VHHs AND FUSION PROTEINS FOR LOADING OF AAV INTO EVS

Abstract

The present invention relates to Variable Heavy domain of Heavy chains (VHHs) which are particularly suitable for the releasable loading of adeno-associated virus (AAV) into extracellular vesicles (EVs). The present disclosure also relates to fusion proteins, comprising an AAV binding polypeptide and a tetraspanin EV polypeptide, which allow for increased AAV loading into EVs.

Description

[0001] VHHs AND FUSION PROTEINS FOR LOADING OF AAV INTO EVS

[0002] TECHNICAL FIELD

[0003] The present invention relates to Variable Heavy domain of Heavy chains (VHHs) which are particularly suitable for the releasable loading of adeno-associated virus (AAV) into extracellular vesicles (EVs). The present disclosure also relates fusion proteins, comprising an AAV binding polypeptide and a tetraspanin EV polypeptide, which allow for increased AAV loading into EVs.

[0004] BACKGROUND

[0005] Adeno-associated virus (AAV) allow cells to be genetically modified through expression of a transgene that is delivered by the AAV. Therapies are currently in development for several genetic disorders, where AAV is employed to deliver a transgene encoding a polypeptide which has reduced expression or function in the disorder. However, a problem with AAV- mediated gene therapy is that, depending on serotype, a large proportion of all potential patients have neutralising antibodies against AAVs, due to previous exposure to the AAV. Neutralising antibodies severely curtail the effectiveness of AAV-mediated gene therapy in these patients. This prevents effective re-dosing with an AAV therapeutic, which is highly desirable, as the expression of the AAV transgene delivered diminishes over time, especially in children whose organs are still growing. Hence, there is also a need to develop AAV- mediated gene therapies that can be used in patients with neutralising antibodies against the AAV.

[0006] AAVs are often given in large doses to ensure sufficient transduction of target cell populations. However, high doses often result in acute liver toxicity and other safety and tolerability concerns, especially when the AAV therapeutic is delivered systemically. Furthermore, both the innate and the adaptive immunological response to AAV are believed to be heavily dose dependent, meaning that large doses are of AAV therapeutics are undesirable in many respects (including from a manufacturing / cost of goods point of view).

[0007] One potential solution is to encapsulate therapeutic AAVs in a delivery vehicle. Extracellular vesicles (EVs) comprise a membrane that encloses an internal space and can be used to deliver therapeutics that they are associated, or “loaded”, with across the cell membrane. When an AAV cargo is loaded into the EV lumen, the EV membrane shields the AAV from neutralising antibodies. EVs have numerous further advantages as therapeutic delivery vehicles including the ability to deliver multiple copies of a cargo to a target cell in a single EV, the potential to achieve cell-type specific targeting, protecting luminally loaded cargo from degradation and preventing luminally loaded cargo from triggering an adverse reaction or immune response.

[0008] However, luminal loading of EVs with AAV presents a challenge. Typically, luminal loading of AAV is achieved through passive, endogenous loading approaches, where the AAV is expressed in an EV producer cell without the expression of further constructs that actively localise and incorporate the AAV into the EV as it forms. Such methods only allow for low amounts of AAV to be loaded into EVs.

[0009] Active endogenous loading approaches for luminally loading AAV into EVs are described in WO 2020 / 215010. However, the approaches described do not allow for the AAV to be released from the EV and to have bioactivity in a target cell. Moreover, a number of these approaches also involve the expression of multiple fusion proteins, thus are complex, and / or involve the modification of the viral capsid, which may affect AAV function.

[0010] Thus, there remains a need for a way of luminally loading EVs with AAVs in a manner that allows for both high levels of AAV loading into the EV and for the AAV to be released and have bioactivity in target cells. In addition, it would be preferable for there to be simplicity of production and no modification of the AAV capsid.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention provides Variable Heavy domain of Heavy chains (VHHs) capable of binding to an AAV, thus allowing for AAV to be luminally loaded into an EV. In particular, the present invention provides VHHs having specific CDR sequences (CDR3 sequences in particular) that allow strong binding to the AAV capsid and thus result in particularly high levels of AAV being loaded into EVs. Moreover, the present invention further provides VHHs having specific CDR sequences (CDR3 sequences in particular) that allow strong binding to the AAV capsid yet also display high release in the EV and / or the target cell. These VHHs allow for both particularly high levels of AAV loading and for the loaded AAV to have bioactivity in a target cell.

[0013] The present invention also provides a fusion protein for loading AAV cargo comprising an AAV binding polypeptide and a tetraspanin EV polypeptide, such as CD63, TSN4, TSN9 or preferably TSN2. These fusion proteins significantly increase the amount of AAV loaded into the EV, as compared with when other types of EV polypeptide are used.

[0014] In a first aspect, the present invention provides a fusion protein comprising or consisting of: (i) an extracellular vesicle (EV) polypeptide and

[0015] (ii) an adeno-associated virus (AAV) binding polypeptide, wherein the EV polypeptide is a tetraspanin.

[0016] In a second aspect, the present invention provides a fusion protein-AAV complex comprising or consisting of an AAV and a fusion protein of the present invention, wherein the AAV is bound to the AAV binding polypeptide of the fusion protein.

[0017] In a third aspect, the present invention provides a method of releasably loading an EV with an AAV, the method comprising

[0018] (i) producing the AAV in a cell;

[0019] (ii) expressing a VHH in the same cell; and

[0020] (iii) culturing the cell under conditions which, in no particular order, the VHH binds the AAV, the cell produces an EV and the AAV bound to the VHH loads into the EV; wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

[0021] In a fourth aspect, the present invention provides the use of a VHH, or a polynucleotide sequence encoding a VHH, for releasable loading of an EV with an AAV, wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

[0022] In a fifth aspect, the present invention provides a VHH-AAV complex comprising or consisting of an AAV releasably bound to a VHH, wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

[0023] In a sixth aspect, the present invention provides a fusion protein-AAV complex comprising or consisting of an AAV and a fusion protein, wherein the fusion protein comprises an EV polypeptide and a VHH, wherein the AAV is releasably bound to the VHH, wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

[0024] In a further aspect, the present invention provides an EV releasably loaded with an AAV using a method of the present invention, preferably wherein the AAV has been released from the VHH into the EV lumen.

[0025] In a further aspect, the present invention provides an EV comprising the fusion protein, fusion-protein AAV complex or VHH-AAV complex of the present invention.

[0026] In a further aspect, the present invention provides a population of EVs comprising a plurality of EVs of the present invention.

[0027] In a further aspect, the present invention provides a composition comprising the VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV or the population of EVs of the present invention and a pharmaceutically acceptable excipient and / or carrier.

[0028] In a further aspect, the present invention provides a method of bioactive delivery of an AAV to a target cell, comprising contacting or incubating the target cell with an EV, a population of EVs or a composition of the present invention.

[0029] In a further aspect, the present invention provides the use of an EV, a population of EVs or a composition of the present invention for the bioactive delivery of an AAV to a target cell.

[0030] In a further aspect, the present invention provides a method of expressing an AAV transgene and / or altering the gene expression in a target cell, comprising contacting or incubating the target cell with an EV, a population of EVs or a composition of the present invention.

[0031] In a further aspect, the present invention provides the use of an EV, a population of EVs or a composition of the present invention for expressing an AAV transgene and / or altering the gene expression in a target cell

[0032] In a further aspect, the present invention provides the VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV, the population of EVs or the composition of the present invention for the preparation of a medicament for treatment or prevention of a disease in a subject.

[0033] In a further aspect, the present invention provides the VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV, the population of EVs or the composition of the present invention for use as a medicament for treatment or prevention of a disease in a subject.

[0034] In a further aspect, the present invention provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV, the population of EVs or the composition of the present invention to a subject suffering from or susceptible to the disease.

[0035] DESCRIPTION OF FIGURES

[0036] Figure 1 shows a Western Blot analysis of density gradient ultracentrifugation (DGUC) fractions from cells expressing different EV polypeptides fused to VHH3 as well as components for the production of AAV. The expression of viral capsid (VP), syntenin (an EV marker) and the VHH is shown. (A) is a negative control where a non-coding “stuffer” sequence is expressed instead of an EV polypeptide fused to VHH3. In (B) BASP1 is used as the EV polypeptide. In (C) TSN2 is used as the EV polypeptide. In (D) PTGFRN is used as the EV polypeptide. In (E) a single-pass transmembrane EV polypeptide is used.

[0037] Figure 2A shows the genome distribution in the different fractions obtained by DGUC from cells expressing different EV polypeptides (BASP1, PTGFRN, TSN2 and single-pass transmembrane EV polypeptide) fused to VHH3 as well as components for the production of AAV. The viral genome was quantified by dPCR and is shown on the graph as a percentage of total viral genomes detected. Figure 2B shows the total viral genome quantified in fraction 6 obtained by DGUC. The number on top of bars shows the fold-change in viral genomes, compared to the EV polypeptide with the lowest quantified viral genomes (BASP1).

[0038] Figure 3A-K show a Western Blot analysis of DGUC fractions from cells expressing TSN2 fused to different VHHs (the VHH tested is shown above each blot) as well as components for the production of AAV. The expression of viral capsid (VP) and the VHH is shown. Figure 3L shows a Western Blot analysis of DGUC fractions from cells expressing a non-coding “stuffer” sequence is expressed instead of TSN2 fused to a VHH as well as components for the production of AAV. Figure 4A-C shows the genome distribution in the different fractions obtained by DGLIC from cells expressing TSN2 fused to different VHHs or expressing a non-coding “stuffer” sequence. Components for the production of AAV were also expressed in the cells. The viral genome was quantified by dPCR and is expressed on the graph as percentage of total viral genomes detected. (A) shows the VHHs that are “strong binders”. (B) and (C) show the VHHs that are medium and weak binders.

[0039] Figure 5A and B show Factor IX activity dose-response curves of EVs that are loaded with AAV, using by expressing a fusion protein comprising TSN2 fused to different VHHs (i.e. VHH1, VHH2, VHH3, VHH4, VHH5 and VHH8) in cells as well as components for the production of AAV, as compared with free AAV (i.e. that is not loaded into an EV). The EVs were obtained from DGLIC fraction 6 and 7. All the AAVs (including those loaded in the EVs) comprise a Factor IX transgene under the control of a liver specific promoter. The assay was performed with (A) 8750 cells / well or (B) 27500 cell / well in 96 well plates. The inset on top of each graph represents magnified portion between 1x103and 1x105MOIs. Figure 5C shows intracellular quantification of viral genomes after the activity Factor IX activity assay was performed in the cells that were dosed with either free AAV or EVs that were loaded with AAV through the expression of a fusion protein comprising TSN2 fused to either VHH2, VHH3 or VHH4 in the EV producer cells.

[0040] Figure 6 shows a neutralisation assay measuring the Factor IX activity induced in cells that are dosed at 7x103MOI with either free AAV (i.e. that is not loaded into an EV) or EVs that were loaded with AAV by expression a fusion protein comprising TSN2 fused to a VHH in the EV producer cells, where the AAV or the EV was pre-incubated with increasing concentrations of an antibody against the AAV capsid (i.e. an anti-AAV9 antibody). All the AAVs (including those loaded in the EVs) comprise a Factor IX transgene under the control of a liver specific promoter. (A) shows cells dosed with either free AAV or EVs that were loaded through the expression of a fusion protein comprising TSN2 fused to VHH4 in the EV producer cells. (B) shows cells dosed with either free AAV or EVs that were loaded through the expression of a fusion protein comprising TSN2 fused to either VHH2, VHH3 or VHH4 in the EV producer cells. The left panel shows the standard activity measured with increasing concentrations of anti-AAV9 antibody. The right panel shows the results normalised by relative factor IX activity in absence of anti-AAV9 antibody. (C) shows the percentage protection from 2000 ng / ml neutralising antibodies that was observed in cells dosed with either free AAV or EVs that were loaded through the expression of a fusion protein comprising TSN2 fused to either VHH2, VHH3 or VHH4 in the EV producer cells. DETAILED DESCRIPTION

[0041] The present inventors sought to take advantage of the benefits associated with EV-mediated delivery of an AAV cargo. In particular, the potential to overcome the problems associated with neutralising antibodies, by luminally loading AAV into EVs.

[0042] Prior to the present invention, when EV-mediated delivery of AAV was employed, only low levels of bioactivity (i.e. AAV transgene expression) could be detected in target cells. This was believed to be due to a relatively low level of loading being achieved when passive approaches were used to endogenously load AAV into the EV lumen and due to the AAV cargo not being released when active approaches were used to endogenously load AAV into the EV lumen.

[0043] The present inventors have identified specific VHHs that allow for both high levels of AAV to be luminally loaded into EVs and for the AAV cargo to have bioactivity in target cells. It is believed that these VHHs have the ability to bind AAV at a relatively high affinity in the cytoplasm of EV producer cells, as the EV is forming, but release the AAV once the EV is taken up into the target cell, thus allowing for the AAV to have functional effects in a target cell.

