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WO2025068572A2 - Polypeptides comprenant des intéines pour ve modifiées - Google Patents

Polypeptides comprenant des intéines pour ve modifiées Download PDF

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Publication number
WO2025068572A2
WO2025068572A2 PCT/EP2024/077371 EP2024077371W WO2025068572A2 WO 2025068572 A2 WO2025068572 A2 WO 2025068572A2 EP 2024077371 W EP2024077371 W EP 2024077371W WO 2025068572 A2 WO2025068572 A2 WO 2025068572A2
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Prior art keywords
residue
intein
polypeptide
sequence
protein
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WO2025068572A3 (fr
Inventor
David Carter
Justin HEAN
Xiuna YANG
Zubair NIZAMUDEEN
David Wood
Hongyu Yuan
Xiuming LIANG
Samir El Andaloussi
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Evox Therapeutics Ltd
Ohio State University
Ohio State Innovation Foundation
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Evox Therapeutics Ltd
Ohio State University
Ohio State Innovation Foundation
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Publication of WO2025068572A2 publication Critical patent/WO2025068572A2/fr
Publication of WO2025068572A3 publication Critical patent/WO2025068572A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain

Definitions

  • Polypeptides comprising Inteins for Engineered EVs
  • 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).
  • inteins self-cleaving proteins
  • 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.
  • 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.
  • 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.
  • “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.
  • inteins 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • a newly developed intein variant e.g. the 129 intein
  • the present invention provides newly developed extein sequences for the 129 intein that further reduce intein mediated cleavage under these conditions.
  • the present invention provides a fusion protein comprising:
  • an extracellular vesicle (EV) polypeptide comprising 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.
  • the present invention further provides a fusion protein comprising:
  • 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.
  • 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:
  • 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
  • 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
  • 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
  • the -3 residue of the N-terminal extein sequence is a Phenylalanine residue
  • 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
  • the -3 residue of the N-terminal extein sequence is a Threonine residue.
  • 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:
  • 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.
  • the present invention provides a fusion protein comprising:
  • 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.
  • 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.
  • variant of SEQ ID NO. 1 further comprises a Glutamine residue at position 167.
  • 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.
  • 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 +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.
  • 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
  • the +5 residue of the C-terminal extein sequence is Ser.
  • 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
  • the EV polypeptide is fused to the N-terminus of the intein.
  • 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.
  • 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 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.
  • the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the C-terminus of the intein.
  • 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.
  • the protein capable of binding to a cargo is associated with a cargo.
  • the present invention provides a polynucleotide encoding the polypeptide of the present invention or fusion protein of the present invention.
  • 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.
  • 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.
  • 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.
  • the EV is an exosome, a microvesicle and/or a related extracellular vesicle.
  • 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.
  • 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.
  • 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.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an EV of the present invention, and a pharmaceutically acceptable excipient and/or carrier.
  • 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.
  • 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.
  • 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.
  • the disease is a genetic disorder, a liver disorder, a neurological disorder, a cardiac disorder, Phenylketonuria, heart failure or ALS.
  • 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).
  • C-terminal extein cargos maltose binding protein (MBP), thioredoxin (TrxA), and p-galactosidase (p-gal)
  • MBP maltose binding protein
  • TrxA thioredoxin
  • p-gal p-galactosidase
  • 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).
  • 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.
  • Figure 4 shows the I29 intein improves EV loading efficiency, compared with the AI-CM intein.
  • construct (1) comprises the I29 intein
  • construct (2) comprises the AI-CM intein
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • Figure 8 shows EVs comprising the I29 intein have increased bioactivity in target cells, as compared with EVs comprising the AI-CM intein.
  • the target cells (HSCs) are dosed with 1 x 10 8 EVs and in (B) the target cells (HSCs) are dosed with 1 x 10 9 EVs.
  • 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.
  • 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
  • TSN2-GGGSG-AI-SM- GGGGS where “DISM” (AI-SM) is a slow cleaving variant of the AI-CM intein
  • 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.
