Pro (SEQ ID NO: 6) positioned +1 to +3 relative to the C-terminal end of SEQ ID NO: 4 or SEQ ID NO: 23.

[0161] In one embodiment, the EV polypeptide is fused (optionally via a linker) to the N-terminus of the intein or the polypeptide comprising or consisting of an intein and optionally an extein as described herein. In one embodiment, the EV polypeptide of the fusion protein comprises the extein sequence. In a preferred embodiment, the EV polypeptide comprises an N-terminal extein sequence of the present disclosure at its C-terminus. In an alternative embodiment, the EV polypeptide of the fusion protein is fused to the extein sequence. In a preferred embodiment, the EV polypeptide is fused to the N-terminus of an N-terminal extein sequence of the present disclosure.

[0162] As used herein, the term “fused” or “fusion” includes where one polypeptide is fused immediately to or into another polypeptide with no intervening amino acid residues. As used herein the term “fused” or “fusion” also includes where one polypeptide is fused to or into another polypeptide wherein further amino acid residues are present between the two polypeptide sequences, for instance the two polypeptide sequences may be fused together via a linker or may comprise a further polypeptide between them.

[0163] As used herein the terms “EV polypeptide” and “EV protein” are interchangeable. As used herein an EV polypeptide is essentially any protein, region, domain, motif, or sequence or stretch of amino acids that is capable of transporting a fusion protein into an EV produced by a given EV-producing cell. Generation of fusion proteins of EV polypeptide with a further protein of interest has been shown to be an effective way to load an EV with the protein of interest. In a preferred embodiment, the EV polypeptide of the present disclosure is an exosomal polypeptide. As used herein the terms “exosomal polypeptide” and “exosomal protein” are interchangeable. As used herein an exosomal polypeptide is essentially any protein, region, domain, motif, or sequence or stretch of amino acids that is capable of transporting a fusion protein into an exosome produced by a given exosome-producing cell. The use of EV polypeptides has the effect of driving loading of the fusion protein into EVs, such that production of EVs comprising the fusion protein is increased by virtue of the pressure exerted on the EV-producing cell to express and translate the delivered polynucleotide cargo.

[0164] In one example, the EV polypeptide is a transmembrane EV polypeptide. In a preferred example the EV polypeptide is a single pass transmembrane protein or multi-pass transmembrane protein such as a tetraspanin. The EV polypeptide may be of a human sequence or derived from a human sequence. In a preferred example the EV polypeptide is selected from the group consisting of the following non-limiting examples: CD9, CD53, CD63, CD81 , CD54, CDSO, FLOT1 , FLOT2, CD49d, CD71, CD133, CD138, CD235a, AAAT, AT1 B3, AT2B4, ALIX, Annexin, BASI, BASP1 , BSG, Syntenin-1 , Syntenin-2, TSP2, TSP3, Lamp2, Lamp2a, Lamp2b, TSN1 , TSN2 (also referred to herein as TSPAN2), TSN3, TSN4, TSNS TSN6, TSN7, TSN8, TSN31 , TSN10, TSN11 , TSN12, TSN13, TSN14, TSN15, TSN16, TSN17, TSN18, TSN19, TSN2, TSN4, TSN9, TSN32, TSN33, TNFR, TfR1 , syndecan-1 , syndecan-2, syndecan-3, syndecan-4, CD37, CD82, CD151 , CD224, CD231 , CD102, N0TCH1 , N0TCH2, N0TCH3, N0TCH4, DLL1 , DLL4, JAG1 , JAG2, CD49d / ITGA4, ITGBS, ITGB6, ITGB7, CD11a, CD11 b, CD11c, CD18 / ITGB2, CD41 , CD49b, CD49c, CD49e, CD51 , CD61 , CD104, CLIC1 , CLIC4, interleukin receptors, CO2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD53, CD86, CD110, CD111 , CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD 274, CD362, COL6A1 , AGRN, EGFR, FPRP, GAPDH, GLUR2, GLUR3, GP130, GPI anchor proteins, GTR1 , HLAA, HLA-DM, HSPG2, ITA3, Lactadherin, L 1CAM, LAMB1 , LAMC1 , LIMP2, MYOF, ARRDC1 , ATP282, ATP283, ATP284, BSG, IGSF2, IGSF3, IGSF8, ITGB1 , ITGA4, ATP1A2, ATP1A3, ATP1A4, ITGA4, SLC3A2, ATP transporters, ATP1A1 , ATP183, ATP281 , LFA-1 , LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, a member of the myristoylated alanine rich Protein Kinase C substrate (MARCKS) protein family such as MARCKSL 1 , matrix metalloproteinase-14 (MMP14), PTGFRN, BASP1 , MARCKS, MARCKSL 1 , PRPH2, R0M1 , SLIT2, SLC3A2, SSEA4, STX3, TCRA, TCRB, TCRD, TCRG, TFR1 , UPK1A, UPK1 B, VTI1A, VTI1 B, PTTG1-IP, VSVG, HIV gag proteins and any other EV polypeptide, and any combinations, derivatives, domains, variants, mutants, or regions thereof. Mutations, truncations, linkers or additions may be introduced into the wildtype sequence of the EV polypeptide to alter its function, for instance a preferred mutant according to the invention is a mutation of the tetraspanin CD63 which replaces the tyrosine in position 235 with alanine (denoted CD63 / Y235A). In a preferred embodiment, the EV polypeptide is selected from TSN2, PTGFRN, PTTG1-IP, CD63, Lamp2b, CD81 , Syntenin-1 , Syntenin-2, Lamp2, Lamp2a, TSN3, BASP1 , MARCKS, a HIV gag protein and VSVG, which can in some circumstances function as an EV polypeptide. In a more preferred embodiment, the EV polypeptide is selected from TSN2, CD63, PTGFRN, PTTG1-IP, a HIV gag protein or Lamp2b.

[0165] In one embodiment, the EV polypeptide is a polypeptide comprising a myristoylation site. As used herein the term “myristoylation site” refers to an amino acid sequence to which a myristic acid molecule is covalently attached when the polypeptide is expressed in a cell, preferably a mammalian cell. In one embodiment, the myristoylation site is Gly-X-X-X- Ser / Thr, where “X” represents any amino acid.

[0166] In another aspect, the present disclosure provides a conjugated protein, comprising or consisting of an intein or a polypeptide comprising or consisting of an intein and optionally an extein sequence of the present disclosure, conjugated to a moiety for loading the polypeptide into the EV.

[0167] In one embodiment, the moiety for loading the polypeptide into the EV is conjugated to the extein sequence. In a preferred embodiment, the EV loading moiety is conjugated to an N- terminal extein sequence of the present disclosure.

[0168] The term “moiety for loading the polypeptide into the EV” is used interchangeably with “EV loading moiety” and “EV localisation moiety” herein. The term loading or loaded when used in relation to an EV, as used herein, refers to the association of a particular molecule with an EV. Preferably the molecule remains associated with the EV following one or more EV isolation or purification steps. The term “EV loaded with” is used interchangeably with the term “EV associated with” and the term “EV comprising”.

[0169] In one embodiment, the moiety for loading the polypeptide into the EV is a membrane anchoring moiety and / or a hydrophobic moiety, preferably a myristic acid, a lipid moiety, cholesterol moiety or a vitamin A moiety.

[0170] In one embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a protein cargo, preferably a therapeutic protein cargo, or a protein-RNA cargo such as a ribonucleoprotein (RNP), e.g. a Cas9, Cas12, Cas12a, base editor, prime editor or similar RNP. In an alternative embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a protein capable of binding to a cargo, preferably a therapeutic cargo.

[0171] In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused (optionally via a linker) to the C-terminus of the intein or the polypeptide comprising or consisting of an intein and optionally an extein as described herein.

[0172] In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex comprises the extein. In a preferred embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex comprises the C-terminal extein sequence as described herein, preferably at its N-terminus. In an alternative embodiment, protein cargo or protein capable of binding to a cargo is fused to the extein sequence. In a preferred embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the C- terminus of the C-terminal extein sequence as described herein. In one embodiment, the intein is positioned such that the protein cargo, protein capable of binding to a cargo or RNP complex is released from the EV polypeptide upon cleavage of the intein-extein bond. In a preferred embodiment, the intein is positioned such that the protein cargo, protein capable of binding to a cargo or RNP complex is released from both the intein and the EV polypeptide upon cleavage of the intein-extein bond. In one embodiment, the intein is positioned between the EV polypeptide or the EV loading moiety and the protein cargo, protein capable of binding to a cargo or RNP complex.

[0173] In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the EV polypeptide or conjugated to the EV loading moiety, such that the protein cargo, protein capable of binding to a cargo or RNP complex is localised to the lumen of the EV.

[0174] In a one embodiment of the fusion protein of the present disclosure, the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure is fused to a domain or terminus of the EV polypeptide that is localised to and / or displayed in the lumen of the EV. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused (via the intein) to a domain or terminus of the EV polypeptide that is localised to and / or displayed in the lumen of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused, via the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure, to the N-terminus or C-terminus of a tetraspanin EV polypeptide, such as TSN2 or CD63, preferably TSN2. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused, via the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure, to the C-terminus of PTTG1-IP.

[0175] In a one embodiment of the conjugated protein of the present disclosure comprising a protein cargo, a protein capable of binding to a cargo or an RNP complex, the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure is conjugated to a part of the of the EV loading moiety that is localised to and / or displayed in the lumen of the EV. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is conjugated (via the intein) to a part of the EV loading moiety that is localised to and / or displayed in the lumen of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In one embodiment the cargo is a therapeutic cargo protein that may be selected from: enzymes, receptors such as decoy receptors, membrane proteins, transporters, cytokines, antigens, neoantigens, immune effector molecules, ribonuclear proteins, 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. In more detail the therapeutic protein cargos (POIs) according to the present invention include: antibodies, intrabodies, nanobodies, single chain variable fragments (scFv), affibodies, bi- and multispecific antibodies or binders including bispecific T-cell engagers (BiTEs), receptors, ligands, transporters, enzymes for e.g. ERT or gene editing, tumour suppressors, viral or bacterial inhibitors, cell component proteins, DNA repair inhibitors, nucleases, proteinases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins (for instance pseudomonas exotoxins), structural proteins, neurotrophic factors such as NT3 / 4, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and its individual subunits such as the 2.5S beta subunit, ion channels, membrane transporters, proteostasis factors, proteins involved in cellular signaling, translation- and transcription related proteins, nucleotide binding proteins, protein binding proteins, lipid binding proteins, glycosaminoglycans (GAGs) and GAG-binding proteins, metabolic proteins, cellular stress regulating proteins, inflammation and immune system regulating proteins such as cytokines and inhibitors of such cytokines (cytokines may include: CXCL8, GMCSF, interleukins including: IL-1 family, IL-2, IL-4, IL-6, IL-6-like, IL-9, IL-10, IL12, IL-13, IL-17, Interferons including INF-alpha / beta / gamma, TNF family members, CD40 and CD40L, TRAIL, and TGF- beta family) mitochondrial proteins, and heat shock proteins, etc. The cargo protein may be a reporter protein such as green fluorescent protein (GFP) or nanoLuc.

[0176] In a preferred embodiment the protein cargo is a gene editing technology, preferably a CRISPR-associated (Cas) protein, a base editor or a prime editor. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) components according to the present invention include CRISPR components that are derived from any bacterial source. The CRISPR components may come from class 1 or class 2, specifically the Cas type may be Cas type I, II, III, IV, V or VI. The specific Cas protein may be Cas9, Cas12 (Cas12a or Cas12b), C2c2, Cpf 1 , Casio, Cas13 (cas13a, Cas13b or Cas13c), Cas3, a Cas14 protein, a CasX protein, or a CasY protein, CasMINI, or SuperFi-Cas9. The CRISPR protein may be a CRISPR nuclease, a CRISPR nickase, or a nuclease deficient CRISPR variant. Yet another alternative may be any other type of CRISPR effector such as the single RNA guided endonuclease Cpf1. The inclusion of Cpf1 is a particularly preferred embodiment of the present invention, as it cleaves target DNA via a staggered double-stranded break. Cpf1 may be obtained from species such as Acidami nococcus or Lachnospiraceae. In yet another exemplary embodiment, the Cas polypeptide may also be fused to a transcriptional activator (such as the P3330 core protein), to specifically induce gene expression.

[0177] In a more preferred embodiment, the Cas protein is a Cas9 a Cas12, such as Cas12a, a Cas3. Optionally, the Cas protein is associated with a guide RNA. As used herein, the term protein cargo also encompasses protein-RNA cargo, cargo such as a ribonucleoprotein (RNP) complex.

[0178] In a preferred embodiment the cargo is a protein-RNA cargo such as a ribonucleoprotein (RNP) complex. In a preferred embodiment, the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor. In a preferred embodiment the RNP complex is loaded in the lumen of the EV, preferable an exosome, a microvesicle and / or a related extracellular vesicle.

[0179] In one embodiment, the protein capable of binding to a cargo is a nucleic acid (NA) binding protein, a protein binding protein or a viral binding protein.

[0180] As used herein, the term “NA-binding protein” refers to a protein or protein domain capable of binding a nucleic acid or polynucleotide. In one embodiment, the NA-binding protein is capable of binding DNA, or preferably RNA, most preferably a guide RNA.

[0181] A "nucleic acid" refers to a polynucleotide and includes polyribonucleotides and polydeoxyribonucleotides. Nucleic acids according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, e.g., cytosine, thymine, and uracil, and adenine and guanine, respectively. (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) and G. Michael Blackburn, Michael J. Gait, David Loakes and David M. Williams, Nucleic Acids in Chemistry and Biology 3rdedition, (RSC publishing 2006) which are herein incorporated in their entirety for all purposes). Indeed, the present invention contemplates any deoxyribonucleotide or ribonucleotide component, and any chemical variants thereof. The polymers or oligomers may be heterogeneous or homogeneous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or doublestranded form, including homoduplex, heteroduplex, and hybrid states. An "oligonucleotide" or "polynucleotide" is a nucleic acid ranging from at least 2, at least 8, at least 15 or at least 25 nucleotides in length, but may be up to 50, 100, 1000, 5000, 10000, 15000, or 20000 nucleotides long or a compound that specifically hybridises to a polynucleotide. Polynucleotides include sequences of DNA or RNA or mimetics thereof, which may be isolated from natural sources, recombinantly produced or artificially synthesised. A further example of a polynucleotide as employed in the present invention may be a peptide nucleic acid (PNA; see U.S. Patent No. 6,156,501 , which is hereby incorporated by reference in its entirety.) The invention also encompasses situations in which there is a non-traditional base pairing, such as Hoogsteen base pairing, which has been identified in certain tRNA molecules and postulated to exist in a triple helix. "Polynucleotide" and "oligonucleotide" are used interchangeably herein. It will be understood that when a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C), this also includes the corresponding RNA sequence (e.g., A, U, G, C) in which "U" replaces "T".

[0182] As used herein, "polynucleotide" includes, for instance, cDNA, RNA, DNA / RNA hybrid, antisense RNA, siRNA, mRNA, gRNA, sgRNA, pegRNA, ribozyme, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified to contain non-natural or derivatised, synthetic, or semi-synthetic nucleotide bases. Also, contemplated are alterations of a wild type or synthetic gene, including but not limited to deletion, insertion, substitution of one or more nucleotides, or fusion to other polynucleotide sequences.

[0183] In one embodiment the NA-binding protein is Ago2, Dicer, Drosha, DGCR8, hnRNPAI, hnRNPA2B1, DDX4, ADAD1 , DAZL, ELAVL4, IGF2BP3, SAMD4A, TDP43, FUS, FMR1, FXR1, FXR2, EIF4A13, the MS2 coat protein, as well as any domains, parts or derivates, thereof. More broadly, particular subclasses of RNA-binding proteins and domains, e.g. mRNA binding proteins (mRBPs), pre-rRNA-binding proteins, tRNA-binding proteins, small nuclear or nucleolar RNA-binding proteins, non-coding RNA-binding proteins, miRNA- binding proteins, shRNA-binding proteins and transcription factors (TFs). Furthermore, various domains and derivatives may also be used as the NA-binding domain to transport an NA cargo into EVs. Non-limiting examples of RNA-binding domains include small RNA- binding domains (RBDs) (which can be both single-stranded and double-stranded RBDs (ssRBDs and dsRBDs) such as DEAD, KH, GTP_EFTU, dsrm, G-patch, IBN_N, SAP, TUDOR, RnaseA, MMR-HSR1, KOW, RnaseT, MIF4G, zf-RanBP, NTF2, PAZ, RBM1CTR, PAM2, Xpo1, Piwi, CSD, and Ribosomal_L7Ae. Such RNA-binding domains may be present in a plurality, alone or in combination with others, and may also form part of a larger RNA- binding protein construct as such, as long as their key function (i.e. the ability to transport an NA cargo of interest, e.g. an mRNA or a short RNA) is maintained. In a preferred embodiment, the NA-binding protein is a PUF or a Cas protein or protein domain. Such PUF and Cas binding domains and their use in EV loading is described in WO2019 / 092145 A1 , which is incorporated by reference in its entirety.

[0184] As used herein, the term “protein binding protein” refers to a protein or protein domain capable of binding another protein. In one embodiment the protein binding domain is an Fc domain, also known as an Fc-binding protein. Fc binders and their use in EV engineering is described in WO 2018 / 015535 A1 and WO 2018 / 011191 A1 , which are both incorporated by reference in its entirety. In a preferred embodiment, the Fc binding polypeptide is bound to an antibody, VHH or nanobody against a viral capsid or envelop protein, preferably an AAV capsid, more preferably an AAV7, AAV8 orAAV9 capsid, most preferably an AAV8 orAAV9 capsid.

[0185] As used herein, the term “viral binding protein is” refers to a protein or a protein domain that is capable of binding to a viral particle, preferably an AAV or a lentivirus. Generally, any protein capable of binding to a viral coat / envelope protein or capable of binding to a viral genome is within the scope of the invention. Exemplary viral binding proteins include: AAVR GPR108, syndecans and albumin. In a preferred embodiment, the viral binding protein is an antibody, or more preferably a nanobody or VHH, that is capable of binding to a viral capsid or envelop protein, preferably an AAV capsid, more preferably an AAV7, AAV8 or AAV9 capsid, most preferably an AAV8 or AAV9 capsid.

[0186] In one embodiment, the protein capable of binding to a cargo is associated with a cargo that it is capable of binding.

[0187] In a preferred embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a viral binding protein as described herein, wherein the viral binding protein is associated with a viral particle. In a preferred embodiment, the viral particle is an AAV or a lentivirus, most preferably an AAV.

[0188] In some embodiments, the AAV vector comprises a capsid from human AAV serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12. In some embodiments, the AAV vector comprises an AAV viral genome comprising inverted terminal repeat (ITR) sequences from human AAV serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rAAV10. In some embodiments, the AAV capsid and the AAV ITR are from the same serotype or from different serotypes. In a preferred embodiment the viral particle is a therapeutic AAV.

[0189] In some embodiments, the lentiviral vector is derived from human immunodeficiency virus, a simian immunodeficiency virus or a feline immunodeficiency virus. In some embodiments, the lentiviral vector is non-replicating. In some embodiments, the lentiviral vector is nonintegrating.