[0044] Furthermore, the present inventors surprisingly further observed that tetraspanin EV polypeptides, such as CD63, TSN4, TSN9 or preferably TSN2, are advantageous for the endogenous active loading of AAV cargo. The amount of AAV loaded into EVs is many fold higher when an AAV binding polypeptide is fused to a tetraspanin EV polypeptide, such as CD63, TSN4, TSN9 or preferably TSN2, as compared with when other types of EV polypeptide are used. It is believed that the subcellular localisation of these tetraspanins in EV producer cells may allow for increased contact with the AAV that is also being produced as or before the EV is forms, thus allowing for a larger proportion of fusion proteins comprising these tetraspanin EV polypeptides to bind AAV, resulting in increased loading.

[0045] In one aspect, the present invention relates to a VHH. VHHs are derived from heavy chain only antibodies, which are produced by members of the Camelidae family. Heavy chain antibodies are homodimers, made up of two heavy chains, with each heavy chain consisting of three domains: the VHH (variable-heavy) domain, the CH2 (constant heavy 2) domain and the CH3 (constant heavy 3) domain. The VHH domain is around 12-14 kDa in size and is found at the N-terminal end of the antibody. The VHH domain is responsible for antigen recognition and binding. VHH antibodies are single-domain antibodies, thus do not need to form dimers in order to function. VHHs typically comprise four Framework Regions (FR1, FR2, FR3 and FR4) and three Complementary-Determining Regions (CDR1 , CDR2 and CDR3). From its N-terminus to its C-terminus, VHHs are typically structures as followed: FR1, CDR1, FR2, CRR2, FR3, CDR3, FR4. The Framework Regions are relatively conserved across VHHs. The Framework Regions typically form beta-sheet structures and function as the scaffold of the VHH domain, supporting the positioning of the CDRs. The CDRs are highly variable regions that interact directly with the target antigen allowing for antigen binding. CDR3 is typically the most variable of the CDRs and contributes most significantly to the specificity of antigen binding.

[0046] As used herein, the term “VHH” includes VHHs as described herein that have undergone further modifications, for instance to improve their stability and / or solubility and / or reduce their immunogenicity or toxicity. Thus, the term “VHH” as used herein includes Nanobodies™.

[0047] In one embodiment, the VHH is capable of binding to an AAV. In one embodiment, the VHH is capable of binding to an AAV capsid.

[0048] In one embodiment, the VHH is a VHH specific for an AAV. In one embodiment, the VHH is a VHH specific for an AAV capsid.

[0049] In one embodiment, the VHH is an anti-AAV VHH. In one embodiment, the VHH is an anti- AAV capsid VHH.

[0050] In a preferred embodiment, the VHH of the present invention is capable of binding the AAV and / or the AAV capsid in the cytoplasm of an EV producer cell as described herein.

[0051] In a preferred embodiment, the AAV capsid is an AAV9 capsid.

[0052] As used herein, the term “AAV” or “adeno-associated virus” refers to a virus, comprising an AAV capsid and an AAV vector, preferably a recombinant AAV vector, encapsulated by the AAV capsid. The AAV may be produced using any conventional method known in the art. Such methods are disclosed in WO 2019 / 217 513, which is incorporated herein by reference in its entirety. For instance, an AAV comprising a transgene in its genome may be produced by expressing an AAV transfer plasmid (also referred to as an AAV vector plasmid) comprising the transgene flanked by AAV inverted terminal repeat (ITR) sequences, in a host cell alongside a plasmid encoding for the AAV rep and cap genes. In a preferred embodiment, additional “helper” functions, such as E2A, E4 and VA RNA genes are also expressed. In some embodiments, the ITR sequences are from human AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or any other natural, recombinant or engineered AAV. In a preferred embodiment of the present invention, the AAV comprises an AAV9 capsid. In a further preferred embodiment, the AAV comprises a transgene, preferably the transgene is a heterologous transgene that allows the AAV to act as a gene replacement therapy. In a preferred embodiment, the transgene encodes a polynucleotide, polypeptide or protein that is linked to a disease or disorder. In a preferred embodiment, the transgene encodes a polynucleotide, polypeptide or protein that displayed reduced expression and / or function in a disease or disorder.

[0053] As used herein, the term “AAV capsid” refers to a protein shell that encapsulates the AAV vector. The AAV capsid is formed of structural VP proteins (a mixture of VP1 , VP2 and VP3) that determine the cellular tropicity of the AAV virus. The VP protein-encoding sequences (Cap genes) vary between AAV serotypes. In a preferred embodiment of the present invention, the AAV capsid of the present invention is comprised in an AAV. In a preferred embodiment of the present invention, the AAV capsid of the present invention is an AAV9 capsid.

[0054] As used herein, the term “AAV9 capsid” refers to a capsid derived from AAV9 (i.e. a capsid from an AAV9 serotype, preferably a human AAV9 serotype) or a derivative, variant, mutant, domain or region thereof. In a preferred embodiment, the AAV9 capsid of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 preferably over its entire length.

[0055] As used herein, "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. “100% sequence identity” or “sequence identity” as used herein refers to two or more referenced entities that are the same when they are "aligned" sequences. For example, when two polynucleotide sequences are identical, they have the same nucleic acid base sequence. Similarly, when two amino acid sequences are identical, they have the same amino acid sequence. An "aligned" sequence refers to multiple polynucleotide or amino acid sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence. In a preferred embodiment, sequence identity is calculated over the entire length of the sequence.

[0056] As used herein, the term “EV producer cell”, which may also be referred to as an “EV source cell” or a “parental cell”, refers to any cell that is capable of producing an EV. Generally, EVs may be derived from essentially any cell source. In a preferred embodiment, the EV producer cell is any cell that produces an EV under the conditions in which the cell is able to survive or preferably grow. In a more preferred embodiment, the EV producer cell is any cell that produces an EV under its usual, or optimum, conditions for growth. In one embodiment, the EV producer cell is capable of producing a micovesicle or preferably an exosome. In one embodiment, the EV producer cell is a eukaryotic cell, preferably a mammalian cell, more preferably a human cell. In one embodiment, the EV producer cell is an ex vivo cell or preferably an in vitro cell such as a primary cell or more preferably a cell-line. In a preferred embodiment, the EV producer cell-line is a HEK cell, more preferably a HEK293 cell, more preferably a Gibco™ Viral Production Cell 1.0 (VPC 1.0 cells) or most a preferably Gibco™ Viral Production Cell 2.0 (referred to as VPC 2.0 cells herein). In a preferred embodiment, the EV producer cell is a suspension cell-line. In a preferred embodiment, the EV producer cell is grown or cultured in serum-free conditions. In one embodiment, the cytoplasm of the EV producer cell has a pH around 7.0 to 7.5, preferably pH 7.1-7.2.

[0057] In one embodiment, the present invention is a VHH produced by immunising a Camelidae family member with an AAV capsid, preferably an AAV9 capsid. In an alternative embodiment, the present invention is a VHH produced by immunising a Camelidae family member with an AAV, preferably the AAV comprises an AAV9 capsid. In more preferred embodiments, the AAV9 capsid comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1. Preferably the Camelidae family member is an alpaca.

[0058] In one embodiment, the VHH of the present invention comprises three Complementary- Determining Regions (CDR1 , CDR2 and CDR3). Preferably the VHH also comprises four Framework Regions (FR1 , FR2, FR3 and FR4). In one embodiment, the VHH of the present invention is structured, from its N-terminus to its C-terminus, as follows: FR1, CDR1 , FR2, CRR2, FR3, CDR3, FR4.

[0059] In one embodiment, the VHH of the present invention comprises a CDR1 , a CDR2 and a CDR3, wherein the CDR3 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 58, SEQ ID NO 60, SEQ ID NO 61 , SEQ ID NO 63, SEQ ID NO 64, SEQ ID NO 65, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69 or SEQ ID NO 70.

[0060] In one embodiment, the VHH of the present invention comprises a CDR1 , a CDR2 and a CDR3, wherein the CDR1 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21 , SEQ ID NO 23, SEQ ID NO 24 or SEQ ID NO 25.

[0061] In one embodiment, the VHH of the present invention comprises a CDR1 , a CDR2 and a CDR3, wherein the CDR2 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 41 , SEQ ID NO 42 or SEQ ID NO 43.

[0062] In a preferred embodiment, the VHH of the present invention comprises a CDR1, a CR2 and a CDR3 that comprise or consist of amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to:

[0063] (i) SEQ ID NO 16, SEQ ID NO 34 and SEQ ID NO 58 respectively;

[0064] (ii) SEQ ID NO 17, SEQ ID NO 35 and SEQ ID NO 60 respectively;

[0065] (iii) SEQ ID NO 17, SEQ ID NO 35 and SEQ ID NO 61 respectively;

[0066] (iv) SEQ ID NO 19, SEQ ID NO 37 and SEQ ID NO 63 respectively;

[0067] (v) SEQ ID NO 20, SEQ ID NO 37 and SEQ ID NO 64 respectively;

[0068] (vi)SEQ ID NO 20, SEQ ID NO 38 and SEQ ID NO 64 respectively;

[0069] (vii)SEQ ID NO 20, SEQ ID NO 37 and SEQ ID NO 65 respectively;

[0070] (viii) SEQ ID NO 21, SEQ ID NO 39 and SEQ ID NO 67 respectively;

[0071] (ix)SEQ ID NO 23, SEQ ID NO 41 and SEQ ID NO 68 respectively;

[0072] (x) SEQ ID NO 24, SEQ ID NO 42 and SEQ ID NO 69 respectively; or (xi) SEQ ID NO 25, SEQ ID NO 43 and SEQ ID NO 70 respectively.

[0073] In a preferred embodiment, the VHH of the present invention is a strong binder.

[0074] As used herein the term “strong binder” refers to a VHH of the present invention that binds to the AAV, preferably an AAV comprising an AAV9 capsid, at a relatively high affinity. In one embodiment, a strong binder VHH of the present invention is a VHH of the present invention that, when expressed in an EV producer cell alongside the AAV to which the VHH is capable of binding, loads more than 7%, preferably more than 10%, more preferably more than 15% of total produced AAV viral genome into EVs. In one embodiment, loading is assessed by quantifying the percentage of the total produced viral genome that is detected in the EV fractions following density gradient ultracentrifugation, as outlined in the examples. In one embodiment, a strong binder VHH of the present invention is a VHH of the present invention that, when expressed in an EV producer cell alongside components for the production of an AAV to which the VHH is capable of binding, loads a higher proportion of the AAV produced as compared with a VHH having an amino acid sequence of SEQ ID NO 74, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79 and / or SEQ ID NO 80, preferably wherein in loading is quantified as the percentage of the total produced viral genome that is detected in the EV fractions following density gradient ultracentrifugation, as outlined in the examples.

[0075] In one embodiment, the strong binder VHH of the present invention comprises a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 60 . Preferably wherein the amino acid residue at position number 9 of SEQ ID NO 60 is Y.

[0076] In an alternative embodiment, the strong binder VHH of the present invention comprises a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67 to 70.

[0077] In one embodiment, the strong binder VHH of the present invention comprises a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 17. Preferably wherein the amino acid residue at position number 2 of SEQ ID NO 17 is I and / or the amino acid residue at position number 4 is S. In an alternative embodiment, the strong binder VHH of the present invention comprises a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 21, 23, 24 or 25.

[0078] In one embodiment, the strong binder VHH of the present invention comprises a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 35. Preferably the amino acid residue at position number 1 of SEQ ID NO 35 is I, at position number 3 is S, at position number 6 is S and / or at position number 7 is T.

[0079] In an alternative one embodiment, the strong binder VHH of the present invention comprises a CDR2 comprising or consisting of 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 39, 41, 42 or 43.

[0080] In one embodiment, the strong binder VHH of the present invention comprises a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 40, 45, 54, 59, 62 or 66.

[0081] In one embodiment, the strong binder VHH of the present invention comprises:

[0082] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 60, preferably the amino acid residue at position number 9 of SEQ ID NO 60 is Y;

[0083] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 17, preferably the amino acid residue at position number 2 of SEQ ID NO 17 is I and / or the amino acid residue at position number 4 is S; and

[0084] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 54 or preferably SEQ ID NO 35, preferably the amino acid residue at position number 1 of SEQ ID NO 35 is I, at position number 3 is S, at position number 6 is S and / or at position number 7 is T. In an alternative embodiment, the strong binder VHH of the present invention comprises:

[0085] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 69;

[0086] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 24; and

[0087] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 62 or preferably SEQ ID NO 42.

[0088] In an alternative embodiment, the strong binder VHH of the present invention comprises:

[0089] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 68;

[0090] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 23; and

[0091] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 59 or preferably SEQ ID NO 41.

[0092] In an alternative embodiment, the strong binder VHH of the present invention comprises:

[0093] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 70;

[0094] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 23; and

[0095] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 66 or preferably SEQ ID NO 43.

[0096] In an alternative embodiment, the strong binder VHH of the present invention comprises:

[0097] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67; (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21 ; and

[0098] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 40, SEQ ID NO 45 or preferably SEQ ID NO 39.