  • the target cells are dosed with 1 x 1O 10 EVs and in (C) and (D) the target cells are dosed with 1 x 10 9 EVs.
  • 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.
  • the target cells are HEK-SL cells and in (C) and (D) the target cells are B16F10-SL cells.
  • the present invention relates to polypeptides comprising inteins that are particularly suitable in the production and use of engineered extracellular vesicles.
  • 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.
  • polypeptides comprising the AI-CM intein alongside these newly developed extein sequences allow for the production of EVs with improved bioactivity.
  • 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.
  • a newly developed intein variant e.g. the I29 intein
  • 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.
  • the I29 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.
  • 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.
  • an acidic pH e.g. pH 6.0 to 6.2
  • 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.
  • 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.
  • 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.
  • 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.
  • all polypeptides and proteins identified herein can be freely combined in fusion proteins using conventional strategies for fusing polypeptides.
  • 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.
  • 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.
  • any and all features for instance any and all members of a Markush group
  • any and all other features for instance any and all members of any other Markush group
  • 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.
  • 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.
  • 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.
  • 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.
  • Standard methods in the art may be used to determine homology.
  • 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.
  • 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.
  • 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.
  • 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.
  • intein refers to an in-frame intervening sequence in a protein as described by Perler (Perler, Davis et al. 1994).
  • intein encompasses modified or mutated inteins and mini-inteins.
  • extein refers to an external sequence to the C-terminal and/or the N-terminal of the intein.
  • intein includes split inteins and contiguous inteins.
  • contiguous intein refers to an intein expressed from a single gene sequence.
  • 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.
  • the intein of the present disclosure is a contiguous intein as defined herein.
  • intein 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.
  • the intein of the present disclosure is a cleaving intein as defined herein.
  • the intein of the present invention has, or is capable of, C-terminal cleavage activity.
  • 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.
  • 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.
  • 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.
  • 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
  • the first (most N-terminal) amino acid i.e. position
  • the term “rate of cleavage” or “cleavage rate” refers to the intein’s cleavage activity (i.e. ability to self-cleave).
  • 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.
  • the rate of cleavage refers to the rate of C-terminal cleavage (i.e. mediating separation between the C-terminal extein and the 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.
  • 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.
  • I29 intein 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.
  • H439Q I29 intein 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.
  • the present disclosure provides a polypeptide sequence comprising or consisting of an intein and at least one extein sequence.
  • 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.
  • N-terminal and/or C-terminal extein may be contiguous with the intein, and so there are no intervening sequences.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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 represent particularly preferable EVs, but other EVs may also be advantageous in various circumstances.
  • the EV is an exosome or microvesicle or any other type of extracellular vesicle related to such exosomes and/or microvesicles.
  • 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”.
  • 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.
  • iPSCs induced pluripotent stem cells
  • 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 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.
  • HIVECs human umbilical cord endothelial cells
  • HEK human embryonic kidney
  • 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.
  • HIVECs human umbilical cord endothelial cells
  • HEK human embryonic kidney
  • endothelial cell lines such as microvascular or lymphatic endothelial cells
  • erythrocytes erythroid progenitors
  • chondrocytes mesenchymal stromal cells (MSCs) of different origin
  • the source cells are immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DCs).
  • immune cells such as B cells, T cells, NK cells, macrophages, monocytes, dendritic cells (DCs).
  • DCs dendritic cells
  • 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 GibcoTM Viral Production Cell 2.0, Viral Production Cells 2.0, VPC 2.0 cells and 2.0-HEK293F cells) or HEKVPC1 cells (GibcoTM 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • engineered EV is used interchangeably with “modified EV” and “genetically engineered EV” herein.
  • 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.
  • 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.
  • EVs may be present in concentrations such as 10 5 , 10 8 , 1O 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 18 , 10 25 ,10 3 ° 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.
  • 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.
  • individual EVs when present in a plurality constitute an EV population.
  • the present invention pertains both to individual EVs and populations comprising EVs, as will be clear to the skilled person.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • the intein sequence may also comprise a Glycine at position 24.
  • Percent (%) sequence identity 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.
