Surface marker binding domain insert in VP3 capsid protein of AAV and method for producing recombinant aav

Abstract

The disclosure relates to an adeno-associated virus (AAV) capsid fusion protein comprising a surface marker binding domain (SMBD), in particular, a single-domain antibody (sdAb) or variable immunoglobulin domain of a heavy chain antibody (VHH), which is inserted in the GH2-GH3 loop of the AAV VP3 capsid protein. Also, an AAV vector particle comprising a SMBD inserted into the VP3 AAV capsid protein is contemplated. Further, nucleotide sequences encoding such fusion proteins and vectors including such nucleotide sequences are contemplated. Further encompassed is a recombinant adeno-associated virus (AAV) which comprises said AAV capsid fusion protein. Moreover the disclosure relates to uses of the fusion proteins, nucleic sequences and corresponding AAVs for use introducing a defined nucleic sequence into a target cell or for improving the transduction efficiency of the AAV targeted cells. The disclosure also relates to methods for producing recombinant AAV, in particular a recombinant AAV that comprises an insert of a surface marker binding domain (SMBD) in one type of the VP capsid protein selected from VP1, VP2 and VP3, but not in the other two types of VP capsid proteins.

Description

[0001] Surface Marker Binding domain insert in VP3 capsid protein of AAV and method for producing recombinant AAV

[0002] FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to an adeno-associated virus (AAV) capsid fusion protein comprising a surface marker binding domain (SMBD), in particular, a single-domain antibody (sdAb) or variable immunoglobulin domain of a heavy chain antibody (VHH), which is inserted in the GH2-GH3 loop of the AAV VP3 capsid protein. Also, an AAV vector particle comprising a SMBD inserted into the VP3 AAV capsid protein is contemplated. Further, nucleotide sequences encoding such fusion proteins and vectors including such nucleotide sequences are contemplated. Further encompassed is a recombinant adeno-associated virus (AAV) which comprises said AAV capsid fusion protein. Moreover, the disclosure relates to uses of the fusion proteins, nucleic sequences and corresponding AAVs for introducing a defined nucleic sequence into a target cell or for improving the transduction efficiency of the AAV targeted cells. The disclosure also relates to methods for producing recombinant AAV, in particular a recombinant AAV that comprises an insert of a surface marker binding domain (SMBD) in one type of the VP capsid protein selected from VP1, VP2 and VP3, but not in the other two types of VP capsid proteins.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Adeno-associated viruses (AAVs) are of increasing importance for gene and tumor therapy. AAVs are used as vectors, since they are non-pathogenic, show low immunogenicity, and confer longterm expression of the transferred gene.

[0006] A challenge in the use of AAVs as vectors in gene therapy is their broad tropism, i.e. the parallel infection of several tissues and cell types. Moreover, neutralizing antibodies that occur in about 80 % of the population impede the efficient binding and transduction of target cells.

[0007] Current techniques of improving target specificity of AAVs include the chemical conjugation of two Fab fragments (Bartlett et al. 1999), the insertion of peptides into the capsid proteins, the fusion of DARPins to the N-terminus of the VP2 capsid protein (Munch et al. 2013), and the use of AAV serotypes that are not recognized by neutralizing antibodies. However, there is still a need to increase the AAV portfolio for efficient cell targeting and to improve AAVs for therapy, in particular to improve tissue specificity of AAVs.

[0008] Also, previously described methods for the preparation of sdAb (Nb)-modified AAV capsids were based on the incorporation of the sdAbs into the GH2 / GH3 or GH12 / GH13 loop of VP1 or VP2. The disadvantage of this method is that the incorporation of the modified VP proteins turned out to be less efficient compared to the wild-type VP proteins, so that the resulting capsid population either does not possess the typical stoichiometry between VP1:VP2:VP3 of 1 : 1 : 10 or a high proportion of the capsids are totally VPl-Nb-less or VP2-Nb-less. In most cases, the sdAb-bearing capsid population has to be enriched via Protein A Sepharose chromatography and typically only about 2% of particles comprising the VPl-Nb is recovered. As this inefficient process is associated with relatively high losses, it makes the production of the capsids uneconomical for clinical applications.

[0009] Therefore, there is a need to provide an improved method for producing recombinant AAV with engineered capsid proteins that comprise an insert of a SMBD, such as a sdAb or VHH.

[0010] SUMMARY OF THE DISCLOSURE

[0011] A first aspect of the disclosure relates to an adeno-associated virus (AAV) capsid fusion protein comprising a VP3 AAV capsid protein and a surface marker binding domain (SMBD), wherein the SMBD is inserted in the GH2-GH3 loop of the VP3 AAV capsid protein.

[0012] Providing a new site for target-specific inserts, such as target specific SMBD (for example a sdAb or a VHH), allows the combination with other capsid modifications, including but not limited to point mutations, peptide insertions, ligand insertions, and / or capsid shuffling. Thereby, simultaneous targeting of two different molecules, in particular cell surface molecules, such as two different receptors that are expressed by the target tissue or target cell can be achieved.

[0013] Surprisingly, insertion of the SMBD (e.g. sdAb or VHH) sequence into the GH2-GH3 loop of the VP3 AAV capsid protein did not impair the structure and function of the VP3 AAV capsid fusion protein, such that AAV comprising said fusion protein comprising the SMBD (e.g. sdAb or VHH) in the GH2-GH3 loop of VP3 can be successfully assembled. This is surprising, since the sequence of the inserted SMBD (e.g. sdAb or VHH) has a considerable length of over 100 aa. It was not clear whether an insert of such length would not disturb the structural and functional integrity of the AAV capsid protein, in particular VP3, and the correspondingly assembled AAV.

[0014] A second aspect of the present disclosure relates to an AAV vector particle comprising

[0015] (a) a VP AAV capsid protein with an inserted surface marker binding domain (SMBD); and

[0016] (b) a wild-type VP AAV capsid protein that does not comprise an inserted SMBD; wherein the VP AAV capsid protein is VP1, VP2 and / or VP3.

[0017] In some embodiments, the AAV vector particle comprises:

[0018] (a) a VP3 AAV capsid protein with an inserted surface marker binding domain (SMBD), wherein the VP3 AAV capsid protein with the inserted SMBD comprises in the following order:

[0019] (i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein;

[0020] (ii) a second sequence segment which comprises a first peptide linker;

[0021] (iii) a third sequence segment which comprises the SMBD;

[0022] (iv) a fourth sequence segment which comprises a second peptide linker; and

[0023] (v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein; and

[0024] (b) a wild-type VP3 AAV capsid protein that does not comprise an inserted SMBD.

[0025] The AAV vector particles of the present disclosure are particularly useful to provide for efficient and target specific transduction into host cells.

[0026] Further aspects of the present disclosure relate to nucleotide sequences encoding such fusion proteins and vectors including such nucleotide sequences. Also, a recombinant adeno-associated virus (AAV) which comprises the AAV capsid fusion protein of the disclosure is contemplated. Moreover, the disclosure relates to uses of the fusion proteins, nucleic sequences and corresponding AAVs for introducing a defined nucleic sequence into a target cell and / or improving the transduction efficiency of the AAV targeted cells.

[0027] The disclosure furthermore relates to methods for producing recombinant AAV: In one aspect, the disclosure relates to a method for the preparation of a recombinant AAV, wherein the recombinant AAV comprises an SMBD inserted into a VP capsid protein selected from VP1, VP2 and / or VP3, wherein the method comprises transfecting into a host cell the following vectors:

[0028] (a) one or more vectors comprising a nucleic acid molecule encoding the AAV helper functions;

[0029] (b) a vector comprising a nucleic acid molecule encoding a VP1, VP2 and / or VP3 capsid protein with an inserted SMBD; and

[0030] (c) a vector comprising a nucleic acid molecule encoding a wild-type AAV VP1, VP2 and / or VP3 capsid protein; and wherein the ratio of vectors comprising the nucleic acid molecules according to (b) and (c) is from about 1 : 1 to about 1 :20, from 1 : 1 to 1 : 10 or from 1 : 1 to 1 :5.

[0031] To the knowledge of the present inventors, mixtures of unmodified and modified vectors have not been tried before nor has a respective method for the preparation of a recombinant AAV been disclosed before. The method of the present disclosure surprisingly provides for efficient assembly of AAV capsids and improved transduction efficiency of the AAV targeted cells.

[0032] In some embodiments, the method for the preparation of a recombinant AAV, wherein the recombinant AAV comprises a SMBD inserted into a VP3 capsid protein, and wherein the method comprises transfecting into a host cell the following vectors:

[0033] (a) one or more vectors encoding the AAV helper functions;

[0034] (b) a vector comprising a nucleic acid molecule encoding a VP3 AAV capsid protein with an inserted SMBD, wherein the inserted SMBD comprises in the following order:

[0035] (i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein;

[0036] (ii) a second sequence segment which comprises a first peptide linker;

[0037] (iii) a third sequence segment which comprises the SMBD;

[0038] (iv) a fourth sequence segment comprising a second peptide linker; and

[0039] (v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein; and (c) one or more vector(s) comprising a nucleic acid molecule encoding a wild-type VP1, VP2 and / or VP3 AAV capsid protein.

[0040] FIGURE LEGENDS

[0041] Figure 1. (A) SDS-PAGE gel of purified wildtype and DPP6-sdAb containing AAV9 capsids, which shows an example of a modified AAV9 capsid which was produced with a method using a packaging plasmid which expresses wildtype VP2 and VP3 and a second plasmid which expresses VP1 with a sdAb recognizing DPP6 inserted into the GH2 / GH3 loop. In the purified vector preparation, a very weak VPl-Nb band is visible, and a ratio of 0.2 / 2 / 10 was determined by densitometric analysis for VPl-Nb / VP2 / VP3. Lane M corresponds to a marker, lane 1 corresponds to the purified preparation of AAV5 reference and lane 2 corresponds to the purified preparation of rAAV9 containing VPl-Nb fusion protein (B) Parental HEK293 cells and HEK293 cells pretransfected with a plasmid which expresses the human DPP6 protein in VP1 were transduced in triplicates with a Multiplicity of Infection (MOI) of 100.000 of GFP-encoding AAV9 capsids produced using a packaging plasmid which expresses wildtype VP2 and VP3 and a second plasmid which expresses a fusion protein of VP1 with a sdAb recognizing DPP6 inserted into the GH2 / GH3 loop. Thus, GFP-encoding AAV9 capsids consisted of wildtype VP2 and VP3 and a fusion protein of VP1 with a sdAb recognizing DPP6 inserted into the GH2 / GH3 loop. Cells were analyzed for GFP expression after 72h by fluorescence microscopy.

[0042] Figure 2. (A) Plasmid map of the packaging plasmid pRep2-CAP9_wt for expression of the AAV2 Rep proteins and the wildtype capsid proteins VP1, VP2 and VP3 of AAV9. (B) Plasmid map of the packaging plasmid pRep2-CAP9-DPP6-Nb with a DPP6 specific sdAb inserted into the GH2 / GH3 loop of the AAV9 CAP gene. The plasmid expresses the modified capsid proteins VPl- Nb, VP2-Nb and VP3- Nb. (C) Plasmid map of the packaging plasmid pRep2-CAP9-DPP6-Nb- Delta VP1+VP2. Same as B but with mutated start codons of VP1 and VP2, such that only VP3- Nb is expressed from the plasmid.

[0043] Figure 3. SDS-PAGE gel of wildtype and DPP6-sdAb containing AAV9 capsids. Capsid protein corresponding to 5.0E+10 vg was loaded per lane. The visualization of VP protein was done using a stain-free protein detection system. Lane 1 : wt control AAV9-CMV-eGFP. Lanes 2 and 3: AAV9-DPP6-Nb-Delta VPl+VP2-CMV-eGFP packaged using molar ratios of AAV9 wildtype and AAV9-Nb containing packaging plasmids of 3: 1 (lane 2) and 4: 1 (lane 3). Lanes 4-6: AAV9- DPP6-Nb-CMV-eGFP packaged with molar ratios of AAV9 wildtype and AAV9-Nb containing packaging plasmids of 2: 1 (lane 4), 3: 1 (lane 5) and 4: 1 (lane 6).

[0044] Figure 4. Functional testing of DPP6 sdAb-presenting AAV9 capsids by fluorescence microscopy. Parental HEK293 cells and HEK293 cells pre-transfected with a plasmid which expresses the human DPP6 protein were transduced with AAV9-CMV-eGFP, AAV9-DPP6-Nb 1 :2, AAV9- DPP6-Nb 1 :3 and AAV9-DPP6-Nb 1 :4 with a multiplicity of infection (MOI) of 10.000. Transduction efficiencies were monitored 72h after transduction via visualization of eGFP expressing cells by fluorescence microscopy. Almost no eGFP positive cells were detectable in parental HEK 293 cells transduced with either vector while strong eGFP fluorescence was found for all DPP6-Nb presenting capsids in pretreated cells but not for the AAV9 wildtype vector.

[0045] Figure 5. Functional analysis of wildtype and aCD38-sdAb containing AAV2RAcapsids which were manufactured with the method of the disclosure, i.e. AAV2RA-aCD38-Nb-CMV-eGFP packaged with molar ratios of AAV2RA-Nb containing packaging plasmids and AAV2RA wildtype of 2: 1 , 1 : 1, 1 :2, 1 :3 and 1 :4. The aCD38-sdAb containing AAV2RAcapsids assembled at ratios of 1 : 1 - 1 :4 result in much higher transduction, i.e. GFP expression detected by flow cytometry, in HEK293 cells expressing CD38 (top row) than in control HEK cells expressing P2X7 (bottom row). The aCD38-sdAb containing AAV2RAcapsids also result in much higher transduction than wildtype AAV2RAcapsids (left panels).

[0046] Figure 6. SDS-PAGE gel of purified AAV5 wildtype and DPP6-sdAb containing AAV5 capsids, wherein the DPP6-sdAb AAV5 capsid comprises VP3 with a sdAb recognizing DPP6 inserted into the GH2 / GH3 loop. The method used molar ratios of wildtype pRep2CAP5 and pRep2CAP5- DPP6-Nb-Delta VP1, Delta VP2 packaging plasmids of 4: 1. Lane 1 shows a marker; Lane 2 shows wt control AAV5 capsids with no modifications and Lane 3 shows AAV5-DPP6-Nb-CMV-eGFP packaged with molar ratios of AAV5 wildtype and AAV5-Nb containing packaging plasmids of 4: 1.

[0047] Figure 7. AAV particles separated on a 10 % SDS-PAGE gel with VP proteins visualized in the gel by silver staining. Molar ratios of wildtype pRep2CAP9 and pRep2CAP9-DPP6-Nb-Delta VP1, Delta VP2 packaging plasmids of 3 : 1 and 4: 1, were used (see lanes 4 and 5, top bands in the gel). Lane 1 : BioRad Precision Plus protein standard, lane 2: AAV5 reference, lane 3: AAV9 wt, lane 4: AAV9 wildtype and AAV9-DPP6-VP3-Nb-Delta VP1+VP2 packaging plasmid with molar ratio of 3:1, lane 5: AAV9 wildtype and AAV9-DPP6-Nb-Delta VP1+VP2 packaging plasmid with molar ratio of 4: 1.

[0048] DETAILED DESCRIPTION OF THE DISCLOSURE

[0049] Before the disclosure is described in detail with respect to some of its some embodiments, the following general definitions are provided.