[0099] In a most preferred embodiment, the VHH of the present invention is a strong binder with high release.

[0100] As used herein, the term “high release VHH” refers to a VHH that, in the EV lumen and / or the target cell, releases the AAV capsid that it is capable of binding or is specific for. In one embodiment, the high release VHH of the present invention allows for the AAV that it loads to have bioactivity in a target cell.

[0101] As used herein, the term or “releasably bound” or “releasable binding”, when used in relation to the binding between a VHH and an AAV capsid, refers to binding that occurs in the cytoplasm of the EV producer cell, but that releases (i.e. does not remain bound) in the EV lumen and / or the target cell. In one embodiment, releasable binding allows for the loaded AAV to have bioactivity in a target cell.

[0102] As used herein, the term “target cell” refers to a cell which an EV is capable of entering i.e. a cell that is capable of EV uptake. In one embodiment, the target cell has a genetic abnormality that is linked to a disease or a disorder. In one embodiment, the genetic abnormality is a deficiency in the RNA or polypeptide encoded for by the transgene of the AAV. In an alternative embodiment the genetic abnormality is reduced function (including no function) of the RNA or polypeptide encoded for by the transgene. In one embodiment, the target cell is a cell of the liver, preferably a hepatocyte, a neuronal cell, a cell of the brain, a muscle cell, a cell of the eye, a cell of the lung, a cell of the liver, a cell of the kidneys, a cell of the heart, a cell of the stomach, a cell of the intestines, a cell of the pancreas, a red blood cell, a white blood cell including a B cell or a T cell, a cell of the lymph nodes, a cell of the bone marrow or a cell of the spleen. In a preferred embodiment, the target cell is a neuronal cell preferably a cell of the CNS or the brain, a muscle cell, a cell of the heart preferably a cardiomyocyte, a cell of the lung, a cell of the immune system, a cell of the liver preferably a hepatocyte. The target cell may be in vivo, ex vivo or in vitro. As used herein the term “bioactivity” or “bioactive delivery” when used in relation to the AAV in a target cell refers to expression of the AAV transgene. In a preferred embodiment, bioactivity in the target cell is a significant increase in the expression, or the function, of the RNA or polypeptide encoded for by the AAV transgene. In a preferred embodiment, bioactivity of a VHH may be measured by its ability to express a factor IX transgene in an assay as described in the examples. In a preferred embodiment, the strong binder with high release VHH of the present invention, when used to luminally load an EV with an AAV that it is capable of binding, preferably an AAV comprising an AAV9 capsid, comprising a transgene, allows for an increase in the expression of the transgene as compared with a VHH having an amino acid sequence selected from SEQ ID NO 74, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79, SEQ ID NO 80, SEQ ID NO 83, SEQ ID NO 84 and / or SEQ ID NO 85. In a preferred embodiment, the strong binder with high release VHH of the present invention, when used to luminally load an EV with a an AAV that it is capable of binding, preferably an AAV comprising an AAV9 capsid, comprising a transgene, allows for an increase in the expression of the transgene as compared with an EV that is passively endogenously loaded with the virus (i.e. wherein the producer cell does not express a further construct that actively localises and incorporates the AAV to the EV as it forms e.g. a fusion protein comprising an EV polypeptide and an AAV binding protein).

[0103] In one embodiment, the strong binder with high release VHH of the present invention comprises a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67. Preferably the amino acid residue at position number 2 of SEQ ID NO 67 is K, at position number 3 is N, at position number 8 is Y, at position number 11 is T, at position number 12 is H and / or at position number 15 is T.

[0104] In one embodiment, the strong binder with high release VHH of the present invention comprises a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21.

[0105] In one embodiment, the strong binder with high release VHH of the present invention comprises a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 39. Preferably the amino acid residue at position number 4 of SEQ ID NO 39 is S, at position 5 is G, at position 6 is D and at position 7 is S. In an alternative embodiment, the strong binder with high release VHH of the present invention comprises a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toa sequence selected from SEQ ID NO 40 or SEQ ID NO 45. Preferably the amino acid residue at position number 5 of SEQ ID NO 40 or SEQ ID NO 45 is S, at position 6 is G, at position 7 is D and at position 8 is S.

[0106] In a preferred embodiment, the strong binder with high release VHH of the present invention comprises:

[0107] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67, preferably wherein the amino acid residue at position number 2 of SEQ ID NO 67 is K, at position number 3 is N, at position number 8 is Y, at position number 11 is T, at position number 12 is H and / or at position number 15 is T;

[0108] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21 ; and

[0109] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 40, SEQ ID NO 45 or preferably SEQ ID NO 39, preferably wherein a. the amino acid residue at position number 4 of SEQ ID NO 39 is S, at position 5 is G, at position 6 is D and at position 7 is S or b. the amino acid residue at position number 5 of SEQ ID NO 40 or SEQ ID NO 45 is S, at position 6 is G, at position 7 is D and at position 8 is S.

[0110] In one embodiment, the VHH of the present invention comprises a FR1, a FR2, a FR3 and FR4, wherein the FR1, the FR2, the FR3 and the FR4 are capable of forming beta-sheet structures. In a preferred embodiment, the FR1, the FR2, the FR3 and the FR4 are capable of forming beta-sheet structures when the VHH is expressed in an EV producer cell as defined herein.

[0111] In one embodiment, the FR1 , FR2, FR3 and FR4 of the VHH function as the scaffold of the VHH domain, supporting the positioning of the CDRs. In one embodiment, the FR1 of the VHH of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 3 to 14, preferably SEQ ID NO 4 or 10 to 14 most preferably SEQ ID NO 10 or 11.

[0112] In one embodiment, the FR2 of the VHH of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 27 to 32, preferably SEQ ID NO 28, 31 or 32, most preferably SEQ ID NO 31.

[0113] In one embodiment, the FR3 of the VHH of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 44 or SEQ ID NO 46 to 53, preferably SEQ ID NO 46, 51 , 52, 53, 55 to 56, most preferably SEQ ID NO 55 or 56.

[0114] In one embodiment, the FR4 of the VHH of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid having SEQ ID NO 72 or preferably having SEQ ID NO 73.

[0115] In one embodiment, the strong binder VHH of the present invention comprises or consists of:

[0116] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 60, preferably the amino acid residue at position number 9 of SEQ ID NO 60 is;

[0117] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 17, preferably the amino acid residue at position number 2 of SEQ ID NO 17 is I and / or the amino acid residue at position number 4 is S; and

[0118] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 35, preferably the amino acid residue at position number 1 of SEQ ID NO 35 is I, at position number 3 is S, at position number 6 is S and / or at position number 7 is T;

[0119] (iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 4;

[0120] (v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 28;

[0121] (vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 46; and

[0122] (vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 72.

[0123] In an alternative embodiment, the strong binder VHH of the present invention comprises or consists of:

[0124] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 69;

[0125] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 24;

[0126] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 42;

[0127] (iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 13;

[0128] (v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 32;

[0129] (vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 52; and

[0130] (vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73.

[0131] In an alternative embodiment, the strong binder VHH of the present invention comprises or consists of:

[0132] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 68; (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 23; and

[0133] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 41 ;

[0134] (iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 12;

[0135] (v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 31 ;

[0136] (vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 51 ; and

[0137] (vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73.

[0138] In an alternative embodiment, the strong binder VHH of the present invention comprises or consists of:

[0139] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 70;

[0140] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 23; and a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 43;

[0141] (iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 14;

[0142] (v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 31 ;

[0143] (vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 53; and

[0144] (vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73.

[0145] In one embodiment, the strong binder with high release VHH of the present invention comprises or consists of: (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67, preferably wherein the amino acid residue at position number 2 of SEQ ID NO 67 is K, at position number 3 is N, at position number 8 is Y, at position number 11 is T, at position number 12 is H and / or at position number 15 is T;

[0146] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21 ; and

[0147] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 39, preferably wherein the amino acid residue at position number 4 of SEQ ID NO 39 is S, at position 5 is G, at position 6 is D and at position 7 is S;

[0148] (iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 10;

[0149] (v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 31;

[0150] (vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 55; and

[0151] (vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73.

[0152] In one embodiment, the strong binder with high release VHH of the present invention comprises or consists of:

[0153] (i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67, preferably wherein the amino acid residue at position number 2 of SEQ ID NO 67 is K, at position number 3 is N, at position number 8 is Y, at position number 11 is T, at position number 12 is H and / or at position number 15 is T;

[0154] (ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21 ; and

[0155] (iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 39, preferably wherein the amino acid residue at position number 4 of SEQ ID NO 39 is S, at position 5 is G, at position 6 is D and at position 7 is S;

[0156] (iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 11;

[0157] (v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 31;

[0158] (vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 56; and

[0159] (vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73.

[0160] In one embodiment, the VHH of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 74 to 85.

[0161] In one embodiment, the strong binder VHH of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 75 or 81 to 85.

[0162] In one embodiment, the strong binder with high release VHH of the present invention comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 81 or 82.

[0163] The VHH may or may not further comprise a Tag, for instance a HisTag or preferably a Flag Tag. In one embodiment, the Tag is a Flag Tag that comprises or consists of an amino acid sequence having SEQ ID NO 86. In a preferred embodiment the Tag is to the C-terminus of the FR4. In a further preferred embodiment, the VHH comprises a linker sequence between the Tag and the FR4. In one embodiment, the linker sequence comprises or consists of an amino acid sequence selected from SEQ ID NO 87 to 98, preferably SEQ ID NO 87.

[0164] In a further aspect, the present invention provides a complex (referred to herein as a VHH- AAV complex), wherein the complex comprises or consists of an AAV bound to a VHH of the present invention, preferably wherein the AAV comprises an AAV9 capsid. In a preferred embodiment, the VHH comprised in the complex is a strong binder VHH of the present invention. In a most preferred embodiment, the AAV is releasably bound to a strong binder high release VHH of the present invention.

[0165] In a further aspect, the present invention provides to a fusion protein that comprises or consists of an EV polypeptide and a VHH of the present invention. In a preferred embodiment, the VHH comprised in the fusion protein is a strong binder VHH of the present invention. In a most preferred embodiment, the VHH comprised in the fusion protein is a strong binder high release VHH of the present invention.

[0166] In a further aspect, the present invention provides a complex (referred to herein as a fusion protein-AAV complex), wherein the complex comprises or consists of an AAV and a fusion protein of the present invention, preferably wherein the AAV comprises an AAV9 capsid, further wherein the AAV is bound to the VHH of the fusion protein. In a preferred embodiment, the VHH comprised in the fusion protein of the complex is a strong binder VHH of the present invention. In a most preferred embodiment, the AAV is releasably bound to a strong binder high release VHH of the present invention.

[0167] In a further aspect, the present invention a provides complex (referred to herein as a fusion protein-AAV complex), wherein the complex comprises or consists of an AAV and a fusion protein, preferably wherein the AAV comprises an AAV9 capsid, wherein the fusion protein that comprises or consists of an EV polypeptide and a VHH of the present invention, further wherein the AAV is bound to the VHH of the fusion protein. In a preferred embodiment, the VHH comprised in the complex is a strong binder VHH of the present invention. In a most preferred embodiment, the AAV is releasably bound to a strong binder high release VHH of the present invention.

[0168] As used herein, the term “fusion” or “fused” includes where one polypeptide is fused immediately to or into another polypeptide with no intervening amino acid residues and 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 as described herein, spacer and / or scaffold sequence or may comprise a further polypeptide between them.

[0169] As used herein the term “EV polypeptide”, or “EV protein”, refers to any protein, region, domain, motif, or sequence or stretch of amino acids that is naturally localised to an EV that is produced by a given EV producer cell or that when expressed in a cell can be made to localise to an EV. In a preferred embodiment, the EV polypeptide is capable of transporting a protein to which it is fused to an EV. Preferably, the protein to which the EV polypeptide is fused is a VHH of the present invention.

[0170] In a preferred embodiment, the EV polypeptide is of eukaryotic origin, preferably mammalian origin, more preferably human origin.

[0171] In a preferred embodiment, the EV polypeptide is a micovesicle polypeptide or more preferably an exosomal polypeptide. The term “micovesicle polypeptide” as used herein refers to any protein, region, domain, motif, or sequence or stretch of amino acids that is naturally localised to a micovesicle that is produced by a given EV producer cell. In a preferred embodiment, the micovesicle polypeptide is capable of transporting a fusion protein into a micovesicle produced by a given EV producer cell as described herein. The term “exosomal polypeptide” as used herein refers to any protein, region, domain, motif, or sequence or stretch of amino acids that is naturally localised to an exosome that is produced by a given EV producer cell. In a preferred embodiment, the exosomal polypeptide is capable of transporting a fusion protein into an exosome produced by a given EV producer cell as described herein.