  • sequence identity 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the C- terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the +1 amino acid residue has a molecular weight of 131 Da or more, 149 Da or more or 155 Da or more.
  • 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.
  • 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.
  • 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.
  • 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.
  • +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.
  • +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.
  • +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.
  • +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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the N- terminal extein sequence increases the rate of cleavage of the intein-extein bond, as compared with SEQ ID NO.
  • 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.
  • 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.
  • 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.
  • 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.
  • the N-terminal extein sequence comprises a -1 amino acid residue that has a hydrophilic side chain.
  • 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.
  • -1 amino acid residue or “-1 residue” refers to the amino acid of the extein closest to the N-terminal of the intein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intein of the present disclosure comprises or consists of a variant of SEQ ID NO. 1.
  • 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.
  • a parent molecule e.g., a protein or peptide disclosed herein
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the intein sequence may also retain a Glycine at position 24.
  • 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.
  • the variant of SEQ ID NO. 1 further comprises a H167Q mutation.
  • 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.
  • 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.
  • the variant of SEQ ID NO. 1 comprises the following specific residues, relative to SEQ ID NO.
  • 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.
  • 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.
  • 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.
  • the variant further comprises a Leucine at position 67 and/or a Glycine at position 150.
  • the variant further comprises a H167Q mutation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 may be verified by the methods outlined in the examples.
  • the C-terminal extein sequence comprises Glu, Phe, His, Leu, Met, Gly or Pro as the +1 amino acid residue.
  • the C-terminal extein sequence of the present disclosure comprises Phe or Gly as the +1 amino acid residue.
  • the C-terminal extein sequence of the present disclosure comprises Gly as the +1 amino acid residue.
  • the C-terminal extein sequence comprises Ser as the +2 amino acid residue and/or Pro as the +3 amino acid residue.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intein comprises or consists of the variant of SEQ ID NO.
  • 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.
  • 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.
  • 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.
  • 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.
  • the intein comprises or consists of the variant of SEQ ID NO.
  • 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.
  • 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.
  • the intein comprises or consists of the variant of SEQ ID NO.
  • 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.
  • 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.
  • the intein comprises or consists of the variant of SEQ ID NO.
  • 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.
  • 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.
  • 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.
  • the intein may be flanked by any combination of N-terminal and C- terminal sequences illustrated in Fig. 7.
  • 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.
  • the intein may have any properties or features disclosed herein.
  • 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.
  • 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.
  • positions 24, 41 , 67, 108, 111 , 150, and/or 152 are invariant.
  • 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.
  • 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.
  • positions 24, 41, 67, 108, 111 , 150, 152 and/or 167 are invariant.
  • 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.
  • 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.
  • the +1 residue of the C-terminal extein sequence is Gly.
  • 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.
  • 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.
  • 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.
  • 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.
  • polypeptide of this aspect may comprise an N-terminal extein.
  • 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.
  • 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.
  • 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.
  • 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 -
  • polypeptide may comprise a sequence according to SEQ ID NO:
  • SEQ ID NO: 10 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • polypeptide 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.
  • 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.
  • SEQ ID NO: 4 SEQ ID NO: 23 or variant as disclosed herein
  • SEQ ID NO: 10 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the EV polypeptide of the fusion protein comprises the extein sequence.
  • the EV polypeptide comprises an N-terminal extein sequence of the present disclosure at its C-terminus.
  • the EV polypeptide of the fusion protein is fused to the extein sequence.
  • the EV polypeptide is fused to the N-terminus of an N-terminal extein sequence of the present disclosure.
  • fused includes where one polypeptide is fused immediately to or into another polypeptide with no intervening amino acid residues.
  • 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.
  • EV polypeptide and “EV protein” are interchangeable.
  • 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.
  • the EV polypeptide of the present disclosure is an exosomal polypeptide.
  • exosomal polypeptide and “exosomal protein” are interchangeable.
  • 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.
  • the EV polypeptide is a transmembrane EV polypeptide.
  • 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.
  • 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, synde
  • 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).
  • 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.