[0190] In some embodiments the viral vector comprises a viral capsid and a viral genome, the viral genome comprising one or more heterologous transgenes. In preferred embodiments, the heterologous transgene encodes a polypeptide or protein. The protein encoded with in the viral genome may be any one of the protein cargos according to the invention allowing the viral cargo to act as a gene replacement therapy.

[0191] In an alternative preferred embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises an NA binding protein as described herein, wherein the NA binding protein is associated with a nucleic acid cargo. The nucleic acid cargo molecule may be selected from the group comprising shRNA, siRNA, saRNA, gRNA, sgRNA, pegRNA, miRNA, an anti-miRNA, mRNA, modified mRNA, gRNA, pri- miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA, IncRNA, ribozymes, minicircle DNA, plasmid DNA, RNA / DNA vectors, trans-splicing oligonucleotides, spliceswitching oligonucleotides, CRISPR guide strands, morpholinos (PMO) antisense oligonucleotides (ASO), peptide-nucleic acids (PNA), a viral genome and viral genetic material (for instance a naked AAV genome), but essentially any type of nucleic acid molecule can be delivered by the EVs of the present invention. Both single-stranded and double-stranded nucleic acid molecules are within the scope of the present invention, and the nucleic acid molecule may be naturally occurring (such as RNA or DNA) or may be a chemically synthesised RNA and / or DNA molecule which may comprise chemically modified nucleotides such as 2’-0-Me, 2’-O-Allyl, 2’-O-MOE, 2’-F, 2’-CE, 2’-EA 2’-FANA, LNA, CLNA, ENA, PNA, phosphorothioates, tricyclo-DNA, thionucleotides, phosphoramidate, PNA, PMO, etc.

[0192] In a preferred embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises an RNA binding protein as described herein, wherein the RNA binding protein is associated with an RNA In a preferred embodiment the RNA is a therapeutic RNA. In a preferred embodiment, the RNA is a guide RNA. The guide RNA may or may not be associated with a Cas protein, such as a Cas9, a Cas12 (such as Cas12a) or a Cas 3.

[0193] In a further alternative preferred embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a DNA binding protein as described herein, wherein the DNA binding protein is associated with a DNA, preferably a recombinant AAV vector. In a preferred embodiment the DNA is a therapeutic recombinant AAV vector.

[0194] In one embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a purification moiety.

[0195] As used herein, the term “purification moiety” refers to any molecule including protein, protein domains and protein tags that can be used to purify EVs, preferably exosomes, microvesicles and / or a related extracellular vesicles. In one embodiment, the purification moiety allows for the EVs to be purified by affinity purification. Affinity purification of EVs is described in WO 2018 / 153581 A1 , WO2019 / 081474 A1 and WO2019 / 238626 A1 , which are incorporated by reference in their entirety.

[0196] In a one embodiment, the purification moiety is fused or conjugated to the EV polypeptide or the EV loading moiety, such that the purification moiety is displayed on the surface of the EV. In one embodiment, the purification moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In an alternative embodiment, purification moiety is conjugated to a part of the EV loading moiety that is localised to and / or displayed on the surface of an EV or exosome. In one embodiment, the purification moiety is fused or conjugated, optionally via the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure, to loop 1 or loop 2 of a tetraspanin EV polypeptide, such as TSN2 or CD63, preferably TSN2. In one embodiment, the purification moiety is fused or conjugated, optionally via the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure, to the N- terminus of PTTG1-IP.

[0197] In one embodiment of the fusion protein of the present disclosure or the conjugated protein of the present disclosure, the intein is positioned such that the purification moiety is released from the EV polypeptide upon cleavage of the intein-extein bond. In one embodiment, the intein is positioned between the EV polypeptide or the EV loading moiety and the purification moiety. In one embodiment, the purification moiety comprises the extein as described herein. In an alternative embodiment, the purification moiety is fused to the extein as described herein.

[0198] In one embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises the purification moiety in addition to a protein cargo as described herein, a protein capable of binding a cargo as described herein or an RNP complex. In one embodiment, the fusion protein comprises two inteins or polypeptides comprising an intein and extein sequence of the present disclosure. Preferably one of the inteins or polypeptides is positioned between the EV polypeptide or the EV loading moiety and the protein cargo, protein capable of binding a cargo or RNP complex and the second intein or polypeptide is positioned between the EV polypeptide or the EV loading moiety and the purification moiety.

[0199] In one embodiment, the purification moiety is a chitin-binding tag.

[0200] In a further embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure further comprises a targeting moiety.

[0201] As used herein, the term “targeting moiety” refers to molecule associated with the EV that enables targeted delivery of the EV to a cell, tissue, organ, and / or compartment of interest. The targeting moiety may be capable of binding to a moiety present of the target cell or of a cell type present in the target tissue or organ. In a preferred embodiment, the targeting moiety targets the liver, the heart, the brain or neuronal tissue, hepatocytes, cardiomyocytes, cardiac smooth muscle cells, sensory neurons, Purkinje neurons, motor neurons, interneurons or glia cells. Targeting can be achieved by a variety of means, for instance the use of targeting peptides. Such targeting peptides may be anywhere from a few amino acids in length to several 100s of amino acids in length, e.g. anywhere in the interval of 3-100 amino acids, 3-30 amino acids, 5-25 amino acids, e.g. 7 amino acids, 12 amino acids, 20 amino acids, etc. Targeting peptides of the present invention may also include full length proteins such as receptors, receptor ligands, etc. In one embodiment, the targeting moiety is a protein, peptide, an antibody, a VHH, a nanobody or any other derivatives of an antibody including monoclonal antibodies, single chain variable fragments (scFvs), nanobodies and other antibody domains.

[0202] In a preferred embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a targeting moiety in addition to the protein cargo as described herein, the protein capable of binding to a cargo as described herein, the RNP complex and / or the purification moiety as described herein.

[0203] In a preferred embodiment, the targeting moiety is fused or conjugated to the EV polypeptide or the EV loading moiety, such that the targeting moiety is displayed on the surface of the EV. In one embodiment, the targeting moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In an alternative embodiment, targeting moiety is conjugated to a part of the EV loading moiety that is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In one embodiment, the targeting moiety is fused or conjugated to loop 1 or loop 2 of a tetraspanin EV polypeptide, such as TSN2 or CD63, preferably TSN2. In one embodiment, the targeting moiety is fused or conjugated to the N- terminus of PTTG1-IP.

[0204] In a further embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure further comprises a multimerization domain.

[0205] As used herein, the term “multimerization domain” refers to a protein or protein domain that enable dimerization, trimerization, or any higher order of multimerization of the fusion polypeptides. This increases the sorting and trafficking of the fusion polypeptides into EVs and may also contribute to increase the yield of vesicles produced by EV-producing cells. In a preferred embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a multimerization domain in addition to a protein cargo as described herein, a protein capable of binding to a cargo a protein cargo as described herein and / or a purification moiety a protein cargo as described herein. In one embodiment the multimerization domain is a homo-multimerization domains or a hetero- multimerization domains. In one embodiment, the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or any higher order of multimerization domain. In one embodiment, the multimerization domain is selected from a 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 trimerisation domain, HPV45 oncoprotein E7 C-terminal dimer domain, and any combination thereof. In a most preferred embodiment, the multimerization domain is a fold-on domain or a leucine zipper In a preferred embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a multimerization domain together with a targeting moiety.

[0206] In a further embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure further comprises an endosomal escape domain or endosomal escape moiety.

[0207] The terms “endosomal escape domain” and “endosomal escape moiety” refer to molecules that allow for components of the EV lumen to be delivered outside of the endo-lysosomal system of a target cell and preferably into the cytoplasm and / or nucleus. The presence of endosomal escape domains advantageously assists to drive endosomal escape and thereby enhance the bioactive delivery of the EV per se. Use of endosomal escape strategies is particularly important in the treatment of diseases where the cargo carried within the EV is required to be delivered into the cytosol of the recipient cell or within any other compartment that is outside of the endo-lysosomal system. In one embodiment, the endosomal escape domain is HA2, VSVG, GALA, B18, HIV TAT PDT (peptide / protein transduction domain), HIV Gp-120, KALA, GALA and INF-7 (derived from the N-terminal domain of influenza virus hemagglutinin HA-2 subunit) or an endosomal escape moiety that acts by causing membrane fusion such as viral glycoproteins, Diphtheria toxin T domain, proton sponge type endosomal escape moieties such as peptides or lipids with histidine or imidazole moieties and cell penetrating peptides (CPPs) as described herein and other moieties that enable endosomal escape. In a preferred embodiment, the endosomal escape domain comprises VSVG or a viral glycoprotein.

[0208] In one embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises an endosomal escape moiety in addition to a protein cargo as described herein, a protein capable of binding to a cargo as described herein or an RNP complex. In a most preferred embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is luminally loaded.

[0209] In a further embodiment, the fusion protein of the present disclosure or the conjugated protein of the present disclosure further comprises a pharmacokinetic and / or pharmacodynamic effector moiety.

[0210] The term “Pharmacokinetic Effector Moiety” as used herein relates to any molecule, including any small molecule, protein, peptide, antibody or nanobody, or fragment or domain thereof, capable of affecting the pharmacokinetics of the EV. The term “Pharmacodynamic Effector Moiety” as used herein relates to any to any molecule, including any small molecule, protein, peptide, antibody or nanobody, or fragment or domain thereof, capable of affecting the pharmacodynamics of the EV. In a preferred embodiment, the pharmacokinetic or pharmacodynamic effector moiety is an albumin binding domain.

[0211] The term “albumin binding domain” (ABD) shall be understood to relate to any protein, peptide, antibody or nanobody, or fragment or domain thereof capable of binding to albumin. ABDs may be derived from any species, preferably the ABD has specific binding affinity for human serum albumin. In one aspect, the ABD is an antibodies or a nanobodies raised against albumin, an ABD derived from PAB protein from Peptostreptococcus magnus or protein G from group C and G streptococci. Alternatively, the albumin binding domain may be an antibody, scFv nanobody, heavy chain antibody (hcAb), single domain antibody (sdAb) such as VHH or VNAR, or a fragment thereof which is capable of binding to albumin. sdAbs and antibody fragments are particularly preferred due to their small size which allows for other additional domains to be introduced into the fusion protein and simple construct generation and expression.

[0212] In a preferred embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to the EV polypeptide or the EV loading moiety, such that the pharmacokinetic and / or pharmacodynamic effector moiety is displayed on the surface of the EV. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In an alternative embodiment pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to a part of the EV loading moiety that is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to loop 1 or loop 2 of a tetraspanin EV polypeptide, such as TSN2 or CD63, preferably TSN2. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to the N-terminus of PTTG1-IP.

[0213] In one embodiment, the, fusion proteins of the present disclosure or the conjugated proteins of the present disclosure further comprise a linker, spacer and / or scaffold sequence. Such sequences allow flexibility and enable optimal display of a cargo protein, a protein capable of binding to a cargo, a multimerization domain, a targeting moiety, an endosomal escape domain, a pharmacokinetic effector moiety, pharmacodynamic effector moiety or any other loaded molecule on the surface of the EV or luminally according to what is required. Linkers according to the invention are useful in providing increased flexibility, improving pharmacokinetics (PK), increasing expression and improving biological activity of the fusion polypeptides, and also to the corresponding polynucleotide constructs, and may also be used to ensure avoidance of steric hindrance and maintained functionality of the fusion polypeptides. Exemplary linkers according to the present invention include glycine or serine linkers which increase stability or flexibility such as (GGGGS)n (n=1 , 2, 4) (SEQ ID NOs: 7, 18, and 19) or (Gly)6 (SEQ ID NO. 14), (Gly)8 (SEQ ID NO. 15), rigid linkers such as (EAAAK)n (n=1-3 - SEQ ID NOs: 16, 20, and 21 , and A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO 17), bending linkers (XP)n or cleavable linkers such as disulphide, protease sensitive sequences.

[0214] A further aspect of the present disclosure relates to a polynucleotide encoding a polypeptide comprising an intein and an extein sequence of the present disclosure, a fusion protein of the present disclosure or a conjugated protein of the present disclosure.

[0215] Another aspect of the present disclosure relates to an EV comprising a polypeptide comprising an intein and an extein sequence of the present disclosure, a fusion protein of the present disclosure, a conjugated protein of the present disclosure or a polynucleotide of the present disclosure.

[0216] A further aspect of the present disclosure relates to an EV comprising a polypeptide comprising an intein and an extein sequence of the present disclosure, a fusion protein of the present disclosure or a conjugated protein of the present disclosure, wherein the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein has been cleaved. In a preferred embodiment, the intein has catalysed the cleavage of the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein of the present disclosure. In a further preferred embodiment, the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein of the present disclosure has been cleaved at an intein-extein bond. In a most preferred embodiment, the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein has been cleaved at the intein-extein bond at the intein C-terminus.

[0217] A further aspect of the present disclosure relates to relates to an EV comprising a fusion protein of the present invention comprising a cargo protein, a protein capable of binding a cargo, an RNP complex and / or a purification moiety or a conjugated protein of the present invention, wherein the cargo protein, protein capable of binding a cargo, an RNP complex and / or purification moiety has been released from the EV polypeptide. In a preferred embodiment, the cargo protein, protein capable of binding a cargo, RNP complex and / or purification moiety has been released from the EV polypeptide and the intein.

[0218] In a preferred embodiment, the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein has been cleaved to release the cargo protein, protein capable of binding a cargo, RNP complex and / or purification moiety. In a further preferred embodiment, the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein has been cleaved at an intein-extein bond. In a most preferred embodiment, the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein has been cleaved at the intein-extein bond at the intein C-terminus.

[0219] The present disclosure also relates to a population of EVs of the present disclosure. In one embodiment, the average number of the polypeptides comprising an intein and an extein sequence of the present disclosure, fusion proteins of the present disclosure or the conjugated proteins of the present disclosure per EV in a population of EVs as described herein is above one per EV, but it may also be below one per EV. In a preferred aspect, 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, in the population of EVs, comprise a polypeptide comprising an intein and an extein sequence of the present disclosure, a fusion protein of the present disclosure or a conjugated protein of the present disclosure.

[0220] In one embodiment, the EV of the present disclosure comprises a cargo, preferably a therapeutic cargo.

[0221] When the cargo is a protein cargo, the cargo may be comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein.

[0222] In one embodiment, the cargo is associated with a protein capable of binding to a cargo. The protein capable of binding to a cargo may be comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein. In one embodiment, the cargo is an RNP complex. The RNP complex may be comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein.

[0223] In one embodiment, the EV comprises the cargo, or an additional cargo, independently of the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure. In one embodiment, the cargo, or the additional cargo, is not fused to the intein or the polypeptide comprising an intein and optionally an extein sequence of the present disclosure and / or does not comprise or is not fused to an extein sequence of the polypeptide comprising an intein and an extein sequence of the present disclosure.

[0224] In one embodiment, the cargo or the additional cargo is loaded by overexpression of the cargo in EV producer cells, preferably by expression of a further fusion protein comprising an EV polypeptide and the cargo or by expression of a further conjugated protein comprising the cargo conjugated to a moiety for loading the polypeptide into the EV. Alternatively, the cargo is loaded by exogenous loading techniques.

[0225] As used herein, the term “exogenous loading technique” is used interchangeably with “exogenous loading method” and refers to the loading of EVs that have been released from EV producer cells. Preferably the EVs have been purified from the EV producer cells and / or other contaminants present in conditioned media from the EV producer cells. The terms “exogenous loading method” and “exogenous loading technique” are used interchangeably herein. In one embodiment, the exogenous loading method is electroporation, transfection with transfection reagents such as cationic transfection agents such as lipofectamine (RTM) and co-incubation of the EV(s), or any combination of these methods. In one embodiment, co-incubation involves conjugating the polypeptide to be loaded to a membrane anchoring moiety such as myristic acid, vitamin A, a lipid or cholesterol tail, a moiety capable of forming chemical bonds with a protein present on the EV surface or a cell penetrating peptide (CPP), as described herein, and incubating the conjugated peptide with the EV(s). In another embodiment, co-incubation involves forming a non-covalent complex between a membrane anchoring moiety such as myristic acid, vitamin A, a lipid or cholesterol tail, a moiety capable of forming chemical bonds with a protein present on the EV surface or a cell penetrating peptide (CPP), as described herein, and incubating the non-covalent complex with the EV(s). As used herein the term “Cell-penetrating peptide (CPP)” refers to a peptide that are capable of penetrating the plasma cell membrane. CPPs are typically positively charged and are usually less than 50 amino acids but may also be longer, are typically highly cationic and rich in arginine and / or lysine amino acids and have the ability to gain access to the interior of virtually any cell type, exemplary CPPs may be transportan, transportan 10, penetratin, MTS, VP22, CADY peptides, MAP, KALA, PpTG20, proline-rich peptides, MPG peptides, PepFect peptides, Pep-1 , L-oligomers, calcitoninpeptides, arginine-rich CPPs such as polyArg, tat and combinations thereof). CPP-mediated EV loading is described in WO 2018 / 011153 A1, which is incorporated by reference in its entirety.

[0226] In one embodiment the cargo is a gene editing technology, such as a Cas protein, a primer editor or a base editor, optionally associated with a guide RNA. In a preferred embodiment, the Cas protein is a Cas9, a Cas12, such as Cas12a, or a Cas3.

[0227] In a preferred embodiment the cargo is a protein-RNA cargo such as a ribonucleoprotein (RNP) complex. In a more preferred embodiment, the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor.

[0228] In an alternative embodiment, the cargo is a viral particle. In a preferred embodiment, the viral particle is an AAV or a lentivirus, most preferably an AAV.

[0229] In a further alternative embodiment, the cargo is a polynucleotide. In one embodiment, the cargo is DNA, preferably a recombinant AAV vector. In another embodiment, the cargo is an RNA, preferably a guide RNA, optionally the guide RNA is associated with a Cas protein, preferably a Cas9, a Cas 12, such as Cas12a, or a Cas 3a.

[0230] In a one embodiment, the cargo is luminally loaded. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to a domain or terminus of the EV polypeptide which is localised to and / or displayed to the lumen an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In an alternative embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to a part of the EV loading moiety that is localised to and / or displayed in the lumen of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the N- terminus or the C-terminus of a tetraspanin EV polypeptide, such as TSN2 or CD63, preferably TSN2. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the C-terminus of PTTG1-IP. In the context of the present disclosure luminal loading of the cargo may have the advantage of shielding the cargo from immune cells and / or neutralising antibodies, providing a less immunogenic therapeutic product, reduced degradation and / or improved stability of the cargo and / or more efficient delivery of the cargo to target cells.

[0231] In an alternative embodiment, the cargo is surface loaded. In one embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV or exosome. In an alternative embodiment, the protein cargo, protein capable of binding to a cargo or RNP complex is fused to a part of the EV loading moiety that is localised to and / or displayed on the surface of an EV or exosome.

[0232] As used herein, the term “luminally loaded” refers to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% of the loaded molecule being present in the EV lumen.

[0233] In one embodiment, the EV of the present disclosure comprises a purification moiety, or an additional purification moiety, as described herein. The purification moiety may be comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein.

[0234] In one embodiment, the EV comprises the purification moiety, or an additional purification moiety, independently of the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure. In one embodiment, the purification moiety is not fused to the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure and / or does not comprise or is not fused to an extein sequence of the polypeptide comprising an intein and an extein sequence of the present disclosure.