[0172] In a preferred embodiment, the EV polypeptide is a transmembrane EV polypeptide. In a more preferred embodiment, the EV polypeptide is a single pass transmembrane protein or a multi-pass transmembrane protein, most preferably a tetraspanin. Single pass transmembrane proteins may be advantageous, since they allow for surface and / or luminal loading to be achieved relatively easily, through engineering of the terminus that is localised to the EV surface and / or engineering of the terminus that is localised to the EV lumen. Multipass transmembrane proteins, such as tetraspanins, may be advantageous since they allow for further opportunities to engineering i.e. at both their termini, but also in their loops.

[0173] In one embodiment 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, TSN3, TSN4, TSNS, TSN5, TSN6, TSN7, TSN8, TSN31, TSN10, TSN11 , TSN12, TSN13, TSN14, TSN15, TSN16, TSN17, TSN18, TSN19, TSN2, TSN4, TSN9, TSN32, TSN33, TNFR, TfR1, syndecan-1 , syndecan-2, syndecan-3, syndecan-4, CD37, CD82, CD151 , CD224, CD231, CD102, NOTCH1 , NOTCH2, NOTCH3, NOTCH4, DLL1 , DLL4, JAG1 , JAG2, CD49d / ITGA4, ITGBS, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18 / ITGB2, CD41, CD49b, CD49c, CD49e, CD51 , CD61 , CD104, CLIC1, CLIC4, interleukin receptors, CO2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD53, CD86, CD110, CD111 , CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD 274, CD362, COL6A1 , AGRN, EGFR, FPRP, GAPDH, GLUR2, GLUR3, GP130, GPI anchor proteins, GTR1 , HLAA, HLA-DM, HSPG2, ITA3, Lactadherin, L 1CAM, LAMB1 , LAMC1 , LIMP2, MYOF, ARRDC1 , ATP282, ATP283, ATP284, BSG, IGSF2, IGSF3, IGSF8, ITGB1 , ITGA4, ATP1A2, ATP1A3, ATP1A4, ITGA4, SLC3A2, ATP transporters, ATP1A1 , ATP183, ATP281 , LFA-1 , LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, a member of the myristoylated alanine rich Protein Kinase C substrate (MARCKS) protein family such as MARCKSL 1 , matrix metalloproteinase-14 (MMP14), PTGFRN, BASP1 , MARCKS, MARCKSL 1 , PRPH2, R0M1 , SLIT2, SLC3A2, SSEA4, STX3, TCRA, TCRB, TCRD, TCRG, TFR1 , UPK1A, UPK1 B, VTI1A, VTI1 B, PTTG1-IP, VSVG, HIV gag proteins, a myristoylation site preferably having the sequence is Gly-X-X-X-Ser / Thr where “X” represents any amino acid and any other EV polypeptide, and any combinations, derivatives, domains, variants, mutants, or regions thereof. Mutations, truncations, linkers or additions may be introduced into the wildtype sequence of the EV polypeptide to alter its function, for instance a preferred mutant according to the invention is a mutation of the tetraspanin CD63 which replaces the tyrosine in position 235 with alanine (denoted CD63 / Y235A). In a preferred embodiment, the EV polypeptide is selected from TSN2, PTGFRN, PTTG1-IP, CD63, Lamp2b, CD81 , Syntenin-1 , Syntenin-2, Lamp2, Lamp2a, TSN3, BASP1 , MARCKS, a HIV gag protein and VSVG. In a more preferred embodiment, the EV polypeptide is selected from TSN2, CD63, PTGFRN, a HIV gag protein or Lamp2b.

[0174] In a preferred embodiment, the EV polypeptide is a tetraspanin, preferably CD63, TSN4, TSN9 or most preferably TSN2.

[0175] As used herein “TSN4”, otherwise referred to as “TSPAN4”, shall be understood to mean “tetraspanin-4”, as well as derivatives, domains, variants, mutants, or regions thereof. The TSN4 protein, is a multi-pass transmembrane protein with four transmembrane regions.

[0176] As used herein “TSN9”, otherwise referred to as “TSPAN9”, shall be understood to mean “tetraspanin-9”, as well as derivatives, domains, variants, mutants, or regions thereof. The TSN9 protein, is a multi-pass transmembrane protein with four transmembrane regions.

[0177] As used herein “TSN2”, otherwise referred to as “TSPAN2”, shall be understood to mean “tetraspanin-2”, as well as derivatives, domains, variants, mutants, or regions thereof. The TSN2 protein, is a multi-pass transmembrane protein with four transmembrane regions. In a preferred embodiment, TSN2 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 99. In a further preferred embodiment, TSN2 comprises or consists of an amino acid sequence encoded for by a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 100.

[0178] In a further preferred embodiment, the VHH is fused to a domain of the EV polypeptide that is displayed in the lumen of the EV. In the context of the present disclosure luminal loading of the cargo may have the advantage of shielding the AAV cargo from immune cells and / or neutralising antibodies, providing a less immunogenic therapeutic product, reduced degradation and / or improved stability of the AAV cargo and / or more efficient delivery of the AAV cargo to target cells. In one embodiment, the VHH of the present invention is fused to the C-terminus of a single-pass EV polypeptide. In a preferred embodiment, the VHH of the present invention is fused to the N-terminus or preferably the C-terminus of a tetraspanin EV polypeptide, such as CD63, TSN4, TSN9 or preferably TSN2.

[0179] In a further aspect, the present invention relates to fusion protein comprising or consisting of:

[0180] (i) an EV polypeptide and

[0181] (ii) an AAV binding polypeptide, wherein the EV polypeptide is a tetraspanin, preferably CD63, TSN4, TSN9 or most preferably TSN2.

[0182] In a further aspect, the present invention relates to a complex, comprising or consisting of an AAV and a fusion protein of the present invention, further wherein the AAV is bound to the AAV binding polypeptide of the fusion protein.

[0183] In a further aspect, the present invention provides a complex, wherein the complex comprises or consists of an AAV and a fusion protein, wherein the fusion protein that comprises or consists of EV polypeptide and an AAV binding polypeptide, wherein the EV polypeptide is a tetraspanin, preferably CD63, TSN4, TSN9 or most preferably TSN2, further wherein the AAV binding polypeptide of the fusion protein in bound, preferably releasably, to the capsid of the AAV.

[0184] As used herein, the term “AAV binding polypeptide” refers to any polypeptide that is capable of binding to any AAV in the cytoplasm of an EV producer cell as described herein. In a preferred embodiment, the AAV comprises an AAV7, AAV8 or preferably an AAV9 capsid. In a preferred embodiment, the AAV binding polypeptide is the AAV-receptor or an anti- AAV7, AAV8 or AAV9 capsid VHH. In a more preferred embodiment, the AAV binding polypeptide is a VHH of the present invention, preferably a strong binder VHH of the present invention, most preferably a strong binder high release VHH of the present invention.

[0185] In a further preferred embodiment, the AAV binding polypeptide is fused to a domain of the EV polypeptide that is displayed in the lumen of the EV. In one embodiment, the AAV binding polypeptide of the present invention is fused to the N-terminus or preferably the C- terminus of the tetraspanin.

[0186] In one embodiment, the fusion proteins and complexes of the present invention further comprises a targeting moiety.

[0187] As used herein, the term “targeting moiety” refers to molecule associated with the EV that enables targeted delivery of the EV to a cell, tissue, organ, and / or compartment of interest. The targeting moiety of the present invention may be obtainable from either humans or from non-human animals. The targeting moiety may be capable of binding to a moiety present of the target cell or of a cell type present in the target tissue or organ. In a preferred embodiment, the targeting moiety targets a cell of the liver, the heart, the brain, the CNS or muscle, a neuronal cell, a hepatocyte, a cardiomyocyte, a cardiac smooth muscle cell, a sensory neuron, a motor neuron, an interneuron or a glia cells. Targeting can be achieved by a variety of means, for instance the use of targeting peptides. Such targeting peptides may be anywhere from a few amino acids in length to several 100s of amino acids in length, e.g. anywhere in the interval of 3-100 amino acids, 3-30 amino acids, 5-25 amino acids, e.g. 7 amino acids, 12 amino acids, 20 amino acids, etc. Targeting peptides of the present invention may also include full length proteins such as receptors, receptor ligands, etc. In one embodiment, the targeting moiety is a protein, peptide, an antibody, a VHH, a nanobody or any other derivatives of an antibody including monoclonal antibodies, single chain variable fragments (scFvs), nanobodies and other antibody domains. In one embodiment, the targeting moiety is rabies virus glycoprotein (RVG), nerve growth factor (NGF), melanotransferrin, the FC5 Peptide or Muscle Specific Peptide (MSP).

[0188] In a preferred embodiment, the targeting moiety is fused or conjugated to a domain or terminus of the EV polypeptide, which is displayed on the surface of an EV, preferably an exosome. In one embodiment, the targeting moiety is fused or conjugated into loop 1 or loop 2 of a tetraspanin EV polypeptide or to the to the N-terminus of a single pass EV polypeptide. In a most preferred embodiment, the targeting moiety is fused or conjugated into loop 1 or loop 2 of CD63, TNS3, TSN9 or most preferably TSN2.

[0189] As used herein, the term “loop 1” when used in relation to a tetraspanin refers to the domain between the first and the second transmembrane domains of the tetraspanin which is displayed on the surface of an EV. In one embodiment, loop 1 of TSN2 is defined by residues 35-54 of SEQ ID NO 99.

[0190] As used herein, the term “loop 2” when used in relation to tetraspanin refers to the domain between the third and the fourth transmembrane domains of the tetraspanin, which is displayed on the surface of an EV. In one embodiment, loop 2 of TSN2 is defined by residues 112-188 of SEQ ID NO 100.

[0191] As used herein the term “first transmembrane domain” when used in reference to a tetraspanin refers to the transmembrane domain most proximal to the N-terminus of the tetraspanin and the term “fourth transmembrane domain” when used in reference to a tetraspanin refers to the transmembrane domain most proximal to the C-terminus of the tetraspanin.

[0192] In an alternative embodiment, the fusion proteins and complexes of the present invention further comprise an Fc-binding protein.

[0193] As used herein, the term “Fc-binding protein” or “Fc binding polypeptide” refers to any protein, polypeptide, or sequence of amino acids which can bind an Fc domain, preferably wherein the Fc domain is comprised in a protein, most preferably wherein the protein is a targeting moiety, purification moity or a pharmacokinetic and / or pharmacodynamic effector moiety as described herein. The Fc binding polypeptide may be derived from either a human or a non-human (e.g. mammal sources, bacteria, etc.). The Fc binding polypeptides of the present invention have high affinity for Fc domains of various antibody isotypes, subtypes, and species (for instance IgG (as non-limiting examples in the case of IgG, lgG1 , lgG2, lgG3, lgG4, lgG2a, lgG2d, and / or lgG2c), IgA, IgM, IgM, IgD, etc.), and can be fused to EV polypeptides. In one embodiment, the Fc polypeptide is Protein A, Protein G, Protein A / G, Z domain, ZZ domain, human FCGRI, human FCGRIIA, human FCGRIIB (as a non-limiting example the accession number 31994), human FCGRI IC (as a non-limiting example the accession number 31995), human FCGRIIIA (as a non-limiting example the accession number P08637), human FCGR3B (as a non-limiting example the accession number 075015), human FCAMR, human FCERA, human FCAR, mouse FCGRI, mouse FCGRIIB, mouse FCGRIII, mouse, mouse FCGRn, and various combinations, derivatives, or alternatives thereof.

[0194] In one embodiment, the Fc-binding protein is bound to an Fc containing protein. In a preferred embodiment, the Fc-containing protein is, or is fused to, a targeting moiety, purification moity or a pharmacokinetic and / or pharmacodynamic effector moiety as described herein.

[0195] As used herein, the term “Fc- containing protein”, “Fc domain-containing protein” or “Fc- containing polypeptide” refers to any protein, polypeptide, or sequence of amino acids which comprises an Fc domain, either naturally or as a result of engineering of the protein to introduce an Fc domain. Fc stands for "fragment crystallizable" or “fragment constant”, which is the name of the tail regions of antibodies. Fc domains can also be created and used on other proteins, not only antibodies. In one embodiment, the Fc domain-containing protein is an antibodies, an antibody derivatives, Fc-modified decoy receptors (such as CD24-Fc or CD52-Fc) and / or signal transducers such as interleukin decoy receptors for IL1 , IL2, IL3, IL4, IL5, IL6 (such as the signal transducer gp130 (as a non-limiting example the accession number P40189)), IL7, IL8, IL9, IL10, IL1 1 , IL12, IL13, IL14, IL15, IL17 (such as IL17R, with as a non-limiting example the accession number Q96F46), IL23 (such as IL23R, with as a non-limiting example the accession number Q5VWK5), etc., Fc domain-containing bi- and multi-specific binders, any type of Fc domain-containing receptors or ligands. Suitable Fc domains that may be fused with a protein that natively lacking an Fc domain include the following non-limiting examples: human IGHM (as a non-limiting example the accession number P01871 ), human IGHA1 (as a non-limiting example the accession number P01876), human IGHA2 (as a non-limiting example the accession number P01877), human IGKC (as a non-limiting example the accession number P01834), human IGHG1 (as a non-limiting example the accession number P01857), human IGHG2 (as a non-limiting example the accession number P01859), human IGHG3 (as a non-limiting example the accession number P01860), human IGHG4 (as a non-limiting example the accession number P01861 ), human IGHD (as a non-limiting example the accession number P01880), human IGHE (as a non-limiting example the accession number P01854).