[0050] The present disclosure, as illustratively described in the following, may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0051] The present disclosure will be described with respect to particular embodiments and with reference to certain figures but the disclosure is not limited thereto but only by the claims.

[0052] Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present disclosure, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.

[0053] For the purposes of the present disclosure, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. an antibody is defined to be obtainable from a specific source, this is also to be understood to disclose an antibody which is obtained from this source.

[0054] Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.

[0055] The terms “about” or “approximately” in the context of the present disclosure denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5% of a given value. Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following and / or in the context of which the terms are used.

[0056] The term “SMBD” refers to surface marker binding domain. Typically, the SMBD (e.g. sdAb or VHH) is specific for a cell surface molecule, in particular for a cell surface molecule which is specific and / or cell specific, also termed cell surface markers. This means that the marker is not ubiquitously expressed and thereby allows to target specific tissue or cell types while it does not bind to other tissue and cell types. The terms “cell surface molecule” and “cell surface marker” refer to proteins or carbohydrates that are present on the surface of a cell. In a specific embodiment, the cell surface marker is a membrane protein. “Binding to a cell surface marker” as used herein, also means that the SMBD (VHH) is specific for a protein, such as an antibody or an antibody fragment that binds to a specific cell surface marker. Typically, the SMBD as used in the context of the present disclosure is capable of directly binding to a specific cell membrane structure, thereby connecting a viral particle expressing the inserted protein to a target cell that is to be transduced by the viral particle. The structure can be, for example, a protein, a glycan, or a lipid.

[0057] The term “fusion protein” denotes proteins in which at least two polypeptides that originally coded for separate proteins or protein domains are combined in a single molecule. The at least two polypeptides are typically connected by translation of the coding sequence in a cell or cell-free extract into a single polypeptide. In the present case the term refers to a fusion protein in which the AAV capsid protein sequence is fused to a SMBD (e.g. sdAb or VHH). In other words, the SMBD (e.g. sdAb or VHH) is integrated into the AAV capsid protein sequence at specific positions which allow the optimal presentation of the SMBD (e.g. sdAb or VHH) without impairing the structure of the capsid protein or the SMBD, and in particular without impairing the structure and function of the AAV in the assembled state.

[0058] The abbreviation “AAV” stands for adeno-associated virus. AAV are small non-enveloped viruses which belong to the family of Parvoviridae . The AAV genome consists of single stranded DNA of about 4.7 kilo bases. It comprises T-shaped inverted terminal repeats (ITRs) of 145 bases on each end and carries two open reading frames (ORF): rep and cap, which encode for functional and capsid proteins. The ORF rep encodes for four Rep proteins (Rep78, Rep68, Rep52 and Rep40). Rep 78 and Rep 68 have site-specific, single-strand endonuclease, DNA helicase, and ATP activities, respectively, which are responsible for DNA replication. Rep52 and Rep40 are responsible for packaging of DNA flanked by ITRs into capsids. The ORF cap encodes for three capsid proteins: VP1, VP2 and VP3. Additionally, the VP1 mRNA encodes for the assemblyactivating protein (AAP) in a different reading frame.

[0059] The term “AAV capsid protein” relates to the three AAV proteins VP1, VP2 and VP3, which together form the capsid of the AAV in the assembled state. Exemplary sequences of AAV capsid proteins for different AAV serotypes are set out in SEQ ID NO: 1 to 39. In a preferred embodiment, the capsid protein is VP-3 (also “VP3”). The skilled person understands that also fragments and variants of said proteins are encompassed, such as sequences which are 70%, 80%, 90% or 95% identical to the sequences of SEQ ID NOs: 1 to 39, in particular fragments and variants which maintain the necessary structure and function for AAV assembly and cell targeting. Thus, the term AAV capsid protein encompasses functional variants and fragments of the AAV capsid protein.

[0060] The determination of percent identity between multiple sequences is preferably accomplished using the AlignX application of the Vector NTI AdvanceTM 10 program (Invitrogen Corporation, Carlsbad CA, USA). This program uses a modified Clustal W algorithm (Thompson et al., 1994. Nucl Acids Res. 22: pp. 4673-4680; Invitrogen Corporation; Vector NTI AdvanceTM 10 DNA and protein sequence analysis software. User’s Manual, 2004, pp.389-662). The determination of percent identity is performed with the standard parameters of the AlignX application.

[0061] Wherever herein reference is made to a variant or fragment sequence having a certain identity to another sequence, it is understood that said variant or fragment remains the function of the original sequence.

[0062] The term “membrane protein” refers to proteins that are attached to or integrated into the membrane of a cell. The term includes integral membrane proteins being permanently anchored to the membrane and peripheral membrane proteins that are temporarily attached to the lipid bilayer or to integral membrane proteins thereof. The integral membrane proteins may have one or several transmembrane domains. The peripheral membrane proteins may be integrated to the membrane via one or several a-helixes, one or several hydrophobic loops, may be covalently bound to a membrane lipid or interact by electrostatic or ionic interactions with membrane lipids or transmembrane proteins. The membrane protein may be selected from the group consisting of multi-span homo trimer membrane protein, type-II single span membrane protein, GPI-anchor membrane protein, integrins, type I membrane proteins, such as members of the immunoglobulin superfamily, e.g. CD4, receptors, such as cytokine and growth factor receptors, Fc receptors, Tolllike receptors, C-type lektin-like receptors or G-protein coupled receptors.

[0063] The term “carbohydrate” particularly refers to mono-, di-, oligo- and polysaccharides and also refers to glycolipids.

[0064] The term “nanobody” as used in in the context of the present disclosure refers to a single-domain antibody (sdAb), i.e. an antibody fragment consisting of a single monomeric variable antibody domain, or a variable domain of the heavy chain of heavy-chain only antibodies (VHH). The first single-domain antibodies were engineered from heavy-chain antibodies found in camelidae such as llamas; these are called “VHH” fragments. sdABs or VHHs are able to bind selectively to a specific antigen. With a molecular weight of only about 12-15 kDa, single-domain antibodies are typically much smaller than common antibodies. The terms “nanobody” and “VHH” are used interchangeably herein.

[0065] Thus, the term “VHH” as used in in the context of the present disclosure refers to a single variable domain of camelid immunoglobulins, i.e. immunoglobulins derived from, for example, llamas, in particular a camelid variable domain of the heavy chain of heavy-chain only antibodies. Llamas carry a variant immunoglobulin locus that permits the generation of unusual antibodies composed only of heavy chains. These antibodies (designated VHH, sdAb, or nanobody) are the smallest antigen-binding domain generated by adaptive immune systems. With a long complementaritydetermining region 3 (CDR3), VHHs can extend into crevices on proteins that are not accessible to conventional antibodies, including functionally interesting sites such as the active site of an enzyme or the receptor-binding canyon on a virus surface. VHHs offer numerous other advantages compared to conventional antibodies carrying variable domains (VH and VL) of conventional antibodies, including higher stability, solubility, expression yields, and refolding capacity, as well as better in vivo tissue penetration. Since VHH do not bind to VL domains, it is much easier to reformat VHHs into bispecific antibody constructs than constructs comprising conventional VH- VL pairs or single domains based on VH domains. The term VHH encompasses functional variants and fragments of the VHHs.

[0066] The exemplary VHHs described herein were isolated from immunized llama or alpacas. These VHHs derive from VHH frameworks that are frequently observed in both, natural sdAbs and in sdAbs from synthetic libraries. In principle, any sdAb-sequence or variant or fragment thereof, immunoglobulin single variable domain or variant or fragment thereof can be used, provided it exhibits good solubility and specificity for the target molecule.

[0067] The term “GH2 / GH3 loop”, also “GH2-GH3 loop” or “GH2 / 3 loop”, denotes a loop in the capsid surface which connects the P-strands GH2 and GH3 of the AAV VP protein. For example, the GH2-GH3 loop of the capsid protein of AAV5 comprises aa 438 to 456, or more specifically aa 440 to 446, the GH2-GH3 loop of the capsid protein of AAV9 comprises aa 447 to 470, or more specifically aa 450 to 458. The GH2-GH3 loop of the capsid protein of AAV2 comprises aa 445 to 469, or more specifically aa 452 to 460. In other examples, the GH2-GH3 loop of the capsid protein of AAV1 comprises aa 446 to 470, of AAV3 comprises aa 445 to 470, of AAV3b comprises aa 445 to 470, of AAV4 comprises aa 439 to 464, of AAV6 aa 446 to 470, of AAV7 aa 447 to 472, of AAV8 aa 448 to 472, of AAV10 aa 448 to 472, of AAV11 aa 438 to 463, of AAV12 aa 447 to 472, of AAV13 aa 444 to 467, of AAVrhlO aa 448 to 472 and of AAVrh74 aa 448 to 472. The GH2-GH3 loop can e.g., be derived from Boucas et al. J Gene Med 2009; 11 : 1103— 1113, Xie et al. Proc Natl Acad Sci USA 2002; 99(16): 10405-10 (alignment between AAV2 and AAV4) and / or Meyer et al. eLife 2019; 8:e44707. The numbering of the amino acids comprised in the GH2 / GH3 loop given above in this paragraph refers to the numbering of amino acids of VP1 for a respective AAV serotype. Referring to the VP3 numbering for the respective AAV serotypes, the GH2-GH3 loop of the capsid protein of AAV5 comprises aa 247 to 265, or more specifically aa 250-254, of AAV5 VP3 (preferably as set out in SEQ ID NO: 15); the GH2-GH3 loop of the capsid protein of AAV9 comprises aa 246 to 269, or more specifically aa 249 to 255, of AAV9 VP3 (preferably as set out in SEQ ID NO: 27); and the GH2-GH3 loop of the capsid protein of AAV2 comprises aa 243 to 267, or more specifically aa 250 to 257, of AAV2 VP3 (preferably as set out in SEQ ID NO: 6).

[0068] The term “GH12 / 13 loop” also “GH12-GH13 loop” or “GH12 / 13 loop” denotes a loop in the capsid surface which connects the P-strands GH12 and GH13 of the AAV VP protein. For example, the GH12-GH13 loop of the VHH of AAV5 comprises aa 569 to 580, the GH12-G13 loop of the VHH of AAV9 comprises aa 581 to 592. The numbering of the amino acids comprised in the GH12 / GH13 loop given in this paragraph refers to the numbering of amino acids of VP1 for a respective AAV serotype.

[0069] The term “linker” as used throughout this application refers to molecules or molecular segments that connect two other, usually larger, molecules or molecular segments. For example, a linker can be a short amino acid segment within a protein that connects two domains. A linker can be either rigid and thus prevent e.g. undesired interactions between the domains, or a linker can be flexible and allow conformational changes. Typically, in context of the present disclosure, a linker refers to a protein sequences that connects an SMBD insert and a AAV capsid protein.

[0070] A “vector” is any molecule or composition that has the ability to carry a nucleic acid sequence into a suitable host cell where synthesis of the encoded polypeptide can take place. Typically, and preferably, a vector is a nucleic acid that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate a desired nucleic acid sequence (e.g. a nucleic acid of the disclosure). The vector may comprise DNA or RNA and / or comprise liposomes.

[0071] The term “target cell” as used herein, refers to a specific cell type or to a group of specific cell types. The target cell may be of endodermal, ectodermal or mesodermal origin. The cell of endodermal origin may be exocrine secretory epithelial cells or a hormone secreting cell. The cell of ectodermal origin may be a cell of the integumentary system or the nervous system. The cell of the mesodermal origin may be a metabolism or storage cell, such as adipocytes, a barrier function cell, such as a lung cell, gut, exocrine glands and urogenital tract cells; extracellular matrix cells, contractile cells, blood and immune system cells, germ cells, nurse cells or interstitial cells. The target cells may also be for example monocytes, T cells or natural killer cells. The target cell may be a tumor cell.

[0072] As referred herein, the term “helper function” derives from the notion of a helper virus. For replication and transcription of the AAV to occur, certain helper functions need to be provided in trans. This can be done via a helper virus, such as viruses from the families of Adenoviridae and Herpesviridae. Said helper functions may also be provided in the form of one or more polypeptides or nucleic acids encoding such one or more polypeptides. Said helper polypeptides and helper polynucleotides may originate from or be derived from naturally occurring viruses such as an adeno-associated virus or a virus selected from the families of Adenoviridae and Herpesviridae.

[0073] Further exemplary information about helper functions can be found, for example, in McPherson et al., Virology 147, 217-222 (1985).

[0074] Adeno-associated virus (AAV) capsid fusion protein

[0075] A first aspect relates to an adeno-associated virus (AAV) capsid fusion protein comprising a VP3 AAV capsid protein and a surface marker binding domain (SMBD), wherein the SMBD is inserted in the GH2-GH3 loop of the VP3 AAV capsid protein.

[0076] In other words, the disclosure relates to an adeno-associated virus (AAV) capsid fusion protein, wherein the AAV capsid fusion protein comprises an AAV VP3 capsid protein comprising, in the GH2-GH3 loop of the AAV VP3 capsid protein, an insert comprising a binding domain for a cell surface molecule, in particular a SMBD (e.g. VHH or a sdAb).

[0077] In some embodiments, the disclosure relates to an AAV capsid fusion protein, wherein the AAV capsid fusion protein is VP3 and comprises a SMBD, such as a VHH or sdAb, which is inserted in the GH2-GH3 loop of the AAV VP3 capsid protein.

[0078] In alternative embodiments, the SMBD can alternatively or additionally be inserted into the GH12 / GH13 loop of the VP3 AAV capsid protein. In yet alternative embodiments, there is only a single SMBD inserted into the VP3 AAV capsid protein and this single SMBD is inserted in the GH2-GH3 loop of the AAV VP3 capsid protein. In other words, the SMBD inserted into the GH2- GH3 loop of the AAV VP3 capsid protein is the only SMBD inserted into the AAV VP3 capsid protein.

[0079] As the VP3 capsid protein forms the major part of the capsid, insertion of the SMBD in the VP3 capsid protein, allows for an about 10-fold increase in SMBD display compared to SMBD inserts in the VP1 or VP2 capsid proteins. By inserting the SMBD into the VP3 capsid protein, binding to target cells and / or target antigens is enhanced. At the same time, insertion of a SMBD into the VP3 protein advantageously does not impair capsid formation.

[0080] Specifically, the inventors showed that by using such constructs, the transduction of targetexpressing cells could be increased (see Figures 4 and 5), i.e. the AAV transduction efficiency of cells expressing a cell surface molecule to which the SMBD entity, such as the VHH or sdAb entity, of the AAV capsid fusion protein specifically binds, was improved. Also, the inventors found that the thermal stability of SMBD-carrying AAV serotypes, such as sdAb-carrying AAV serotypes, was comparable to background AAV serotypes without the SMBD.

[0081] AAV vector particle

[0082] A second aspect of the present disclosure relates to an AAV vector particle comprising (a) a VP AAV capsid protein with an inserted surface marker binding domain (SMBD); and

[0083] (b) a wild-type VP AAV capsid protein that does not comprise an inserted SMBD; wherein the VP AAV capsid protein is VP1, VP2 and / or VP3.

[0084] In some embodiments, the VP AAV capsid protein is VP3.

[0085] In some embodiments, the AAV vector particle comprises or consists of

[0086] (a) a VP3 AAV capsid protein with an inserted SMBD;

[0087] (b) a wild-type VP3 AAV capsid protein that does not comprise an inserted SMBD;

[0088] (c) a wild-type VP1 AAV capsid protein;

[0089] (d) a wild-type VP2 AAV capsid protein; and

[0090] (e) a nucleic acid molecule comprising an expression cassette encoding a transgene, optionally flanked with ITR sequences, optionally wherein the capsid proteins (a) to (d) assemble to form the AAV vector particle and further optionally wherein the nucleic acid (e) is enclosed in the AAV vector particle.