[0235] In one embodiment, the purification moiety is loaded by overexpression of the purification moiety in EV producer cells, preferably by expression of a further fusion protein comprising an EV polypeptide and the purification moiety or by expression of a further conjugated protein comprising the purification moiety conjugated to a moiety for loading the polypeptide into the EV. Alternatively, the purification moiety is loaded by exogenous loading techniques. In a preferred embodiment, the purification moiety is surface loaded. In one embodiment, the purification moiety is fused to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In an alternative embodiment, purification moiety is fused to a part of the EV loading moiety that is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In the context of the present disclosure surface loading of the purification moiety may have the advantage of allowing for purification of the EVs following intein induced cleavage.

[0236] As used herein, the term “surface loaded” refers to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% at least 98% or 100% of the loaded molecule being displayed on the outer EV membrane.

[0237] In one embodiment, the EV of the present disclosure comprises a targeting moiety as described herein.

[0238] In one embodiment, the targeting moiety is comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein.

[0239] In one embodiment, the EV comprises the targeting moiety, or an additional targeting moiety, independently of the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure. In one embodiment, the targeting moiety is not fused to the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure and / or does not comprise or is not fused to an extein sequence of the polypeptide comprising an intein and an extein of the present disclosure.

[0240] In one embodiment, the targeting moiety is loaded by overexpression of the targeting moiety in EV producer cells, preferably by expression of a further fusion protein comprising an EV polypeptide and the targeting moiety or by expression of a further conjugated protein comprising the targeting moiety conjugated to a moiety for loading the polypeptide into the EV. Advantageous EV polypeptides in the context include TSN2, CD63, LAMP2B, PTTG1-IP, PTGFRN and BASP1 , as well as derivatives, domains, variants, mutants, or regions thereof. Alternatively, the targeting moiety is loaded by exogenous loading techniques. In a preferred embodiment, the targeting moiety is surface loaded. In one embodiment, the targeting moiety is fused to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. In an alternative embodiment, targeting moiety is fused or conjugated to a part of the EV loading moiety that is localised to and / or displayed on the surface of an EV, preferably an exosome, microvesicle and / or a related extracellular vesicle. In one embodiment, the targeting moiety is fused or conjugated to loop 1 or loop 2 of a tetraspanin EV polypeptide, such as TSN2 or CD63, preferably TSN2. In one embodiment, the targeting moiety is fused or conjugated to the N-terminus of PTTG1-IP. In the context of the present disclosure surface loading of the targeting moiety may have the advantage of allowing for more efficient targeting.

[0241] In one embodiment, the EV of the present disclosure comprises a multimerization domain as described herein.

[0242] In one embodiment, the multimerization domain is comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein.

[0243] In one embodiment, the EV comprises the multimerization domain, or an additional multimerization domain, independently of the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure. In one embodiment, the multimerization domain, is not fused to the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure and / or does not comprise or is not fused to an extein sequence of the polypeptide comprising an intein and an extein sequence of the present disclosure.

[0244] In one embodiment, the multimerization domain is loaded by expression of a further fusion protein comprising an EV polypeptide and the multimerization domain or by expression of a further conjugated protein comprising the multimerization domain conjugated to a moiety for loading the polypeptide into the EV. Alternatively, the polypeptide comprising the multimerization domain is loaded by exogenous loading techniques. In a preferred embodiment, the EV comprises the multimerization domain together in the same fusion protein as a targeting moiety. In one embodiment, the EV of the present disclosure comprises an endosomal escape domain as described herein.

[0245] In one embodiment, the endosomal escape domain is comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein.

[0246] In one embodiment, the EV comprises the endosomal escape domain, or an additional endosomal escape domain, independently of the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure. In one embodiment, the endosomal escape domain, is not fused to the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure and / or does not comprise or is not fused to an extein sequence of the polypeptide comprising an intein and an extein sequence of the present disclosure.

[0247] In one embodiment, the endosomal escape domain is loaded by overexpression of the endosomal escape domain in EV producer cells, optionally by expression of a further fusion protein comprising an EV polypeptide and the endosomal escape domain or by expression of a further conjugated protein comprising the endosomal escape domain conjugated to a moiety for loading the polypeptide into the EV. Alternatively, the endosomal escape domain is loaded by exogenous loading techniques.

[0248] In one embodiment, the EV of the present disclosure comprises a pharmacokinetic and / or pharmacodynamic effector moiety as described herein, preferably an albumin binding domain as described herein.

[0249] In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is comprised in the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure, as described herein.

[0250] In one embodiment, the EV comprises the pharmacokinetic and / or pharmacodynamic effector moiety, or an additional pharmacokinetic and / or pharmacodynamic effector moiety domain, independently of the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is not fused to the intein or the polypeptide comprising an intein and an extein sequence of the present disclosure and / or does not comprise or is not fused to an extein sequence of the polypeptide comprising an intein and an extein sequence of the present disclosure.

[0251] In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is loaded by overexpression of the pharmacokinetic and / or pharmacodynamic effector moiety in EV producer cells, preferably by expression of a further fusion protein comprising an EV polypeptide and the pharmacokinetic and / or pharmacodynamic effector moiety or by expression of a further conjugated protein comprising the pharmacokinetic and / or pharmacodynamic effector moiety conjugated to a moiety for loading the polypeptide into the EV. Alternatively, the pharmacokinetic and / or pharmacodynamic effector moiety domain is loaded by exogenous loading techniques.

[0252] In a preferred embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused to the EV polypeptide or conjugated to EV loading moiety, such that the pharmacokinetic and / or pharmacodynamic effector moiety is surface loaded. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed to the surface of an EV, preferably an exosome, microvesicle and / or a related extracellular vesicle. In an alternative embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to a part of the EV loading moiety that is localised to and / or displayed to the surface of an EV, preferably an exosome, microvesicle and / or a related extracellular vesicle. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to loop 1 or loop 2 of a tetraspanin EV polypeptide, such as CD63 or TSN2, preferably TSN2. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to the N-terminus of PTTG1-IP. In the context of the present disclosure surface loading of the pharmacokinetic and / or pharmacodynamic effector moiety may have the advantage of allowing for more efficient pharmacokinetic and / or pharmacodynamic effect.

[0253] In a preferred embodiment, the EV is an exosome, a microvesicle, and / or any form of related extracellular vesicle.

[0254] A further aspect of the present invention relates to a cell comprising the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure, the conjugated protein of the present disclosure, the polynucleotide sequence of the present disclosure or the EV of the present disclosure.

[0255] In one embodiment, the cell is an EV producer cell as described herein. In an alternative embodiment, the cell is a target cell.

[0256] The term “target cell” as defined herein are the cells to which the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure, the conjugated protein of the present disclosure, the polynucleotide of the present disclosure or the EV of the present disclosure have been delivered. In one embodiment the target cells are cells that present a moiety, such as a protein or a receptor, on their surface that is capable of interacting with a targeting moiety that is present on the EV. In a preferred embodiment, the target cells are cells of the liver, the heart, the brain or neuronal tissue, hepatocytes, cardiomyocytes, cardiac smooth muscle cells, sensory neurons, Purkinje neurons, motor neurons, interneurons or glia cells.

[0257] Further aspects of the present invention relate to methods of preparing an EV or population of EVs according to the present invention.

[0258] In one aspect, the method for producing EVs comprises: (i) introducing into an EV-producing cell a polynucleotide construct of the present invention; and (ii) expressing a construct in the EV-producing cell, thereby generating EVs comprising a polypeptide of the present invention or a fusion protein of the present invention.

[0259] In an alternative aspect, the method for producing EVs comprises: (i) culturing EV producer cells and collecting EVs from said cells; and (ii) loading said EVs with the polypeptide comprising an intein and an extein sequence of the present disclosure, the fusion protein of the present disclosure or the conjugated protein of the present disclosure by one or more exogenous loading method as described herein.

[0260] In one embodiment, the methods for producing EVs of the present invention may comprise a further step of introducing into the same EV producing cell a (or an additional) polynucleotide construct encoding a (or a further) protein cargo as described herein, protein capable of binding a cargo as described herein, purification moiety, targeting moiety as described herein, endosomal escape moiety as described herein, pharmokinetic moiety as described herein and / or pharmoeffector moiety as described herein, optionally in the form of a fusion protein with an EV polypeptide. In one embodiment, the methods for producing EVs of the present invention may comprise a further step of loading said EVs with a (or an additional) protein cargo as described herein, protein capable of binding a cargo as described herein, purification moiety, targeting moiety as described herein, endosomal escape moiety as described herein, pharmokinetic moiety as described herein and / or pharmoeffector moiety as described herein, by one or more exogenous loading method as described herein.

[0261] In one embodiment of the method of preparing an EV or population of EVs according to the present invention, the method comprises an additional step of incubating the EV of the present disclosure or population of EVs of the present disclosure at temperature that is sufficient to allow for the intein to catalyse the cleavage of the polypeptide comprising an intein and an extein sequence, fusion protein or the conjugated protein. In one embodiment, the temperature is room temperature. In an alternative embodiment, the temperature is 32- 42°C, 34-40°C, 36-38°C, or preferably 37°C.

[0262] In a preferred embodiment, the EV or population of EVs is incubated for a time-period that is sufficient to allow for the intein to catalyse the cleavage of the polypeptide comprising an intein and an extein sequence, fusion protein or the conjugated protein. Such cleavage may be verified by the methods outlined in the examples.

[0263] In one embodiment, the EV or population of EVs is incubated for a time-period that is sufficient to allow for the intein to catalyse the cleavage of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% of the polypeptides comprising an intein and an extein sequence. In one embodiment, the EV or population of EVs is incubated at room temperature for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours or at least 24 hours.

[0264] In one embodiment, the methods for producing EVs of the present invention may comprise a step of purifying the EVs. Purification of EVs is achieved by any method including but not limited to: techniques comprising liquid chromatography (LC), high-performance liquid chromatography (HPLC), bead-eluate chromatography, ionic exchange chromatography, spin filtration, tangential flow filtration (TFF), hollow fiber filtration, centrifugation, immunoprecipitation, flow field fractionation, dialysis, microfluidic-based separation, etc., or any combination thereof. In an advantageous embodiment, the purification of the EVs is carried out using a sequential combination of filtration (preferably ultrafiltration (UF) tangential flow filtration (TFF) or hollow fibre filtration) and affinity chromatography, optionally also including size exclusion LC or bead-eluate LC. Combining purification steps normally enhances the purity of the resulting samples and, in turn leads to superior therapeutic activity. Further, as compared to UC, which is routinely employed for purifying exosomes, sequential filtration-chromatography is considerably faster and possible to scale to higher manufacturing volumes, which is a significant drawback of the current UC methodology that dominates the prior art. Another advantageous purification method is TFF, which offers scalability and purity, and which may be combined with any other type of purification technique.

[0265] A further aspect of the present invention relates to an in vitro, in vivo or ex vivo method of delivering a therapeutic cargo to a target cell as described herein, comprising contacting a target cell with an EV of the present disclosure. In one embodiment the target cell has a genetic abnormality and / or is linked to a disease or a disorder.

[0266] Another aspect of the present invention relates to a pharmaceutical composition comprising an EV of the present disclosure and a pharmaceutically acceptable excipient and / or carrier.

[0267] The term "pharmaceutically acceptable" is used herein to refer to a material may be administered to a subject without causing any undesirable biological effects.

[0268] The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. In one embodiment, the pharmaceutically acceptable excipient is any substance approved by a regulatory agency such as the FDA or EMEA or listed in the U.S. Pharmacopeia for use in animals, including humans. In one embodiment the pharmaceutically acceptable carrier comprises an aqueous solution, optionally an aqueous solution comprising 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), wherein the pharmaceutically acceptable carrier optionally comprises a monosaccharide, disaccharide, polyvinylpyrrolidone, polyvinyl alcohol, dihydric alcohol, polyhydric alcohol (optionally sorbitol, polyethylene glycol or propylene glycol) and / or a detergent, optionally a polyoxyethylenesorbitan (Tween). In one embodiment, the pharmaceutically acceptable carrier is any substance that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the therapeutic cargo. In a preferred embodiment the excipients and carriers are generally safe and non-toxic. The pharmaceutical compositions of the present disclosure may be formulated by any known method of formulation. In one aspect, the pharmaceutical compositions of the present disclosure may be formulated as Oral formulations, including Tablet, Capsule, Sustained release, liquid; Intravenous Formulations; Parenteral Formulations; Topical Formulations; cutaneous administration including cream, ointment, gel, paste, powder; Modified release Formulations including sustained release formulation and Liquid or lyophilized formulations.

[0269] Another aspect of the present invention relates to an EV of the present disclosure or a pharmaceutical composition of the present disclosure for the preparation of a medicament for treatment or prevention of a disease in a subject.

[0270] "Treat" or "treatment" is defined herein as a therapeutic treatment which refers to a treatment administered to a subject who exhibits signs or symptoms of pathology for the purpose of diminishing or eliminating those signs or symptoms. The signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective. In a preferred aspect, "Treat" or "treatment" refers to the reduction or amelioration of the progression, severity, and / or duration of a disease (or symptom related thereto). Ameliorate as used herein refers to the action of lessening the severity of symptoms, progression, or duration of a disease.

[0271] Another aspect of the present invention relates to an EV of the present disclosure or a pharmaceutical composition of the present disclosure for use as a medicament for the treatment or prevention of a disease in a subject.

[0272] A further aspect of the present invention relates to a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the EV of the present disclosure or a pharmaceutical composition of the present disclosure to a subject suffering from or susceptible to the disease.

[0273] As used herein, the term "effective amount" refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0274] As used herein, a "subject" refers to an animal that is the object of treatment, observation or experiment. "Animal" includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles, and in particular, mammals. "Mammal," as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals. In one aspect, the subject is a mammal, such as mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes. In a preferred embodiment, the subject is a human.

[0275] In a preferred embodiment of the in vitro, in vivo or ex vivo method of delivering a therapeutic cargo to a target cell of the present disclosure, the preparation of a medicament according to the present disclosure, the use as a medicament according to the present disclosure, or the method of treatment of the present disclosure; the EV comprises a polypeptide comprising an intein and an extein sequence of the present disclosure, a fusion protein of the present disclosure of or a conjugated protein of the present disclosure that has been cleaved. In a more preferred embodiment, the EV is an EV of the present disclosure, wherein the protein cargo, protein capable of binding a cargo, RNP complex and / or the purification moiety has been released from the EV polypeptide by the intein induced cleavage of the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein.

[0276] In a preferred embodiment the disease described herein is Crohn’s disease, ulcerative colitis, ankylosing spondylitis, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, sarcoidosis, idiopathic pulmonary fibrosis, psoriasis, tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS), deficiency of the interleukin-1 receptor antagonist (DIRA), endometriosis, autoimmune hepatitis, scleroderma, myositis, stroke, acute spinal cord injury, vasculitis, Guillain-Barre 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 the associated underlying etiology, graft-vs-host disease, Duchenne muscular dystrophy and other muscular dystrophies, In-born errors of metabolism including: Disorders of carbohydrate metabolism e.g., G6PD deficiency galactosemia, hereditary fructose intolerance, fructose 1 ,6-diphosphatase deficiency and the glycogen storage diseases, Disorders of organic acid metabolism (organic acidurias) such as alkaptonuria, 2-hydroxyglutaric acidurias, methylmalonic or propionic acidemia, multiple carboxylase deficiency, Disorders of amino acid metabolism such as phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, Aminoacidopathies e.g., hereditary tyrosinemia, nonketotic hyperglycinemia, and homocystinuria, Hereditary tyrosinemia, Fanconi syndrome, Primary Lactic Acidoses e.g., pyruvate dehydrogenase, pyruvate carboxylase and cytochrome oxidase deficiencies, Disorders of fatty acid oxidation and mitochondrial metabolism such as short, medium, and long- chain acyl-CoA dehydrogenase deficiencies also known as Beta-oxidation defects, Reye’s syndrome, Medium-chain acylcoenzyme A dehydrogenase deficiency (MCADD.), MELAS, MERFF, pyruvate dehydrogenase deficiency, Disorders of porphyrin metabolism such as acute intermittent porphyria, Disorders of purine or pyrimidine metabolism such as Lesch-Nyhan syndrome, Disorders of steroid metabolism such as lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia, Disorders of mitochondrial function such as Kearns-Sayre syndrome, Disorders of peroxisomal function such as Zellweger syndrome and neonatal adrenoleukodystrophy, congenital adrenal hyperplasia or SmithLemli-Opitz, Menkes syndrome, neonatal hemochromatosis, Urea cycle disorders such as N-Acetylglutamate synthase deficiency, carbamoyl phosphate synthetase deficiency, ornithine transcarbamoylase deficiency, citrullinemia (deficiency of argininosuccinic acid synthase), argininosuccinic aciduria (deficiency of argininosuccinic acid lyase), argininemia (deficiency of arginase), hyperornithinemia, hyperammonemia, homocitrullinuria (HHH) syndrome (deficiency of the mitochondrial ornithine transporter), citrullinemia II (deficiency of citrin, an aspartate glutamate transporter), lysinuric protein intolerance (mutation in y+L amino acid transporter 1 , orotic aciduria (deficiency in the enzyme uridine monophosphate synthase UMPS), all of the lysosomal storage diseases, for instance Alpha-mannosidosis, Betamannosidosis, Aspartylglucosaminuria, Cholesteryl 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 II, Krabbe Disease, Lysosomal acid lipase deficiency, Metachromatic Leukodystrophy, MPS I -Hurler Syndrome, MPS I - Scheie Syndrome, MPS I Hurler-Scheie Syndrome, MPS II - Hunter Syndrome, MPS IIIA - Sanfilippo Syndrome Type A, MPS I II B - Sanfilippo Syndrome Type B, MPS 111 B - Sanfilippo Syndrome Type C, MPS 111 B - Sanfilippo Syndrome Type D, MPS IV Morquio Type A, MPS IV - Morquio Type B, MPS IX - Hyaluronidase Deficiency, MPS VI - Maroteaux-Lamy, MPS VII - Sly Syndrome, Mucolipidosis I - Sialidosis, Mucolipidosis I IIC, 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, Salla Disease, Schindler Disease and Wolman Disease, etc. cystic fibrosis, primary ciliary dyskinesia, pulmonary alveolar proteinosis, ARC syndrome, Ret syndrome, neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, GBA associated Parkinson’s disease, Huntington’s disease and other trinucleotide repeat-related diseases, dementia, ALS, cancer-induced cachexia, anorexia, diabetes mellitus type 2, and various cancers. Virtually all types of cancer are relevant disease targets for the present invention, for instance, Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, cerebellar or cerebral, Basal-cell carcinoma, Bile duct cancer, Bladder cancer, Bone tumor, Brainstem glioma, Brain cancer, Brain tumor (cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma), Breast cancer, Bronchial adenomas / carcinoids, Burkitt's lymphoma, Carcinoid tumor (childhood, gastrointestinal), Carcinoma of unknown primary, Central nervous system lymphoma, Cerebellar astrocytoma / Malignant glioma, Cervical cancer, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Chronic myeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Extracranial germ cell tumor, Extragonadal Germ cell tumor, Extrahepatic bile duct cancer, Eye Cancer (Intraocular melanoma, Retinoblastoma), Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal stromal tumor (GIST), Germ cell tumor (extracranial, extragonadal, or ovarian), Gestational trophoblastic tumor, Glioma (glioma of the brain stem, Cerebral Astrocytoma, Visual Pathway and Hypothalamic glioma), Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias ((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), Lymphomas, AIDS-related lymphoma, Burkitt lymphoma, cutaneous T-Cell lymphoma, Hodgkin lymphoma, Non-Hodgkin, Medulloblastoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma / Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic / Myeloproliferative Diseases, Myelogenous Leukemia, Chronic Myeloid Leukemia (Acute, Chronic), Myeloma, Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Neuroblastoma, Oral Cancer, Oropharyngeal cancer, Osteosarcoma / malignant fibrous histiocytoma of bone, Ovarian cancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian low malignant potential tumor, Pancreatic cancer, Pancreatic islet cell cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, Pineoblastoma and supratentorial primitive neuroectodermal tumors, Pituitary adenoma, Pleuropulmonary blastoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma (Ewing family of tumors sarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma), Sezary syndrome, Skin cancer (nonmelanoma, melanoma), Small intestine cancer, Squamous cell, Squamous neck cancer, Stomach cancer, Supratentorial primitive neuroectodermal tumor, Testicular cancer, Throat cancer, Thymoma and Thymic carcinoma, Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter, Urethral cancer, Uterine cancer, Uterine sarcoma, Vaginal cancer, Vulvar cancer, Waldenstrom macroglobulinemia, and / or Wilm’s tumor. In a preferred embodiment, the disease is a genetic disorder, a liver disorder, a neurological disorder or a cardiac disorder. In a most preferred embodiment, the disease is Phenylketonuria, a neurological disorder, a genetic disorder, heart failure orALS.