[0196] In a preferred embodiment, the Fc-binding protein is fused or conjugated to a domain or terminus of the EV polypeptide, which is displayed on the surface of an EV, preferably an exosome. In one embodiment, the Fc-binding protein is fused or conjugated into loop 1 or loop 2 of a tetraspanin EV polypeptide or to the to the N-terminus of a single pass EV polypeptide. In a most preferred embodiment, the Fc-binding protein is fused or conjugated into loop 1 or loop 2 of CD63, TNS3, TSN9 or most preferably TSN2.

[0197] In one embodiment of the fusion proteins and complexes of the present invention, the fusion protein further comprises a linker, spacer and / or scaffold sequence. Such sequences allow flexibility and enable optimal display of the VHH and, if present, the targeting moiety. Linkers also improve pharmacokinetics (PK), increase expression and improve the 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. In one embodiment, the fusion protein comprises a glycine or serine linkers which increase stability or flexibility such SEQ ID NOs 88 to 92, rigid linkers such as SEQ ID NOs: 93 to 96, bending linkers (XP)n or cleavable linkers such as disulphide, protease sensitive sequences. In a preferred embodiment, the fusion protein comprises a linker having an amino acid sequence selected from any one of SEQ ID NO 87 to 98. In a more preferred embodiment, where the EV polypeptide is TSN2, the fusion protein comprises a linker having SEQ ID NO 88 between the C-terminus of TSN2 and the N- terminus of the VHH of the present invention.

[0198] In a further embodiment the fusion proteins and complexes of the present invention further comprise a multimerization domain. In a preferred embodiment, the fusion protein or complex of the present invention comprises a multimerization domain together with a targeting moiety.

[0199] As used herein, the term “multimerization domain” refers to a protein or protein domain that enable dimerization, trimerization, or any higher order of multimerization of the fusion polypeptides. This increases the sorting and trafficking of the fusion polypeptides into EVs and may also contribute to increase the yield of vesicles produced by EV-producing cells. In a preferred embodiment, the fusion protein of the present disclosure comprises a multimerization domain in addition to a targeting moiety. In one embodiment the multimerization domain is a homo-multimerization domains or a hetero-multimerization domains. In one embodiment, the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or any higher order of multimerization domain. In one embodiment, the multimerization domain is selected from a leucine zipper, fold-on domain, fragment X, collagen domain, 2G12 IgG homodimer, mitochondrial antiviralsignaling protein CARD filament, Cardiac phospholamban transmembrane pentamer, parathyroid hormone dimerization domain, Glycophorin A transmembrane, HIV Gp41 trimerisation domain, HPV45 oncoprotein E7 C-terminal dimer domain, and any combination thereof. In a most preferred embodiment, the multimerization domain is a fold-on domain or a leucine zipper.

[0200] In a further aspect, the present invention provides a polynucleotide comprising or consisting of a sequence encoding for a VHH or a fusion protein of the present invention. In one embodiment the polynucleotide is a DNA sequence. In an alternative embodiment the polynucleotide is an RNA sequence, preferably an mRNA sequence. In a preferred embodiment, the RNA sequence comprises a polyA tail.

[0201] In a further aspect, the present invention provides an expression vector comprising a VHH of the present invention or a fusion protein of the present invention. In one embodiment, the VHH is under the control of a constitutive promoter. In a preferred embodiment, the VHH is under the control of a U6 promoter.

[0202] In a further aspect, the present invention provides an EV comprising a VHH, a fusion protein, a complex, a polynucleotide or a vector of the present invention. In a preferred embodiment, the VHH, the VHH comprised in the fusion protein or complex or the VHH encoded for by the polynucleotide or vector is a strong binder VHH of the present invention. In a most preferred embodiment, the VHH, the VHH comprised in the fusion protein or complex or the VHH encoded for by the polynucleotide or vector is a strong binder high release VHH of the present invention.

[0203] In one embodiment, the EV of the present invention further comprises an AAV as described herein. In a preferred embodiment the AAV is comprised in the lumen of the EV. In a preferred embodiment, wherein the EV comprises a strong binder high release VHH of the present invention, the AAV has been released by the VHH (i.e. is not bound or no longer bound to the VHH). In a further preferred embodiment wherein the EV comprises a strong binder high release VHH of the present invention, the EV is releasably loaded with the AAV.

[0204] As used herein, the term “extracellular vesicle” or “EV” refers to any type of vesicle that is obtainable from a cell in any form, as well as an extracellular vesicle mimics and cellular membrane vesicles obtained through membrane extrusion, sonication or other techniques and hybridosomes obtained by fusing EVs with liposomes or nanoparticles. Essentially, an EV 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 or that has native therapeutic or pharmacological effects. Typically, an EV comprises a lipid-based membrane that encloses an internal space (i.e., lumen). The size of EVs may vary considerably, but an EV typically comprises has a radius of below 1000 nm.

[0205] The EVs of the present invention are genetically modified EVs. As used herein the term “modified” when used in relation to an EV can mean an EV that has been modified either using genetic or chemical approaches. As used herein the term “genetically modified” or “genetically engineered” when used in relation to an EV refers to an EV that is derived from a genetically modified cell that expresses and / or modifies the expression of proteins in the lumen, extravesicular membrane and / or displayed on the surface of the EV. Genetically modified EVs do not occur in nature.

[0206] In a preferred embodiment the EV of the present invention is derived from a cell, preferably a eukaryotic cell, more preferably a mammalian cell, more preferably a human cell. In a preferred embodiment, the EV is derived from an EV producer cell as described herein.

[0207] In a preferred embodiment, the EV of the present invention is a microvesicle (e.g. any vesicle shed from the plasma membrane of a cell) or more preferably an exosome (e.g. any vesicle derived from the endo-lysosomal pathway or from any other cellular pathway producing exosomes). In one embodiment, an exosome of the present invention has a radius of between 30 and 300 nm, preferably in between 40 and 250 nm, most preferably between 40 and 160 nm.

[0208] In one embodiment, the EV of the present invention may comprise one or more further fusion proteins. In one embodiment, the EV further comprises:

[0209] (i) a fusion protein that comprises an EV polypeptide and a purification moiety;

[0210] (ii) a fusion protein that comprises an EV polypeptide and a targeting moiety;

[0211] (iii) a fusion protein that comprises an EV polypeptide a pharmacokinetic or pharmacodynamic effector moiety; and / or

[0212] (iv) a fusion protein that comprises an EV polypeptide and an Fc-binding protein; In one embodiment wherein the EV comprises a further fusion protein comprising an Fc- binding protein, the Fc-binding protein is bound to an Fc-containing protein as described herein. Preferably, the Fc-containing protein is a purification moiety, a targeting moiety or a pharmacokinetic or pharmacodynamic effector moiety as described herein.

[0213] In one embodiment, the purification moiety, the targeting moiety, the Fc-binding protein, the pharmacokinetic effector moiety and / or the pharmacodynamic effector moiety are each comprised on separate further fusion proteins. In a preferred embodiment, the further fusion protein(s) comprise a different EV polypeptide to the one comprised in the fusion protein of the present invention.

[0214] In one embodiment the further fusion protein(s) also comprise a multimerization domain. In a preferred embodiment, the further fusion protein comprising the targeting moiety also comprises a multimerization domain.

[0215] In one embodiment, the fusion protein of the present invention or the EV of the present invention may further comprise a fusogen. As used herein, the term “fusogen” refers to any protein or agent that is capable of fusing lipid bilayers. In one embodiment, the fusogen is capable of merging two separate lipid bilayers into a single continuous lipid bilayer, preferably in a target cell as defined herein. In one embodiment, the lipid bilayers are cell membranes. In a preferred embodiment, the fusogen is an endosomal escape domain. The inclusion of a fusogen in the fusion protein of the present invention or in the EV of the present invention may enhance bioactivity in a target cell. In one embodiment, where the EV of the present invention further comprises a fusogen, the EV comprises a further fusion protein, wherein the fusion protein comprises an EV polypeptide and the fusogen. In one embodiment, where the EV of the present invention further comprises a fusogen, the EV comprises the fusogen in one of the further fusion proteins described herein. In a preferred embodiment the fusogen is VSVG.

[0216] As used herein, the term “purification moiety” refers to any molecule including protein, protein domains and protein tags that can be used to purify EVs, preferably exosomes. In one embodiment, the purification moiety allows for the EVs to be purified by affinity purification. Affinity purification of EVs is described in WO 2018 / 153581 A1, WO2019 / 081474 A1 and WO2019 / 238626 A1 , which are incorporated by reference in their entirety. In a one embodiment, the purification moiety is fused or conjugated to the EV polypeptide, such that the purification moiety is displayed on the surface of the EV. In one embodiment, the purification moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is displayed on the surface of an EV. In one embodiment, the purification moiety is fused or conjugated, optionally via the polypeptide comprising an intein and an extein sequence of the present disclosure, to loop 1 or loop 2 of a tetraspanin EV polypeptide or to the N- terminus of single-pass transmembrane EV polypeptide.

[0217] In one embodiment, the targeting moiety of the further fusion protein is fused or conjugated to the EV polypeptide, such that the targeting moiety is displayed on the surface of the EV. In one embodiment, the targeting moiety of the further fusion protein is fused or conjugated to a domain or terminus of the EV polypeptide which is displayed on the surface of an EV. In one embodiment, the targeting moiety of the further fusion protein is fused or conjugated to loop 1 or loop 2 of a tetraspanin EV polypeptide or to the N- terminus of single-pass transmembrane EV polypeptide. In one, embodiment, the (additional) fusion protein further comprises a multimerization domain as described herein.

[0218] As used herein, the term “Pharmacokinetic Effector Moiety” relates to any molecule, including any small molecule, protein, peptide, antibody or nanobody, or fragment or domain thereof, capable of affecting the pharmacokinetics of the EV. The term “Pharmacodynamic Effector Moiety” as used herein relates to any to any molecule, including any small molecule, protein, peptide, antibody or nanobody, or fragment or domain thereof, capable of affecting the pharmacodynamics of the EV. In one embodiment, the pharmacokinetic or pharmacodynamic effector moiety is an albumin binding domain. In a one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to the EV polypeptide, such that the pharmacokinetic and / or pharmacodynamic effector moiety is displayed on the surface of the EV. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to a domain or terminus of the EV polypeptide which is displayed on the surface of an EV. In one embodiment, the pharmacokinetic and / or pharmacodynamic effector moiety is fused or conjugated to loop 1 or loop 2 of a tetraspanin EV polypeptide or to the N- terminus of single-pass transmembrane EV polypeptide.

[0219] In a further aspect, the present invention provides a population of EVs comprising a plurality of EVs of the present invention.

[0220] As used herein the term “population of EVs” or “EV population” refers to any plurality of EVs comprising a plurality of EVs of the present invention. In one embodiment of a population of EVs of the present invention, at least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and / or at least 95% of all the EVs present in the population are EVs of the present invention. In a preferred embodiment of a population of EVs of the present invention, at least 5%, at least 10%, at least 20%, at least 50%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and / or at least 95% of all the EVs present in the population comprise an AAV.

[0221] In a further aspect, the present invention provides a method for producing an EV or a population of EVs comprising the following steps: (i) introducing into an EV producer cell as described herein, a (first) polynucleotide or a vector construct of the present invention; and

[0222] (ii) expressing the (first) polynucleotide or vector construct in the EV-producing cell, thereby generating EVs comprising a VHH of the present invention or a fusion protein of the present invention.

[0223] In a preferred embodiment of the method of the present invention, the method further comprises a step of introducing into the same EV producing cell constructs for the production of an AAV and expressing said constructs, alongside the (first) polynucleotide or vector construct of the present invention, in the EV producer cell, thereby generating EVs that further comprise an AAV. In a preferred embodiment, the constructs for the production of an AAV are expressed in the EV producer cell at the same time as the polynucleotide or a vector construct of the present invention. In one embodiment, the constructs for the production of the AAV comprise AAV rep components, AAV cap components (preferably AAV9 cap components), an AAV transgene (e.g. a transgene flanked by AAV ITRs) and preferably helper components such as E2A, E4 and VA RNA.

[0224] In a preferred embodiment of the methods of the present invention, wherein the VHH is a strong binder high release VHH of the present invention, the method is for producing an EV that is releasably loaded with an AAV, preferably wherein the AAV comprises an AAV9 capsid.