[0091] It can be preferred in the afore-mentioned embodiments that the SMBD is inserted in the GH2- GH3 loop or the GH12-13 of the VP3 AAV capsid protein. It can further be preferred that all of the mentioned VP1, VP2 and VP3 AAV capsid proteins are from the same AAV serotype, e.g. from AAV9, AAV2 or AAV5. Alternatively, the mentioned AAV capsid proteins may be from different AAV serotypes. It can further be preferred that the underlying VP1, VP2 and VP3 sequences are selected from the group of sequences consisting of (i) SEQ ID NO: 1 to 3; (ii) SEQ ID NO: 4 to 6; (iii) SEQ ID NO: 7 to 9; (iv) SEQ ID NO: 10-12; (v) SEQ ID NO: 13-15; (vi) SEQ ID NO: 16-18; (vii) SEQ ID NO: 19-21; (viii) SEQ ID NO: 22-24; (ix) SEQ ID NO: 25-27; (x) SEQ ID NO: 28-30; (xi) SEQ ID NO: 31-33; (xii) SEQ ID NO: 34-36; (xiii) SEQ ID NO: 37-39; or from sequences that are at least 70%, at least 80%, at least 90% or at least 95% identical to the sequences of (i) to (xiii).

[0092] In some embodiments, the AAV vector particle comprises:

[0093] (a) a VP3 AAV capsid protein with an inserted surface marker binding domain (SMBD), wherein the VP3 AAV capsid protein with the inserted SMBD comprises in the following order:

[0094] (i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein; (ii) a second sequence segment which comprises a first peptide linker;

[0095] (iii) a third sequence segment which comprises the SMBD;

[0096] (iv) a fourth sequence segment which comprises a second peptide linker; and

[0097] (v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein; and

[0098] (b) a wild-type VP3 AAV capsid protein that does not comprise an inserted SMBD.

[0099] In some embodiments, the VP3 AAV capsid protein with an inserted surface marker binding domain (SMBD) is a VP3 AAV capsid protein as disclosed herein.

[0100] In specific embodiments of the AAV vector particle, the SMBD is inserted in the GH2-GH3 loop or the GH12-13 of the VP3 AAV9 capsid protein. For example, in some embodiments, the AAV serotype is AAV9 and wherein the SMBD is inserted in the GH2-GH3 loop, in particular at any position in the region of aa 249 to 255 or aa 249 to 254 of AAV9 VP3 as set out in SEQ ID NO: 27, preferably wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 250 to 253 or amino acids 250 to 252 are modified and / or deleted. In a preferred embodiment, the AAV serotype is AAV9 and the SMBD is inserted at position 254 of AAV9 VP3 as set out in SEQ ID NO: 27. In another preferred embodiment, the AAV serotype is AAV9 and the SMBD is inserted at position 249 (i.e. immediately after aa 249) of AAV9 VP3 as set out in SEQ ID NO: 27 while aa 250 to 252 are modified and / or deleted. Thus, the AAV serotype may be AAV9 and the SMBD may be inserted at position 250 of AAV9 VP3 as set out in SEQ ID NO: 27 such that aa 250 to 252 are modified and / or deleted. In some embodiments, the SMBD is inserted in the GH12-GH13 loop of a AAV9 capsid protein, the SMBD is inserted in particular at any position in the region of aa 384 to 390 of AAV9 as set out in SEQ ID NO: 27, preferably wherein 0-7 amino acids of this region, more preferably amino acids 385 to 389, even more preferably amino acids 386 to 387 are modified and / or deleted.

[0101] In other embodiments, the SMBD is inserted in the GH2-GH3 loop or the GH12-13 of the VP3 AAV2 capsid protein. In specific embodiments, the AAV serotype is AAV2 and the SMBD is inserted in the GH2 / 3 loop of AAV2 at any position in the region of aa 452 to 460 of AAV2 VP1 as set out in SEQ ID NO: 4, wherein 0-12 amino acids of this region are modified and / or deleted. For example, in an AAV vector of the disclosure, the AAV serotype is AAV2 and the SMBD is inserted in the region of aa 452 to 460 of AAV2 VP1 as set out in SEQ ID NO: 4, preferably, wherein the amino acids 453 to 459 are modified and / or deleted. In other specific embodiments, the AAV serotype is AAV2 and the SMBD is inserted in the GH2 / 3 loop of AAV2 at any position in the region of aa 250 to 257 or aa 250 to 258 of AAV2 VP3 as set out in SEQ ID NO: 6, wherein 0-12 amino acids of this region are modified and / or deleted. For example, in an AAV vector of the disclosure, the AAV serotype is AAV2 and the SMBD is inserted in the region of aa 250 to 257 or aa 250 to 258 of AAV2 VP3 as set out in SEQ ID NO: 6, preferably, wherein the amino acids 251 to 257 or amino acids 252 to 257 are modified and / or deleted.

[0102] In other embodiments, the SMBD is inserted in the GH2-GH3 loop or the GH12-13 of the VP3 AAV5 capsid protein. In specific embodiments, the AAV serotype is AAV5 and the SMBD is inserted in the GH2 / 3 loop of AAV5 at any position in the region of 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15, wherein 0-12 amino acids of this region are modified and / or deleted. In other specific embodiments, the AAV serotype is AAV5 and the SMBD is inserted in the GH2 / 3 loop of AAV5 at any position in the region of aa 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15, wherein 0-12 amino acids of this region are modified and / or deleted. For example, in an AAV vector of the disclosure, the AAV serotype is AAV5 and the SMBD is inserted in the region of aa 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15, preferably, wherein the amino acids 251 to 253 are modified and / or deleted.

[0103] In further embodiments of the AAV vector of the disclosure, the SMBD is a VHH that specifically binds to DPP6, and the AAV serotype is AAV9. In particular embodiments, the AAV capsid fusion protein comprises the sequence of SEQ ID NO: 47 or 49 or a sequence at least 80% identical thereto.

[0104] In other embodiments of the AAV vector of the disclosure, the SMBD is a VHH that specifically binds to CD38, in particular nanobody MU1053, and the AAV serotype is AAV2. In particular, the AAV capsid fusion protein comprises the sequence of SEQ ID NO: 51 or a sequence at least 80% identical thereto. In some embodiments, the SMBD is a VHH that specifically binds CD38, and is selected from the group consisting of MU1053, MU375 and MU551.

[0105] The AAV capsid protein

[0106] There are three different AAV capsid proteins, namely VP1, VP2 and VP3. Exemplary sequences of AAV capsid proteins for different AAV serotypes are set out in SEQ ID NO: 1 to 39. The skilled person understands that also fragments and variants of said proteins are encompassed, such as sequences which are 70%, 80%, 90% or 95% identical to the sequences of SEQ ID NOs: 1 to 39, in particular fragments and variants which maintain the necessary structure and function for AAV assembly and cell targeting. Thus, the term AAV capsid protein encompasses functional variants and fragments of the AAV capsid protein.

[0107] Based on the differences in the amino acid sequences of the capsid proteins, different AAV serotypes are distinguished, i.e., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO. Thus, in a specific embodiment the AAV serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrhlO or variants thereof or a combination thereof. In one embodiment the AAV serotype is selected from the group consisting of AAV2 and AAV9. In a specific embodiment, the AAV serotype is AAV9. In another specific embodiment, the AAV serotype is AAV9.

[0108] In some embodiments, the AAV capsid protein is a AAV9 VP3 capsid protein. In another embodiment, the AAV capsid protein is a AAV2 VP3 capsid protein.

[0109] The sequences of the capsid proteins of the various serotypes are set out in the following:

[0110] The skilled person understands that also fragments or variants of said proteins are encompassed, in particular sequences which are 70%, 80%, 90% or 95% identical to the sequences of SEQ ID Nos: 1 to 39, in particular fragments and variants which maintain the necessary structure and function for AAV assembly and cell targeting.

[0111] In some embodiments, the amino acid sequence of the fusion protein may comprise one or more phenotypically silent substitutions.

[0112] “Phenotypically silent substitutions” are also named “conservative amino acid substitutions". The concept of "conservative amino acid substitutions" is understood by the skilled artisan, and preferably means that codons encoding positively-charged residues (H, K, and R) are substituted with codons encoding positively-charged residues, codons encoding negatively- charged residues (D and E) are substituted with codons encoding negatively-charged residues, codons encoding neutral polar residues (C, G, N, Q, S, T, and Y) are substituted with codons encoding neutral polar residues, and codons encoding neutral non-polar residues (A, F, I, L, M, P, V, and W) are substituted with codons encoding neutral non-polar residues. These variations can spontaneously occur, be introduced by random mutagenesis, or can be introduced by directed mutagenesis. Those changes can be made without destroying the essential characteristics of these polypeptides. The ordinarily skilled artisan can readily and routinely screen variant amino acids and / or the nucleic acids encoding them to determine if these variations substantially reduce or destroy the ligand binding capacity by methods known in the art.

[0113] Exemplary variants of the VP proteins may be modified to avoid binding to physiological binding molecules, for example in AAV9 the mutation W503A may be introduced. The W503A mutation inhibits binding of AAV9 to terminal galactose (Adachin et al. 2014 ). The present inventors could show that the W503A mutation does not reduce specific transduction of HEK cells expressing the cognate membrane protein, e.g. CD73, with AAV9WAvia an inserted sdAb (CD73 -specific SB 116). The inventors found that the transduction is enhanced about 200fold compared to AAV9WAwithout a sdAb insert. Thus, AAV transduction via binding of the sdAb to its target is independent of galactose binding. Another example are mutations reducing binding to heparin sulfate proteoglycan, such as AAV2RA, wherein AAV2 carries two mutations at positions 585 and 588, wherein arginine residues R585 and R588 are replaced by alanine residues. These arginine —> alanine mutations reduce binding to cell surface heparan sulfate proteoglycan (HSPG). (Boucas et al. 2009; Munch et al. 2013). The skilled person is also aware of other variants, which are for example modified by the insertion of peptides in the region comprising R585 and R588. Other variations are chimeric AAVs, in which the capsid proteins stem from different (e.g. VP1 stemming from AAV and VP2 und VP3 stemming from AAV2), or AAVs in which all capsid proteins comprise sequence sections stemming from different AAV serotypes. An example of a capsid protein of the disclosure carrying two mutations at positions 585 and 588 is set out in SEQ ID NO: 51.

[0114] In some embodiments, the capsid protein is VP-3 (also “VP3”). In other embodiments, the capsid protein is VP3 of either AAV2, AAV5 or AAV9 serotype.

[0115] The SMBD

[0116] In embodiments of the present disclosure, the SMBD is capable of directly binding to a specific cell membrane structure, thereby connecting a viral particle expressing the inserted protein to a target cell that is to be transduced by the viral particle. The structure to which the SMBD binds can be, for example, a protein, a glycan, or a lipid.

[0117] Examples of SMBD, include, but are not limited to, sdAb or VHH, immunoglobulin single variable domain (ISVD), a single chain variable fragment (scFv), a single chain Fab fragment (Fab), Fab', F(ab')2, Fv), or a synthetic or natural binding domain for a cell surface molecule, such as a darpin or the extracellular domain of a membrane protein. In other words, in embodiments of the present disclosure, the SMBD is selected from the group consisting of an antibody fragment, a VHH, an immunoglobulin single variable domain, scFv, Fab, Fab', F(ab')2, Fv and a synthetic or natural binding domain for a cell surface molecule. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv and scFv fragments; as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAbs), such as sdAbs, VHH and VNAR fragments, single-chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments or antibodies, including but not limited to Fab-Fv or Fab-Fv-Fv constructs. In some embodiments, the SMBD is a sdAB or a VHH.

[0118] Typically, the SMBD insert as used herein may have a length of at least 50 aa, preferably of 50 aa to 800 aa, 50 aa to 700 aa, 50 aa to 600 aa, 50 aa to 500 aa, 50 aa to 200 aa, 50 aa to 150 aa, 80 aa to 140 aa, 90 aa to 130aa, 100 aa to 120 aa.

[0119] Stated differently, the SMBD preferably has a length of at least 100 amino acids, more preferably 100 to 200 amino acids, more preferably 110 to 190 amino acids, even more preferably 120 to 180 amino acids, still more preferably 130 to 170 amino acids, even more preferably 140 to 160 amino acids, most preferably 145 to 155 amino acids.

[0120] The inventors found that surprisingly these rather large domains can be included into the AAV capsid protein at the GH12 / 13 loop and / or the GH2 / 3 loop. In some embodiments, these domains are inserted into the GH2 / 3 loop of the VP3 capsid protein, wherein the capsid is able to assemble fully intact AAVs with enhanced cell targeting properties.

[0121] Typically, the SMBD (e.g. sdAb or VHH) is exposed on the surface of the AAV capsid when the capsid fusion protein is incorporated in the AAV. By the integration in the insertion sites as described herein, e.g. the GH12-GH13 loop and / or the GH2-GH3 loop exposure of the SMBD (e.g. sdAb or VHH) on the surface of the AAV capsid may be achieved.

[0122] The SMBD (e.g. sdAb or VHH) may be directed against any membrane protein or membrane glycolipid capable of binding a sdAb or VHH, respectively. In one embodiment the SMBD (e.g. sdAb or VHH) may be directed against a protein, such as an antibody or an antibody fragment, that specifically binds to a cell surface marker.

[0123] In the following, some examples for cell surface marker molecules (antigens) capable of binding the SMBD integrated in the capsid proteins of the present disclosure, such as the GH12 / 13 or GH2 / 3 loop of one of the capsid proteins VP1, VP2, and / or VP3, in particular the GH2 / 3 loop of the AAV VP3 capsid protein of the present disclosure, are given:

[0124] An antigen capable of binding the SMBD may be, for example, a type-II single span membrane protein.

[0125] In a specific embodiment, the antigen capable of binding the SMBD may be, for example, human diepeptidyl peptidase like 6 (also “DPP6”, SEQ ID NO: 45). DPP6 is a single-pass type II membrane protein that is a member of the peptidase S9B family of serine proteases.

[0126] Thus, in embodiments of the present disclosure, the SMBD is a single domain antibody (VHH) that specifically binds a dipeptidyl aminopeptidase-like protein 6 (DPP6). In some embodiments, the SMBD is a VHH that specifically binds to DPP6, and the AAV serotype is AAV9. In another specific embodiment, the type-II single span membrane protein may be CD38. CD38 (cluster of differentiation 38, SEQ ID NO: 44) is prominently expressed by plasma cells and NK cells. CD38 is overexpressed on the surface of many tumor cells, such as multiple myeloma cells. Hence, also encompassed are the fusion proteins comprising a CD38-specific VHH for use as a medicament. More specifically, contemplated are fusion proteins comprising CD38 specific VHHs for the treatment of cancer, such as multiple myeloma.

[0127] Thus, in embodiments of the present disclosure, the SMBD is a VHH that specifically binds CD38, preferably, wherein the CD38-specific VHH is selected from the group consisting of MU1053, MU375 and MU551. In some embodiments, the SMBD is a VHH that specifically binds to CD38, and the AAV serotype is AAV2.