[0277] The EVs and pharmaceutical compositions of the present disclosure may be administered to an animal subject, preferably a human via various different administration routes. In one embodiment, the administration route is auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra- amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracerebroventricular, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratym panic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above administration routes, which typically depends on the disease to be treated, the cargo molecule(s) comprised, the characteristics of the EVs and / or the EV population as such. In one embodiment, the EVs or pharmaceutical compositions of the present disclosure are administered systemically. In a preferred embodiment, the EVs or pharmaceutical compositions of the present disclosure are administered locally. In a more preferred embodiment, the EVs or pharmaceutical compositions of the present disclosure are administered locally to the liver, CNS, brain or heart.

[0278] The dosages of EVs when applied in vivo may naturally vary considerably depending on the disease to be treated, the administration route, the activity and effects of the cargo of interest, any targeting moieties present on the EVs, the pharmaceutical formulation, etc.

[0279] It is envisaged that any dosage regime would be applicable to the engineered EVs of the invention. The dosage regime chosen will depend on the cargo being delivered by the EVs and the disease to be treated and any additional therapies being administered which will be determined by the skilled physician.

[0280] It is envisaged that the EVs of the present invention will be administered multiple times, i.e. more than 1 time but normally more than 2 times or potentially for chronic, long-term treatment (i.e. administered tens to hundreds to thousands of times). Preferably, if the cargo is an antigen that is being administered as a vaccine, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks. Similarly, if the cargo is e.g. a viral particle, such as an AAV, the EVs comprising the cargo in question will likely be administered more than once, normally multiple times as part of a chronic treatment regimen.

[0281] The invention is further defined in the following numbered paragraphs:

[0282] 1. A polypeptide comprising an intein and an extein sequence.

[0283] 2. The polypeptide of paragraph 1, wherein the intein is capable of catalysing cleavage of the polypeptide.

[0284] 3. The polypeptide of paragraph 1 or 2, wherein the extein sequence is C-terminal to the intein.

[0285] 4. The polypeptide of paragraph 1 or 2, wherein the extein sequence is N-terminal to the intein.

[0286] 5. The polypeptide of paragraph 1 or 2, wherein the polypeptide comprises a first extein sequence to the N-terminus of the intein and a second extein sequence to the C- terminus of the intein.

[0287] 6. The polypeptide of paragraph 1 to 5, wherein the intein is capable of catalysing cleavage of an intein-extein bond.

[0288] 7. The polypeptide of paragraph 1 to 6, wherein the intein is capable of catalysing cleavage of the C-terminal intein-extein bond.

[0289] 8. The polypeptide of paragraph 1 to 7, wherein the intein is a cleaving intein.

[0290] 9. The polypeptide of paragraph 1 to 8, wherein the intein comprises mutations that reduce its splicing activity.

[0291] 10. The polypeptide of paragraph 1 to 8, wherein the intein has low or no, or substantially no splicing activity.

[0292] 11. The polypeptide of paragraph 1 to 10, wherein the intein is capable of mediating the release of the intein from the C-terminal extein and / or the N-terminal extein.

[0293] 12. The polypeptide of paragraph 11 , wherein the intein is capable of mediating the release of the intein from the C-terminal extein. The polypeptide of paragraph 1 to 12 wherein the intein is a contiguous intein. The polypeptide of paragraph 1 to 13, wherein the intein is capable of catalysing cleavage of the polypeptide in the lumen of an extracellular vesicle. The polypeptide of paragraph 14, wherein the extracellular vesicle is an exosome, a microvesicle and / or a related extracellular vesicle. The polypeptide of paragraph 1 to 13, wherein the intein is capable of catalysing cleavage of the polypeptide at pH 5.5 to 6.7, pH 5.8 to 6.4 or preferably pH 6.0 to 6.2 The polypeptide of paragraph 1 to 13, wherein the intein is capable of catalysing cleavage of the polypeptide at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 1 to 13, wherein the intein is capable of catalysing cleavage of the polypeptide at room temperature. The polypeptide of paragraph 1 to 18, wherein the intein comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1. The polypeptide of paragraph 19, wherein the amino acid residue at position 150 of SEQ ID NO: 1 is Glycine. The polypeptide of paragraph 19 or 20, wherein the amino acid residue at position 67 of SEQ ID NO: 1 is Leucine. The polypeptide of paragraph 19 to 21, wherein the amino acid residue at position 24 of SEQ ID NO: 1 is Glycine. The polypeptide of paragraph 19 to 22, wherein the N-terminal extein sequence is capable of increasing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2, in the lumen of an EV. The polypeptide of paragraph 19 to 23, wherein the C-terminal extein sequence is capable of increasing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO 3, in the lumen of an EV. The polypeptide of paragraph 23 or 24, wherein the EV is an exosome, a microvesicle and / or a related extracellular vesicle. The polypeptide of paragraph 19 to 25, wherein the N-terminal extein sequence is capable of increasing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2, at pH 5.5 to 6.7, pH 5.8 to 6.4, pH 6.0 to 6.2, preferably at pH 6.2. The polypeptide of paragraph 19 to 26, wherein the C-terminal extein sequence is capable of increasing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO 3, at pH 5.5 to 6.7, pH 5.8 to 6.4, pH 6.0 to 6.2, preferably at pH 6.2. The polypeptide of paragraph 23 to 27, wherein the N-terminal extein sequence and / or the C-terminal extein sequence is capable of increasing the rate of cleavage of the intein-extein bond at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 19 to 28, wherein the N-terminal extein sequence and / or the C-terminal extein sequence is capable of increasing the rate of cleavage of the intein-extein bond at room temperature. The polypeptide of paragraph 19 to 29, wherein the N-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2, in the cytoplasm of a cell. The polypeptide of paragraph 19 to 30, wherein the C-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO 3, in the cytoplasm of a cell. The polypeptide of paragraph 30 or 31 , wherein the cell is a mammalian cell, preferably a mammalian cell in culture, most preferably an EV producer cell. The polypeptide of paragraph 19 to 32, wherein the N-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2, at pH 8.5 or pH 7.2. The polypeptide of paragraph 19 to 33, wherein the C-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO 3, at pH 8.5 or pH 7.2. The polypeptide of paragraph 30 to 34, wherein the N-terminal extein sequence and / or the C-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 30 to 35, wherein the N-terminal extein sequence and / or the C-terminal extein sequence does not significantly increase the rate of cleavage of the intein-extein bond at room temperature. The polypeptide of paragraph 19 to 36, wherein the C-terminal extein sequence comprises a +1 amino acid residue that has a Molecular weight of 131 Da, 149 Da or more , or preferably 155 Da or more. The polypeptide of paragraph 19 to 37, wherein the +1 residue of the C-terminal extein is His, Met, Glu, Leu or Phe. The polypeptide of paragraph 38, wherein the +1 residue of the C-terminal extein is His or Met. The polypeptide of paragraph 39, wherein the +1 residue of the C-terminal extein is His. The polypeptide of paragraph 39, wherein the +1 residue of the C-terminal extein is Met. The polypeptide of paragraph 19 to 41 , wherein the +2 residue of the C-terminal extein is Ser, the +3 residue of the C-terminal extein is Pro, the +4 residue of the C- terminal extein is Pro and / or the +5 residue of the C-terminal extein is Phe. The polypeptide of paragraph 41 , wherein the +1 residue of the C-terminal extein is Met, the +2 residue of the C-terminal extein is Ser, the +3 residue of the C-terminal extein is Pro, the +4 residue of the C-terminal extein is Pro and the +5 residue of the C-terminal extein is Phe. The polypeptide of paragraph 19 to 43, wherein the -1 residue of the N-terminal extein is an amino acid with a hydrophilic side chain. The polypeptide of paragraph 19 to 44, wherein the -1 residue of the N-terminal extein is Ala, Asp, Gly, Gin, His, Glu, Lys, Pro, Thr, Arg, Cys, Trp or Met, preferably Ala, Asp, Gly, Gin, or His. The polypeptide of paragraph 19 to 45, wherein the -2 residue of the N-terminal extein is Glu, the -3 of the N-terminal extein is Ser, the -4 residue of the N-terminal extein is lie and / or the -5 residue of the N-terminal extein is Arg. The polypeptide of paragraph 45, wherein the -1 residue of the N-terminal extein is Ala, Asp, Gly, Gin or His, the -2 residue of the N-terminal extein is Glu, the -3 of the N-terminal extein is Ser, the -4 residue of the N-terminal extein is lie and the -5 residue of the N-terminal extein is Arg. The polypeptide sequence of paragraph 2 to 18, wherein the intein consists or comprises of a variant of SEQ ID NO 1. The polypeptide sequence of paragraph 48, wherein the variant of SEQ ID NO. 1 exhibits a reduced rate of cleavage of the intein-extein bond, as compared to an intein having SEQ ID NO 1 , in the cytoplasm of a cell. The polypeptide of paragraph 49, wherein the cell is a mammalian cell, preferably a mammalian cell in culture, most preferably an EV producer cell. The polypeptide of paragraph 48 to 50, wherein the variant of SEQ ID NO. 1 exhibits a reduced the rate of cleavage of the intein-extein bond as compared to SEQ ID NO 1 , at pH 7.0-7.5, preferably at pH 7.2. The polypeptide of paragraph 48 to 51 , wherein the variant of SEQ ID NO. 1 exhibits a reduced rate of cleavage of the intein-extein bond, as compared to SEQ ID NO 1, at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 50 to 52, wherein the variant of SEQ ID NO. 1 is capable of catalysing the intein-extein bond in the lumen of an EV. The polypeptide of paragraph 48 to 53, wherein the variant of SEQ ID NO. 1 is capable of catalysing the intein-extein bond at pH 5.5 to 6.7, pH 5.8 to 6.4 or preferably pH 6.0 to 6.2 The polypeptide of paragraph 48 to 54, wherein the variant of SEQ ID NO. 1 is capable of catalysing the intein-extein bond at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 48 to 55, wherein the variant of SEQ ID NO. 1 is capable of catalysing the intein-extein bond at room temperature. The polypeptide of paragraph 48 to 56, wherein the variant of SEQ ID NO. 1 does not exhibit a significantly reduced rate of cleavage of the intein-extein bond in the lumen of an EV. The polypeptide of paragraph 48 to 57, wherein the variant of SEQ ID NO. 1 does not exhibit a significantly reduced rate of cleavage at pH 5.5 to 6.7, pH 5.8 to 6.4 or preferably pH 6.0 to 6.2. The polypeptide of paragraph 57 or 58, wherein the variant of SEQ ID NO. 1 does not exhibit a significantly reduced rate of cleavage at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 57 to 59, wherein the variant of SEQ ID NO. 1 does not exhibit a significantly reduced rate of cleavage at room temperature. The polypeptide of paragraph 48 to 56, wherein the variant of SEQ ID NO. 1 exhibits a cleavage rate of the intein-extein bond in the lumen of an EV that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited by SEQ ID NO 1. The polypeptide of paragraph 48 to 57, wherein the variant of SEQ ID NO. 1 exhibits a cleavage rate of the intein-extein bond at pH 5.5 to 6.7, pH 5.8 to 6.4 or preferably pH 6.0 to 6.2 that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited by SEQ ID NO 1. The polypeptide of paragraph 61 or 62, wherein the variant of SEQ ID NO. 1 exhibits a cleavage rate of the intein-extein bond at 34-40°C, 36-38°C or preferably at 37°C that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited by SEQ ID NO 1. The polypeptide of paragraph 61 to 63, wherein the variant of SEQ ID NO. 1 exhibits a cleavage rate of the intein-extein bond at room temperature that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited by SEQ ID NO 1. The polypeptide of paragraph 48 to 60, wherein the variant of SEQ ID NO. 1 comprises mutations at:

[0294] (i) position 41, 108, 111 and / or 152 of SEQ ID NO. 1 ; or

[0295] (ii) position 41 , 108, 111 and 152 of SEQ ID NO. 1; optionally the variant of SEQ ID NO. 1 further comprises a mutation at position 167 of SEQ ID NO. 1. The polypeptide of paragraph 48 to 65, wherein the variant of SEQ ID NO. 1 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4 or SEQ ID NO: 23. The polypeptide of paragraph 66, wherein the variant of SEQ ID NO. 1 comprises a Glycine at position 150. The polypeptide of paragraph 66 or 67, wherein the variant of SEQ ID NO. 1 comprises a Leucine at position 67. The polypeptide of paragraph 66 to 68, wherein the variant of SEQ ID NO. 1 comprises a Glycine at position 24. The polypeptide of paragraph 66 to 69, wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41. The polypeptide of paragraph 66 to 70, wherein the variant of SEQ ID NO. 1 comprises a Valine residue at position 108. The polypeptide of paragraph 66 to 71 , wherein the variant of SEQ ID NO. 1 comprises a Proline residue at position 111. The polypeptide of paragraph 66 to 72, wherein the variant of SEQ ID NO. 1 comprises a Valine residue at position 152. The polypeptide of paragraph 66 to 73, wherein the variant of SEQ ID NO. 1 comprises a Glutamine residue at position 167. The polypeptide of paragraph 48 to 74, wherein the N-terminal extein sequence is capable of reducing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2 and / or SEQ ID NO. 5, in the cytoplasm of a cell. The polypeptide of paragraph 48 to 75, wherein the C-terminal extein sequence is capable of reducing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO 3, in the cytoplasm of a cell. The polypeptide of paragraph 75 or paragraph 76, wherein the cell is a mammalian cell, preferably a mammalian cell in culture, most preferably an EV producer cell. The polypeptide of paragraph 48 to 77, wherein the N-terminal extein sequence is capable of reducing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO. 2 and / or SEQ ID NO. 5 at pH 7.0-7.5, preferably at pH 7.3. The polypeptide of paragraph 48 to 78, wherein the C-terminal extein sequence is capable of reducing the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO 3 at pH 7.0-7.5, preferably at pH 7.3. The polypeptide of paragraph 75 to 79, wherein the N-terminal extein sequence and / or the C-terminal extein sequence is capable of reducing the rate of cleavage at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 48 to 80, wherein the N-terminal extein sequence does not prevent cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. The polypeptide of paragraph 48 to 81 , wherein the C-terminal extein sequence does not prevent cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. The polypeptide of paragraph 81 or 82, wherein the N-terminal extein sequence does not prevent cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2. The polypeptide of paragraph 81 to 83, wherein the C-terminal extein sequence does not prevent cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2. The polypeptide of paragraph 81 to 84, wherein the N-terminal extein sequence and / or the C-terminal extein sequence does not prevent cleavage of the intein-extein bond at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 81 to 85, wherein the N-terminal extein sequence and / or the C-terminal extein sequence does not prevent cleavage of the intein-extein bond at room temperature. The polypeptide of paragraph 48 to 86, wherein the N-terminal extein sequence does not significantly reduce cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. The polypeptide of paragraph 48 to 87, wherein the C-terminal extein sequence does not significantly reduce cleavage of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle. The polypeptide of paragraph 87 or 88, wherein the N-terminal extein sequence does not significantly reduce cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2. The polypeptide of paragraph 87 to 89, wherein the C-terminal extein sequence does not significantly reduce cleavage of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2. The polypeptide of paragraph 87 to 90, wherein the N-terminal extein sequence and / or the C-terminal extein sequence does not significantly reduce cleavage of the intein-extein bond at 34-40°C, 36-38°C or preferably at 37°C. The polypeptide of paragraph 87 to 91 , wherein the N-terminal extein sequence and / or the C-terminal extein sequence does not significantly reduce cleavage of the intein-extein bond at room temperature. The polypeptide of paragraph 48 to 86, wherein the cleavage rate of the intein-extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the N-terminal extein sequence is SEQ ID NO. 2 and / or SEQ ID NO. 5. The polypeptide of paragraph 48 to 86 or 93, wherein the cleavage rate of the intein- extein bond in the lumen of an EV, preferably in the lumen of an exosome, a microvesicle and / or a related extracellular vesicle, that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the C-terminal extein sequence is SEQ ID NO. 3. The polypeptide of paragraph 48 to 86, 93 or 94, wherein the cleavage rate of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the N-terminal extein sequence is SEQ ID NO. 2 and / or SEQ ID NO. 5. The polypeptide of paragraph 48 to 86 or 93 to 95, wherein the cleavage rate of the intein-extein bond at a pH of 5.0 to 6.8, pH 6.0 to 6.5, preferably 6.2, is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the C-terminal extein sequence is SEQ ID NO. 3. The polypeptide of paragraph 93 to 96, wherein the cleavage rate of the intein-extein bond at 34-40°C, 36-38°C, or preferably at 37°C, is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the N-terminal extein sequence is SEQ ID NO. 2 and / or SEQ ID NO. 5 and / or is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the C-terminal extein sequence is SEQ ID NO. 3. The polypeptide of paragraph 93 to 97, wherein the cleavage rate of the intein-extein bond at room temperature, is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the N- terminal extein sequence is SEQ ID NO. 2 and / or SEQ ID NO. 5 and / or is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or 100% of the cleavage rate exhibited wherein the C-terminal extein sequence is SEQ ID NO. 3. The polypeptide of paragraph 48 to 98, wherein the +1 residue of the C-terminal extein is Glu, Phe, His, Leu, Met, Gly or Pro, preferably the C-terminal extein is Gly. The polypeptide of paragraph 48 to 99, wherein the C-terminal extein comprises:

[0296] (i) Gly as the +1 residue, Ser as the +2 residue and Pro as the +3 residue;

[0297] (ii) Gly as the +1 residue, Gly as the +2 residue, Gly as the +3 residue, Gly as the +4 residue and Ser as the +5 residue;

[0298] (iii) Thr as the +1 residue, Arg as the +2 residue and Met as the +3 residue; or