[0225] In one embodiment of the methods of the present invention, the method further comprises a step of introducing into the same EV producing cell a further, polynucleotide construct, wherein the further polynucleotide construct encoding for a fusion protein, wherein the fusion protein comprises an EV polypeptide and a purification moiety, a targeting moiety, a pharmacokinetic moiety and / or pharmoeffector moiety and / or an Fc-binding protein, and expressing the further polynucleotide construct in the EV-producing cell, thereby generating EVs that further comprise a purification moiety, a targeting moiety, and / or a pharmacokinetic and / or pharmacodynamic effector moiety and / or an Fc-binding protein. In a preferred embodiment, the further polynucleotide construct is expressed in the EV producer cell at the same time as the polynucleotide or a vector construct of the present invention. In a preferred embodiment, wherein the first polynucleotide or a vector construct encodes for a fusion protein of the present invention (i.e. a fusion protein comprising an AAV binding protein or a VHH of the present invention), the fusion protein encoded for by the second, polynucleotide construct comprises a different EV polypeptide to the one comprised in the fusion protein encoded for by the first polynucleotide or a vector construct. In one embodiment, the methods for producing EVs of the present invention may comprise a step of purifying the EVs. Purification of EVs is achieved by any method including but not limited to: techniques comprising liquid chromatography (LC), high-performance liquid chromatography (HPLC), bead-eluate chromatography, ionic exchange chromatography, spin filtration, tangential flow filtration (TFF), hollow fiber filtration, centrifugation (preferably density gradient ultracentrifugation), immunoprecipitation, flow field fractionation, dialysis, microfluidic-based separation, etc., or any combination thereof. In an advantageous embodiment, the purification of the EVs is carried out using a sequential combination of filtration (preferably ultrafiltration (UF) tangential flow filtration (TFF) or hollow fibre filtration) and affinity chromatography, optionally also including size exclusion LC or bead-eluate LC. Combining purification steps normally enhances the purity of the resulting samples and, in turn leads to superior therapeutic activity. Further, as compared to UC, which is routinely employed for purifying exosomes, sequential filtration-chromatography is considerably faster and possible to scale to higher manufacturing volumes, which is a significant drawback of the current UC methodology that dominates the prior art. Another advantageous purification method is TFF, which offers scalability and purity, and which may be combined with any other type of purification technique.

[0226] In one aspect, the VHHs, fusion proteins, complexes, polynucleotides and vectors of the present invention are for use in the loading of an EV with an AAV. In a preferred embodiment, the VHH, the VHH comprised in the fusion protein or complexes or the VHH encoded for by the polynucleotide or vector is a strong binder VHH of the present invention.

[0227] In one aspect, the VHHs, fusion proteins, complexes, polynucleotides and vectors of the present invention are for use in the releasable loading of an EV with an AAV. In a preferred embodiment, the VHH, the VHH comprised in the fusion protein or complexes or the VHH encoded for by the polynucleotide or vector is a strong binder high release VHH of the present invention.

[0228] In one aspect, the present invention provides a method of loading an EV with an AAV, preferably wherein the AAV comprises an AAV9 capsid, the method comprising expressing in an EV producer cell

[0229] (i) the AAV and

[0230] (ii) a VHH, a fusion protein, a complex, a polynucleotide and / or a vector of the present invention under conditions which, in no particular order, the VHH binds the AAV and which the AAV bound to the VHH loads into the EV. In a preferred embodiment, the VHH, the VHH comprised in the fusion protein or complexes or the VHH encoded for by the polynucleotide or vector is a strong binder VHH of the present invention.

[0231] In one aspect, the present invention provides a method of loading an EV with an AAV, preferably wherein the AAV comprises an AAV9 capsid, the method comprising

[0232] (i) producing the AAV in a cell;

[0233] (ii) expressing a VHH, a fusion protein, a complex, a polynucleotide and / or a vector of the present invention in the same cell; and

[0234] (iii) culturing the cell under conditions which, in no particular order, the VHH binds the AAV, the cell produces an EV and the AAV bound to the VHH loads into the EV.

[0235] In one embodiment of the methods or uses of the present invention, the present invention provides the use of a VHH, a fusion protein, a complex, a polynucleotide and / or a vector of the present invention for loading an EV with an AAV, preferably wherein the AAV comprises an AAV9 capsid.

[0236] In a preferred embodiment of the methods or uses of the present invention, the VHH, the VHH comprised in the fusion protein or complexes or the VHH encoded for by the polynucleotide or vector is a strong binder VHH of the present invention.

[0237] In a more preferred embodiment of the methods or uses of the present invention, the loading is releasable loading and the VHH, the VHH comprised in the fusion protein or complexes or the VHH encoded for by the polynucleotide or vector is a strong binder high release VHH of the present invention.

[0238] In one embodiment of the methods of the present invention, the VHH is expressed by transfecting or transducing the cell with a polynucleotide or vector of the present disclosure that encodes for the VHH.

[0239] In one embodiment of the methods of the present invention, the AAV is expressed or produced by transfecting or transducing the cell with constructs for producing an AAV, including one or more constructs encoding AAV rep components, AAV cap components, AAV cap components (preferably AAV9 cap components), an AAV transgene (e.g. a transgene flanked by AAV ITRs) and preferably helper components such as E2A, E4 and VA RA. As used herein “cell culture” or “culturing” when used in relation to a cell, refers to the process of growing cells under controlled conditions. In one embodiment, the cells are grown outside their natural environment. The conditions required to grow different cell types are known to the skilled person. In one embodiment, the cell is cultured in a cell culture medium, at around 37°C, in around 5% CO2. In a preferred embodiment, the cell is cultured in serum-free conditions. In one embodiment, culturing is adherent cell culture. In a preferred embodiment, the cell is cultured in suspension.

[0240] In a preferred embodiment of the methods or uses of the present invention, the cell is an EV producer cell as described herein.

[0241] In a further preferred embodiment, the cell is both an EV producer cell as described herein and a cell that the VHH binds the AAV, the cell produces an EV and the AAV bound to the VHH loads into the EV when cultured under conditions in which the cell is able to survive or preferably grow. In a more preferred embodiment, the cell is both an EV producer cell as described herein and a cell that the VHH binds the AAV, the cell produces an EV and the AAV bound to the VHH loads into the EV when cultured under conditions that are normal, common or optimum conditions for the cell’s growth.

[0242] In a further aspect, the present invention provides an EV or a population of EVs directly obtained by the methods of the present invention.

[0243] In one embodiment, the present invention provides the use of a VHH, a fusion protein, a complex, a polynucleotide and / or a vector of the present invention for releasably loading an EV with an AAV, preferably wherein the AAV comprises an AAV9 capsid. In a preferred embodiment, the VHH, the VHH comprised in the fusion protein or complexes or the VHH encoded for by the polynucleotide or vector is a strong binder high release VHH of the present invention.

[0244] As used herein, the term “EV loading” or “loading” refers to the incorporation of a cargo into the extracellular vesicle. EV loading may be exogenous or, in a preferred embodiment of the present invention, endogenous. As used herein, the term “exogenous” when used in relation to loading refers to the incorporation of the cargo into EVs after they have been secreted or released by EV producer cells. As used herein, the term “endogenous” refers to the incorporation of the cargo into EVs during their biogenesis in EV producer cells. In the present invention, the endogenous EV loading is active. As used herein, the term “active” when used in relation to endogenous loading refers to when the incorporation of the cargo in the producer cell into the EV involves an EV polypeptide that is either fused to the cargo or is fused to a polypeptide that binds to the cargo. Active loading increases EV loading as compared to passive endogenous loading, where the cargo is simply expressed in the EV producer cells. This increase in loading may include a higher proportion of the EVs produced being loaded with the cargo and / or the loaded EVs comprising a higher amount of the cargo.

[0245] In a preferred embodiment of the present invention, the loading is luminal loading.

[0246] As used herein, the term “releasable loading” refers to the loading of an EV with a cargo by association with an EV polypeptide or EV localisation moiety, wherein the cargo is subsequently released from the EV polypeptide or EV localisation moiety. Preferably the cargo is released in the EV lumen and / or in the target cell. In a preferred embodiment of the present invention, the AAV is loaded into the EV by active, endogenous methods. In a more preferred embodiment, the AAV is loaded into the EV by expression of a fusion protein comprising an EV polypeptide and a strong binder high release VHH of the present invention. In a further preferred embodiment, the loading is luminal.

[0247] In one embodiment, the VHHs, fusion proteins, complexes, polynucleotides and vectors of the present invention are for use in the bioactive, EV-mediated delivery of an AAV to a target cell. In a preferred embodiment, the VHH, the VHH comprised in the fusion protein or complexes or the VHH encoded for by the polynucleotide or vector is a strong binder high release VHH of the present invention.

[0248] In one aspect, the present invention further provides a composition comprising a VHH, a fusion protein, a complex, a polynucleotide, a vector, an EV or a population of EVs of the present invention and a pharmaceutically acceptable excipient and / or carrier.

[0249] The term "pharmaceutically acceptable" is used herein to refer to a material may be administered to a subject without causing any undesirable biological effects.

[0250] The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. In one embodiment, the pharmaceutically acceptable excipient is any substance approved by a regulatory agency such as the FDA or EMEA or listed in the U.S. Pharmacopeia for use in animals, including humans. In one embodiment the pharmaceutically acceptable carrier comprises an aqueous solution comprising a dual phosphate system as a pH buffer, wherein the pharmaceutically acceptable carrier optionally comprises a monosaccharide, disaccharide, polyvinylpyrrolidone, polyvinyl alcohol, dihydric alcohol, polyhydric alcohol (optionally sorbitol, polyethylene glycol or propylene glycol) and / or a detergent, optionally a polyoxyethylenesorbitan (Tween). In one embodiment, the pharmaceutically acceptable carrier is any substance that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the therapeutic cargo. In a preferred embodiment the excipients and carriers are generally safe and non-toxic.

[0251] The pharmaceutical compositions of the present invention may be formulated by any known method of formulation. In one aspect, the pharmaceutical compositions of the present disclosure may be formulated as Oral formulations, including Tablet, Capsule, Sustained release, liquid; Intravenous Formulations; Parenteral Formulations; Topical Formulations; cutaneous administration including cream, ointment, gel, paste, powder; Modified release Formulations including sustained release formulation and Liquid or lyophilized formulations.

[0252] In one aspect, the present invention provides a method of delivering an AAV to a target cell as described herein, comprising contacting or incubating the target cell with an EV, a population of EVs or a composition of the present invention.

[0253] In one aspect, the present invention provides the use of an EV, a population of EVs or a composition of the present invention for the delivery of an AAV to a target cell as described herein.

[0254] In one aspect, the present invention provides a method of expressing a transgene and / or altering the gene expression in a target cell as described herein, comprising contacting or incubating the target cell with an EV, a population of EVs or a composition of the present invention.

[0255] In one aspect, the present invention provides the use of an EV, a population of EVs or a composition of the present invention for expressing a transgene and / or altering the gene expression in a target cell as described herein.

[0256] In one embodiment of the methods or uses of the present invention, the AAV comprises an AAV9 capsid. In a preferred embodiment of the methods or uses of the present invention, the VHH comprised in the EV, population of EVs or composition of the present invention is a strong binder high release VHH of the present invention and the delivery is bioactive delivery as described herein

[0257] In one aspect, the present invention further provides a VHH, a fusion protein, a complex, a polynucleotide, a vector, an EV, a population of EVs or a composition of the present invention for the preparation of a medicament for treatment or prevention of a disease in a subject.

[0258] In one aspect, the present invention further provides a VHH, a fusion protein, a complex, a polynucleotide, a vector, an EV, a population of EVs or a composition of the present invention for use as a medicament for treatment or prevention of a disease in a subject.

[0259] In one aspect, the present invention further provides a method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of a VHH, a fusion protein, a complex, a polynucleotide, a vector, an EV, a population of EVs or a composition of the present invention to a subject suffering from or susceptible to the disease.

[0260] The terms "Treat" or "treatment" as used herein refer to therapeutic treatment of a subject who exhibits signs or symptoms of pathology for the purpose of diminishing or eliminating those signs or symptoms. The signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective. In a preferred aspect, "Treat" or "treatment" refers to the reduction or amelioration of the progression, severity, and / or duration of a disease (or symptom related thereto). Ameliorate as used herein refers to the action of lessening the severity of symptoms, progression, or duration of a disease.

[0261] As used herein, the term "effective amount" refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results.

[0262] It will be clear to the skilled artisan that when describing medical and scientific uses and applications of the EVs, the present invention normally relates to a plurality of EVs, i.e. a population of EVs which may comprise thousands, millions, billions or even trillions of EVs. As can be seen from the experimental section below, EVs may be present in concentrations such as 105108, 1010, 1011, 1012, 1013, 1014, 1015, 1018, 1025,1030EVs (often termed “particles”) per unit of volume (for instance per ml or per litre), or any other number larger, smaller or anywhere in between. In the same vein, the term “population” shall be understood to encompass a plurality of entities which together constitute such a population. In other words, individual EVs when present in a plurality constitute an EV population. Thus, the present invention pertains both to individual EVs and populations comprising EVs. 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 AAV cargo, any targeting moieties present on the EVs, the pharmaceutical formulation, etc.