[0128] Other specific examples for antigens capable of binding the SMBD are:

[0129] CD73 (SEQ ID NO: 53), is a cell membrane protein that belongs to the 5 '-nucleotidase family. CD73 is a glycosylphosphatidylinositol (GPI) anchored purine salvage enzyme expressed on the surface of human T and B lymphocytes. CD73 is expressed in a wide range of cancer types, including breast cancer, colorectal cancer, glioblastoma, melanoma, prostate cancer, ovarian cancer, and non-small-cell lung cancer (NSCLC).

[0130] Thus, in embodiments of the present disclosure, the SMBD is a VHH that specifically binds CD73, preferably, wherein the CD73-specific VHH is SB116.

[0131] Programmed death-ligand 1 (PD-L1; SEQ ID NO: 54) is the primary ligand of Programmed cell death -1 (PD-1) and is constitutively expressed on antigen presenting cells, mesenchymal stem cells and bone marrow-derived mast cells. As well as on wide range of tumor cells, including lung cancer, breast cancer and melanoma.

[0132] Thus, in embodiments of the present disclosure, the SMBD is a VHH specific that specifically binds PD-L1, preferably, wherein the PD-L1 specific VHH is B3.

[0133] P2X7 (SEQ ID NO: 55) is a trimeric ion channel receptor that is gated by extracellular adenosine 5'-triphosphate. P2X7 is present on various cells types including stem, blood, glial, neural, ocular, bone, dental, exocrine, endothelial, muscle, renal and skin cells. P2X7 is thought to relate to inflammatory molecule release, cell proliferation and death, metabolic events, and phagocytosis.

[0134] Thus, in embodiments of the present disclosure, the SMBD is a VHH specific for P2X7, preferably, wherein the P2X7-specific VHH is lc81.

[0135] The linker

[0136] One or several linkers may be used in order to facilitate the fixing of the SMBD (e.g. sdAb or VHH) to the capsid surface and to allow flexible movement of the antigen-binding tip of the VHH away from the capsid surface, i.e. toward the cognate molecule on the surface of the target cell.

[0137] Thus, in some embodiments the AAV capsid fusion protein comprises at least one linker linking the SMBD (e.g. sdAb or VHH) sequence with the VP protein sequence.

[0138] In some embodiments of the present disclosure, the linkers are flexible. For example, in the AAV vector particle of the present disclosure, the first and second peptide linkers are flexible.

[0139] Typically the length of such a linker (in case of several linkers the length of each linker) is 1 to 100, 2 to 50, 3 to 40, 4 to 30, such as 5 amino acids. For example, in the AAV vector particle of the present disclosure, the first or second linker has a length of 1 to 5 amino acids. Typically, the SMBD (e.g. sdAb or VHH) is flanked by an N-terminal linker and by a C-terminal linker. The N- terminal and the C-terminal linker may be the same or different. In some embodiments, AAV capsid fusion protein comprises an N-terminal linker, but not a C-terminal linker. The N-terminal linker may have a length of 1 to 50, preferably 10 to 35, more preferably 15 to 30, most preferably 20 to 25 aa. In particular, the N-terminal linker may facilitate flexible movement of the antigenbinding tip of the VHH away from the capsid surface, i.e. toward the cognate molecule on the surface of the target cell.

[0140] The C-terminal linker may have a length of 1 to 20, preferably 1 to 10, more preferably 2 to 7 amino acids. This linker may fix the base of the SMBD (e.g. sdAb or VHH) near the capsid surface.

[0141] The skilled person is aware of alternative linkers that are suitable for combining two functional polypeptides (Els-Konrath et al. 2001). In a specific embodiment the N-terminal linker comprises the sequence as set out in SEQ ID NO: 40 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto.

[0142] In one embodiment the C-terminal linker comprises a sequence selected from the group consisting of SEQ ID NO: 41 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto, SEQ ID NO: 42 or a sequence at least 80% identical thereto and SEQ ID NO: 43 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto. In one embodiment the C-terminal linker comprises the sequence set out in SEQ ID NO: 41 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto.

[0143] Thus, in a specific embodiment the N-terminal linker comprises the sequence as set out in SEQ ID NO: 40 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto and the C-terminal linker comprises the sequence set out in SEQ ID NO: 41 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto.

[0144] The insert and insertion sites

[0145] The term “inserted”, in particular the expression “wherein the SMBD (e.g. sdAb or VHH) is inserted in the region” as used herein, means, that the SMBD (e.g. sdAb or VHH) sequence (including optional linkers) can be inserted after any amino acid in said region.

[0146] By the insertion of the SMBD (e.g. sdAb or VHH), amino acids from said region may be deleted or modified. Modified, means, that they are substituted without being part of the SMBD (e.g. sdAb or VHH) sequence. Such modifications may be used for example for introduction of restriction sites.

[0147] In one embodiment 1, 2, 3, 4, 5, 6 or 7 amino acids may be modified or may be deleted from the GH2-GH3 loop. In particular 1, 2, 3, 4, 5, 6 or 7 amino acids may be deleted from the GH2-GH3 loop. In one embodiment, the two amino acids flanking the insertion site are deleted upon insertion of the VHH.

[0148] In another embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids may be modified or may be deleted from the GH12-GH13 loop. In particular, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids may be deleted from the GH12-GH13 loop. In one embodiment two amino acids are deleted at the insertion site in the GH12-GH13 loop. In one embodiment, the sequence is introduced between two consecutive amino acids without deletion of amino acids.

[0149] GH2-GH3 insert

[0150] In some embodiments, the SMBD (e.g. sdAb or VHH) is inserted in the GH2-GH3 loop of an AAV capsid protein, such as the GH2 / 3 loop of one of the capsid proteins VP1, VP2, and / or VP3, in particular the GH2 / 3 loop of the AAV VP3 capsid protein.

[0151] Insertion of the SMBD into the GH2 / 3 loop is advantageous, because the incorporation of the SMBD in the GH2 / GH3 loop of VP3 results in higher transduction efficiency, compared with the incorporation of the SMBD into the GH2 / GH3 or GH12 / GH13 loop of VP1 or VP2.

[0152] Providing the specific serotypes of the AAV capsid fusion protein bypasses the need to generate mosaic AAV capsids, where the fusion protein is derived of different AAV serotype as VP2 and VP3. In other words, AAV comprising all three VP proteins, VP1, VP2 and VP3 from the same serotype, in particular from serotype AAV2, serotype AAV5 or serotype AAV9, can be provided.

[0153] Some embodiments relate to an AAV capsid fusion protein comprising a SMBD (e.g. sdAb or VHH) which is inserted in the GH2-GH3 loop of the AAV9 VP3 capsid protein.

[0154] In one embodiment, the SMBD (e.g. sdAb or VHH) is inserted at any position in the region of aa 246 to 269 or more specifically in the region of aa 249 to 255 or aa 249 to 254 of AAV9 VP3 sequence as set out in SEQ ID NO: 27, preferably wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 250 to 252 or amino acids 250 to 253 are modified and / or deleted. In one embodiment, the SMBD (e.g. sdAb or VHH) is inserted at aa 250 or in the region of aa 250 to 252 of AAV9 VP3 sequence as set out in SEQ ID NO: 27 or a sequence at least 80% identical thereto.

[0155] An exemplary embodiment relates to an AAV capsid fusion protein, wherein the SMBD is a human DPP6 sdAb and which is inserted into the GH2 / 3 loop of the AAV9 VP3 capsid protein. In a particularly preferred aspect of this embodiment, the AAV capsid fusion protein comprises the sequence of SEQ ID NO: 47 or 49 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto. This construct is also denoted AAV9-DPP6-Nb herein. Figure 4 shows that specific transduction of cells expressing DPP6 is achieved with this construct. Also, transduction efficiency with this construct is higher compared to wild-type AAV9.

[0156] In other embodiments, the SMBD (e.g. sdAb or VHH) is inserted in the GH2-GH3 loop of AAV2 VP3 capsid protein. For example, the SMBD (e.g. sdAb or VHH) is inserted in the GH2-GH3 loop of AAV2 VP3 capsid protein at any position in the region of aa 243 to 267, or more specifically in the region of aa 250 to 257 or aa 250 to 258 of AAV2 VP3 as set out in SEQ ID NO: 6, optionally, wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted. For example, amino acids 251 to 257 or amino acids 252 to 257 as set out in SEQ ID NO: 6 can be deleted. Specific embodiments relate to an AAV capsid fusion protein, wherein the SMBD (e.g. sdAb or VHH) is inserted at any position in the region of aa 250 to 257 or aa 250 to 258 of AAV2 VP3 as set out in SEQ ID NO: 6 or a sequence at least 80% identical thereto.

[0157] An exemplary embodiment refers to an AAV capsid fusion protein wherein the SMBD (e.g. sdAb or VHH) is specific for CD38 (in particular, the CD38-specific VHH is MUI 053) and which is inserted into the GH2 / 3 loop of the AAV2 VP3 capsid protein. In a particularly preferred aspect of this embodiment, the capsid fusion protein comprises the sequence of SEQ ID NO: 51 or a sequence at least 60%, 70%, 80 %, 90% 95%, 97%, or 99% identical thereto. Figure 5 shows that this construct enhances transduction (the construct is denoted “AAV2RA-1053 (aCD38)” in Figure 5).

[0158] In other embodiments, the SMBD (e.g. sdAb or VHH) is inserted in the GH2-GH3 loop of AAV5 VP3 capsid protein. For example, the SMBD (e.g. sdAb or VHH) is inserted in the GH2-GH3 loop of AAV5 VP3 capsid protein at any position in the region of aa 247 to 265, or more specifically in the region of aa 250 to 254 of AAV2 VP3 as set out in SEQ ID NO: 15, optionally, wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted. For example, amino acids 251 to 253 as set out in SEQ ID NO: 15 can be deleted. Specific embodiments relate to an AAV capsid fusion protein, wherein the SMBD (e.g. sdAb or VHH) is inserted at any position in the region of aa 250 to 254 of AAV2 VP3 as set out in SEQ ID NO: 15 or a sequence at least 80% identical thereto.

[0159] The considerations described here can be used to insert any SMBD (e.g. sdAb or VHH) or similar ligand into the GH2-GH3 and / or GH12-GH13 loops of any other AAV-capsid, including other serotypes and variants, including but not limited to mutated and reshuffled AAV capsids. Structure-based alignment can be used as a guide to select an insertion site within the indicated amino acid sequences between the two flanking P-strands, i.e. GH2-GH3 and GH12-GH13, i.e., it is likely that the position of the VHH / ligand insertion can be moved by one or more residues without compromising AAV assembly or transduction efficiency.

[0160] GH12-GH13 insert

[0161] Other embodiments relate to an AAV capsid fusion protein, wherein the SMBD (e.g. sdAb or VHH) is inserted in the GH12-GH13 loop of a AAV capsid protein selected from VP1, VP2, and / or VP3.

[0162] In another example, the SMBD (e.g. sdAb or VHH) may be inserted at any position in the region of aa 384 to 390 of AAV9 VP1 as set out in SEQ ID NO: 27, preferably whereby 0 to 7 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 385 to 389 are modified and / or deleted. In one embodiment, the SMBD (e.g. sdAb or VHH) is inserted at any position in the region of aa 386 to 387 of AAV9 VP3 as set out in SEQ ID NO: 27. In a specific embodiment, the insert is between aa 384 to 390 of AAV9 VP1 as set out in SEQ ID NO: 27.

[0163] Nucleotide sequences

[0164] Another aspect of the disclosure relates to a nucleotide sequence encoding the fusion protein as described herein.

[0165] The skilled person understands that also the nucleic acid encoding the fusion protein may be modified. Useful modifications in the overall nucleic acid sequence include codon optimization of the sequence. Alterations may be made which lead to conservative substitutions within the expressed amino acid sequence. These variations can be made in complementarity determining and non-complementarity determining regions of the amino acid sequence of the fusion protein that do not affect function. Usually, additions and deletions should not be performed in the CDR3 region. In one embodiment the sequence the AAV capsid fusion protein was codon optimized for expression in HEK cells.

[0166] In one embodiment, the AAV capsid fusion protein expression is regulated by a CMV promotor. In a specific embodiment, the vector is condon optimized for expression (in particular for expression in HEK cells) and the AAV capsid fusion protein expression is regulated by a CMV promotor. In the following, the nucleotide sequences to the corresponding amino acids are set out for the constructs as disclosed herein.

[0167] Vectors

[0168] In a further aspect, the disclosure relates to a vector comprising nucleotide sequence as described above.

[0169] The vector may be a plasmid, shuttle vector, phagemide, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector, MVA vector, and AAV vector, preferably the vector is a plasmid. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known to those of ordinary skill in the art. A vector preferably is an expression vector that includes a nucleic acid according to the present disclosure operably linked to sequences allowing for the expression of said nucleic acid.

[0170] In other embodiments, the vector comprises the nucleotide sequence coding for the fusion protein comprising a VP1 capsid protein, in which the cell surface marker specific VHH is integrated. The vector may further comprise one or several mutations that inhibit the production of mRNA coding for VP2 and VP3.

[0171] For the production of an AAV, an additional vector could be used, which is mutated, so that VP1 is not expressed, e.g. by a mutation of the VP1 start codon and which therefore codes for VP2 and VP3 but not for VP1.

[0172] The skilled person understands that this principle could be also applied for the production of AAVs in which the SMBD, e.g. a sdAb or VHH, is integrated into two different VP capsid protein types, such as VP2 and VP3. Accordingly, in general, a first vector comprises the nucleotide sequence coding for a fusion protein comprising the one type of capsid protein in which the cell surface marker specific VHH is integrated, while the expression of the two other types of capsid proteins is inhibited. A second vector comprises the nucleotide sequence coding for the two other types of capsid proteins, while the expression of the capsid protein, which is modified is inhibited.

[0173] Recombinant AAV

[0174] Herein, the terms “AAV vector particle” and “recombinant AAV” (“rAAV”) may be used interchangeably.

[0175] Another aspect of the disclosure relates to an AAV comprising an AAV capsid fusion protein as described herein.

[0176] In some embodiments, the capsid protein into which the SMBD (e.g. sdAb or VHH) is integrated is of the same serotype as the capsid proteins into which the SMBD (e.g. sdAb or VHH) protein is not integrated.

[0177] In other embodiments, the capsid protein into which the SMBD (e.g. sdAb or VHH) is integrated is of a different serotype than the capsid proteins into which the SMBD (e.g. sdAb or VHH) protein is not integrated.

[0178] The inventors showed that surprisingly the capsid protein comprising a SMBD (e.g. sdAb or VHH) could be used in combination with capsid proteins of a different serotype without compromising the transfection efficiency. Surprisingly such mosaic viruses showed a strong transfection efficiency. The usage of a SMBD (e.g. sdAb or VHH) comprising capsid protein of a different serotype allows to easily adapt the whole repertoire of AAV virus serotypes without the need of modifying the capsid proteins for each serotype. Thereby an efficient system for the fast generation of different AAV serotypes specific for a cell surface protein is provided.