  • the EV polypeptide is selected from TSN2, CD63, PTGFRN, PTTG1-IP, a HIV gag protein or Lamp2b.
  • the EV polypeptide is a polypeptide comprising a myristoylation site.
  • 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.
  • the myristoylation site is Gly-X-X-X- Ser/Thr, where “X” represents any amino acid.
  • 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.
  • the moiety for loading the polypeptide into the EV is conjugated to the extein sequence.
  • the EV loading moiety is conjugated to an N- terminal extein sequence of the present disclosure.
  • moiety for loading the polypeptide into the EV is used interchangeably with “EV loading moiety” and “EV localisation moiety” herein.
  • loading or loaded when used in relation to an EV 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.
  • EV loaded with is used interchangeably with the term “EV associated with” and the term “EV comprising”.
  • 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.
  • 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.
  • RNP ribonucleoprotein
  • 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.
  • 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.
  • the protein cargo, protein capable of binding to a cargo or RNP complex comprises the extein.
  • 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.
  • protein cargo or protein capable of binding to a cargo is fused to the extein sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • enzymes 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.
  • receptors such as decoy receptors, membrane proteins, transporters
  • therapeutic protein cargos 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
  • 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 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.
  • 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.
  • the Cas polypeptide may also be fused to a transcriptional activator (such as the P3330 core protein), to specifically induce gene expression.
  • the Cas protein is a Cas9 a Cas12, such as Cas12a, a Cas3.
  • the Cas protein is associated with a guide RNA.
  • the term protein cargo also encompasses protein-RNA cargo, cargo such as a ribonucleoprotein (RNP) complex.
  • the cargo is a protein-RNA cargo such as a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor.
  • the RNP complex is loaded in the lumen of the EV, preferable an exosome, a microvesicle and/or a related extracellular vesicle.
  • the protein capable of binding to a cargo is a nucleic acid (NA) binding protein, a protein binding protein or a viral binding protein.
  • NA nucleic acid
  • NA-binding protein refers to a protein or protein domain capable of binding a nucleic acid or polynucleotide.
  • the NA-binding protein is capable of binding DNA, or preferably RNA, most preferably a guide RNA.
  • 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 3 rd edition, (RSC publishing 2006) which are herein incorporated in their entirety for all purposes).
  • 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.
  • 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.
  • 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.
  • 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.
  • Non-traditional base pairing such as Hoogsteen base pairing
  • 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”.
  • 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.
  • 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 miRNA-binding proteins
  • shRNA-binding proteins transcription factors
  • various domains and derivatives may also be used as the NA-binding domain to transport an NA cargo into EVs.
  • 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.
  • RBDs small RNA- binding domains
  • 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.
  • the NA-binding protein is a PUF or a Cas protein or protein domain.
  • 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.
  • protein binding protein refers to a protein or protein domain capable of binding another protein.
  • 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.
  • 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.
  • viral binding protein refers to a protein or a protein domain that is capable of binding to a viral particle, preferably an AAV or a lentivirus.
  • a viral particle preferably an AAV or a lentivirus.
  • 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.
  • 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.
  • the protein capable of binding to a cargo is associated with a cargo that it is capable of binding.
  • 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.
  • the viral particle is an AAV or a lentivirus, most preferably an AAV.
  • the AAV vector comprises a capsid from human AAV serotype AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV12.
  • 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.
  • the AAV capsid and the AAV ITR are from the same serotype or from different serotypes.
  • the viral particle is a therapeutic AAV.
  • 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.
  • the viral vector comprises a viral capsid and a viral genome, the viral genome comprising one or more heterologous transgenes.
  • 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.
  • 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
  • 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.
  • 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,
  • 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
  • the RNA is a therapeutic RNA.
  • 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.
  • 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.
  • the DNA is a therapeutic recombinant AAV vector.
  • the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a purification moiety.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fusion protein comprises two inteins or polypeptides comprising an intein and extein sequence of the present disclosure.
  • 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.
  • the purification moiety is a chitin-binding tag.