[0299] (iv) Thr as the +1 residue, Arg as the +2 residue and His as the +3 residue. The polypeptide of paragraph 48 to 99, wherein the +2 residue of the C-terminal extein is Ser and / or the +3 residue of the C-terminal extein is Pro. The polypeptide of paragraph 100, wherein the 1 residue of the C-terminal extein is Gly, the +2 residue of the C-terminal extein is Ser and the +3 residue of the C- terminal extein is Pro. The polypeptide of paragraph 48 to 99, wherein the +2 residue of the C-terminal extein is Gly, the +3 residue of the C-terminal extein is Gly, the +4 residue of the C- terminal extein is Gly and / or the +5 residue of the C-terminal extein is Ser. The polypeptide of paragraph 100, wherein the 1 residue of the C-terminal extein is Gly, wherein the +2 residue of the C-terminal extein is Gly, the +3 residue of the C- terminal extein is Gly, the +4 residue of the C-terminal extein is Gly and the +5 residue of the C-terminal extein is Ser. The polypeptide of paragraph 48 to 104, wherein the N-terminal extein comprises

[0300] (i) Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue;

[0301] (ii) Ser as the -1 residue, Ala as the -2 residue and Phe as the -3 residue;

[0302] (iii) Met as the -1 residue, Phe as the -2 residue and Phe as the -3 residue;

[0303] (iv) Phe as the -1 residue, Ala as the -2 residue and Phe as the -3 residue; or

[0304] (v) Met as the -1 residue, Arg as the -2 residue and Thr as the -3 residue. The polypeptide of paragraph 48 to 104, wherein the N-terminal extein comprises Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue. The polypeptide of paragraph 48 to 104 comprising:

[0305] (i) a C-terminal extein comprising Gly as the +1 residue, Gly as the +2 residue, Gly as the +3 residue, Gly as the +4 residue and Ser as the +5 residue and an N-terminal extein comprising Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue;

[0306] (ii) a C-terminal extein comprising Gly as the +1 residue, Ser as the +2 residue and Pro as the +3 residue and an N-terminal extein comprising Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue;

[0307] (iii) a C-terminal extein comprising Gly as the +1 residue, Ser as the +2 residue and Pro as the +3 residue and an N-terminal extein comprising Ser as the -1 residue, Ala as the -2 residue and Phe as the -3 residue;

[0308] (iv) a C-terminal extein comprising Gly as the +1 residue, Ser as the +2 residue and Pro as the +3 residue and an N-terminal extein comprising Met as the -1 residue, Phe as the -2 residue and Phe as the -3 residue;

[0309] (v) a C-terminal extein comprising Gly as the +1 residue, Ser as the +2 residue and Pro as the +3 residue and an N-terminal extein comprising Phe as the -1 residue, Ala as the -2 residue and Phe as the -3 residue;

[0310] (vi) a C-terminal extein comprising Gly as the +1 residue, Ser as the +2 residue and Pro as the +3 residue and an N-terminal extein comprising Met as the -1 residue, Arg as the -2 residue and Thr as the -3 residue;

[0311] (vii) a C-terminal extein comprising Thr as the +1 residue, Arg as the +2 residue and Met as the +3 residue and an N-terminal extein comprising Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue;

[0312] (viii) a C-terminal extein comprising Thr as the +1 residue, Arg as the +2 residue and His as the +3 residue and an N-terminal extein comprising Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue;

[0313] (ix) a C-terminal extein comprising Thr as the +1 residue, Arg as the +2 residue and Met as the +3 residue and an N-terminal extein comprising Met as the -1 residue, Arg as the -2 residue and Thr as the -3 residue.

[0314] (x) a C-terminal extein comprising Thr as the +1 residue, Arg as the +2 residue and His as the +3 residue and an N-terminal extein comprising Met as the -1 residue, Arg as the -2 residue and Thr as the -3 residue. A fusion protein comprising or consisting of the polypeptide of paragraph 1 to 107 and an EV polypeptide. The fusion protein of paragraph 108, wherein the EV polypeptide comprises the N- terminal or C-terminal extein. The fusion protein of paragraph 109, wherein the EV polypeptide comprises the N- terminal extein. The fusion protein of paragraph 110, wherein the EV polypeptide comprises the N- terminal extein at its C-terminus. The fusion protein of paragraph 108, wherein the EV polypeptide is fused to the N- terminal or C-terminal extein. The fusion protein of paragraph 112, wherein the EV polypeptide is fused to the N- terminal extein. The fusion protein of paragraph 113, wherein the EV polypeptide is fused to the N- terminus of the N-terminal extein. The fusion protein of paragraph 108 or 114, wherein the EV polypeptide is an exosomal polypeptide. The fusion protein of paragraph 108 to 115, wherein the EV polypeptide is a transmembrane protein. The fusion protein of paragraph 116, wherein the transmembrane protein is a multipass transmembrane protein, such as tetraspanin, or a single pass transmembrane protein. The fusion protein of paragraph 108 to 116, wherein the EV polypeptide is TSN2, PTGFRN, PTTG1-IP, CD63, Lamp2b, CD81, Syntenin-1 , Syntenin-2, Lamp2, Lamp2a, TSN3, BASP1, MARCKS, a HIV gag protein or VSVG. The fusion protein of paragraph 118, wherein the EV polypeptide is TSN2, CD63, PTGFRN, PTTG1-IP, a HIV gag protein or Lamp2b. The fusion protein of paragraph 118, wherein the EV polypeptide is TSN2 or PTTG1- IP. The fusion protein of paragraph 108 to 116, wherein the EV polypeptide is a polypeptide comprising a myristoylation site. A conjugated protein comprising or consisting of the polypeptide of paragraph 1 to 107 conjugated to a moiety for loading the polypeptide into the EV. The conjugated protein of paragraph 122, wherein the moiety for loading the polypeptide into the EV is conjugated to the N-terminal extein. The conjugated protein of paragraph 123 wherein the moiety for loading the polypeptide into the EV is conjugated to the N-terminus of the N-terminal extein. The conjugated protein of paragraph 122 to 124, wherein the moiety for loading the polypeptide into the EV is a membrane anchoring moiety and / or a hydrophobic moiety. The conjugated protein of paragraph 122 to 125, wherein the moiety for loading the polypeptide into the EV is a myristic acid. The fusion protein or conjugated protein of paragraph 108 to 126, comprising a protein cargo or a protein capable of binding to a cargo. The fusion protein or conjugated protein of paragraph 127, wherein the protein cargo or protein capable of binding to a cargo comprises the N-terminal or the C-terminal extein. The fusion protein or conjugated protein of paragraph 128, wherein the protein cargo or protein capable of binding to a cargo comprises the C-terminal extein. The fusion protein or conjugated protein of paragraph 129, wherein the protein cargo or protein capable of binding to a cargo comprises the C-terminal extein at its N- terminus. The fusion protein or conjugated protein of paragraph 127, wherein the protein cargo or protein capable of binding to a cargo is fused to the N-terminal or C-terminal extein. The fusion protein or conjugated protein of paragraph 131, wherein the protein cargo or protein capable of binding to a cargo is fused to the C-terminal extein. The fusion protein or conjugated protein of paragraph 132, wherein the protein cargo or protein capable of binding to a cargo is fused to the C-terminus of the C-terminal extein. The fusion protein or conjugated protein of paragraph 127 to 133, wherein the intein is positioned such that the protein cargo or a protein capable of binding to a cargo is released from the EV polypeptide upon cleavage of the intein-extein bond. The fusion protein or conjugated protein of paragraph 127 to 134, wherein the intein is positioned such that the protein cargo or a protein capable of binding to a cargo is released from the EV polypeptide and the intein upon cleavage of the intein-extein bond. The fusion protein or conjugated protein of paragraph 127 to 135, wherein the intein is positioned between the EV polypeptide or the EV loading moiety and the protein cargo or protein capable of binding to a cargo. The fusion protein of paragraph 127 to 136, wherein the protein cargo or protein capable of binding to a cargo is fused, via the polypeptide comprising an intein and an extein sequence, to a domain or terminus of the EV polypeptide which is localised to and / or displayed in the lumen of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein of paragraph 127 to 137, wherein the protein cargo or protein capable of binding to a cargo is fused, via the polypeptide comprising an intein and an extein sequence, to the C-terminus of PTTG1-IP. The fusion protein of paragraph 127 to 137, wherein the protein cargo or protein capable of binding to a cargo is fused, via the polypeptide comprising an intein and an extein sequence, or to the N-terminus or C-terminus of the EV polypeptide, wherein the EV polypeptide is a tetraspanin such as TSN2 or CD63, preferably TSN2. The conjugated protein of paragraph 127 to 136, wherein the protein cargo or protein capable of binding to a cargo is conjugated, via the polypeptide comprising an intein and an extein sequence, to a part of the moiety for loading a polypeptide into the EV which is localised to and / or displayed in the lumen of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein or conjugated protein of paragraph 127 to 140, wherein the cargo is a therapeutic cargo. The fusion protein or conjugated protein of paragraph 127 to 141 , wherein the cargo is a Cas protein or a ribonucleoprotein (RNP) complex. The fusion protein or conjugated protein of paragraph 142, wherein:

[0315] (i) the Cas protein is a Cas9 or a Cas12, such as Cas12a;

[0316] (ii) the Cas protein is a Cas9 or a Cas12, such as Cas12a, a Cas3, a base editor or a prime editor; or

[0317] (iii) the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor. The fusion protein or conjugated protein of paragraph 142 or 143, wherein the Cas protein is associated with a guide RNA. The fusion protein or conjugated protein of paragraph 127 to 140, wherein the protein capable of binding to a cargo is a nucleic acid binding protein, a protein binding protein or a viral binding protein. The fusion protein or conjugated protein of paragraph 145, wherein the protein capable of binding to a cargo is an AAV capsid binding protein. The fusion protein or conjugated protein of paragraph 146, wherein the AAV capsid binding protein is an AAV8 or an AAV9 capsid binding protein. The fusion protein or conjugated protein of paragraph 145 to 147, wherein the viral binding protein is associated with a viral particle. The fusion protein or conjugated protein of paragraph 148, wherein the viral particle is an AAV. The fusion protein or conjugated protein of paragraph 145, wherein the protein capable of binding to a cargo is an RNA binding protein. The fusion protein or the conjugated protein of paragraph 150, wherein the RNA binding protein is associated with an RNA. The fusion protein or the conjugated protein of paragraph 151 wherein the RNA is a guide RNA. The fusion protein or the conjugated protein of paragraph 152, wherein the guide RNA is associated with a Cas protein. The fusion protein or the conjugated protein of paragraph 153, wherein:

[0318] (i) the Cas protein is a Cas9 or a Cas12, such as Cas12a;

[0319] (ii) the Cas protein is a Cas9 or a Cas12, such as Cas12a, a Cas3, a base editor or a prime editor; or

[0320] (iii) the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor. The fusion protein or conjugated protein of paragraph 145, wherein the protein capable of binding to a cargo is a DNA binding protein. The fusion protein or the conjugated protein of paragraph 155, wherein the DNA binding protein is associated with a DNA cargo. The fusion protein or the conjugated protein of paragraph 156, wherein the DNA cargo is a recombinant AAV vector. The fusion protein or conjugated protein of paragraph 108 to 157, comprising a purification moiety. The fusion protein or conjugated protein of paragraph 158, wherein the purification moiety comprises the N-terminal or C-terminal extein. The fusion protein or conjugated protein of paragraph 159, wherein the purification moiety comprises the C-terminal extein. The fusion protein or conjugated protein of paragraph 158, wherein the purification moiety is fused to the N-terminal or C-terminal extein. The fusion protein or conjugated protein of paragraph 161, wherein the purification moiety is fused to the C-terminus of the C-terminal extein. The fusion protein or conjugated protein of paragraph 158 to 162, wherein the intein is positioned such that the purification moiety is released from the EV polypeptide upon cleavage of the intein-extein bond. The fusion protein or conjugated protein of paragraph 158 to 163, wherein the intein is positioned between the EV protein or the moiety for loading the polypeptide and the purification moiety. The fusion protein of paragraph 158 to 164, wherein purification moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein of paragraph 158 to 165, wherein the purification moiety is fused or conjugated, via the polypeptide comprising an intein and an extein sequence, to the N-terminus of PTTG1-IP or to loop 1 or loop 2 of the EV polypeptide, wherein the EV polypeptide is a tetraspanin such as TSN2 or CD63, preferably TSN2. The conjugated protein of paragraph 158 to 165, wherein the purification moiety is conjugated to a part of the moiety for loading a polypeptide into the EV which is localised to and / or displayed in the lumen of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein or conjugated protein of paragraph 108 to 167, comprising a targeting moiety. The fusion protein of paragraph 168, wherein the targeting moiety is fused or conjugated to the EV polypeptide. The fusion protein of paragraph 169, wherein the targeting moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein of paragraph 169 or 170, wherein the targeting moiety is fused or conjugated to the N-terminus of PTTG1-IP or to loop 1 or loop 2 of the EV polypeptide, wherein the EV polypeptide is a tetraspanin such as TSN2 or CD63, preferably TSN2. The conjugated protein of paragraph 168, wherein the targeting moiety is conjugated to the moiety for loading a polypeptide into the EV. The conjugated protein of paragraph 172, wherein the targeting moiety is conjugated to a part of the moiety for loading a polypeptide into the EV which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein or conjugated protein of paragraph 168 to 173, wherein the targeting moiety targets the liver, the heart, the brain or neuronal tissue, hepatocytes, cardiomyocytes, cardiac smooth muscle cells, sensory neurons, motor neurons, interneurons and / or glia cells. The fusion protein or conjugated protein of paragraph 168 to 174, wherein the targeting moiety is capable of binding a moiety present of the surface of a cell of the liver, a cell of the heart, a brain cell, a neuronal cell, a hepatocyte, a cardiomyocyte, a cardiac smooth muscle cell, a sensory neuron, a motor neuron, an interneuron or a glia cell. The fusion protein or conjugated protein of paragraph 108 to 175, further comprising a multimerization domain. The fusion protein of or the conjugated protein of paragraph 176 wherein the multimerization domain is a fold-on domain or a leucine zipper. The fusion protein or conjugated protein of paragraph 108 to 177, further comprising an endosomal escape domain. The fusion protein of paragraph 178, wherein the endosomal escape domain is fused or conjugated to the EV polypeptide. The fusion protein of paragraph 179, wherein the endosomal escape domain is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed in an EV membrane, preferably an exosome membrane. The conjugated protein of paragraph 178, wherein the endosomal escape domain is conjugated to the moiety for loading a polypeptide into the EV. The conjugated protein of paragraph 181, wherein the endosomal escape domain is conjugated to a part of the moiety for loading a polypeptide into the EV which is localised to and / or displayed in an EV membrane, preferably an exosome membrane. The fusion protein or conjugated protein of paragraph 178 to 182, wherein the endosomal escape domain is VSVG. The fusion protein or conjugated protein of paragraph 108 to 183, further comprising a pharmacokinetic and / or pharmacodynamic effector moiety. The fusion protein of paragraph 184, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to the EV polypeptide. The fusion protein of paragraph 185, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein of paragraph 184 to 186, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to the N-terminus of PTTG1-IP or to loop 1 or loop 2 of the EV polypeptide, wherein the EV polypeptide is a tetraspanin such as TSN2 or CD63, preferably TSN2. The conjugated protein of paragraph 184, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is conjugated to the moiety for loading a polypeptide into the EV. The conjugated protein of paragraph 188, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is conjugated to a part of the moiety for loading a polypeptide into the EV which is localised to and / or displayed on the surface of an EV, preferably an exosome, a microvesicle and / or a related extracellular vesicle. The fusion protein of paragraph 184 to 189, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is an albumin binding domain. A polynucleotide encoding the polypeptide of paragraph 1 to 107 or the fusion protein or the conjugated protein of paragraph 108 to 190. An EV, comprising the polypeptide of paragraph 1 to 107, the fusion protein or the conjugated protein of paragraph 108 to 190, or the polynucleotide of paragraph 191 An EV comprising the polypeptide of paragraph 1 to 107 or the fusion protein or the conjugated protein of paragraph 108 to 190, wherein the intein has catalysed the cleavage of the polypeptide comprising an intein and an extein sequence. The EV of paragraph 193, wherein the polypeptide comprising an intein and an extein sequence has been cleaved at an intein-extein bond. The EV of paragraph 194, wherein the polypeptide comprising an intein and an extein sequence has been cleaved at the intein-extein bond at the intein C-terminus. An EV comprising the fusion protein or the conjugated protein of paragraph 108 to 190, wherein the protein cargo or protein capable of binding to a cargo has been released from the EV polypeptide. An EV comprising the fusion protein or the conjugated protein of paragraph 108 to 190, wherein the protein cargo or protein capable of binding to a cargo has been released from the EV polypeptide and the intein. An EV comprising the fusion protein or the conjugated protein of paragraph 158 to 190, wherein the purification moiety has been released from the EV polypeptide. An EV comprising the fusion protein or the conjugated protein of paragraph 158 to 190, wherein purification moiety has been released from the EV polypeptide and the intein. An EV comprising the fusion protein or the conjugated protein of paragraph 108 to

[0321] 190, wherein the polypeptide comprising an intein and an extein sequence has been cleaved to release the protein cargo, protein capable of binding to a cargo or the purification moiety. The EV of paragraph 196 to 200, wherein the intein has catalysed the cleavage of the polypeptide comprising an intein and an extein sequence. The EV of paragraph 201, wherein the polypeptide comprising an intein and an extein sequence has been cleaved at an intein-extein bond. The EV of paragraph 202, wherein the polypeptide comprising an intein and an extein sequence has been cleaved at the intein-extein bond at the intein C-terminus. The EV of paragraph 192 to 203, wherein the EV comprises a cargo. The EV of paragraph 204, wherein the cargo, or a protein capable of binding to the cargo, is comprised in or fused to the fusion protein or the conjugated protein. The EV of paragraph 205, wherein the EV comprises a second cargo. The EV of paragraph 206, wherein the second cargo, or a protein capable of binding to the second cargo, is fused to a second EV polypeptide or EV localisation moiety. The EV of paragraph 204 to 207, wherein the cargo is a therapeutic cargo. The EV of paragraph 204 to 208, wherein the cargo is a protein cargo or an RNP complex. The EV of paragraph 209, wherein:

[0322] (i) the protein cargo is a Cas protein;

[0323] (ii) the Cas protein is a Cas9 or a Cas12, such as Cas12a, a Cas3, a base editor or a prime editor; or

[0324] (iii) the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor. The EV of paragraph 210, wherein the Cas protein is Cas9 or Cas12, such as Cas12a. The EV of paragraph 210 or 211 , wherein the Cas protein is associated with a guide RNA. The EV of paragraph 204 to 208, wherein the cargo is a viral particle. The EV of paragraph 213, wherein the viral particle is an AAV. The EV of paragraph 214, wherein the AAV is anAAV8 or an AAV9. The EV of paragraph 204 to 208, wherein the cargo is an RNA. The EV of paragraph 216, wherein the RNA is a guide RNA. The EV of paragraph 217, wherein the guide RNA is associated with a Cas. The EV of paragraph 218, wherein the Cas is:

[0325] (i) a Cas9 or a Cas12, such as Cas12a; or

[0326] (ii) a Cas9 or a Cas12, such as Cas12a, a Cas3, a base editor or a prime. The EV of paragraph 204 to 208, wherein the cargo is a DNA. The EV of paragraph 220, wherein the DNA cargo is a recombinant AAV vector. The EV of paragraph 204 to 221 , wherein the cargo is luminally loaded. The EV of paragraph 192 to 222, wherein the EV comprises a purification moiety. The EV of paragraph 223, wherein the purification moiety is comprised in or fused to the fusion protein or the conjugated protein. The EV of paragraph 223, wherein the purification moiety is comprised in or fused to a second fusion protein of paragraphs 108 to 190. The EV of paragraph 223, wherein the purification moiety is fused to a second EV polypeptide or EV localisation moiety. The EV of paragraph 223 to 226, wherein the purification moiety is surface loaded. The EV of paragraph 192 to 227, wherein the EV comprises a targeting moiety. The EV of paragraph 228, wherein the targeting moiety is comprised in or fused to the fusion protein or the conjugated protein. The EV of paragraph 228, wherein the targeting moiety is fused to a second EV polypeptide or EV localisation moiety. The EV of paragraph 228 or 230, wherein the targeting moiety is capable of binding a moiety present of the surface of a cell of the liver, a cell of the heart, a brain cell, a neuronal cell, a hepatocyte, a cardiomyocyte, a cardiac smooth muscle cell, a sensory neuron, a motor neuron, an interneuron or a glia cell. The EV of paragraph 228 to 231 , wherein the targeting moiety is surface loaded. The EV of paragraph 192 to 232, wherein the EV comprises an endosomal escape domain. The EV of paragraph 233, wherein the endosomal escape domain is VSVG. The EV of paragraph 233 or 234, wherein the endosomal escape domain is loaded into the EV membrane. The EV of paragraph 192 to 235, wherein the EV comprises a pharmacokinetic and / or pharmacodynamic effector moiety. The EV of paragraph 236, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is comprised in or fused to the fusion protein or the conjugated protein. The EV of paragraph 236, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is fused to a second EV polypeptide or EV localisation moiety. The EV of paragraph 236 to 238, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is an albumin binding domain. The EV of paragraph 236 or 239, wherein the pharmacokinetic and / or pharmacodynamic effector moiety is surface loaded. The EV of paragraph 192 to 240, wherein the EV is an exosome, a microvesicle and / or a related extracellular vesicle. A population of EVs comprising an EV of paragraph 192 to 241. A population of EVs, wherein 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 the EVs in the population of EVs comprise or consist of an EV of paragraph 192 to 241. A cell comprising the polypeptide of paragraph 1 to 107, the fusion protein or the conjugated protein of paragraph 108 to 190, the polynucleotide of paragraph 191 , or the EV of paragraph 192 to 241. The cell of paragraph 244, wherein the cell is an EV producer cell, preferably wherein the EV producer cell is a HEK cell, more preferably a HEK 293 cell, most preferably a HEKVPC2 cell or HEKVPC1 cell. A cell comprising the EV of paragraph 192 to 241. The cell of paragraph 246, wherein the cell is a target cell, preferably wherein the target cell is a cell of the liver, a cell of the heart, a brain cell, a neuronal cell, a hepatocyte, a cardiomyocyte, a cardiac smooth muscle cell, a sensory neuron, a motor neuron, an interneuron or a glia cell. A method for producing an EV or a population of EVs comprising the following steps:

[0327] (i) introducing into an EV-producing cell a polynucleotide construct of paragraph 191 ; and (ii) expressing a construct in the EV-producing cell, thereby generating EVs comprising a polypeptide of paragraph 1 to 107 or a fusion protein or conjugated protein of paragraph 108 to 190. A method for producing an EV or a population of EVs comprising the following steps:

[0328] (i) culturing EV producer cells and collecting EVs from said cells and

[0329] (ii) loading said EVs with the polypeptide comprising an intein and an extein sequence of 1 to 107, the fusion protein or the conjugated protein of paragraph 108 to 190 by one or more exogenous loading method as described herein. The method of paragraph 248 or 249 comprising a further step of introducing into the same EV producing cell a, or an additional, polynucleotide construct encoding a, or a further, protein cargo, protein capable of binding a cargo, purification moiety, targeting moiety, endosomal escape moiety, pharmokinetic moiety and / or pharmoeffector moiety, optionally in the form of a fusion protein with an EV polypeptide. The method of paragraph 248 to 250 comprising a further step of loading into the EVs a, or an additional, polynucleotide construct encoding a, or a further, protein cargo, protein capable of binding a cargo, purification moiety, targeting moiety, endosomal escape moiety, pharmokinetic moiety and / or pharmoeffector moiety, by one or more exogenous loading method as described herein. The method of paragraph 248 to 251 , wherein the method comprises an additional step of incubating the EV of the present disclosure or population of EVs of the present disclosure at temperature that is sufficient to allow for the intein to catalyse the cleavage of the polypeptide comprising an intein and an extein sequence. The method of paragraph 252 wherein the temperature is room temperature. The method of paragraph 252 wherein the temperature is 32-42°C, 34-40°C, 36- 38°C, or preferably 37°C. The method of paragraph 252 to 254 wherein the EV or population of EVs is incubated for a time-period that is sufficient to allow for the intein to catalyse the cleavage of the polypeptide comprising an intein and an extein sequence. The method of paragraph 255, wherein the EV or population of EVs is incubated for a time-period that is sufficient to allow for the intein to catalyse the cleavage of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or 100% of the polypeptides comprising an intein and an extein sequence. The method of paragraph 255 or 256 wherein the EV or population of EVs is incubated at room temperature for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours or at least 24 hours. The method of paragraph 248 to 257, wherein the method comprises a step of purifying the EVs. The method of paragraph 249 to 258, wherein the exogenous loading method is electroporation. The method of paragraph 249 to 258, wherein the exogenous loading method is coincubation of the one or more EVs with the polypeptide of paragraph 1 to 107 or the fusion protein or the conjugated protein of paragraph 108 to 190, wherein the polypeptide of paragraph 1 to 107 or the fusion protein or the conjugated protein of paragraph 108 to 190 is conjugated to a membrane anchoring moiety. The method of paragraph 260, wherein the membrane anchoring moiety is a hydrophobic moiety. The method of paragraph 261 , wherein the membrane anchoring moiety is vitamin A, a lipid or a cholesterol. The method of paragraph 248 to 262, wherein the EV producer cells are cells grown in culture. The method of paragraph 248 to 263, wherein the EV producer cells are a cell line, preferably HEK cells, more preferably HEK293 cells, most preferably HEKVPC2 or HEKVPC1 cells. 265. An in vitro, in vivo or ex vivo method of delivering a cargo, preferably a therapeutic cargo, to a target cell, comprising contacting a target cell with an EV of paragraph 192 to 241 or a population of EVs of paragraph 242 or 243.

[0330] 266. A pharmaceutical composition comprising an EV of paragraph 192 to 241 or a population of EVs of paragraph 242 or 243, and a pharmaceutically acceptable excipient and / or carrier.

[0331] 267. The EV of paragraph 192 to 241 , the population of EVs of paragraph 242 or 243 or the pharmaceutical composition of paragraph 266 for the preparation of a medicament for treatment or prevention of a disease in a subject.

[0332] 268. The EV of paragraph 192 to 241, the population of EVs of paragraph 242 or 243 or the pharmaceutical composition of paragraph 266 for use as a medicament for treatment or prevention of a disease in a subject.

[0333] 269. A method for treating or preventing a disease comprising administering a therapeutically or prophylactical ly effective amount of the EV of paragraph 192 to 241 , the population of EVs of paragraph 242 or 243 or the pharmaceutical composition of paragraph 266 to a subject suffering from or susceptible to the disease.

[0334] 270. The use or the method of paragraph 265 to 269, wherein the disease is a genetic disorder, a liver disorder, a neurological disorder or a cardiac disorder.

[0335] 271. The use or the method of paragraph 265 to 270, wherein the disease is Phenylketonuria, heart failure or ALS.

[0336] 272. The use or the method of paragraph 265 to 271 , wherein the EV, population of EVs or pharmaceutical composition is locally administered.

[0337] 273. The use or the method of paragraph 272, wherein the EV, population of EVs or pharmaceutical composition is locally administered to the liver, the CNS, the brain or the heart.

[0338] The invention is further described in the following numbered embodiments: A fusion protein comprising

[0339] (i) a polypeptide comprising an intein and

[0340] (ii) an EV polypeptide; wherein the intein is capable of catalysing cleavage of the polypeptide; further wherein the intein comprises or consists of variant of SEQ ID NO 1 , wherein the variant of SEQ ID NO 1 comprises an Arginine residue at position 41; a Valine residue at position 108; a Proline residue at position 111 and a Valine residue at position 152. A polypeptide comprising an intein, wherein the intein is capable of catalysing cleavage of the polypeptide; further wherein the intein comprises or consists of variant of SEQ ID NO 1 , wherein the variant of SEQ ID NO 1 comprises an Arginine residue at position 41; a Valine residue at position 108; a Proline residue at position 111 and a Valine residue at position 152; wherein the polypeptide further comprises:

[0341] (i) a C-terminal extein sequence, wherein the +1 residue of the C-terminal extein sequence is Glu, Phe, His, Leu, Met, Gly or Pro; and / or

[0342] (ii) an N-terminal extein sequence, wherein the N-terminal extein sequence comprises Gly as the -1 residue, Ser as the -2 residue, Gly as the -3 residue, Gly as the -4 residue and Gly as the -5 residue. The polypeptide of embodiment 2, wherein:

[0343] (i) the +1 residue of the C-terminal extein sequence is Gly; and / or

[0344] (ii) the +2 residue of the C-terminal extein sequence is Ser and / or the +3 residue of the C-terminal extein is Pro; and / or

[0345] (iii) the +1 residue of the C-terminal extein sequence is Gly, the +2 residue of the C-terminal extein sequence is Ser, and the +3 residue of the C-terminal extein sequence is Pro. The polypeptide of embodiment 2 or embodiment 3, wherein the +1 residue of the C- terminal extein sequence is Gly, the +2 residue of the C-terminal extein sequence is Gly, the +3 residue of the C-terminal extein sequence is Gly, the +4 residue of the C- terminal extein sequence is Gly and the +5 residue of the C-terminal extein sequence is Ser. The polypeptide of embodiment 2 to 4, wherein the C-terminal extein sequence reduces the rate of cleavage in the cytoplasm of a cell and / or at a pH of 7.0-7.5 and at a temperature of 34-40°C, as compared with where the C-terminal extein sequence is SEQ ID NO. 3. The polypeptide of embodiment 2 to 5, wherein the N-terminal extein sequence is GGGSG (SEQ ID NO: 10), FAS (SEQ ID NO: 11), FFM (SEQ ID NO: 12), FAF (SEQ ID NO: 13) or TRM (SEQ ID NO: 9). The polypeptide of embodiment 2 to 6, wherein the N-terminal extein sequence reduces the rate of cleavage in the cytoplasm of a cell and / or at a pH of 7.0-7.5 and at a temperature of 34-40°C, as compared with where the N-terminal extein sequence is SEQ ID NO. 2 and / or SEQ ID NO. 5. A fusion protein comprising:

[0346] (i) the polypeptide of any one of embodiments 2 to 7 and

[0347] (ii) an EV polypeptide. The polypeptide or fusion protein of embodiment 1 to 8, wherein the intein exhibits a reduced rate of cleavage in the cytoplasm of a cell and / or at a pH of 7.0-7.5 and at a temperature of 34-40°C, as compared to an intein according to SEQ ID NO 1. The polypeptide or fusion protein of embodiment 1 to 9, wherein the intein comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4. The fusion protein of embodiment 8 to 10, wherein the EV polypeptide comprises the N-terminal extein or the EV polypeptide is fused to the N-terminus of the N-terminal extein. The fusion protein of embodiment 1 or embodiments 8 to 11 , wherein the intein is comprised in or fused to a domain or terminus of the EV polypeptide that is localised to and / or displayed in the lumen of the EV. The fusion protein of embodiment 1 or embodiments 8 to 12, wherein the fusion protein further comprises a protein cargo or a protein capable of binding to a cargo. The fusion protein of embodiments 8 to 12, wherein the fusion protein further comprises a protein cargo or a protein capable of binding to a cargo, further wherein the protein cargo or protein capable of binding to a cargo comprises the C-terminal extein or wherein the protein cargo or protein capable of binding to a cargo is fused to the C-terminus of the C-terminal extein. The fusion protein of embodiment 13 or 14, wherein the protein capable of binding to a cargo is associated with a cargo. A polynucleotide encoding the polypeptide or fusion protein of any of the preceding embodiments. An EV comprising the polypeptide, fusion protein or polynucleotide of any of the preceding embodiments. An EV comprising the polypeptide or fusion protein of any of the preceding embodiments, wherein the intein has catalysed the cleavage of the polypeptide comprising the intein sequence. An EV comprising the fusion protein of embodiment 13 to 15, wherein the protein cargo or protein capable of binding to a cargo has been released from the EV polypeptide. The EV of embodiment 17 to 19, wherein the EV is an exosome. A method of preparing an EV of embodiment 17 to 20, comprising:

[0348] (i) introducing into an EV-producing cell a polynucleotide of embodiment 16;

[0349] (ii) expressing the construct in the EV-producing cell, thereby generating EVs comprising a polypeptide or fusion protein of embodiments 1 to 15. The method of embodiment 21 , comprising an additional step of collecting the EVs of step (ii) and incubating the EVs at a temperature that is sufficient to allow for the intein to catalyse the cleavage of the polypeptide comprising the intein sequence. A cell comprising the polypeptide, fusion protein, polynucleotide or EV of embodiments 1 to 20. 24. A pharmaceutical composition comprising an EV of embodiments 17 to 20, and a pharmaceutically acceptable excipient and / or carrier.

[0350] 25. The EV of embodiments 17 to 20 or the pharmaceutical composition of embodiment 24 for use as a medicament for treatment or prevention of a disease in a subject, optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure orALS syndrome.

[0351] 26. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the EV of embodiments 17 to 20 or the pharmaceutical composition of embodiment 24 to a subject suffering from or susceptible to the disease; optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure orALS.

[0352] Examples

[0353] Example 1: Characterisation of AI-CM Extein sequences

[0354] Different C-terminal extein sequences were screened by engineering 5-amino acid residues into constructs comprising the AI-CM intein.

[0355] The constructs comprised a CBD (chitin binding domain) tag fused to the N-terminus of the AI-CM intein, via an N-terminal extein sequence of Arg-lle-Ser-Glu-Phe (SEQ ID NO. 2). This N-terminal extein sequence is commonly used for cloning the inteins into fusions with N-terminal polypeptides, including affinity tags.

[0356] 5-amino acid residues were fused to the C-terminus of the AI-CM intein. The residues were fused to different C-extein polypeptides: maltose binding protein (MBP), thioredoxin (TrxA) or P-galactosidase (P-gal). The C-terminal extein residues were based on the +1 to +5 C- terminal extein residues that occur in the natural Mtu RecA sequence (SEQ ID NO. 3), however the +1-cysteine residue was replaced with His, Met, Glu, Leu, and Phe. Figure 1a is a schematic representative of the different constructs tested.

[0357] The constructs were expressed in E. coli and the polypeptides were purified on a chitin resin. Clarified E. coli lysate was added to the column and the flow-through was collected and reapplied to the column. Next, the column was washed with a high-salt wash buffer (20 mM AMPD / PIPE, 500 mM NaCI, pH 8.5). The column was then washed with cleaving buffer (20 mM AMPD / PIPE, 200 mM NaCI, pH 6.2) to alter the environmental conditions of the column from pH 8.5 to pH 6.2. Finally, the column was sealed, cleaving buffer was added and the column was incubated for a period of time to allow sufficient cleavage of the intein. After completion of the cleavage reaction, the purified tag-less protein was collected in the elution fraction.

[0358] Samples were taken at different time points and the percentage of the precursor in the samples was tracked using SDS-PAGE. The precursor percentage was calculated by d [precursor] k[premrsor] = - - — - dividing the intensity of the precursor band by the sum of the intensities of the precursor and cleaved product bands. The cleavage reaction was assumed to follow first-order kinetics, and the rate constant and half-life of the reaction can be determined using the corresponding formulas:

[0359] This method may be used to asses the cleavage rate of any polypeptide comprising an intein and extein sequence.

[0360] Figure 1b shows the rate constant, K, of the cleavage reactions at pH 6.2 for the polypeptides comprising the different +1 C-terminal extein residues and the different C- extein polypeptides. When the +1 residue was histidine the intein consistently exhibited the fastest cleaving rate, while phenylalanine and leucine offered relatively slower cleaving rates. The relative cleaving rate of the intein remained consistent for the five different amino acids at the +1 position regardless of the C-extein polypeptide.

[0361] A regression analysis was conducted on various chemical properties of the side chain residues, including molecular weight, volume, hydrophobicity, polarity, charge, and isoelectric point. The analysis revealed that the volume and molecular weight of the side chain residues significantly influenced the intein cleavage rate and exhibited a positive correlation. Generally, side chain residues with larger volume or higher molecular weight at +1 position contributed to faster C-terminal cleavage rates (data not shown).

[0362] Different N-terminal extein sequences were also screened by engineering 5-amino acid residues into constructs comprising the AI-CM intein. The constructs comprised SEQ ID NO. 3 as the +1 to +5 residues of the C-terminal extein, with Streptokinase as the C-extein polypeptide. The 5-amino acid residue N-terminal extein sequences consist of the sequence Arg-lle-Ser-Glu-X, with “X" representing the variable residue at position -1 and were inserted between the C-terminal end of the CBD tag and the N-terminus of the AI-CM intein.

[0363] Cleaving rates were determined in line with the methods outlined above. Figure 1c shows the rate constant, k, of the cleavage reactions of the polypeptides with the different +1 N- terminal extein residues at pH 6.2 and at pH 8.5. The fastest cleaving at pH 6.2 was achieved with Ala, Asp, Gly, Gin or His residues at the -1 position. Subsequently, regression analysis was conducted to correlate the cleaving rate with the chemical properties of the side chains of the -1 residues. It was found that there is a strong correlation between the cleaving rate of the AI-CM intein and the hydrophobicity index of the side chains. -1 residues with hydrophilic side chains generally provide faster C-terminal cleavage for the AI-CM intein (data not shown).

[0364] Example 2 - The AI-CM intein is associated with premature cleavage

[0365] The EV loading efficiency of the AI-CM intein was assessed through the expression of three different florescent fusion proteins in HEK293CTS cells. Figure 2a is a schematic of the three different fusion proteins. All three fusion proteins comprise the same exosomal protein, CD63, and the same florescent proteins (mScarlet and eGFP). Construct A is the control fusion protein that does not comprise the AI-CM intein. Construct B comprises eGFP to the N-terminal of the AI-CM intein and mScarlet to the C-terminal. Construct C comprises mScarlet to the N-terminal of the AI-CM intein and eGFP to the C-terminal.

[0366] The constructs were transfected into HEK293CTS cells and the secreted exosomes were purified and lysed using a commercial CD63 pulldown kit. Flow cytometry was then employed to detect the fluorescence signals in the exosomes and the loading efficacy was determined by comparing and analysing the fluorescence signals on the different sides of the intein.