[0179] For example, the capsid protein into which the SMBD (e.g. sdAb or VHH) is integrated is of the serotype AAV2, while the remaining capsid proteins are of a different serotype, for example a serotype selected from the group consisting of AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAVrhlO. In one embodiment, the capsid protein into which the SMBD (e.g. sdAb or VHH) is integrated is of the serotype AAV2, or a variation thereof such as AAV2RA, and the other capsid proteins are of a different serotype, e.g. AAV8 or AAV9. For example, in one embodiment, a fusion protein of the VP3 capsid protein into which the SMBD, e.g. a sdAb or a VHH, is integrated, is of the serotype AAV2 and the capsid proteins VP1 and VP2 (not comprising an SMBD insert) are of another serotype (such as serotype AAV8 or AAV9). The skilled person understands that the modular system combining capsid proteins comprising VHH domains of one serotype with capsid proteins (not comprising a VHH domain) can be transferred to all serotypes.

[0180] AAV capsid protein assembly and method for preparing recombinant AAV

[0181] In a further aspect, the present disclosure relates to methods for producing recombinant AAV.

[0182] During assembly of the AAV capsid, the capsid proteins VP1, VP2, and VP3 are incorporated approximately at a ratio of 1 : 1 : 10 into the AAV capsids.

[0183] Previously described methods for the preparation of sdAb (Nb)-modified AAV capsids were based on the incorporation of the sdAbs into the GH2 / GH3 or GH12 / GH13 loop of VP1 or VP2. Typically, the VHH is integrated into AAV capsid protein VP2 or VP1. In previous methods, the VHH was preferably integrated into VP1. VP1 - in contrast to VP2 - is required for efficient infection due to the N-terminal phospholipase A2 domain (Girod et al. 2002; Warrington et al. 2004). The fact that VP1 is encoded by a separate messenger RNA, while VP2 and VP3 are encoded by a common mRNA, facilitates the assembly of VHH-comprising chimeric capsids of different AAV serotypes.

[0184] The disadvantage of this previous method is that the incorporation of the modified VP proteins turned out to be less efficient compared to the wild-type VP proteins, so that the resulting capsid population either does not possess the typical stoichiometry between VP1:VP2:VP3 of 1 : 1 : 10 or a high proportion of the capsids are totally VPl-Nb-less or VP2-Nb-less. In most cases, the sdAb- bearing capsid population has to be enriched via Protein A Sepharose chromatography and typically only about 2% of particles containing the VPl-Nb is recovered. As this inefficient process is associated with relatively high losses, it makes the production of the capsids uneconomical for clinical applications. However, as the VP3 capsid protein forms the major part of the capsid, insertion of the SMBD in the VP3 capsid protein allowing for an about 10-fold increase in SMBD display compared to SMBD inserts in the VP1 or VP2 capsid proteins, is desirable.

[0185] In a specific aspect, the disclosure relates to a method for the preparation of a recombinant AAV, wherein the recombinant AAV comprises an SMBD inserted into a VP capsid protein selected from VP1, VP2 and / or VP3, wherein the method comprises transfecting into a host cell the following vectors: a) one or more vectors comprising a nucleic acid molecule encoding the AAV helper functions;

[0186] (b) a vector comprising a nucleic acid molecule encoding a VP1, VP2 and / or VP3 capsid protein with an inserted SMBD; and

[0187] (c) a vector comprising a nucleic acid molecule encoding a wild-type AAV VP1, VP2 and / or VP3 capsid protein; preferably encoding a wild-type AAV VP1 capsid protein, a wild-type AAV VP2 capsid protein and a wild-type AAV VP3 capsid protein; and optionally wherein vector (c) comprises a nucleic acid molecule encoding at least the wild-type VP capsid protein of the VP capsid protein that is encoded by the nucleic acid molecule of vector

[0188] (b) in the form of a VP capsid protein with an inserted SMBD (this means that if e.g. vector (b) comprises a nucleic acid molecule encoding a VP3 capsid protein with an inserted SMBD, vector

[0189] (c) comprises a nucleic acid molecule encoding at least the wild-type VP3 capsid protein; as another example, if e.g. vector (b) comprises a nucleic acid molecule encoding a VP2 capsid protein with an inserted SMBD, vector (c) comprises a nucleic acid molecule encoding at least the wild-type VP2 capsid protein), and wherein the ratio of vectors comprising the nucleic acid molecules according to (b) and (c) is from about 1 : 1 to about 1 :20, from 1 : 1 to 1 : 10 or from 1 :1 to 1 :5, preferably from 1 : 1 to 1 :4, or wherein the ratio of vectors comprising the nucleic acid molecules according to (b) and (c) is 1 :2, 1 :3 or 1 :4.

[0190] The inventors surprisingly found that providing both mutated and wildtype VP AAV capsid protein is favorable to produce AAV vector particles. It is believed that this allows for better structural stability and increased production efficiency compared to methods that are based on providing a VP AAV capsid either mutated or wildtype. It can be preferred that vector (b) comprises a nucleic acid molecule encoding a VP1, VP2 or VP3 capsid protein with an inserted SMBD (i.e. only one VP capsid protein has an inserted SMBD, preferably VP3) and that vector (c) comprises a nucleic acid molecule encoding a wild-type AAV VP1, VP2 and VP3 capsid protein (i.e. all the VP capsid proteins are encoded, each as wild-type).

[0191] In the method of the disclosure, thus, a) nucleic acids encoding helper functions, b) the nucleic acids encoding the modified and unmodified VPs, and optionally a packaging plasmid encoding an ITR-flanked transgene expression cassette are used.

[0192] The inventors believe that providing VP3, either alone or in combination with VP1 and / or VP2, both in modified form including an SMBD and in wildtype form (unmodified) is advantageous in view of the abundance of VP3 in the AAV vector particle. Thus, it is believed that a more stable and consistent platform for nanobody insertion can be achieved, without significantly disrupting the overall capsid integrity. Furthermore, VP3 insertion is beneficial in view of higher production yields of AAV vector particles, and it is believed that it allows safe use of the AAV vector particle, e.g. in gene therapy.

[0193] In some embodiments, the SMBD is inserted in (a) the VP3 capsid protein, and in (b) the VP1 and / or VP2 capsid proteins.

[0194] In some embodiments, the SMBD is inserted in the VP1 capsid protein, but not in the VP2 and / or VP3 capsid proteins. In other some embodiments, the SMBD is inserted in the VP2 capsid protein, but not in the VP1 and / or VP3 capsid proteins. In yet other some embodiments, the SMBD is inserted in the VP3 capsid protein, but not in the VP1 and / or VP2 capsid proteins.

[0195] This way, expression of both recombinant AAV capsid protein, e.g. the AAV VP3 capsid protein comprising the SMBD insert in its GH2 / 3 loop, and wild-type or otherwise recombinant AAV capsid proteins, e.g. VP1 and VP2, that do not comprise a SMBD insert at the same time is facilitated. Thereby, assembly of the AAV capsid and preparing recombinant AAV is improved, since the typical stoichiometry of VP1 :VP2:VP3 of 1 : 1 : 10 is provided and the number of SMBD inserts per capsid is increased compared to using separate packaging plasmids, such as one encoding the two wild-type VP proteins (such as VP1 and VP2) and a second encoding the Nb- modified VP protein (VP3-Nb). In particular, the incorporation rates of the sdAb-modified capsids and the AAV particles production yields are higher compared to using separate vectors for the VP capsid protein comprising the SMBD insert and the VP capsid proteins not carrying the SMBD insert.

[0196] By inserting the SMBD into the VP3 capsid protein, binding to target cells and / or target antigens is enhanced. At the same time, insertion of a SMBD into the VP3 protein advantageously does not impair capsid formation.

[0197] In the present disclosure, the incorporation of the sdAb in the GH2 / GH3 loop of VP3 results in higher efficiency, compared with the incorporation of the sdAbs into the GH2 / GH3 or GH12 / GH13 loop of VPl or VP2.

[0198] In some embodiments, the method for the preparation of a recombinant AAV vector particle, wherein the AAV vector particle comprises a SMBD inserted into a VP3 capsid protein, comprises transfecting into a host cell the following vectors:

[0199] (a) one or more vectors encoding the AAV helper functions;

[0200] (b) a vector comprising a nucleic acid molecule encoding a VP3 AAV capsid protein with an inserted SMBD, wherein the inserted SMBD comprises in the following order:

[0201] (i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein;

[0202] (ii) a second sequence segment which comprises a first peptide linker;

[0203] (iii) a third sequence segment which comprises the SMBD;

[0204] (iv) a fourth sequence segment comprising a second peptide linker; and

[0205] (v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein; and

[0206] (c) one or more vector(s) comprising a nucleic acid molecule encoding a wild-type VP1,

[0207] VP2 and / or VP3 AAV capsid protein.

[0208] In a preferred embodiment, the SMBD is inserted in a VP3 capsid protein, but not in the VP1 and / or VP2 capsid proteins. In other some embodiments, the SMBD is inserted in a VP3 capsid protein, and wherein the SMBD is further inserted in the VP1 and / or VP2 capsid proteins.

[0209] In some embodiments, the ratio of vectors encoding the nucleic acid molecules according to (b) and (c) is from about 1 : 1 to about 1 :20, from 1 : 1 to 1 : 10 or from 1 : 1 to 1 :5. For example, the ratio of transgene-expression plasmid and / or the helper-plasmid to Rep / Cap- plasmid is in the range from 1 :2 to 1 :20, preferably from 1 :2 to 1 : 10 and more preferably from 1 :2 to 1 :5.

[0210] The methods of the present disclosure may in some embodiments comprises further transfecting into the host cell

[0211] (d) a vector comprising a nucleic acid molecule encoding a transgene expression cassette, optionally flanked with ITR sequences.

[0212] In typical embodiments of the methods according to the present disclosure, the vector is selected from a plasmid, shuttle vector, phagemide, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector, MVA vector, and AAV vector, preferably the vector is a plasmid.

[0213] In embodiments of the claimed methods, the AAV capsid protein is selected from the group consisting of AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAVrhlO capsid protein or variants thereof or a combination thereof, preferably wherein AAV capsid protein is AAV2 capsid protein or AAV9 capsid protein.

[0214] In the same or further embodiments of the claimed method, the SMBD is selected from the group of an antibody fragment, a VHH, an immunoglobulin single variable domain, scFv, Fab, Fab', F(ab')2, Fv and a synthetic or natural binding domain for a cell surface molecule.

[0215] In a specific embodiment, the SMBD is a sdAb, preferably a human DPP6 sdAb. In other words, in a specific embodiment, the SMBD is a VHH that specifically binds DPP6.

[0216] In another specific embodiment, the SMBD is a VHH, preferably the VHH is specific for CD38, CD73, PD-L1, or P2X7. For example, the SMBD can be a VHH specific for CD38, preferably, wherein the CD38-specific VHH is MU1053, MU375 and MU551. In another example, the SMBD is a VHH specific for CD73, preferably, wherein the CD73-specific VHH is SB116. In yet another example, the SMBD is a VHH specific for PD-L1, preferably, wherein the PD-L1 specific VHH is B3. In the same or further embodiments, the SMBD is inserted in the GH2 / 3 loop of the VP capsid protein. In other embodiments, the SMBD is inserted in the GH12 / 13 loop of the VP capsid protein. In yet other embodiments, the SMBD is inserted in the GH12 / 13 loop and in the GH2 / 3 loop of the VP capsid protein.

[0217] In exemplary specific embodiment of the claimed method, the capsid protein is of AAV serotype AAV9 and the SMBD is inserted at any position in the region of aa 249 to 255 or aa 249 to 254 of AAV9 VP3 as set out in SEQ ID NO: 27, preferably wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 250 to 252 or amino acids 250 to 253 are modified and / or deleted.

[0218] In another specific embodiment of the claimed method, the capsid protein is of AAV serotype AAV2 and the SMBD is inserted at any position in the region of aa 250 to 257 or aa 250 to 258 of AAV2 VP3 as set out in SEQ ID NO: 6, wherein 0-9 amino acids of this region are modified and / or deleted. For example, in an AAV vector of the disclosure, the AAV serotype is AAV2 and the SMBD is inserted in the region of aa 250 to 257 or aa 250 to 258 of AAV2 VP3 as set out in SEQ ID NO: 6, preferably, wherein the amino acids 251 to 257 or amino acids 252 to 257 are modified and / or deleted.

[0219] In an exemplary specific embodiment of the claimed method, the capsid protein is of AAV serotype AAV5 and the SMBD is inserted at any position in the region of aa 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15, preferably wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 251 to 253 are modified and / or deleted.

[0220] In some embodiments, the methods further comprise a step of transfecting into the host cell a transgene-expression plasmid.

[0221] In embodiments of the method according to the present disclosure, the host cell is selected from hamster BHK21 cells, HEK cells, HEK293 cells and derivatives. Other target protein-ex pressing cells may be mouse mammary carcinoma cells, lymphoma cell lines, and primary bone marrow cells endogenously expressing CD38 or P2X7. Some embodiments of the methods of the present disclosure further comprise the step of incubating the transfected host cells under incubation conditions suitable for producing AAV vector particles.

[0222] In some exemplary embodiments, the incubation conditions comprise a temperature in the range of 25 to 40°C, preferably in the range of 30 to 40 °C, more preferably in the range of 32 to 40°C, even more preferably in the range of 35 to 39°C, most preferably in the range of 36 to 38 °C. In other exemplary embodiments, the incubation conditions comprise an atmosphere having a CO2- content of 5% - 10%, preferably 6% to 9%, more preferably 7% to 9%. In further exemplary embodiments, the incubation conditions comprise an atmosphere having a CO2-content of 5% - 10%, preferably 6% to 9%, more preferably 7% to 9%. In yet other exemplary embodiments, the incubation conditions comprise an incubation period in the range of 60 to 80 hours, preferably 65 to 75 hours, more preferably 68 to 74 hours, even more preferably 70-72 hours. This way, the transfection is facilitated, and the transfection efficiency can be increased under optimized conditions.

[0223] In the same or further embodiments, the methods of the present disclosure further comprise the step of harvesting the recombinant AAV capsid.

[0224] In some embodiments, harvesting comprises lysing the host cells under lysing conditions to obtain a lysate. In specific embodiments, lysing comprises incubating the cells und lysing-incubating conditions. In some embodiments, harvesting comprises separating the recombinant AAV capsid from the lysate, such as sterile filtrating, centrifugation and / or affinity chromatography.

[0225] Thus, the present disclosure represents an improvement on the previous methods, both in terms of the incorporation rates of the SMBD-modified capsids and the AAV particles production yields.

[0226] Uses

[0227] Another aspect of the disclosure relates to use of the fusion proteins, the nucleotide sequences, the vectors, the AAVs as described herein for improving the transduction efficiency of the AAV targeted cells.

[0228] Another aspect of the disclosure relates to use of the fusion proteins, the nucleotide sequences, the vectors, the AAVs as described herein for introducing a defined nucleic sequence into a target cell. A further aspect of the disclosure relates to the fusion proteins, the nucleotide sequences, the vectors, the AAVs as described herein for use as a medicament.

[0229] Thus, one embodiment relates to the fusion proteins, the nucleotide sequences, the vectors, the (recombinant) AAVs as described herein for use in gene therapy. Another embodiment relates to the use of the fusion proteins, the AAV vector particles, the nucleotide sequences, or the vectors according to the present disclosure in gene therapy.

[0230] Another aspect of the present disclosure relates to a method of delivering a transgene to a cell of a subject, said method comprising administering to the subject an AAV vector particle according to the present disclosure, wherein said AAV vector particle comprises said transgene. The subject is preferably a human. In principle, the transgene can be any gene that encodes for a protein of interest or an RNA of interest (e.g. an RNAi or a micro-RNA), wherein the gene is to be expressed in the cell of the subject and wherein this expression is beneficial (in particular disease-treating) in the subject. As the transgene is typically to be delivered to a specific (class of) cell(s) of the subject, wherein such specific (class of) cell(s) are characterized by a specific cell surface marker, the AAV vector particle of the present aspect comprises a SMBD directed to or capable of binding the afore-mentioned surface marker, such that the AAV vector particle is specifically targeted to such specific (class of) cell(s) of the subject.