  • the fusion protein of the present disclosure or the conjugated protein of the present disclosure further comprises a targeting moiety.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fusion protein of the present disclosure or the conjugated protein of the present disclosure further comprises a multimerization domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • the multimerization domain is a fold-on domain or a leucine zipper
  • the fusion protein of the present disclosure or the conjugated protein of the present disclosure comprises a multimerization domain together with a targeting moiety.
  • 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.
  • 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.
  • 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.
  • 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.
  • the endosomal escape domain comprises VSVG or a viral glycoprotein.
  • 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.
  • the protein cargo, protein capable of binding to a cargo or RNP complex is luminally loaded.
  • the fusion protein of the present disclosure or the conjugated protein of the present disclosure further comprises a pharmacokinetic and/or pharmacodynamic effector moiety.
  • 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.
  • the pharmacokinetic or pharmacodynamic effector moiety is an albumin binding domain.
  • albumin binding domain 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmacokinetic and/or pharmacodynamic effector moiety is fused or conjugated to the N-terminus of PTTG1-IP.
  • the, fusion proteins of the present disclosure or the conjugated proteins of the present disclosure further comprise a linker, spacer and/or scaffold sequence.
  • a linker, spacer and/or scaffold sequence 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polypeptide comprising an intein and an extein sequence, fusion protein or conjugated protein has been cleaved at an intein-extein bond.
  • 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.
  • the present disclosure also relates to a population of EVs of the present disclosure.
  • 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.
  • 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.
  • the EV of the present disclosure comprises a cargo, preferably a therapeutic cargo.
  • the cargo 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cargo is loaded by exogenous loading techniques.
  • 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.
  • exogenous loading method and “exogenous loading technique” are used interchangeably herein.
  • 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.
  • 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).
  • 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
  • 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).
  • a membrane anchoring moiety such as myristic acid, vitamin A, a lipid or cholesterol tail
  • CPP cell penetrating peptide
  • 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.
  • 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.
  • the Cas protein is a Cas9, a Cas12, such as Cas12a, or a Cas3.
  • the cargo is a protein-RNA cargo such as a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor.
  • the cargo is a viral particle.
  • the viral particle is an AAV or a lentivirus, most preferably an AAV.
  • the cargo is a polynucleotide.
  • the cargo is DNA, preferably a recombinant AAV vector.
  • 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.
  • the cargo is luminally loaded.
  • 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.
  • 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.
  • 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.
  • the protein cargo, protein capable of binding to a cargo or RNP complex is fused to the C-terminus of PTTG1-IP.
  • 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.
  • the cargo is surface loaded.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the purification moiety is loaded by exogenous loading techniques.
  • the purification moiety is surface loaded.
  • 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.
  • 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.
  • surface loading of the purification moiety may have the advantage of allowing for purification of the EVs following intein induced cleavage.
  • 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.
  • the EV of the present disclosure comprises a targeting moiety as described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the targeting moiety is loaded by exogenous loading techniques.
  • the targeting moiety is surface loaded.
  • 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.
  • 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.
  • 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.
  • the targeting moiety is fused or conjugated to the N-terminus of PTTG1-IP.
  • surface loading of the targeting moiety may have the advantage of allowing for more efficient targeting.
  • the EV of the present disclosure comprises a multimerization domain as described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the polypeptide comprising the multimerization domain is loaded by exogenous loading techniques.
  • the EV comprises the multimerization domain together in the same fusion protein as a targeting moiety.
  • the EV of the present disclosure comprises an endosomal escape domain as described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • the endosomal escape domain is loaded by exogenous loading techniques.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmacokinetic and/or pharmacodynamic effector moiety domain is loaded by exogenous loading techniques.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmacokinetic and/or pharmacodynamic effector moiety is fused or conjugated to the N-terminus of PTTG1-IP.
  • surface loading of the pharmacokinetic and/or pharmacodynamic effector moiety may have the advantage of allowing for more efficient pharmacokinetic and/or pharmacodynamic effect.
  • the EV is an exosome, a microvesicle, and/or any form of related extracellular vesicle.
  • 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.