[0263] As used herein, a "subject" refers to an animal that is the object of treatment, observation or experiment. "Animal" includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles, and in particular, mammals. "Mammal," as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals. In one aspect, the subject is a mammal, such as mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes. In a preferred embodiment, the subject is a human. In a more preferred embodiment, the subject is a child. Preferably the child is under 21 years of age, under 18 years of age under 16 years of age, or more preferably, is pubescent or pre-pubescent. Most preferably the child’s organs are still growing.

[0264] The VHH, fusion protein, complex, polynucleotide, vector, EV, population of EVs or composition of the present invention may be administered to a human or animal subject via various different administration routes, for instance auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracerebroventricular, intracisternal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratym panic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above administration routes, which typically depends on the disease to be treated. In a preferred embodiment, the VHH, fusion protein, complex, polynucleotide, vector, EV, population of EVs or composition of the present invention is administered via intravenous, intrathecal, intracerebroventricular, intraparenchymal, intra-cisterna magna or intra-coronary routes.

[0265] In one embodiment of the uses or methods of the present invention, the disease is a genetic disorder. As used herein, the term “genetic disorder” refers to a disorder or a disease caused by one or more abnormalities in the DNA. Preferably the abnormality in the DNA causes reduced or no expression of a gene or causes the gene product to have reduced or no function. More preferably, the genetic disorder can be treated, or symptoms may be improved, by expressing a copy of the gene that does not comprise the abnormality. In a preferred embodiment, the genetic disorder is a liver disorder, a neurological disorder or a cardiac disorder.

[0266] In one embodiment of the uses or methods of the present invention, the subject is administered with two or more doses of the VHH, fusion protein, complex, polynucleotide, vector, EV, population of EVs or composition of the present invention. In one embodiment, the doses are at least several weeks, preferably several years apart.

[0267] In one embodiment of the uses or methods of the present invention, the subject has immunity to the AAV comprised in the complex, EV, population of EVs or composition of the present invention. In one embodiment, the subject has been previously exposed to an AAV capsid, preferably the AAV capsid comprised in complex, EV, population of EVs or composition administered to the subject. In one embodiment, this exposure is due to previous administration of an AAV comprising an AAV capsid preferably the AAV capsid comprised in complex, EV, population of EVs or composition administered to the subject.

[0268] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where features, aspects, embodiments, or alternatives of the present invention are described in terms of Markush groups, a person skilled in the art will recognise that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. The person skilled in the art will further recognise that the invention is also thereby described in terms of any combination of individual members or subgroups of members of Markush groups. Additionally, it should be noted that embodiments and features described in connection with one of the aspects and / or embodiments of the present invention also apply mutatis mutandis to all the other aspects and / or embodiments of the invention. Any accession numbers or SEQ ID NOs mentioned herein in connection with peptides, polypeptides and proteins shall only be seen as examples and for information only, and all peptides, polypeptides and proteins shall be given their ordinary meaning as the skilled person would understand them. Thus, as above- mentioned, the skilled person will also understand that the present invention encompasses not merely the specific SEQ ID NOs and / or accession numbers referred to herein but also variants and derivatives thereof. All accession numbers referred to herein are UniProtKB accession numbers, and all proteins, polypeptides, peptides, nucleotides and polynucleotides mentioned herein are to be construed according to their conventional meaning as understood by a skilled person.

[0269] EXAMPLES

[0270] Example 1 - Identification of VHHs to Capsid 9

[0271] An alpaca was immunised with an AAV comprising an AAV9 capsid. Peripheral blood mononuclear cells (PBMCs) were isolated from the alpaca.

[0272] 12 VHH sequences of interest were identified:

[0273] • VHH1 having SEQ ID NO 84

[0274] • VHH2 having SEQ ID NO 81

[0275] • VHH3 having SEQ ID NO 83

[0276] • VHH4 having SEQ ID NO 82

[0277] • VHH5 having SEQ ID NO 85

[0278] • VHH6 having SEQ ID NO 77

[0279] • VHH7 having SEQ ID NO 78

[0280] • VHH8 having SEQ ID NO 74

[0281] • VHH9 having SEQ ID NO 75

[0282] • VHH10 having SEQ ID NO 79

[0283] • VHH11 having SEQ ID NO 76 and

[0284] • VHH12 having SEQ ID NO 80.

[0285] Example 2 - Active loading using a VHH offers an improvement over passive loading and EV polypeptide TSN2 results in the highest levels of loading

[0286] The present inventors carried out initial tests with VHH3 (having SEQ ID NO 83) to assess whether this VHH could be used to load AAV into the lumen of EVs.

[0287] AAV9 component constructs, including an AAV Factor IX transgene construct, and a helper virus construct were expressed in EV producer cells (Gibco™ VPC 2.0 suspension cells) in a 30 mL culture.

[0288] A fusion protein comprising an EV polypeptide fused to VHH was also expressed in the EV producer cells. The following fusion proteins were tested:

[0289] - BASP1-VHH3 (i.e. VHH3 fused to the C-terminus of BASP1 via a linker having SEQ ID NO 97) - TSN2-VHH3 (i.e. VHH3 fused to the C-terminus of TSN2 having SEQ ID NO 99 via a linker having SEQ ID NO 88)

[0290] - PTGFRN-VHH3 (i.e. VHH3 fused to the C-terminus of PTGFRN via a linker having SEQ ID NO 98)

[0291] - Single pass transmembrane EV polypeptide (i.e. VHH3 fused to the C-terminus of a single pass transmembrane EV polypeptide via a linker having SEQ ID NO 88).

[0292] - A “stuffer control” was also included, where the fusion protein is absent and replaced by a non-coding sequence. In this control, only “passive” luminal loading of the AAV occurs.

[0293] Density gradient ultracentrifugation (DGLIC) was performed on the cultures and the expression of capsids (viral protein) across the different fractions obtained was evaluated by western blot.

[0294] Figure 1a shows the expression of viral capsid (VP) and syntenin (an EV marker) in different fractions from the EV producer cells in which the stuffer control is expressed. The viral capsid protein is not observed in the fractions where there is high expression of syntenin and is only observed in the denser fractions (fractions 8-12, iodixanol concentrations from 25% to 60%). This indicates that only a very small amount of AAV (i.e. too little to be detected) is passively loaded into the EVs.

[0295] Figures 1b to 1e show the expression of viral capsid (VP) and syntenin (an EV marker) in different fractions from the EV producer cells that express BASP1-VHH3, TSN2-VHH3, PTGFRN-VHH3 and Single pass EV polypeptide P-VHH respectively. In all of these western blots, capsid proteins are partially co-localising with EV fractions (fractions 1-7, iodixanol concentration from 0% to 25%). This indicates that, when the producer cell expresses a VHH against the AAV capsid fused to any EV polypeptide, there is increased EV loading of the AAV.

[0296] Loading of vector genomes was evaluated by digital PCR (dPCR). Figure 2a shows viral genome quantification in the different fractions from the EV producer cells that express BASP1-VHH3, TSN2-VHH3, PTGFRN-VHH3 and single pass EV polypeptide-VHH. This figure further supports that, when the producer cell expresses a VHH against the AAV capsid fused to any EV polypeptide, there is EV loading of the AAV. This figure also indicates that the highest levels of loading are achieved when tetraspanins are used as the EV polypeptide.

[0297] Figure 2b shows the overall viral genome (VG) titre in EV fractions, normalised to that observed from the EV producer cells that express BASP1-VHH3. This data shows that EV producer cells that express the tetraspanin TSN2 fused to VHH3 have over 150-fold higher titres than is achieved when VHH3 is fused to other EV polypeptides.

[0298] Example 3 - VHH1, VHH2, VHH3, VHH4, VHH5 and VHH9 result in the highest levels of loading

[0299] The inventors went on to perform a VHH screen to assess which of the VHHs allow for the highest level of loading. EV producer cells (Gibco™ VPC 2.0 suspension cells) were transfected with TSN2-VHH fusions (i.e. the VHH fused to the C-terminus of TSN2 via a linker having SEQ ID NO 88), along with AAV9 components, including a human factor IX transgene under the control of a liver specific promoter (which avoids loading of the factor IX protein from producer cells), and helper elements.

[0300] Fractions were then obtained from the cultures using density gradient ultracentrifugation (DGLIC). The collected fractions were analysed by Western Blot to assess the presence of capsids (viral proteins).

[0301] Figures 3a-l show the expression of viral capsid (VP) in the in different fractions from the EV producer cells (EV fractions are fractions 1-7). These data indicate that that some VHHs result in higher levels of loading than others.

[0302] Loading of vector genomes was then evaluated by digital PCR (dPCR).

[0303] Figure 4a-c shows viral genome quantification in the different fractions from the EV producer cells. Figure 4a shows the VHHs that load approximately 20% of total produced viral genome in EV fractions (VHH1, VHH2, VHH3, VHH4, VHH5 and VHH9). Figure 4b shows the VHHs that load approximately 5% of total produced viral genome in EV fractions (VHH8 and VHH11). Figure 4c shows the VHHs that load around 1% of total produced viral genome in EV fractions (VHH7, VHH10 and VHH12).

[0304] Example 4 - VHH2 and VHH4 result in the highest level of bioactivity in target cells The inventors went on to investigate which VHH allowed for the highest level of bioactivity in target cells. In order for the AAV cargo to have bioactivity in target cells, as well as allowing for the AAV to be efficiently loaded into EVs in the EV-producer cells the VHH must also allow for the AAV to be released in a target cell.

[0305] EVs purified as described in example 3 from a 1 litre culture of EV producer cells (Gibco™ VPC 2.0 suspension cells) that has been transfected with TSN2-VHH fusions (i.e. VHH1 , VHH2, VHH3, VHH4, VHH5 or VHH8 fused to the C-terminus of TSN2 having SEQ ID NO 99 via a linker having SEQ ID NO 88), along with AAV9 components, a human factor IX transgene under the control of a liver specific promoter (which avoids loading of the factor IX protein from producer cells) and helper elements. Fractions 6 and 7 from each batch were pooled and assayed for Factor IX activity in HLIH7 cells. AAVs that are not encapsulated in EVs were also added to the HLIH7 cells as a control. Samples were tested using two starting initial cell densities (8750 cell / well or 27500 cell / well in 96 well plates), to test ExoAAV performance at different degrees of cell confluency.

[0306] Figures 5A and 5B shows that EVs from producer cells that expressed TSN2-VHH2 and TSN2-VHH4 exhibit exponential dose-response activity trends that are largely higher than free AAV9 control, indicating the EVs comprising these constructs are more potent that the free AAV9 control. Results from assays performed at different cell confluency are very comparable, suggesting no correlation between cell density and transduction.

[0307] Following the Factor IX potency assay, HLIH7 cells treated with EVs from producer cells that expressed TSN2-VHH2, TSN2-VHH3, TSN2-VHH4 or with free AAV were collected and processed to measure intracellular viral genomes to understand the levels of transgene present after transduction.

[0308] Figure 5C shows that all tested EV samples are internalised by cells more efficiently, when compared to free AAV9. Uptake increases linearly in direct correlation with higher MOIs, and no difference is observed between EV samples. The dose-response results in Figures 5A and B show that the EVs comprising TSN2-VHH3 have lower activity than AAV9 control and tends to plateau, while the EVs comprising TSN2-VHH2 or TSN2-VHH4 maintain an exponential trend. This indicates that the explanation for different potency of EV samples is not related to cell uptake but is instead due to differences in AAV release efficiency after cell internalisation (i.e. VHH2 and VHH4 allow for release of the AAV and thus increased bioactivity in the target cell). Example 5 - Encapsulation of AAV cargo in EVs results in protection from neutralising antibodies and increased bioactivity.

[0309] The present inventors went on to perform a neutralisation assay in presence of increasing concentrations of anti-AAV9 neutralising antibodies (ADK9) in order to better understand the degree of protection granted by loading AAV9 into the EV lumen.

[0310] The assay was performed using EVs purified, as described in example 3, from a 30 mL culture of Gibco™ VPC 2.0 producer cells that expressed a fusion protein comprising TSN2 and VHH4 (i.e. VHH4 fused to the C-terminus of TSN2 via a linker having SEQ ID NO 88) together with AAV9 components, a human factor IX transgene under the control of a liver specific promoter and helper elements. AAVs that are not encapsulated in EVs were used as a control.

[0311] The assay used was the same Factor IX potency assay described in example 4 above.

[0312] Figure 6A shows the Factor IX levels expressed as a percentage of Factor IX, compared to calibrator plasma, which Factor IX activity is set conventionally at 100%. Cells treated with 7x103MOI of EVs were able to maintain stable Factor IX activity levels despite increasing antibody concentrations, with activity only declining by 50% at the highest concentration. Conversely, cells treated with free AAV9 show stably declining activity correlated with antibody concentration. This indicates that luminal loading of AAVs into EVs protects the AAV cargo from neutralising antibodies and allows for increased bioactivity.