[0231] Thus, some embodiments refer to the treatment of genetic deficiencies or genetic disorders. In one embodiment the genetic disorder is selected from the group consisting of retinitis pigmentosa and other forms of blindness, hemophilia and other blood clotting diseases, muscular dystrophy, lysosomal storage diseases. Here, the sdAb-displaying AAVs can aid in the delivery of functional replacements and / or functional versions of the damaged gene.

[0232] In particular, one embodiment relates to the fusion proteins, the nucleotide sequences, the vectors, the AAVs as described herein for use in the treatment of cancer. In one embodiment, the cancer is hematopoietic or solid cancer. In exemplary embodiments, the cancer is multiple myeloma, pancreatic carcinoma, lung cancer, prostate cancer, breast cancer, colorectal cancer, glioblastoma, melanoma, ovarian cancer, or non-small-cell lung cancer (NSCLC). In some embodiments, the cancer is multiple myeloma, pancreatic carcinoma, lung cancer, prostate cancer. In one embodiment, the cancer comprises cells expressing a protein selected from the group consisting of P2X7, CD38, CD73 and PD-L1. In a specific embodiment, the cancer cells express a protein selected from the group consisting of CD38 and CD73.

[0233] In particular, one embodiment relates to the fusion proteins, the nucleotide sequences, the vectors, the AAVs as described herein for use in the treatment of neurodegenerative diseases, in particular neurogenerative diseases where cells, such as neurons, astrocytes, microglia in the affected brain region express CD38, CD73 and / or P2X7 or other target antigens recognized by the cognate SMBD (e.g. sdAb or VHH). Here, the SMBD-displaying AAV can enhance the delivery of mediators that promote the survival of neurons, prevent formation and / or the deposit of amyloid plaques. In one embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer, Huntington, Parkinson, Amyotrophic lateral sclerosis or prion's disease.

[0234] Some embodiments refer to the fusion proteins, the nucleotide sequences, the vectors, the AAVs as described herein for use in the treatment of chronic infectious diseases, such as those caused by HIV, hepatitis virus, or human papilloma virus. Thereby, the SMBD (e.g. sdAb or VHH) displaying AAV can enhance the delivery of virus neutralizing antibodies and / or immune cell mediators.

[0235] Examples

[0236] Example 1. Generation of VP1- DPP6 sdAb fusion proteins of AAV9

[0237] A modified AAV9 capsid was produced with a method using:

[0238] (i) a packaging plasmid which expresses wildtype VP2 and VP3 (VP1 expression of wildtype (wt) VP1 is inhibited by mutation of start codon in VP1, referred to also as delta VP1), and

[0239] (ii) a second packaging plasmid which expresses VP1 with a sdAb recognizing DPP6 inserted into the GH2 / GH3 loop at aa 451 (i.e. immediately after aa 451) while deleting amino acids 452 to 454. In this second plasmid, the ACG and ATG start codons of VP2 and VP3 were mutated to GGG (VP3) and ACC (VP2), respectively, such that the expression of unmodified VP2 and VP3 is inhibited and only VPl-sdAb is expressed from the second packaging plasmid.

[0240] A wtAAV5 capsid was also produced as a reference.

[0241] For the production of the modified AAV9, HEK293 cells were transfected with the pHelper plasmid, the transgene plasmid pscAAV-CMV-eYFP and plasmids encoding rep of AAV2 and cap of AAV9 (i.e. for the cap of AAV9 plamids (i) and (ii) as mentioned above). Three days after the transfection, AAVs were harvested from the cell supernatant and cell lysates. The purification was performed by POROS™ CaptureSelect™ AAVX Affinity Resin (Thermo Fisher) and an iodixanol gradient centrifugation.

[0242] As shown in Figure 1A, in the purified vector preparation a very weak VPl-Nb band is visible, and a ratio of 0.2 / 2 / 10 was determined by densitometric analysis for VPl-Nb / VP2 / VP3.

[0243] For the functional analysis, parental HEK293 cells (named “DPP6 negative HEK293 cells” in Figure 1 B) and HEK293 cells pre-transfected with a plasmid which expresses the human DPP6 protein (named “DPP6 positive HEK293 cells” in Figure 1 B) were transduced with eYFP- encoding AAV9 capsids consisting of wildtype VP2 and VP3 and a fusion protein of VP1 with a sdAb recognizing DPP6 inserted in the GH2 / GH3 loop, with a Multiplicity of Infection (MOI) of 100.000. The assay was conducted in triplicates and was analyzed via visualization of eYFP expressing cells by fluorescence microscopy; see Figure IB. The functional analysis resulted in high transduction rates. However, MOI of 100.000 was needed compared the MOI used for vectors shown in Examples 2 / 3 and Figure 4 (MOI of 10.000), which were manufactured with the method of the disclosure.

[0244] Example 2. Generation of VP3- DPP6 sdAb fusion proteins of AAV9

[0245] Cloning of plasmids

[0246] The AAV9 CAP gene, which contains an insertion of a codon optimized human DPP6 sdAb at position 451 (i.e. immediately after aa 451) while aa 452 to 454 were deleted or modified, was synthesized and cloned into pcDNA3 by Geneart (Thermo Fisher) resulting in plasmid pcDNA3.1 - AAV9- VP 1 -DPP6_Nb .

[0247] For the generation of pRep2CAP9-DPP6-Nb, the C-terminal portion of the CAP9 gene was excised from the pRep2CAP9 packaging plasmid (Figure 2A) by BsiWI and Xcml and was replaced by the DPP6 Nanobody containing fragment from pcDNA3. l-AAV9-VPl-DPP6_Nb by Gibson assembly. pRep2-CAP9-DPP6-Nb-Delta VP1+VP2 (Figure 2C) was generated by excision of the N-terminal part of the CAP9 gene of pRep2Cap9-DPP6-Nb (Figure 2B) with Hindlll and BsiWI. This fragment was replaced by Gibson Assembly using PCR fragments of pRep2 CAP9-delta VP1 and pRep2 CAP9-delta VP2 which span the mutated VP1 and VP2 start codons, respectively.

[0248] The plasmid pcDNA3-hDPP6 for expression of the human DPP6 protein was synthesized and cloned into pcDNA3.1 by Geneart (Thermo Fisher).

[0249] Production of rAAV9 and rAAV9 containing VP3-Nb fusion proteins

[0250] The production of recombinant AAVs was performed by triple or quadruple transfection of Viral Production Cells 2.0 (VPCs 2.0 from Gibco™) with three or four plasmids as shown below:

[0251] (a) pRep2CAP9 (also referred to as “wt” or “unmodified” packaging plasmid) + pHelper + pAAV- CMV-eGFP (triple transfection, AAV9wt)

[0252] (b) pRep2CAP9 + pRep2CAP9-DPP6-Nb (also referred to as “VPl-Nb, VP2-Nb and VP3-Nb modified” packaging plasmid) + pHelper + pAAV-CMV-eGFP (quadruple transfection)

[0253] (c) pRep2CAP9 + pRep2CAP9-DPP6-Nb-Delta VP1+VP2 (also referred to as “VP3 modified” packaging plasmid) + pHelper + pAAV-CMV-eGFP (quadruple transfection)

[0254] Figure 2 illustrates the DNA plasmids maps used for the production of sdAb-carrying AAV capsids. In particular, an sdAb against DPP6 was inserted into the GH2 / GH3 loop of AAV9 VP1, VP2 and VP3 capsid proteins (Figure 2B) or inserted into the GH2 / GH3 loop of only AAV9 VP3 capsid protein, and not into VP 1 and VP2 proteins (Figure 2C). The plasmid in Figure 2B expresses the modified capsid proteins VPl-Nb, VP2-Nb and VP3-Nb. The plasmid in Figure 2C comprises mutated start codons of VP1 and VP2, such that the expression of unmodified VP1 and VP2 is inhibited and only VP3-Nb is expressed from the plasmid. Figure 2A shows the plasmid map for pRep2CAP9.

[0255] For the quadruple transfections of plasmids in (b) and (c) above, the pRep2CAP9 (wt plasmid) and pRep2CAP9-DPP6-Nb or pRep2CAP9-DPP6-Nb-Delta VP1+VP2 were transfected into the cells at different ratios. The following ratios of the wt and modified plasmid were assayed:

[0256] Table 1: Ratios of sdAb modified packaging plasmids and wt packaging plasmids were assayed.

[0257] On the day of transfection, the viability was determined using a Vial -cassette™ and Nucleocounter™. The cells reached a density of 4.5 to 6.0 x 106viable cells / mL and a viability of >95% to proceed with the transfection. The cells were diluted to a final density of 3 x io6viable cells / mL with fresh Viral Production Medium supplemented with 4 mM GlutaMAX™. Cells were counted again with a Vial -cassette™ and a Nucleocounter™ to ensure they were at the target density of 3 x 106viable cells / mL.

[0258] The cells were incubated in a 37°C incubator with a humidified atmosphere of 8% CO2on an orbital shaker until the DNA / transfection complexes were ready.

[0259] The molar ratio of pAAV-CMV-eGFP plasmid to pRepCap-plasmid DNA to pHelper plasmid DNA was 1 :3 : 1. A total plasmid DNA of 1.5 pg was used for every mL of culture to be transfected. The plasmid DNA was diluted with Viral -Pl ex™ Complexation Buffer to a final volume of 10% of the culture volume to be transfected. To a new tube, AAV-MAX™ Transfection Booster was added at 3 pL per mL of culture to be transfected followed by AAV-MAX™ Transfection Reagent at 6 pL per mL of culture to be transfected. The pre-mixed AAV-MAX™ Transfection Booster / Reagent was slowly added to the diluted plasmid DNA. The complexes were incubated at room temperature for 20 to 30 minutes, then the solution was slowly pipetted into the shaker flask(s) while swirling. AAV-MAX™ Enhancer was added at 10 pL per mL of culture. The flask(s) were gently swirled to mix the cells. The cells were incubated in a 37°C incubator with a humidified atmosphere of 8% CO2 on an orbital shaker for approximately 72 hours.

[0260] Harvesting the rAAV9 particles

[0261] The rAAV particles were harvested 70 to 72 hours post-transfection by adding AAV-MAX™ Lysis Buffer and MgCl2directly to the culture flask (final concentration of IX and 2 mM, respectively). The flasks were swirled to evenly distribute the lysis buffer. Per flask, Benzonase® nuclease was added to a final concentration of 90 units / mL. The flasks were incubated at 37 °C for at least 2 hours on an orbital shaker.

[0262] Clarification and Downstream Processing of rAAV9 particles

[0263] The cell-lysate was clarified using a Sartoclear Dynamics® Lab kit. Sartoclear Dynamics® Lab kits are designed for the rapid clarification and sterile filtration of cell-culture lysates. The kit eliminates the centrifugation step otherwise needed for clarification.

[0264] To clarify and sterilize the sample, diatomaceous earth was added inside the bottle-top filter and then the cell lysate was added. A vacuum was attached to the nozzle on the filter to draw the lysate through the filter.

[0265] To minimize rAAV degradation, the clarified lysate was processed immediately or stored at -20 °C. AAV particles in the clarified lysate were then captured at a flow rate of 1 ml / min on 1-ml Poros™ CaptureSelect™ AAV9 (Thermo Fisher Scientific) columns using an AKTA™ pure chromatography system (Cytiva). Captured material was eluted with 200 mM citrate buffer at pH 2.0 and collected as 1-ml fractions in tubes containing 850 pl 1 M Tris at pH 8.0 for immediate neutralization of the eluted AAV particles. Peak fractions were pooled and stored at 4 °C. For separation of empty and full particles, affinity-captured AAV samples were loaded onto discontinuous iodixanol gradients from 15 % to 60 % and centrifuged for 3 h at 4 °C and 50,000 rpm in a 50.2 Ti Rotor using an Optima™ L-70i ultracentrifuge (both from Beckman Coulter Life Sciences). AAV particles enriched in full capsids were then collected from the 40 % / 60 % iodixanol interface.

[0266] Particles enriched in full capsids were concentrated and formulated using Amicon™ Ultra- 15 centrifugal concentrators with a 100,000 molecular weight cut-off (Merck). Formulated AAV particles were then sterile-filtered through Acrodisc® PP, PES, 0.2 pm 1 cm2 filter units (Pall). 2- step purified particles were formulated in Dulbecco’s phosphate buffered saline (DPBS, 1.47 mM monopotassium phosphate, 2.7 mM potassium chloride, 8.06 mM disodium phosphate heptahydrate, 137 mM NaCl) containing 0.001 % (w / v) poloxamer 188.

[0267] Quality control

[0268] The titers of vector genome containing AAV particles (vg / ml) were determined by qPCR using ITR-specific primers. To confirm the purity after production, AAV particles corresponding to 1.0 x 1010vg were separated on a 10 % SDS-PAGE using the Bio-Rad stain system for visualization of VP proteins. Finally, AAV samples were aliquoted and frozen at - 80 °C.

[0269] Results

[0270] Table 2 below shows the productivity in vg / L of purified AAV9 particles containing DPP6 sdAb, packaged with the different ratios of wildtype to sdAb-expressing packaging plasmids assayed and shown in Table 1. As shown in Table 2, the productivity increases with decreasing amounts of AAV9-Nb packaging plasmid.

[0271] Table 2: Productivity in vg / L of purified DPP6-sdAb containing AAV9 vectors packaged with different ratios of wildtype to Nb-expressing packaging plasmids. Vector yields increase with decreasing amounts of AAV9-Nb packaging plasmids. The relative productivity is based on the 1 : 1 ratio of wildtype plasmid to Nb-expressing packaging plasmid.

[0272] AAV particles were separated on a 10 % SDS-PAGE gel and the different VP proteins were visualized in the gel. As shown in Figure 3, when using molar ratios of wildtype pRep2CAP9 and pRep2CAP9-DPP6-Nb-Delta VP1, Delta VP2 packaging plasmids of 3: 1 and 4: 1 (number (“Num”) 6 and 7 in Table 2), capsids are composed of VP1, VP3-Nb, VP2 and VP3 at molar ratios of 3:2:2: 10 (see lanes 2 and 3, top bands in the gel). This is compared with the transfection of wildtype pRep2CAP9 and pRep2CAP9-DPP6-Nb (Nb inserted in all of VP1, VP2 and VP3) at molar ratios of both plasmids of 3:1 and 4: 1 (Num 4 and 5 in Table 2), wherein the capsids are composed of VPl-Nb, VP2-Nb, VP1, VP3-Nb, VP2 and VP3. In this case, the incorporation rate of VP1, VP3-Nb and VP2 is comparable to the capsids in lanes 2 and 3, while those of VPl-Nb and VP2-Nb are significantly underrepresented (see lanes 5 and 6). In conclusion, about 10 times more Nb-containing VP proteins become incorporated into the AAV9 capsid using the method of the present disclosure. This was especially observed in cases in which unmodified plasmid and Nb-modified plasmid were used at a ratio of 2: 1, 3 : 1 or 4: 1 and in which the nanobody was inserted only in VP3.