  • the cell is an EV producer cell as described herein. In an alternative embodiment, the cell is a target cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cleavage may be verified by the methods outlined in the examples.
  • 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 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 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.
  • 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.
  • filtration preferably ultrafiltration (UF) tangential flow filtration (TFF) or hollow fibre filtration
  • affinity chromatography optionally also including size exclusion LC or bead-eluate LC.
  • 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.
  • the target cell has a genetic abnormality and/or is linked to a disease or a disorder.
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an EV of the present disclosure and a pharmaceutically acceptable excipient and/or carrier.
  • pharmaceutically acceptable is used herein to refer to a material may be administered to a subject without causing any undesirable biological effects.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • the term "effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • 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.
  • 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.
  • the subject is a human.
  • 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.
  • 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.
  • 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, grafta,
  • cystic fibrosis 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.
  • 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 astrocyto
  • the EVs and pharmaceutical compositions of the present disclosure may be administered to an animal subject, preferably a human via various different administration routes.
  • 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, intrae
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • a polypeptide comprising an intein and an extein sequence.
  • 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.
  • 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 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.
  • 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 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 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 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 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 48 to 53 wherein the variant of SEQ ID NO.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 to 71 wherein the variant of SEQ ID NO. 1 comprises a Proline residue at position 111.
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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, 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 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.
  • 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 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 118 wherein the EV polypeptide is TSN2 or PTTG1- IP.
  • 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 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 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:
  • the Cas protein is a Cas9 or a Cas12, such as Cas12a;
  • the Cas protein is a Cas9 or a Cas12, such as Cas12a, a Cas3, a base editor or a prime editor; or
  • the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor.
  • 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 145, wherein the protein capable of binding to a cargo is an RNA binding protein.
  • the fusion protein or the conjugated protein of paragraph 151 wherein the RNA is a guide RNA.
  • the Cas protein is a Cas9 or a Cas12, such as Cas12a;
  • the Cas protein is a Cas9 or a Cas12, such as Cas12a, a Cas3, a base editor or a prime editor; or
  • 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 156, wherein the DNA cargo is a recombinant AAV vector.
  • 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 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 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 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 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 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 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 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.
  • the EV of paragraph 204 to 208, wherein the cargo is a protein cargo or an RNP complex.
  • the protein cargo is a Cas protein
  • the Cas protein is a Cas9 or a Cas12, such as Cas12a, a Cas3, a base editor or a prime editor; or
  • the RNP comprises Cas9, Cas12 preferably Cas12a, Cas3, a base editor or a prime editor.
  • 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 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 218, wherein the Cas is:
  • 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 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 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 236 to 238, wherein the pharmacokinetic and/or pharmacodynamic effector moiety is an albumin binding domain.
  • 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 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:
  • a method for producing an EV or a population of EVs comprising the following steps:
  • 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 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 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.
  • EV producer cells are a cell line, preferably HEK cells, more preferably HEK293 cells, most preferably HEKVPC2 or HEKVPC1 cells.
  • 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.
  • 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.
  • 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.
  • a fusion protein comprising
  • 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.
  • 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.
  • a fusion protein comprising:
  • an EV polypeptide (ii) an EV polypeptide.
  • 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 embodiment 13 or 14, wherein the protein capable of binding to a cargo is associated with a cargo.
  • 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.
  • step (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.
  • 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.
  • 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.
  • 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).
  • MBP maltose binding protein
  • TrxA thioredoxin
  • P-gal P-galactosidase
  • 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.
  • 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.
  • This method may be used to asses the cleavage rate of any polypeptide comprising an intein and extein sequence.
  • 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.
  • K rate constant
  • 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).
  • 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.
  • Example 2 The AI-CM intein is associated with premature cleavage
  • FIG. 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.
  • 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.
  • This method may be used to asses the loading efficiency of any polypeptide comprising an intein and extein sequence.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • +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).
  • Example 4 The 129 intein is associated with reduced premature cleavage
  • 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).
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • the eGFP signal is approximately less than one-tenth of the mScarlet signal, suggesting that the intein has self-cleaved before loading into the.