[0367] This method may be used to asses the loading efficiency of any polypeptide comprising an intein and extein sequence.

[0368] Figure 2b shows the fluorescence signals (mScarlet single positive (mScarlet SP); eGFP single positive (eGFP SP) or double positive for both mScarlet and eGFP (DP)) detected in exosomes. The majority of exosomes derived from HEK293 CTS cells that express the control fusion protein, shown as “Construct A” in Figure 2a, were double positive for both mScarlet and eGFP (DP), demonstrating that the fusion protein becomes localised to exosomes and that there is limited cleavage in the absence of the AI-CM intein. The majority of exosomes derived from HEK293 CTS cells that express the fusion protein shown as “Construct B” in Figure 2a were eGFP single positive and relatively few exosomes were double positive for both mScarlet and eGFP. The majority of exosomes derived from HEK293 CTS cells that express the fusion protein shown as “Construct C” in Figure 2a were mScarlet single positive and relatively few exosomes were double positive for both mScarlet and eGFP. These results indicate that the AI-CM intein is undergoing self-cleavage prior to the fusion protein becoming localised to exosomes i.e. in the intracellular environment of the HEK293 CTS cells at 37°C and pH 7.3. Thus, the cargo protein at the C-terminal of the intein is pre-cleaved, while the exosomal protein, intein and any cargo protein to the N-terminal of the intein are still loaded into exosomes.

[0369] Example 3 - Characterisation of 129 Extein sequences

[0370] Different C-terminal extein sequences were screened by engineering 3-amino acid residues into constructs comprising the 129 intein.

[0371] The constructs comprised a CBD (chitin binding domain) tag fused to the N-terminus of the 129 intein, via an N-terminal extein sequence of Arg-lle-Ser-Glu-Phe-Ala-Ser (SEQ ID NO. 5). This N-terminal extein sequence was used for producing the yeast surface display library in which the 129 intein was discovered.

[0372] The 3-amino acid residues fused between the C-terminus of the 129 intein and the N- terminus of eGFP. The 3-amino acid residues were based on the +1 to +3 residues of the natural Mtu RecA C-terminal extein sequence (Cys-Ser-Pro), however the +1 cysteine residue was replaced with Glu, Phe, His, Leu, Met, Gly and Pro. Figure 3a is a schematic representative of the different constructs tested.

[0373] The constructs were expressed in E. coli and the polypeptides were purified on a chitin resin. Clarified E. coli lysate was added to the column and the flow-through was collected and reapplied to the column. Next, the column was washed with a high-salt wash buffer (20 mM AMPD / PIPE, 500 mM NaCI, pH 8.5). To conduct cleaving analysis, 1 column volume of cleaving buffer (20 mM AMPD / PIPE, 200 mM NaCI) at pH 6.0 or pH 7.3 was applied to resins, then the resin was incubated at 20°C or 37°C and samples were taken at different time points. The intensity of the precursor and target protein bands at each time point was measured using SDS-PAGE and the percentage of the precursor was calculated by dividing the intensity of the precursor band by the total value of intensities of the precursor band and the intein with the N-exteins band. The experimental half-life was determined by taking the natural logarithm of 2 and dividing it by the absolute value of the rate constant.

[0374] Figure 3b shows the estimated half-life of intein cleavage reactions with various +1 residues (Glu, Phe, His, Leu, Met, Gly and Pro) in different conditions (pH 7.3 and pH 6.0 as well as 37°C and 20°C), as inferred from SDS-PAGE results. When the +1 residue was Gly the I29 intein demonstrates a significantly longer half-life than all the other amino acids under the conditions set to mimic the cytoplasm (37°C, pH 7.3), whilst providing a half-life that is not significantly longer than other amino acids under the designated cleavage conditions within the lumen of exosomes (20°C, pH 6.2).

[0375] Example 4: The 129 intein is associated with reduced premature cleavage

[0376] The EV loading efficiency of the 129 intein was assessed through the expression of three different florescent fusion proteins in a commonly used suspension cell line, HEK Expl-293F. Figure 4a is a schematic of the three different fusion proteins. The constructs comprise the same exosomal protein, CD63, connected to the N-terminal of the intein via the same N- terminal extein sequence of Arg-lle-Ser-Glu-Phe-Ala-Ser (SEQ ID NO. 5). The constructs also all comprise the same C-terminal extein cargo protein (a His-tagged eGFP) connected to the C-terminal of the intein by the same +1 to +3 C-terminal extein residues (GSP (SEQ ID NO: 6)). The three constructs comprise different inteins: construct (1) comprises the I29 intein; construct (2) comprises the AI-CM intein and construct (3) comprises an N440A variant of the I29 intein that is non-cleaving.

[0377] The constructs were transfected into HEK Expl-293F cells and the secreted exosomes were purified using a CD63 Exosome isolation kit (Miltenyi). A western blot was run with the Inlet of magnetically labelled conditioned media (In), the flow-through of magnetic columns (FT), the wash flow through (WS) and the elution of exosomes lysate (EL) as samples. Figure 4b is an image of this western blot and shows the presence of the non-cleaved protein (between 70 and 100kDa) and the cleaved cargo (between 25 and 35 kDa) in the different samples. In the exosome sample derived from cells expressing the non-cleaving 129 intein construct (shown as construct (3) in Figure 4a), only a very faint band was detected between 25 and 35 kDa, indicating that there is limited cleavage of the cargo when this construct is expressed. When comparing samples derived from cells expressing the AI-CM intein construct (shown as construct (2) in Figure 4a) and from cells expressing the 129 intein construct (shown as construct (1) in Figure 4a), more cleaved cargo is detected in the In and FT samples from the cells expressing the AI-CM intein, indicating that this polypeptide results in more cleaved eGFP outside of the exosomes. Conversely, more cleaved cargo is detected in the EL sample from the cells expressing the 129 intein, indicating that more cleaved (bioactive) cargo is loaded into exosomes when the 129 intein is expressed.

[0378] As an alternative approach to assess the extent of premature cleavage, HEK Expl-293F cells were transfected with a CD63-RISEFAS-mScarlet-l-29-GSP-eGFP-His construct, a CD63- RISEFAS-mScarlet-AI-CM-GSP-eGFP-His construct or a CD63- RISEFAS-mScarlet- l-29(N440A)-GSP-eGFP-His construct, the secreted exosomes were purified using a CD63 Exosome isolation kit (Miltenyi) and the flow cytometry was then employed to detect the fluorescence signals in the exosomes. The loading efficacy was determined by comparing and analysing the fluorescence signals on the different sides of the intein.

[0379] Figure 4c shows FACs plots of the exosomes purified from the HEK cells expressing the different constructs, wherein the signal of eGFP is plotted on the x-axis and the signal of mScarlet is plotted on the y-axis. Each point on the graph represents a fluorescence measurement of an individual exosome. The mScarlet signal levels in the three sample groups are similar, indicating similar loading performance of CD63 with the three intein variants. The non-cleaved 129 intein control group shows a basic 1 :1 ratio of mScarlet to eGFP signals, indicating that both fluorescent proteins are loaded into the exosomes since premature cleavage does not occur prior to loading. In the AI-CM intein group, the eGFP signal is approximately less than one-tenth of the mScarlet signal, suggesting that the intein has self-cleaved before loading into the. With the I29 construct, the eGFP signal is approximately half of the mScarlet signal, a significant reduction in premature cleavage and an improvement in loading efficacy compared to the AI-CM intein. This demonstrates the superior efficacy of the I29 intein over the AI-CM intein.

[0380] To investigate the general applicability of the I29 and GSP C-terminal extein sequence combination in reducing premature cleavage during the loading process, alternative constructs were produced, where the C-extein eGFP was replaced with either mScarlet or the Cas9 protein, AsCpfl . The cleavage rates of these constructs were assessed by calculating the percentage of precursor, according to the methods described above. Figure 4d is a graph showing the rate of cleavage of the constructs comprising 129 with GSP C- terminal extein fused to the different C-terminal cargos (eGFP, mScarlet and Cas9 protein AsCpfl) at 37°C, pH 7.3. Although not identical, the cleavage rates of the three C-terminal cargos do not differ significantly. This indicates that the improvement observed in example 4 with the 129 intein (i.e. reduced premature cleavage) is not dependent on the C-terminal cargo. The methods described in this example may be used to assess the amount of premature cleavage and the amount of cleavage in the cytoplasm of a cell and / or in the lumen of an EV mediated by any polypeptide comprising an intein and extein sequence.

[0381] Example 5 - Characterisation of 129 intein mediated cleavage at pH 6.2

[0382] To investigate whether the 129 intein can mediate cleavage in an EV, the CD63-RISEFAS-I- 29-GSP-eGFP-His construct shown in Figure 4a and described above in example 4 was again transfected into HEK Expl-293F cells and the secreted exosomes were purified using a CD63 Exosome isolation kit (Miltenyi). Since the fusion protein loaded in exosomes was expected to undergo intein cleavage and release of the cargo protein under conditions of approximately pH 6.2 and 20°C, the purified exosomes obtained using the CD63 exosomes isolation kit were washed off the magnetic beads using a 1X PBS buffer titrated to pH 6.2 as the elution solution. Subsequently, the eluted exosomes were incubated at 20 °C for 24 hours. Samples were taken at 0 hours, 5 hours and 24 hours.

[0383] A western blot was run with the Inlet of magnetically labelled conditioned media (In), the flow-through of magnetic columns (FT), the wash flow through (WS) and the elution of exosomes lysate incubated at 20 °C for 0 hours (Oh), 5 hours (5h) and 24 hours (24) as samples. Figure 5a is an image of this western blot and shows the presence of the noncleaved protein (between 70 and 100kDa) and the cleaved cargo (between 25 and 35 kDa) in the different samples. A prominent band corresponding to the non-cleaved protein was initially observed in the elution. However, in the subsequent 5-hour and 24-hour samples, the intensity of the band decreased demonstrating that the 129 intein with the GSP linker loaded into exosomes retains its ability to undergo cleavage reactions, with the majority of the precursor undergoing cleavage within 24 hours.

[0384] Example 6 - Characterisation ofAI-CM intein mediated cleavage and 129 intein mediated cleavage with different EV polypeptides

[0385] To further investigate the general applicability of 129 to improve EV loading, constructs were comprising different EV polypeptides were produced. Figure 6a shows the different constructs tested. The constructs comprise either the 129 intein (1-29), the AI-CM intein (dICM) or a non-cleavable control amino acid sequence (AA). The different constructs all comprise the same C-terminal extein sequence (GGGGS (SEQ ID NO: 7)) fused to the C- terminus of the intein and the same C-terminal extein polypeptide of eGFP The different constructs all also comprise the same N-terminal extein sequence (GGGSG (SEQ ID NO: 10)) fused to the N-terminus of the intein. Different EV polypeptides, either CD63 or TSN2, were fused to the N-terminus of this N-terminal extein sequence. To confirm cleavage activity in these constructs, the constructs were transfected into HEK CTS cells and, 72 hours later, conditioned media was collected, and a western blot was run with the different samples. Figure 6b is a JESS image showing the amount of the cleaved GFP cargo and non-cleaved protein present. In the conditioned media derived from cells expressing the non-cleaving amino acid construct, a high proportion of the non-cleaved GFP labelled protein is present (lanes 2 and 5). There is a high amount of cleaved GFP cargo in the samples from cells transfected with constructs comprising either the AI-CM intein (2) (lane 3 and 6) or the 129 intein (3) (lanes 4 and 7). Thus, the 129 intein retains cleavage activity.

[0386] This method may be used to assess the ability of any polypeptide comprising an intein and extein sequence to cleave in the cytoplasm of a cell and / or in the lumen of an EV.

[0387] To assess the extent of premature cleavage, FACs was carried out to quantify proportion of GFP positive particles in conditioned media was quantified. Figure 6c illustrates FACs data and shows that in the conditioned media derived from cells expressing the non-cleaving amino acid construct, a high proportion of the particles detected are GFP positive, indicating that the eGFP cargo is successfully loaded into exosomes. When comparing samples derived from cells expressing the AI-CM intein construct and from cells that express the 129 intein construct, a higher proportion of particles detected are GFP positive in the conditioned media taken from cells expressing the 129 intein construct, indicating that the 129 intein mediates improved cargo loading, likely due to a reduction in premature cleavage that occurs prior to the protein being localised to exosomes. The same trend is observed whether CD63 or TSN2 is used as the exosomal polypeptide, indicating that the advantages of the 129 intein do not appear to be dependent on the EV scaffold polypeptide used.

[0388] To further investigate the impact of using different EV polypeptides in combination with the newly developed 129 intein, the following constructs were produced:

[0389] HIV Gag Protein--GGGSG-I29-GGGGS-Cas9 (bars 1 and 9 in figure 6d) HIV Gag Protein -GGGSG-l29-GSP-Cas9 (bars 2 and 10 in figure 6d) Myristylation site-GGGSG-l29-GGGGS-Cas9 (bars 3 and 11 in figure 6d) Myristylation-GGGSG-l29-GSP-Cas9 (bars 4 and 12 in figure 6d) Single-pass EV polypeptide-GGGSG-l29-GGGGS-Cas9 (bars 5 and 13 in figure 6d)

[0390] Single-pass EV polypeptide-GGGSG-l29-GSP-Cas9 (bars 6 and 14 in figure 6d) - TSN2-GGGSG-I29-GGGGS-Cas9 (bars 7 and 15 in figure 6d) - TSN2-GGGGG-l29-GSP-Cas9 (bars 8 and 16 in figure 6d)

[0391] These constructs, together with a gRNA directed to the mTtr211 gene locus and VSVG (an endosomal escape moiety) were transfected into HEK293 cells. 48 hours after transfection the conditioned media was collected from the HEK293 cells and EVs were purified.

[0392] The purified EVs were added to N2a cells. 48 hours later, the N2a cells were harvested and DNA extraction was performed. PGR of the mTtr211 cut site was performed to asses genome editing. Figure 6d is a bar chart showing the percentage of genome editing at the mTtr211 gene locus for the EVs derived from cells expressing each of the different constructs, when different amounts of EVs were added to the N2a cells. The results offer further evidence that all of the EV scaffolds induce a comparable or increased amount of genome editing as compared with TSN2. Thus, the advantages of the I29 intein appear to be conserved across a range of different scaffolds.

[0393] Example 7 - Characterisation of further 129 Extein sequences

[0394] The results of example 3 demonstrate that the +1 C-terminal extein residue can influence the rate of I29 mediated cleavage. Further investigations into I29 mediated cleavage with different N-terminal and C-terminal extein sequences were caried out by engineering different amino acid linkers into constructs comprising the I29 intein.

[0395] Figure 7a is a schematic representative of the constructs comprising the different-terminal and C-terminal extein sequences tested. The constructs comprise EV polypeptide, TSN2, connected to the N-terminal of the I29 intein via different N-terminal extein sequences (GGGSG (SEQ ID NO: 10), FAS (SEQ ID NO: 11), FFM (SEQ ID NO: 12), FAF (SEQ ID NO: 13) and TRM (SEQ ID NO: 9)). The constructs all also comprise eGFP as the C-terminal extein cargo. Connected to the C-terminus of the I29 intein via different C-terminal extein sequences (GGGGS (SEQ ID NO: 7), GSP (SEQ ID NO: 6), TRM (SEQ ID NO: 9), TRH (SEQ ID NO: 8)).

[0396] To investigate the amount of premature cleavage and EV loading efficiency, the constructs were transfected into HEK CTS cells and, 72 hours later, conditioned media was collected and FACs was carried out to quantify proportion of GFP positive particles in conditioned media.

[0397] Figure 7b is a bar chart showing the proportion of GFP positive particles in the conditioned media taken from cells transfected with each of the constructs shown in Figure 7a (bars 3 to 12) or with a construct comprising a non-cleaving amino acid sequence in the place of the 129 intein (bar 1) or with a construct comprising the TRM-AI-CM-TRH intein / extein in the place of the I29 intein and specified extein (bar 2). In the conditioned media derived from cells expressing the non-cleaving amino acid construct, a high proportion of the particles detected are GFP positive, indicating that the eGFP cargo is successfully loaded into exosomes. In the conditioned media derived from cells expressing the AI-CM intein construct, very few particles were GFP positive, demonstrating that there is very low efficiency of eGFP cargo loading into exosomes, likely as a result of premature cleavage. All of the constructs expressing the I29 intein induced an improvement in loading as compared with the AI-CM intein construct (compare bars 3 to 12 with bar 2), indicating that less premature cleavage occurred. However, the different N-terminal and C-terminal extein sequences influenced the efficiency of EV loading.

[0398] This method may be used to assess the amount of premature cleavage and the amount of cleavage in the cytoplasm of a cell mediated by any polypeptide comprising an intein and extein sequence.

[0399] To investigate the extent to which the constructs retain the ability to mediate cleavage, which is beneficial since it allows for the cargo to have improved bioactivity in a target cell, a western blot was carried out on the conditioned media samples. Figure 7c is a JESS image and shows the amount of the cleaved GFP cargo and non-cleaved protein present in conditioned media taken from cells transfected with the fusion protein constructs comprising the non-cleavable AA sequence (lane 1), the dICM intein sequence (lane 2) or the I29 intein constructs shown in Figure 7a (lanes 3 to 12). In the conditioned media derived from cells expressing the non-cleaving amino acid construct, a high proportion of the non-cleaved GFP labelled protein is present (lane 1). There is a high amount of cleaved GFP cargo in the samples from cells transfected with constructs comprising the dICM intein (lane 2). The constructs comprising the I29 intein showed different amounts of cleavage depending on the N-terminal and C-terminal extein linker sequences. The GGGSG N-terminal extein linker sequence and the GGGGS and GSP C-terminal extein linker sequences give highest proportion of GFP positive particles, while retaining cleavage of GFP.

[0400] Example 8 - The 129 intein allows for EVs loaded with Cre to have increased bioactivity as compared with the AI-CM intein

[0401] The following constructs were expressed in EV producer cells (HEK293 cells) together with a construct encoding an endosomal escape moiety (VSVG):

[0402] TSN2-AI-CM intein-Cre

[0403] TSN2-I29 intein-Cre In the AI-CM intein construct, the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH. In the 129 intein construct, the N-terminal extein sequence is GGGSG and the C-terminal extein sequence is GGGGS.

[0404] EVs were collected from the cells and purified. 1 x 108EVs or 1 x 109EVs were then added to Hematopoietic stem cells (HSCs) that had been isolated from Ai9 reporter mice. Ai9 reporter mice are a Cre reporter tool strain designed to have a / oxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. Ai9 mice express robust tdTomato fluorescence following Cre-mediated recombination.

[0405] The percentage of tdTomato positive cells in the cell population was quantified.

[0406] Figure 8a and 8b show that, as compared with when the AI-CM intein is used, the I29 intein significantly increases the tdTomato positive cell population, especially with a low dose of EVs. This indicates that, as well as allowing for increased loading, the I29 intein allows for the production of EVs that have increased bioactivity in target cells.