[0273] In a separate experiment when using molar ratios of wildtype pRep2CAP9 and pRep2CAP9- DPP6-Nb-Delta VP1, Delta VP2 packaging plasmids of 3: 1 and 4: 1, respectively, AAV capsids were composed of VP1, VP3-Nb, VP2 and VP3 at ratios of (about) 1.3: 1.1 :0.8: 10 and 1.2:0.9:0.8: 10, respectively (see lanes 4 and 5, top bands in the gel in Figure 7). This was compared with the transfection of wildtype pRep2CAP9, wherein the AAV capsids were composed of VP1, VP2 and VP3 at ratios 2:2: 10 (see lane 3 in Figure 7). Titer determination via qPCR provided titers of 2.7E+13 VG / mL and 4.9E13VG / mL for AAV9-DPP6-VP3-Nb-Delta VP1+VP2 preparations using molar ratios of wildtype pRep2CAP9 and pRep2CAP9-DPP6-Nb- Delta VP1, Delta VP2 packaging plasmids of 3 : 1 and 4: 1, respectively. Thus, packaging efficiency decreased with increasing amounts of Nb expressing packaging plasmids. Hence, in case of AAV9 vector particle comprising a DPP6-sdAb inserted only in VP3, producing said AVV vector particles using unmodified plasmid and Nb-modified plasmid at a ratio of 3 : 1 or 4: 1 was observed to be advantageous in view of titer and packaging efficiency.

[0274] Example 3. Functional testing of DPP6 sdAb presenting AAV9 capsids

[0275] 2.5 x 105HEK 293 cells per well were seeded on day one in 6-well plates. After 24h, cells were transfected with pcDNA3-hDPP6 using jetPEI® (Polyplus) transfection reagent.

[0276] After 72h, the cells were passaged and seeded into 12-well plates (2 x 105cells / well). As negative control the same number of wells were seeded with non-transfected HEK 293 cells.

[0277] The next day transfected and non-transfected cells were transduced in duplicates with the AAV9 particles to be tested with a Multiplicity of Transduction (MOI) of 10.000. In particular, the cells were transduced with rAAV particles generated via co-transfection of the following plasmids:

[0278] (a) As a control, triple transfection with pRep2CAP9 + pHelper + pAAV-CMV-eGFP

[0279] (b) Quadruple transfection with pRep2CAP9 + pRep2CAP9-DPP6-Nb + pHelper + pAAV- CMV-eGFP

[0280] (c) Quadruple transfection with pRep2CAP9 + pRep2CAP9-DPP6-Nb-Delta VP1+VP2 + pHelper + pAAV-CMV-eGFP

[0281] Plasmids pRep2CAP9 and pRep2CAP9-DPP6-Nb or pRep2CAP9-DPP6-Nb-Delta VP1+VP2 were transfected in different molar ratios.

[0282] After 72h in a 37°C incubator with a humidified atmosphere of 5% CO2, successful transduction of the cells was monitored by visual inspection of eGFP positive cells under a fluorescence microscope.

[0283] As shown in Figure 4, almost no eGFP positive cells were detectable in parental HEK 293 cells transduced with any vector, while strong eGFP fluorescence was found for all DPP6-sdAb presenting capsids in pre-treated cells but not for the AAV9 wildtype vector. No substantial difference in vector functionality was observed between AAV9-DPP6-Nb 1 :2, AAV9-DPP6-Nb 1 :3, AAV9-DPP6-Nb 1 :4, AAV9-DPP6-Nb-A VP1+VP2_1 :3 and AAV9-DPP6-Nb-A VP1+VP2_1 :4 which is consistent with the comparable VP3-Nb incorporation ratios shown in Fig.3 lanes 4-6. Example 4. Generation of VP3-aCD38 sdAb fusion proteins of AAV2RA

[0284] AAV2RAis a capsid variant of AAV2 carrying two mutations at position 585 and 588 from Arginine to Alanine that reduce binding to cell surface heparan sulfate proteoglycan (HSPG). MU1053 is a sdAb that specifically recognizes human CD38. GFP-encoding AAV2RAthat display MUI 053 in the GH2 / GH3 surface loop were produced in 15 cm cell culture dishes in HEK293 AAV cells by transient transfection. The insertion site of the sdAb into the GH2 / 3 loop of VP3 of AAV2 is between amino acids 453-459, more specifically at amino acid 452 (i.e. immediately after aa 452) while replacing amino acids 453 to 459 (GTTTQSR).

[0285] The pRepCap2 plasmid encoding the AAV2RAcapsid proteins with MUI 053 in the GH2 / GH3 loop was mixed with the parental wild type plasmid encoding the capsid proteins of AAV2RA(without sdAb) in ratios of 2: 1, 1 : 1, 1 :2, 1 :3 and 1 :4, as well as with a GFP-encoding transgene and a helper plasmid.

[0286] The plasmid mixture was transfected into HEK cells using polyethyleneimine. Cells were cultivated for 72h, the supernatants were harvested and cleared by high-speed centrifugation. Free plasmid and genomic DNAs were removed by digestion with benzonase. Cell supernatants (1 pl per well) were added to HEK cells stably transfected with CD38 or P2X7 (2xl04cells / well). P2X7 was used as a control. Cells were incubated for 48h and harvested. Transduction efficiencies were monitored by analyzing cells for GFP expression by flow cytometry.

[0287] The results are presented in Figure 5. The results show much higher GFP-expression of CD38- transfected HEK cells than of P2X7 -transfected cells after incubation with AAV2RAthat carry the CD38-specific sdAb MUI 053 inserted into the GH2 / GH3 loop. The aCD38-sdAb containing AAV2RAcapsids assembled at ratios of 1 : 1 - 1 :4 result in much higher transduction, i.e. GFP expression detected by flow cytometry, in HEK293 cells expressing CD38 (top row) than in control HEK cells expressing P2X7 (bottom row). The aCD38-sdAb containing AAV2RAcapsids also result in much higher transduction than wildtype AAV2RAcapsids (left panels). The transduction of the CD38-expressing target cells was enhanced most when the plasmid encoding the AAV2RAcapsid proteins (wild-type plasmid) was mixed in a threefold molar excess with the plasmid encoding AAV2RAcapsid proteins with the sdAb in the GH2 / GH3 loop. Control transductions were performed with cell supernatants containing AAV2RA(without sdAb). These show comparable low levels of GFP expression. Example 5. Generation of VP3-DPP6 sdAb fusion proteins of AAV5

[0288] Cloning of plasmids

[0289] The plasmid pRep2Cap5_VPl_VP2_start_mut_DPP6Nb, which contains an insertion of a codon optimized human DPP6 sdAb at aa 441 (i.e. immediately after aa 441) while removing aa 442 to 444) of the CAP5 gene, was generated by Gibson Assembly. The CAP5 gene was excised with Swal and Spel. For the reconstruction of the N-terminal and C-terminal CAP5 portions and for the insertion of the DPP6 sdAb, four PCR fragments were amplified using the pRep2Cap5 packaging plasmid and the pRep2CAP5-DPP6-Nb-Delta VP1+VP2 plasmid as PCR templates. pRep2CAP5-DPP6-Nb-Delta VP1+VP2 was generated by excision of the C-terminal part of Rep and the N-terminal part of the CAP5 gene of pRep2Cap5-DPP6-Nb with Hindlll and BsiWI. This fragment was replaced by Gibson Assembly using PCR fragments of pRep2 CAP5-delta VP1 and pRep2 CAP5-delta VP2 which span the mutated VP1 and VP2 start codons, respectively.

[0290] The plasmid pcDNA3-hDPP6 for expression of the human DPP6 protein was synthesized and cloned into pcDNA3.1 by Geneart (Thermo Fisher).

[0291] Production of rAAV5 and rAAV5 containing VP3-Nb fusion proteins

[0292] The production of recombinant AAVs was performed by triple or quadruple transfection of Viral Production Cells 2.0 (VPCs 2.0 from Gibco™) with three or four plasmids as shown below:

[0293] DPP6-modified: pRep2CAP5 + pRep2-Cap5_VPl_VP2_start_mut_DPP6Nb + pHelper + pAAV- CMV-eGFP (quadruple transfection) wt AAV5: pRep2CAP5 + pHelper + pAAV-CMV-eGFP (triple transfection)

[0294] For the quadruple transfections of the plasmid above, the pRep2CAP5 (wt plasmid) and pRep2- Cap5_VPl_VP2_start_mut_DPP6Nb were transfected into the cells at a ratio of 4: 1.

[0295] On the day of transfection, the viability was determined using a Vial -cassette™ and Nucleocounter™. The cells reached a density of 4.5 to 6.0 x 106viable cells / mL and a viability of >95% to proceed with the transfection. The cells were diluted to a final density of 3 x io6viable cells / mL with fresh Viral Production Medium supplemented with 2 mM GlutaMAX™. Cells were counted again with a Vial -cassette™ and a Nucleocounter™ to ensure they were at the target density of 3 x 106viable cells / mL. The cells were incubated in a 37°C incubator with a humidified atmosphere of 8% CO2on an orbital shaker until the DNA / transfection complexes were ready. AAV-MAX™ Enhancer was added at 10 pL per mL of culture. The flask(s) were gently swirled to mix the cells.

[0296] The molar ratio of pAAV-CMV-eGFP plasmid to pRepCap-plasmids DNA to pHelper plasmid DNA was 1 :3 : 1. A total plasmid DNA of 1.5 pg was used for every mL of culture to be transfected. The plasmid DNA was diluted with Viral -Pl ex™ Complexation Buffer to a final volume of 10% of the culture volume to be transfected. To a new tube, AAV-MAX™ Transfection Booster was added at 3 pL per mL of culture to be transfected followed by AAV-MAX™ Transfection Reagent at 6 pL per mL of culture to be transfected and incubated for 10 minutes. The pre-mixed AAV- MAX™ Transfection Booster / Reagent was slowly added to the diluted plasmid DNA. The complexes were incubated at room temperature for 20-25 minutes, then the solution was slowly pipetted into the shaker flask(s) while swirling. The cells were incubated in a 37°C incubator with a humidified atmosphere of 8% CO2 on an orbital shaker for approximately 72 hours.

[0297] Harvesting the rAAV5 particles

[0298] The rAAV5 particles were harvested 70 to 72 hours post-transfection by adding AAV-MAX™ Lysis Buffer and MgCl2directly to the culture flask (final concentration of IX and 2 mM, respectively). The flasks were swirled to evenly distribute the lysis buffer. Per flask, Benzonase® nuclease was added to a final concentration of 250 units / mL. The flasks were incubated at 37 °C for at least 2 hours on an orbital shaker.

[0299] Clarification and Downstream Processing of rAAV5 particles

[0300] The cell-lysate was clarified using a Sartoclear Dynamics® Lab kit. Sartoclear Dynamics® Lab kits are designed for the rapid clarification and sterile filtration of cell-culture lysates. The kit eliminates the centrifugation step otherwise needed for clarification.

[0301] To clarify and sterilize the sample, diatomaceous earth was added inside the bottle-top filter and then the cell lysate was added. A vacuum was attached to the nozzle on the filter to draw the lysate through the filter.

[0302] To minimize rAAV degradation, the clarified lysate was processed immediately or stored at -20 °C. AAV particles in the clarified lysate were then captured at a flow rate of 1 ml / min on 1-ml Poros™ GoPure AAVx (Thermo Fisher Scientific) columns using an AKTA™ pure chromatography system (Cytiva). Captured material was eluted with 200 mM citrate buffer at pH 2.0 and collected as 1-ml fractions in tubes containing 850 pl 1 M Tris at pH 8.0 for immediate neutralization of the eluted AAV particles. Peak fractions were pooled and stored at 4 °C. For separation of empty and full particles, affinity-captured AAV samples were loaded onto discontinuous iodixanol gradients from 15 % to 60 % and centrifuged for 3 h at 4 °C and 50,000 rpm in a 50.2 Ti Rotor using an Optima™ L-70i ultracentrifuge (both from Beckman Coulter Life Sciences). AAV particles enriched in full capsids were then collected from the 40 % / 60 % iodixanol interface.

[0303] Particles enriched in full capsids were concentrated and formulated using Amicon™ Ultra- 15 centrifugal concentrators with a 100,000 molecular weight cut-off (Merck). Formulated AAV particles were then sterile-filtered through Acrodisc® PP, PES, 0.2 pm 1 cm2 filter units (Pall). 2- step purified particles were formulated in Dulbecco’s phosphate buffered saline (DPBS, 1.47 mM monopotassium phosphate, 2.7 mM potassium chloride, 8.06 mM disodium phosphate heptahydrate, 137 mM NaCl) containing 0.001 % (w / v) poloxamer 188.

[0304] Quality control

[0305] The titers of vector genome containing AAV5 particles (vg / ml) were determined by qPCR using ITR-specific primers. To confirm the purity after production, AAV particles corresponding to 1.0 x 1010vg were separated on a 10 % SDS-PAGE using the Bio-Rad stain system for visualization of VP proteins. Finally, AAV samples were aliquoted and frozen at - 80 °C.

[0306] Results

[0307] Table 3 below shows the productivity in vg / L of purified AAV5 particles containing DPP6 sdAb.

[0308] Table 3: Productivity in vg / L of purified DPP6-sdAb containing AAV5 vector packaged with 4: 1 wildtype to Nb-expressing packaging plasmids. AAV particles were separated on a 10 % SDS-PAGE gel and the different VP proteins were visualized in the gel. As shown in Figure 6, when using molar ratios of wildtype pRep2CAP5 and pRep2CAP5-DPP6-Nb-Delta VP1, Delta VP2 packaging plasmids of 4: 1, capsids are composed of VP1, VP3-Nb, VP2 and VP3 at molar ratios of 2:3:2: 10.

[0309] The description further contains the following numbered embodiments:

[0310] 1. An adeno-associated virus (AAV) capsid fusion protein comprising a VP3 AAV capsid protein and a surface marker binding domain (SMBD), wherein the SMBD is inserted in the GH2-GH3 loop of the VP3 AAV capsid protein.

[0311] 2. The AAV capsid fusion protein according to embodiment 1, wherein the AAV capsid fusion protein further comprises at least one linker.

[0312] 3. The AAV capsid fusion protein according to embodiment 2, wherein each linker has a length of 1 to 100, 2 to 50, 3 to 40, 4 to 30, such as 5 amino acids.

[0313] 4. The AAV capsid fusion protein according to any one of embodiments 1 to 3, wherein the SMBD is flanked by an N-terminal and a C-terminal linker.

[0314] 5. The AAV capsid fusion protein according to embodiment 4, wherein the N-terminal linker has a length of 1 to 45, preferably 10 to 35, more preferably 15 to 30, most preferably 20 to 25 aa.

[0315] 6. The AAV capsid fusion protein according to any one of embodiments 4 or 5, wherein the N- terminal linker comprises the sequence as set out in SEQ ID NO: 40 or a sequence at least 80 % identical thereto.

[0316] 7. The AAV capsid fusion protein according to any one of embodiments 4 to 6, wherein the C- terminal linker has a length of 1 to 20, preferably 1 to 10, more preferably 2 to 7 amino acids.

[0317] 8. The AAV capsid fusion protein according to any one of embodiments 4 to 7, wherein the C- terminal linker comprises a sequence selected from the group consisting of

[0318] (i) SEQ ID NO: 41 or a sequence at least 80% identical thereto,

[0319] (ii)SEQ ID NO: 42 or a sequence at least 80% identical thereto, and

[0320] (iii) SEQ ID NO: 43 or a sequence at least 80% identical thereto.