  • 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.
  • FIG. 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • FIG. 6b is a JESS image showing the amount of the cleaved GFP cargo and non-cleaved protein present.
  • a high proportion of the non-cleaved GFP labelled protein is present (lanes 2 and 5).
  • 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.
  • FIG. 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.
  • 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)
  • 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.
  • 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.
  • the advantages of the I29 intein appear to be conserved across a range of different scaffolds.
  • 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.
  • 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.
  • 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).
  • a high proportion of the particles detected are GFP positive, indicating that the eGFP cargo is successfully loaded into exosomes.
  • 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.
  • 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).
  • a high proportion of the non-cleaved GFP labelled protein is present (lane 1).
  • 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.
  • the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH.
  • the N-terminal extein sequence is GGGSG and the C-terminal extein sequence is GGGGS.
  • 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.
  • tdTomato red fluorescent protein variant
  • the percentage of tdTomato positive cells in the cell population was quantified.
  • 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.
  • Example 9 - 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:
  • the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH.
  • 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.
  • the EV-producing cells were co-cultured with HEK-SL reporter cells.
  • Ill HEK-SL reporter cells stably express an “mCherry-F2A-linker-stop-GFP” contract under the control of a CMV promoter.
  • 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.
  • FACs was performed on the HEK-SL cells to assess the percentage of GFP positive cells.
  • 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.
  • EV producer cells (adherent HEK293 cells) were transfected with plasmids encoding VSVG, Ttr gRNA and Scaffold-intein-Cas9 complexed with PEI max reagent.
  • Scaffold-intein-Cas9 constructs were compared:
  • 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.
  • 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.
  • EV producer cells adheredherent HEK293 cells were transfected with plasmids encoding VSVG, Ttr gRNA and Scaffold-intein-Cas9 complexed with PEI max reagent.
  • the following Scaffold-intein-Cas9 constructs were compared:
  • 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.
  • 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.
  • 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.
  • EV producer cells were transfected with Ttr gRNA and the following Scaffold-intein-Cas9 constructs:
  • 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.
  • 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.
  • Example 10- The 129 intein allows for EVs loaded with Cas12 RNPto have increased bioactivity as compared with the AI-CM intein
  • EV producer cells (HEK293 cells) were transfected with the following constructs, together with a construct encoding an endosomal escape moiety (VSVG):
  • the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH.
  • 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.
  • 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.
  • 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:
  • the N-terminal extein sequence is TRM and the C-terminal extein sequence is TRH.
  • 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.
  • 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.
  • SEQ ID NO. 2 N-terminal extein sequence formed by cloning RISEF
  • SEQ ID NO. 5 N-terminal extein sequence formed by cloning RISEFAS
  • SEQ ID NO: 18 linker sequence GGGGSGGGGS
  • SEQ ID NO: 19 linker sequence GGGGSGGGGSGGGGSGGGGS
  • SEQ ID NO.22 extein sequence GGSGGGSG

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Abstract

La présente invention concerne des polypeptides comprenant des intéines (protéines à auto-clivage), qui sont particulièrement appropriées dans la production et l'utilisation de vésicules extracellulaires (VE) modifiées. En particulier, la présente divulgation concerne des protéines de fusion comprenant un variant d'intéine nouvellement développé et un polypeptide de VE ou une fraction de chargement de VE, qui permettent un chargement plus efficace de charges dans des VE. De plus, la présente divulgation concerne des polypeptides comprenant une nouvelle variante d'intéine ainsi que des séquences d'extéine nouvellement développées qui améliorent encore le chargement de VE et/ou la bioactivité des VE chargées produites. La présente invention concerne également des constructions polynucléotidiques codant pour ces polypeptides et des protéines de fusion, ainsi que des VE comprenant les polypeptides et les protéines de fusion aussi bien dans leur forme native que dans leur forme clivée.
PCT/EP2024/077371 2023-09-29 2024-09-27 Polypeptides comprenant des intéines pour ve modifiées Pending WO2025068572A2 (fr)

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