[0313] The inventors then went on to perform a larger-scale neutralisation assay using EVs purified, as described in example 3, from a 1 L culture of Gibco™ VPC 2.0 producer cells that expressed a fusion protein comprising TSN2 and VHH2, -VHH3 and -VHH4 (i.e. VHH2, VHH3 or VHH4 fused to the C-terminus of TSN2 via a linker having SEQ ID NO 88). As above, Factor IX activity was assayed in Huh7 cells when increasing concentrations of anti- AAV9numbers antibodies alongside the EVs or AAV.

[0314] Figure 6B and C show that Factor IX activity of cells treated with all ExoAAV samples remained stable, while AAV9 F.IX activity declined steadily, at increasing antibody doses. This data confirms that luminal loading of AAVs into EVs protects the AAV cargo from neutralising antibodies and allows for increased bioactivity. SEQUENCE LISTING

[0315]

Claims

CLAIMS1. A fusion protein comprising or consisting of:(i) an extracellular vesicle (EV) polypeptide and(ii) an adeno-associated virus (AAV) binding polypeptide, wherein the EV polypeptide is a tetraspanin.

2. A fusion protein-AAV complex comprising or consisting of an AAV and a fusion protein of claim 1, wherein the AAV is bound to the AAV binding polypeptide of the fusion protein.

3. The fusion protein of claim 1 or the fusion protein-AAV complex of claim 2, wherein the tetraspanin is CD63, TSN4, TSN9 or preferably TSN2.

4. The fusion protein or fusion protein-AAV complex of any one of claims 1 to 3, wherein the AAV binding polypeptide is a Variable Heavy domain of Heavy chain (VHH) capable of binding to an AAV.

5. The fusion protein or fusion protein-AAV complex of any one of claims 1 to 4, wherein the AAV binding polypeptide is a VHH, wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO 69, SEQ ID NO 70 or SEQ ID NO 60.

6. The fusion protein or fusion protein-AAV complex of claim 5, wherein the CDR1 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25 or SEQ ID NO 17.

7. The fusion protein or fusion protein-AAV complex of claim 5 or claim 6, wherein the CDR2 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43 or SEQ ID NO 35.

8. A method of releasably loading an EV with an AAV, the method comprising(i) producing the AAV in a cell;(ii) expressing a VHH in the same cell; and(iii) culturing the cell under conditions which, in no particular order, the VHH binds the AAV, the cell produces an EV and the AAV bound to the VHH loads into the EV; wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

9. Use of a VHH, or a polynucleotide sequence encoding a VHH, for releasable loading of an EV with an AAV, wherein the VHH comprises a CDR1, a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

10. A VHH-AAV complex comprising or consisting of an AAV releasably bound to a VHH, wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

11. A fusion protein-AAV complex comprising or consisting of an AAV and a fusion protein, wherein the fusion protein comprises an EV polypeptide and a VHH, wherein the AAV is releasably bound to the VHH, wherein the VHH comprises a CDR1, a CDR2 and a CDR3, wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 67, SEQ ID NO 69 and SEQ ID NO 70.

12. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 11, wherein the VHH comprises a CDR1 , a CDR2 and a CDR3, further wherein the CDR3 comprises or consists of an amino acid sequence having over its entire length at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67.

13. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of claim 12, wherein the CDR3 amino acid sequence comprises:(i) a K as the amino acid residue at position number 2 of SEQ ID NO 67,(ii) an N as the amino acid residue at position number 3 of SEQ ID NO 67,(iii) a Y as the amino acid residue at position number 8 of SEQ ID NO 67,(iv) a T as the amino acid residue at position number 11 of SEQ ID NO 67,(v) an H as the amino acid residue at position number 12 of SEQ ID NO 67 and / or(vi) a T as the amino acid residue at position number 15 of SEQ ID NO 67.

14. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 13, wherein the VHH is capable of binding to an AAV, preferably wherein the AAV comprises an AAV9 capsid or a derivative, variant, mutant, domain or region thereof, preferably wherein the AAV9 capsid comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 over its entire length.

15. The fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 2 to 14, wherein the AAV comprises an AAV9 capsid or a derivative, variant, mutant, domain or region thereof, preferably wherein the AAV9 capsid comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 1 over its entire length.

16. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 12 to 15, wherein the CDR1 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21 over its entire length.

17. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 12 to 16, wherein the CDR2 comprises or consists of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 39 over its entire length.

18. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of claim 17, wherein the CDR2 amino acid sequence comprises:(i) an S as the amino acid residue at position number 4 of SEQ ID NO 39,(ii) a G as the amino acid residue at position number 5 of SEQ ID NO 39,(iii) a D as the amino acid residue at position number 6 of SEQ ID NO 39 and / or(iv) an S as the amino acid residue at position number 7 of SEQ ID NO 39.

19. The method or the use of any one of claims 8, 9, 12 to 18, wherein the AAV is loaded into the lumen of the EV.

20. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 19, wherein the VHH loads more than 7%, preferably more than 10% or more preferably more than 15% of the total produced viral genome into EVs when expressed in an EV producer cell together with an AAV to which the VHH is capable of binding.

21. The fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 20, wherein the AAV is released from the VHH.

22. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 21 , wherein the VHH comprises an FR1 , an FR2, an FR3 and an FR4, wherein the FR1, the FR2, the FR3 and the FR4 form beta-sheet structures.

23. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 22, wherein the VHH comprises(i) an FR1 that comprises or consists of an amino acid sequence having over its entire length at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acidsequence selected from SEQ ID NO 3 to 14, preferably SEQ ID NO 4 or 10 to 14 most preferably SEQ ID NO 10 or 11 ;(ii) an FR2 that comprises or consists of an amino acid sequence having over its entire length at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 27 to 32, preferably SEQ ID NO 28, 31 or 32, most preferably SEQ ID NO 31 ;(iii) an FR3 that comprises or consists of an amino acid sequence having over its entire length at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NO 44 or SEQ ID NO 46 to 53, preferably SEQ ID NO 46, 51 , 52 or 53, most preferably SEQ ID NO 55 or 56; and / or(iv) an FR4 that invention comprises or consists of an amino acid sequence having over its entire length at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid having SEQ ID NO 72 or preferably having SEQ ID NO 73 over its entire length.

24. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 23, wherein the VHH comprises or consists of:(i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67 over its entire length, preferably wherein the amino acid residue at position number 2 of SEQ ID NO 67 is K, at position number 3 is N, at position number 8 is Y, at position number 11 is T, at position number 12 is H and / or at position number 15 is T;(ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21 over its entire length;(iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 39 over its entire length, preferably wherein the amino acid residue at position number 4 of SEQ ID NO 39 is S, at position 5 is G, at position 6 is D and at position 7 is S;(iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 10 over its entire length;(v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 31 over its entire length;(vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO55 over its entire length; and(vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73 over its entire length.

25. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 23, wherein the VHH comprises or consists of:(i) a CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 67 over its entire length, preferably wherein the amino acid residue at position number 2 of SEQ ID NO 67 is K, at position number 3 is N, at position number 8 is Y, at position number 11 is T, at position number 12 is H and / or at position number 15 is T;(ii) a CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 21 over its entire length;(iii) a CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 39 over its entire length, preferably wherein the amino acid residue at position number 4 of SEQ ID NO 39 is S, at position 5 is G, at position 6 is D and at position 7 is S;(iv) an FR1 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 11 over its entire length;(v) an FR2 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 31 over its entire length;(vi) an FR3 having at least 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO56 over its entire length; and(vii) an FR4 having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 73 over its entire length.

26. The fusion protein, fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 4 to 23, wherein the VHH comprises or consists of an amino acid sequence having over its entire length at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO 81 or SEQ ID NO 82.

27. The method, use or VHH-AAV complex of any one of claims 8 to 10 or 12 to 26, wherein the VHH is fused to an EV polypeptide.

28. The method, use or VHH-AAV complex of claim 27 or the fusion protein-AAV complex of any one of claims 11 to 26, wherein the EV polypeptide is a single pass transmembrane protein or a tetraspanin.

29. The method, use or VHH-AAV complex of claim 27 or the fusion protein-AAV complex of any one of claims 11 to 26, wherein the EV polypeptide is selected from TSN4, TSN9, CD63, PTGFRN, Lamp2b or preferably is TSN2.

30. The method, use or VHH-AAV complex of claim any one of claims 27 to 29, the fusion protein-AAV complex of any one of claims 11 to 26, 28 or 29, or the fusion protein of any one of claims 1 to 7 or 12 to 26, wherein the VHH is fused to a domain of the EV polypeptide that is displayed in the lumen of the EV.

31. The method, the use or the VHH-AAV complex of claim any one of claims 27 to 29, the fusion protein-AAV complex of any one of claims 11 to 26, 28 or 29, or the fusion protein of any one of claims 1 to 7 or 12 to 26, wherein the EV polypeptide is a tetraspanin, wherein the VHH is fused to the N-terminus or preferably the C-terminus of the tetraspanin, preferably wherein the tetraspanin is TSN2.

32. The method, the use or the VHH-AAV complex of any one of claim 27 to 31 , the fusion protein-AAV complex of any one of claims 11 to 26 or 28 to 31 or the fusion protein of any one of claims 1 to 7, 12 to 26, 30 or 31 , wherein the EV polypeptide further comprises a targeting moiety, preferably wherein the targeting moiety is fusedor conjugated to a domain of the EV polypeptide that is displayed on the surface of the EV.

33. The method, the use or the VHH-AAV complex of any one of claim 27 to 31 , the fusion protein-AAV complex of any one of claims 11 to 26 or 28 to 31 or the fusion protein of any one of claims 1 to 7, 12 to 26, 30 or 31 , wherein the EV polypeptide further comprises a targeting moiety, wherein the EV polypeptide is a tetraspanin, wherein the targeting moiety is fused or conjugated to loop 1 or loop 2 of the tetraspanin, preferably wherein the tetraspanin is TSN2.

34. The fusion protein-AAV complex, method, use or VHH-AAV complex of any one of claims 2 to 33, wherein the AAV comprises a transgene.

35. The method of any one of claims 8 or 12 to 34, wherein the cell is an EV producer cell.

36. The method of any one of claims 8 or 12 to 35, wherein the cell is(i) a eukaryotic cell, preferably a mammalian cell, more preferably a human cell; and / or(ii) a cell line, preferably a HEK cell line, more preferably a HEK293 cell line.

37. An EV releasably loaded with an AAV using a method of any one of claims 8 or 12 to 36, preferably wherein the AAV has been released from the VHH into the EV lumen.

38. An EV comprising the fusion protein, fusion-protein AAV complex or VHH-AAV complex of any one of claims 1 to 7 or 10 to 34.

39. An EV of claim 38, wherein the AAV has been released from the VHH into the EV lumen.

40. A population of EVs comprising a plurality of EVs of any one of claims 37 to 39.41 . A composition comprising the VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV or the population of EVs of any one of claims 1 to 7, 10 to 34 or 37 to 40 and a pharmaceutically acceptable excipient and / or carrier.

42. A method of bioactive delivery of an AAV to a target cell comprising contacting or incubating the target cell with an EV, a population of EVs or a composition of any one of claims 37 to 41.

43. Use of an EV, a population of EVs or a composition of any one of claims 37 to 41 for the bioactive delivery of an AAV to a target cell.

44. A method of expressing an AAV transgene and / or altering the gene expression in a target cell, comprising contacting or incubating the target cell with an EV, a population of EVs or a composition of any one of claims 37 to 4145. Use of an EV, a population of EVs or a composition of any one of claims 37 to 41 for expressing an AAV transgene and / or altering the gene expression in a target cell46. The use or method of any one of claims 42 to 45, wherein the target cell is in vivo, ex vivo or in vitro.

47. The use or method of any one of claims 42 to 46, wherein the target cell has a genetic abnormality that is linked to a disease or a disorder.

48. The use or method of any one of claims 42 to 47, wherein the target cell is a cell that is deficient in the RNA or polypeptide encoded for by the AAV transgene or wherein the RNA or polypeptide encoded for by the AAV comprises a transgene has reduced function or is non-functional.

49. The VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV, the population of EVs or the composition of any one of claims 1 to 7, 10 to 34 or 37 to 41 for the preparation of a medicament for treatment or prevention of a disease in a subject.

50. The VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV, the population of EVs or the composition of any one of claims 1 to 7, 10 to 34 or 37 to 41 for use as a medicament for treatment or prevention of a disease in a subject.

51. A method for treating or preventing a disease comprising administering a therapeutically or prophylactically effective amount of the VHH-AAV complex, the fusion protein, the fusion protein-AAV complex, the EV, the population of EVs or thecomposition of any one of claims 1 to 7, 10 to 34 or 37 to 41 to a subject suffering from or susceptible to the disease.

52. The uses or methods of claims 49 to 51 , wherein the subject is a human.53 The uses or methods of claims 49 to 52, wherein the disease is a genetic disorder, preferably a genetic liver disorder, a genetic neurological disorder or a genetic cardiac disorder.

54. The uses or methods of claims 49 to 53, wherein the subject is administered with two or more doses of the VHH, fusion protein, complex, polynucleotide, vector, EV, population of EVs or composition.

Images