[0407] Example 9 - The 129 intein allows for EVs loaded with Cas9 RNP to have increased bioactivity as compared with the AI-CM intein

[0408] EV producer cells (HEK293 cells) were transfected with the following constructs, together with a further construct encoding a single guide RNA:

[0409] • CD63-AI-CM intein-Cas9

[0410] • CD63-I29 intein-Cas9

[0411] • TSN2-AI-CM intein-Cas9

[0412] • TSN2-I29 intein-Cas9

[0413] • VSVG-Foldon-AI-CM intein-Cas9

[0414] • VSVG-Foldon-I29intein-Cas9

[0415] In the AI-CM intein constructs, the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH. In the 129 intein construct, the N-terminal extein sequence is GGGSG and the C-terminal extein sequence is GGGGS. A construct encoding an endosomal escape moiety (VSVG) was also expressed in the EV producer cells expressing the first four constructs.

[0416] 24 hours after transfection, the EV-producing cells were co-cultured with HEK-SL reporter cells.

[0417] Ill HEK-SL reporter cells stably express an “mCherry-F2A-linker-stop-GFP” contract under the control of a CMV promoter. In the absence of gene editing, the HEK-SL reporter cells express mCherry only, since the “stop” prevents GFP from being expressed. The guide RNA that was transfected into the EV producer cells includes a spacer sequence that targets the linker and when Cas9 mediated gene editing occurs, the stop is disrupted, leading to expression of GFP.

[0418] 48 hours after co-culture, FACs was performed on the HEK-SL cells to assess the percentage of GFP positive cells.

[0419] Figure 9a indicates that, as compared with when the AI-CM intein is used, the 129 intein significantly increased the proportion of GFP positive cells. This again indicates that the 129 intein allows for the production of EVs that have increased bioactivity in target cells when Cas9 and a guide RNA are the cargo. The 129 intein improves bioactivity with all the different EV polypeptides tested.

[0420] The inventors went on to test gene editing Ttr - an endogenous gene target. EV producer cells (adherent HEK293 cells) were transfected with plasmids encoding VSVG, Ttr gRNA and Scaffold-intein-Cas9 complexed with PEI max reagent. In this case, the following Scaffold-intein-Cas9 constructs were compared:

[0421] • GAG-TRM-AI-CM-TRH-Cas9

[0422] • GAG-GGGSG-I29-GGGGS-Cas9

[0423] • TSN2-GGGSG-I29-GGGGS-Cas9.

[0424] Supernatants were harvested 48 hours post transfection. The supernatant was 0.2 micron filtered, followed by Denarase treatment, concentrated using 500kDA TFF step, purified using 700A Captacore column, then further concentrated using TFF, and then aliquoted and stored in 0.2% HSA solution.

[0425] N2a cells were seeded at 80k cells well in a 48 well plate. EVs were added at a maximum of 100ul volume, starting from 1e11 / ml as the top dose. Cells were harvested 72h after treatment for DNA extraction, PCR and Sanger sequencing followed by TIDE / ICE analysis to quantify frameshift mutations. Figure 9B shows that the 129 intein results in up to six times more editing when using GAG as a scaffold, as compared with when the AI-CM intein is used. High editing was also achieved with the 129 intein when using TSN2 as a scaffold.

[0426] The inventors carried out further experiments to compare gene editing with the two inteins of Ttr (an endogenous cell target) when TSN2 is used as the scaffold. Again, EV producer cells (adherent HEK293) cells were transfected with plasmids encoding VSVG, Ttr gRNA and Scaffold-intein-Cas9 complexed with PEI max reagent. In this experiment, the following Scaffold-intein-Cas9 constructs were compared:

[0427] • TSN2-TRM-AI-CM-TRH-Cas9

[0428] • TSN2-GGGSG-I29-GGGGS-Cas9.

[0429] The supernatant was harvested 48h post-transfection, then 0.2 micron filtered and denarase treated before purification. The supernatants were concentrated using 10kDa spin filters, followed by 700A viral polish columns and then spin filtered again using 10kDa spin filters.

[0430] N2a cells were seeded at 80k cells well in a 48wp. EVs were added at a maximum of 100ul volume, starting from 1e11 / ml as the top dose. Cells were harvested 72h after treatment for DNA extraction, PCR and Sanger sequencing followed by TIDE / ICE analysis to quantify frameshift mutations.

[0431] Figure 9C shows the I29 intein also leads to higher editing, as compared with when the AI- CM intein, when using TSN2 is used as a scaffold.

[0432] The inventors then went on to test the loading of gRNA when different extein combinations were used. In these experiments, EV producer cells were transfected with Ttr gRNA and the following Scaffold-intein-Cas9 constructs:

[0433] • TSN2-GGGSG-I29-GGGGS-Cas9

[0434] • TSN2-GGGSG-I29aa-GGGGS-Cas9

[0435] • TSN2-FAS-l29-GSP-Cas9

[0436] • TSN2-TRM-I29-TRH-Cas9

[0437] • TSN2-GGGSG-AI-SM-GGGGS

[0438] I29aa is a non-cleaving intein mutant, thus was not expected to display any cleavage, including premature cleavage, in EV producer cells. AI-SM is a slow-cleaving mutant of AI- CM, thus was expected to display a low level of cleavage, including premature cleavage in EV producer cells. EVs were collected from the cells and purified and the number of gRNA copies loaded into 1e6 EVs was quantified.

[0439] Figure 9D shows that, surprisingly, the I29 intein results in higher loading of gRNA than the AI-SM intein. This is the case with all the different extein combinations tested (the first column of the graph labelled “I29” is the GGGSG-I29-GGGGS construct). The FAS-I29-GSP and TRM-I29-TRH constructs resulted in the highest levels of gRNA loading.

[0440] Example 10- The 129 intein allows for EVs loaded with Cas12 RNPto have increased bioactivity as compared with the AI-CM intein

[0441] EV producer cells (HEK293 cells) were transfected with the following constructs, together with a construct encoding an endosomal escape moiety (VSVG):

[0442] • TSN2-AI-CM intein-Cas12a

[0443] • TSN2-I29 intein-Cas12a

[0444] • CD63-AI-CM intein-Cas12a

[0445] • CD63-I29 intein-Cas12a

[0446] In the AI-CM intein constructs, the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH. In the I29 intein construct, the N-terminal extein sequence is GGGSG and the C-terminal extein sequence is GGGGS. The EV producer cells were also co-transfected with a guide RNA (two different guide designs were tested “Guide 1” and “Guide 2”). The medium was changed to Opti-MEM 6 hours after transfection. The conditional medium was then harvested 2 days after transfection and EVs were isolated by TFF which were concentrated by 10 kD spin filters. EV concentrations were determined via ZetaView. The indicated doses (1 E10 or 1 E9) of isolated EVs were then incubated with HEK-SL2 reporter cells (described in example 9 above) in 96-well plates for 72 hours. GFP- positive cells were quantified by FACS analysis.

[0447] Figure 10a-d indicate that, as compared with when the AI-CM intein is used, the I29 intein significantly increased the proportion of GFP positive cells. This provides further evidence that the I29 intein allows for the production of EVs that have increased bioactivity in target cells, this time when the EVs are loaded with Cas12 and a guide RNA as the cargo. Again, the I29 intein improves bioactivity no matter which EV polypeptide is used.

[0448] Example 11 - An alternative intein that comprises the same mutations present in the 129 intein allows for EVs loaded with Cas9 RNP to have increased bioactivity as compared with the AI-CM intein EV producer cells (HEK293 cells) were transfected with the following constructs, together with a further construct encoding a single guide RNA:

[0449] • CD63-AI-CM intein-Cas9

[0450] • CD63-I29 intein-Cas9

[0451] • CD63-H439Q 129 intein-Cas9

[0452] • TSN2-AI-CM intein-Cas9

[0453] • TSN2-I29 intein-Cas9

[0454] • TSN2-H439Q 129 intein-Cas9

[0455] In the AI-CM intein constructs, the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH. In the 129 intein constructs and the H439Q 129 intein constructs, the N-terminal extein sequence is GGGSG and the C-terminal extein sequence is GGGGS. A construct encoding an endosomal escape moiety (VSVG) was also expressed in the EV producer cells. EVs were isolated as described above and the indicated doses of isolated EVs were then incubated with either HEK-SL2 reporter cells (described in example 9 above) or B16F10-SL reporter cells for 96 hours. GFP-positive cells were quantified by FACS analysis.

[0456] Figure 11a indicates that, as compared with when the AI-CM intein is used, both the I29 intein and the H439Q I29 intein significantly increased the proportion of GFP positive cells. This indicates that, like the I29 intein, the H439Q I29 intein also allows for the production of EVs that have increased bioactivity in target cells. The H439Q I29 intein improves bioactivity with both different EV polypeptides tested. In addition, the H439Q I29 intein offers a further improvement in bioactivity as compared with the I29 intein.

[0457] SEQUENCE LISTING:

[0458] SEQ ID NO. 1 : AI-CM

[0459] ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLHARPVVSWFDQGTRDVIGLRIA GGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPIPARVQALADALDDKF LHDMLAEELRYSVIREVLPTRRARTFGLEVEELHTLVAEGVVVHN

[0460] SEQ ID NO. 2: N-terminal extein sequence formed by cloning RISEF

[0461] SEQ ID NO. 3: Mtu RecA C-terminal extein sequence

[0462] CSPPF

[0463] SEQ ID NO. 4: I-29

[0464] ALAEGTRIFDPVTGTTHRIEDWGGRKPIHVVAAAKDGTLRARPWSWFDQGTRDVIGLRIA

[0465] GGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPVPAPVQALADALDDK FLHDMLAEELRYSVIREVLPTRRARTFGLVVEELHTLVAEGVVVHN

[0466] SEQ ID NO. 5: N-terminal extein sequence formed by cloning RISEFAS

[0467] SEQ ID NO. 6: extein sequence

[0468] GSP

[0469] SEQ ID NO. 7: extein sequence

[0470] GGGGS

[0471] SEQ ID NO. 8: extein sequence

[0472] TRH

[0473] SEQ ID NO. 9: extein sequence

[0474] TRM

[0475] SEQ ID NO.10: extein sequence

[0476] GGGSG SEQ ID NO. 11: extein sequence

[0477] FAS

[0478] SEQ ID NO. 12: extein sequence

[0479] FFM

[0480] SEQ ID NO. 13: extein sequence

[0481] FAF

[0482] SEQ ID NO. 14: linker sequence GGGGGG

[0483] SEQ ID NO 15: linker sequence GGGGGGGG

[0484] SEQ ID NO 16: linker sequence EAAAK

[0485] SEQ ID NO 17: linker sequence

[0486] A(EAAAK)4ALEA(EAAAK)4A

[0487] SEQ ID NO: 18: linker sequence GGGGSGGGGS

[0488] SEQ ID NO: 19: linker sequence GGGGSGGGGSGGGGSGGGGS

[0489] SEQ ID NO: 20: linker sequence EAAAKEAAAK

[0490] SEQ ID NO: 21 : linker sequence EAAAKEAAAKEAAAK

[0491] SEQ ID NO.22: extein sequence GGSGGGSG

[0492] SEQ ID NO. 23: I-29 ALAEGTRIFDPVTGTTHRIEDVVGGRKPIHVVAAAKDGTLRARPVVSWFDQGTRDVIGLRIA

[0493] GGAILWATPDHKVLTEYGWRAAGELRKGDRVAQPRRFDGFGDSAPVPAPVQALADALDDK

[0494] FLHDMLAEELRYSVIREVLPTRRARTFGLVVEELHTLVAEGVVVQN

Claims

CLAIMS1. A fusion protein comprising(i) an intein and(ii) an extracellular vesicle (EV) polypeptide, wherein the intein comprises or consists of a variant of SEQ ID NO. 1 , wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41 , a Valine residue at position 108, a Proline residue at position 111 and a Valine residue at position 152.

2. A polypeptide comprising an intein, wherein the intein comprises or consists of a variant of SEQ ID NO. 1, wherein the variant of SEQ ID NO. 1 comprises an Arginine residue at position 41, a Valine residue at position 108, a Proline residue at position 111 and a Valine residue at position 152, wherein the polypeptide further comprises:(i) a C-terminal extein sequence, wherein: a. the +1 residue of the C-terminal extein sequence is a Glutamate, Phenylalanine, Histidine, Leucine, Methionine, Glycine or Proline residue, preferably wherein the +1 residue of the C-terminal extein sequence is a Glycine residue, b. the +1 residue of the C-terminal extein sequence is a Glycine residue, the +2 residue of the C-terminal extein sequence is a Serine residue and the +3 residue of the C-terminal extein sequence is a Proline residue, c. the +1 residue of the C-terminal extein sequence is a Glycine residue, the +2 residue of the C-terminal extein sequence is a Glycine residue, the +3 residue of the C-terminal extein sequence is a Glycine residue, the +4 residue of the C-terminal extein sequence is a Glycine residue and the +5 residue of the C-terminal extein sequence is a Serine residue, or d. the +1 residue of the C-terminal extein sequence is a Threonine residue, the +2 residue of the C-terminal extein sequence is Arginine residue and the +3 residue of the C-terminal extein sequence is Histidine residue; and / or(ii) an N-terminal extein sequence, wherein a. the -1 residue of the N-terminal extein sequence is a Glycine residue, the -2 residue of the N-terminal extein sequence is aSerine residue, the -3 residue of the N-terminal extein sequence is a Glycine residue, the -4 residue of the N-terminal extein sequence is a Glycine residue and the -5 residue of the N-terminal extein sequence is a Glycine residue, b. the -1 residue of the N-terminal extein sequence is a Serine residue, the -2 residue of the N-terminal extein sequence is an Alanine residue and the -3 residue of the N-terminal extein sequence is a Phenylalanine residue, or c. the -1 residue of the N-terminal extein sequence is a Methionine residue, the -2 residue of the N-terminal extein sequence is an Arginine residue and the -3 residue of the N-terminal extein sequence is a Threonine residue.

3. The fusion protein of claim 1 or polypeptide of claim 2 wherein the variant of SEQ ID NO. 1 exhibits a reduced rate of cleavage in the cytoplasm of a cell and / or at a pH of 7.0-7.5 and at a temperature of 34-40°C, as compared to an intein according to SEQ ID NO 1.

4. The fusion protein or polypeptide of any one of claims 1 to 3, wherein the variant of SEQ ID NO. 1 has C-terminal cleavage activity.

5. The fusion protein or polypeptide of any one of claims 1 to 4, wherein the intein comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 4 over its entire length.

6. The fusion protein or polypeptide of any one of claims 1 to 5, wherein the intein comprises or consists of an amino acid sequence having 100% sequence identity to SEQ ID NO. 4 over its entire length.

7. The fusion protein or polypeptide of any one of claims 1 to 4, wherein the variant of SEQ ID NO. 1 further comprises a Glutamine residue at position 167.

8. The fusion protein or polypeptide of claim 7, wherein the intein comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 23 over its entire length.

9. The fusion protein or polypeptide of claim 7 or claim 8, wherein the intein comprises or consists of an amino acid sequence having 100% sequence identity to SEQ ID NO. 23 over its entire length.

10. The polypeptide of claim 2 to 10, wherein the polypeptide comprises a C-terminal extein sequence and an N-terminal extein sequence, wherein:(i) the +1 residue of the C-terminal extein sequence is Gly, the +2 residue of the C-terminal extein sequence is Ser and the +3 residue of the C-terminal extein sequence is Pro and the -1 residue of the N-terminal extein sequence is Ser, the -2 residue of the N-terminal extein sequence is Ala and the -3 residue of the N-terminal extein sequence is Phe;(ii) the +1 residue of the C-terminal extein sequence is Gly, the +2 residue of the C-terminal extein sequence is Gly, the +3 residue of the C-terminal extein sequence is Gly, the +4 residue of the C-terminal extein sequence is Gly and the +5 residue of the C-terminal extein sequence is Ser and the -1 residue of the N-terminal extein sequence is Gly, the -2 residue of the N-terminal extein sequence is Ser, the -3 residue of the N-terminal extein sequence is Gly, the - 4 residue of the N-terminal extein sequence is Gly and the -5 residue of the N-terminal extein sequence is Gly; or(iii) the +1 residue of the C-terminal extein sequence is Thr, the +2 residue of the C-terminal extein sequence is Arg and the +3 residue of the C-terminal extein sequence is His and the -1 residue of the N-terminal extein sequence is Met, the -2 residue of the N-terminal extein sequence is Arg and the -3 residue of the N-terminal extein sequence is Thr.

11. A fusion protein comprising:(i) the polypeptide of any one of claims 2 to 10 and(ii) an EV polypeptide.

12. The fusion protein of claim 1 or claim 11 , wherein the fusion protein further comprises:(i) a protein cargo;(ii) a protein capable of binding to a cargo that is associated with the cargo; or(iii) a ribonucleoprotein (RNP) complex, preferably wherein the RNP complex comprises a Cas9, a Cas12 such as Cas12a, a Cas3, a base editor or a prime editor;preferably wherein protein cargo, protein capable of binding to a cargo or the RNP complex is the fused to the C-terminus of the intein.

13. The fusion protein of claim 11, wherein the fusion protein further comprises:(i) a protein cargo;(ii) a protein capable of binding to a cargo that is associated with the cargo; or(iii) a ribonucleoprotein (RNP) complex, preferably wherein the RNP complex comprises a Cas9, a Cas12 such as Cas12a, a Cas3, a base editor or a prime editor; wherein the protein cargo, protein capable of binding to a cargo or RNP complex comprises the C-terminal extein sequence or is fused to the C-terminus of the C- terminal extein sequence.

14. The fusion protein of claim 1 or 11 to 13, wherein the EV polypeptide is the fused to the N-terminus of the intein.

15. The fusion protein of claim 11 or 13, wherein the EV polypeptide comprises the N- terminal extein sequence or wherein the EV polypeptide is fused to the N-terminus of the N-terminal extein sequence.

16. A polynucleotide encoding the polypeptide or fusion protein of any of the preceding claims.

17. An extracellular vesicle (EV) comprising the polypeptide or fusion protein of any of the preceding claims.

18. An EV comprising the polypeptide or fusion protein of any of the preceding claims, wherein the intein has catalysed the cleavage of the polypeptide or fusion protein.

19. An EV comprising the fusion protein of claim 12 to 15, wherein the protein cargo, protein capable of binding to a cargo or RNP complex has been released from the EV polypeptide.

20. The EV of claim 17 to 19, wherein the EV is an exosome, a microvesicle and / or a related extracellular vesicle.

21. A method of preparing an EV of claim 17 to 20, comprising:(i) introducing into an EV-producing cell a polynucleotide of claim 16; and(ii) expressing the polynucleotide in the EV-producing cell, thereby generating EVs comprising a polypeptide or fusion protein of claims 1 to 15; optionally comprising an additional steps of collecting the EVs of step (ii) and incubating the EVs at a temperature that is sufficient to allow for the intein to catalyse the cleavage of the polypeptide comprising the intein sequence or the fusion protein.

22. A cell comprising the polypeptide, fusion protein, polynucleotide or EV of claims 1 to 20.

23. A pharmaceutical composition comprising an EV of claims 17 to 20, and a pharmaceutically acceptable excipient and / or carrier.

24. The EV of claims 17 to 20 or the pharmaceutical composition of claim 23 for use as a medicament for the treatment or prevention of a disease in a subject, optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure or ALS syndrome.

25. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the EV of claims 17 to 20 or the pharmaceutical composition of claim 23 to a subject suffering from or susceptible to the disease; optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure or ALS.

26. The EV of claims 17 to 20 or the pharmaceutical composition of claim 24 for the preparation of a medicament for treatment or prevention of a disease in a subject; optionally wherein the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure or ALS.