[0321] 9. The AAV capsid fusion protein according to any one of embodiments 4 to 8, wherein the C- terminal linker comprises the sequence set out in SEQ ID NO: 41 or a sequence at least 80% identical thereto.

[0322] 10. The AAV capsid fusion protein according to any one of embodiments 1-9, wherein the SMBD is capable of directly binding to a specific cell membrane structure, thereby connecting a viral particle expressing the inserted protein to a target cell that is to be transduced by the viral particle. The AAV capsid fusion protein according to any one of embodiments 1-10, wherein the VP3 AAV capsid protein is selected from the group consisting of AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAVrhlO capsid protein or variants thereof or a combination thereof. The AAV capsid fusion protein according to any one of embodiments 1-11, wherein the VP3 AAV capsid protein is AAV9 capsid protein. The AAV capsid fusion protein according to any one of embodiments 1-11, wherein the VP3 AAV capsid protein is AAV2 capsid protein. The AAV capsid fusion protein according to any one of embodiments 1-11, wherein the VP3 AAV capsid protein is AAV5 capsid protein. The AAV capsid fusion protein according to any one of the preceding embodiments, wherein the SMBD is selected from the group consisting of an antibody fragment, a VHH, an immunoglobulin single variable domain, scFv, Fab, Fab', F(ab')2, Fv and a synthetic or natural binding domain for a cell surface molecule. The AAV capsid fusion protein according to embodiment 15, wherein the SMBD is a VHH that specifically binds a dipeptidyl aminopeptidase-like protein 6 (DPP6). The AAV capsid fusion protein according to embodiment 15, wherein the SMBD is a VHH that specifically binds to CD38, CD73, PD-L1, or P2X7. The AAV capsid fusion protein according to embodiment 17, wherein the SMBD is a VHH that specifically binds CD38, preferably, wherein the CD38-specific VHH is selected from the group consisting of MU1053, MU375 and MU551. The AAV capsid fusion protein according to embodiment 17, wherein the SMBD is a VHH that specifically binds CD73, preferably, wherein the CD73-specific VHH is SB116. The AAV capsid fusion protein according to embodiment 17, wherein the SMBD is a VHH that specifically binds PD-L1, preferably, wherein the PD-L1 specific VHH is B3. The AAV capsid fusion protein according to any one of embodiments 1 to 12 and 15 to 20, wherein the AAV serotype is AAV9 and wherein the SMBD is inserted at any position in the region of aa 249 to 255 of AAV9 VP3 as set out in SEQ ID NO: 27; preferably, wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 250 to 252 are modified and / or deleted. The AAV capsid fusion protein according to any one of embodiments 1 to 12 and 15 to 21, wherein the AAV serotype is AAV9 and wherein the SMBD is inserted at position 250 of AAV9 VP3 as set out in SEQ ID NO: 27, preferably replacing and / or modifying amino acids 250 to 252. The AAV capsid fusion protein according to any one of embodiments 1 to 11, 13 and 15 to 20, wherein the AAV serotype is AAV2 and wherein the SMBD is inserted at any position in the region of aa 250 to 258 or 250 to 257 of AAV2 VP3 as set out in SEQ ID NO: 6, preferably wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted, more preferably, wherein the amino acids 251 to 258 or 251 to 257 are modified and / or deleted. The AAV capsid fusion protein according to any one of embodiments 1 to 11, 13, 15 to 20, and 23, wherein the AAV serotype is AAV2 and wherein the SMBD is inserted at any position in the region of aa 250 to 257 or aa 252 to 257 of AAV2 VP3 as set out in SEQ ID NO: 6, preferably replacing amino acids GTTTQSR. The AAV capsid fusion protein according to any one of embodiments 1 to 11 and 14 to 20, wherein the AAV serotype is AAV5 and wherein the SMBD is inserted at any position in the region of aa 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15; preferably wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted. The AAV capsid fusion protein according to any one of embodiments 1 to 11, 14 to 20 and 25, wherein the AAV serotype is AAV5 and wherein the SMBD is inserted at position aa 250 of AAV5 VP3, preferably replacing amino acids 251 to 253. The AAV capsid fusion protein according to any one of embodiments 1 to 26, wherein the SMBD is a VHH that specifically binds to DPP6, and the AAV serotype is AAV9. The AAV capsid fusion protein according to embodiment 27, wherein the AAV capsid fusion protein comprises the sequence of SEQ ID NO: 47 or a sequence at least 80% identical thereto. The AAV capsid fusion protein according to any one of embodiments 1 to 26, wherein the SMBD is a VHH that specifically binds to CD38, and the AAV serotype is AAV2. 30. The AAV capsid fusion protein according to embodiment 29, wherein the AAV capsid fusion protein comprises the sequence of SEQ ID NO: 51 or a sequence at least 80% identical thereto.

[0323] 31. An AAV vector particle comprising:

[0324] (a) a VP3 AAV capsid protein with an inserted surface marker binding domain (SMBD), wherein the VP3 AAV capsid protein with the inserted SMBD comprises in the following order:

[0325] (i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein;

[0326] (ii) a second sequence segment which comprises a first peptide linker;

[0327] (iii) a third sequence segment which comprises the SMBD;

[0328] (iv) a fourth sequence segment which comprises a second peptide linker; and

[0329] (v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein; and

[0330] (b) a wild-type VP3 AAV capsid protein that does not comprise an inserted SMBD.

[0331] 32. The AAV vector particle of embodiment 31, wherein the first and second peptide linkers are flexible.

[0332] 33. The AAV vector particle according to embodiment 31 or 32, wherein the first or second linker has a length of 1 to 100, 2 to 50, 3 to 40, 4 to 30 amino acids.

[0333] 34. The AAV vector particle according to embodiment 31 or 32, wherein the first or second linker has a length of 1 to 5 amino acids.

[0334] 35. The AAV vector particle according to any one of embodiments 31-34, wherein the SMBD is capable of directly binding to a specific cell membrane structure, thereby connecting a viral particle expressing the inserted protein to a target cell that is to be transduced by the viral particle.

[0335] 36. The AAV vector particle according to any one of embodiments 31-35, wherein the VP3 AAV capsid protein is selected from the group consisting of AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAVrhlO capsid protein or variants thereof or a combination thereof.

[0336] 37. The AAV vector particle according to any one of embodiments 31-36, wherein the VP3 AAV capsid protein is AAV9 capsid protein.

[0337] 38. The AAV vector particle according to any one of embodiments 31-36, wherein the VP3 AAV capsid protein is AAV2 capsid protein.

[0338] 39. The AAV vector particle according to any one of embodiments 31-36, wherein the VP3 AAV capsid protein is AAV5 capsid protein.

[0339] 40. The AAV vector particle according to any one of embodiments 31-39, wherein the SMBD is selected from the group consisting of an antibody fragment, a VHH, an immunoglobulin single variable domain, scFv, Fab, Fab', F(ab')2, Fv and a synthetic or natural binding domain for a cell surface molecule.

[0340] 41. The AAV vector particle according to embodiment 40, wherein the SMBD is a VHH that specifically binds a dipeptidyl aminopeptidase-like protein 6 (DPP6).

[0341] 42. The AAV vector particle according to embodiment 40, wherein the SMBD is a VHH that specifically binds to CD38, CD73, PD-L1, or P2X7.

[0342] 43. The AAV vector particle according to embodiment 42, wherein the SMBD is a VHH that specifically binds CD38, preferably, wherein the CD38-specific VHH is selected from the group consisting ofMU1053, MU375 and MU551.

[0343] 44. The AAV vector particle according to embodiment 42, wherein the SMBD is a VHH specific that specifically binds CD73, preferably, wherein the CD73-specific VHH is SB116.

[0344] 45. The AAV vector particle according to embodiment 42, wherein the SMBD is a VHH specific that specifically binds PD-L1, preferably, wherein the PD-L1 specific VHH is B3.

[0345] 46. The AAV vector particle according to any one of embodiments 31-45, wherein the SMBD is inserted in the GH2-GH3 loop or the GH12-13 of the VP3 AAV9 capsid protein.

[0346] 47. The AAV vector particle according to any one of embodiments 31-46, wherein the AAV serotype is AAV9 and wherein the SMBD is inserted at any position in the region of aa 249 to 254 or aa 249 to 255 of AAV9 VP3 as set out in SEQ ID NO: 27, preferably, wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 250 to 253 or amino acids 250 to 252 are modified and / or deleted. The AAV vector particle according to embodiment 47, wherein the AAV serotype is AAV9 and wherein the SMBD is inserted at position 254 of AAV9 VP3 as set out in SEQ ID NO: 27 or at position 249, preferably replacing and / or modifying aa 250 to 252. The AAV vector particle according to any one of embodiments 31-45, wherein the SMBD is inserted in the GH2-GH3 loop or the GH12-13 of the VP3 AAV2 capsid protein. The AAV vector particle according to any one of embodiments 31-45 and 49, wherein the AAV serotype is AAV2 and wherein the SMBD is inserted at any position in the region of aa 250 to 258 or aa 250 to 257 of AAV2 VP3 as set out in SEQ ID NO: 6, preferably wherein 0-9 amino acids of this region are modified and / or deleted. The AAV vector particle according to embodiment 49 or 50, wherein the AAV serotype is AAV2 and wherein the SMBD is inserted in the region of aa 250 to 258 or aa 250 to 257 of AAV2 VP3 as set out in SEQ ID NO: 6, preferably, wherein the amino acids 251 to 258 or 251 to 257 are modified and / or deleted. The AAV vector particle according to any one of embodiments 49-51, wherein the AAV serotype is AAV2 and wherein the SMBD is inserted between amino acids 251 to 257 as set out in SEQ ID NO: 6, preferably replacing amino acids GTTTQSR. The AAV vector particle according to any one of embodiments 31-45, wherein the SMBD is inserted in the GH2-GH3 loop or the GH12-13 of the VP3 AAV5 capsid protein. The AAV vector particle according to any one of embodiments 31-45 and 53, wherein the AAV serotype is AAV5 and wherein the SMBD is inserted at any position in the region of aa 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15; preferably wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted. The AAV vector particle according to any one of embodiments 31-45, 53 and 54, wherein the AAV serotype is AAV5 and wherein the SMBD is inserted at position 250, preferably replacing or removing amino acids 251 to 253 of AAV5 VP3 as set out in SEQ ID NO: 15. The AAV vector particle according to any one of embodiments 31 to 48, wherein the SMBD is a VHH that specifically binds to DPP6, and the AAV serotype is AAV9. The AAV vector particle according to embodiment 56, wherein the AAV capsid fusion protein comprises the sequence of SEQ ID NO: 47 or a sequence at least 80% identical thereto. The AAV vector particle according to any one of embodiments 31-45 or 49-52, wherein the SMBD is a VHH that specifically binds to CD38, and the AAV serotype is AAV2. The AAV vector particle according to embodiment 58, wherein the AAV capsid fusion protein comprises the sequence of SEQ ID NO: 51 or a sequence at least 80% identical thereto. A nucleotide sequence encoding the AAV capsid fusion protein according to any one of embodiments 1 to 30. A vector comprising the nucleotide sequence according to embodiment 60. An AAV comprising the capsid fusion protein according to any one of embodiments 1 to 30. The AAV according to embodiment 62, wherein the capsid protein into which the SMBD is inserted is of AAV9 serotype or AAV2 serotype or AAV5 serotype. The AAV of embodiment 62 or 63, wherein the VP1 and / or VP2 capsid proteins do not comprise an SMBD insert. Use of the AAV capsid fusion protein according to any one of embodiments 1 to 30, the AAV vector particle according to any one of embodiments 31 to 59, the nucleotide sequence according to embodiment 60, the vector according to embodiment 61, or the AAV according to any one of embodiments 62-64 for introducing a defined nucleic sequence into a target cell. Use of the AAV capsid fusion protein according to any one of embodiments 1 to 30, the AAV vector particle according to any one of embodiments 31 to 59, the nucleotide sequence according to embodiment 60, the vector according to embodiment 61, or the AAV according to any one of embodiments 62-64 for improving the transduction efficiency of the AAV targeted cells. Use of the AAV capsid fusion protein according to any one of embodiments 1 to 30, the AAV vector particle according to any one of embodiments 31 to 59, the nucleotide sequence according to embodiment 60, the vector according to embodiment 61, or the AAV according to any one of embodiments 62-64 in gene therapy. The AAV capsid fusion protein according to any one of embodiments 1 to 30, the AAV vector particle according to any one of embodiments 31 to 59, the nucleotide sequence according to embodiment 60, the vector according to embodiment 61, or the AAV according to any one of embodiments 62-64 for use in gene therapy. A method of delivering a transgene to a cell of a subject, said method comprising administering to the subject an AAV vector particle according to any one of embodiments 31 to 59, wherein said AAV vector particle comprises said transgene. 70. A method for the preparation of a recombinant AAV, wherein the recombinant AAV comprises an SMBD inserted into a VP capsid protein selected from VP1, VP2 and / or VP3, wherein the method comprises transfecting into a host cell the following vectors:

[0347] (a) one or more vectors comprising a nucleic acid molecule encoding the AAV helper functions;

[0348] (b) a vector comprising a nucleic acid molecule encoding a VP1, VP2 and / or VP3 capsid protein with an inserted SMBD; and

[0349] (c) a vector comprising a nucleic acid molecule encoding a wild-type AAV VP1, VP2 and / or VP3 capsid protein; and wherein the ratio of vectors comprising the nucleic acid molecules according to (b) and (c) is from about 1 : 1 to about 1 :20, from 1 : 1 to 1 : 10 or from 1 : 1 to 1 :5. The method of embodiment 70, wherein the SMBD is inserted in the VP1 capsid protein, but not in the VP2 and / or VP3 capsid proteins. The method of embodiment 70, wherein the SMBD is inserted in the VP2 capsid protein, but not in the VP1 and / or VP3 capsid proteins. The method of embodiment 70, wherein the SMBD is inserted in the VP3 capsid protein, but not in the VP1 and / or VP2 capsid proteins. A method for the preparation of a recombinant AAV vector particle, wherein the AAV vector particle comprises a SMBD inserted into a VP3 capsid protein, wherein the method comprises transfecting into a host cell the following vectors:

[0350] (a) one or more vectors encoding the AAV helper functions;

[0351] (b) a vector comprising a nucleic acid molecule encoding a VP3 AAV capsid protein with an inserted SMBD, wherein the inserted SMBD comprises in the following order:

[0352] (i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein;

[0353] (ii) a second sequence segment which comprises a first peptide linker;

[0354] (iii) a third sequence segment which comprises the SMBD; (iv) a fourth sequence segment comprising a second peptide linker; and

[0355] (v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein; and

[0356] (c) one or more vector(s) comprising a nucleic acid molecule encoding a wild-type VP1, VP2 and / or VP3 AAV capsid protein.

[0357] 75. The method of embodiment 74, wherein the SMBD is inserted in a VP3 capsid protein, but not in the VP1 and / or VP2 capsid proteins.

[0358] 76. The method of embodiment 74, wherein the SMBD is inserted in a VP3 capsid protein, and wherein the SMBD is further inserted in the VP1 and / or VP2 capsid proteins.

[0359] 77. The method according to any one of embodiments 70-76, wherein the method comprises further transfecting into the host cell

[0360] (d) a vector comprising a nucleic acid molecule encoding a transgene expression cassette, optionally flanked with ITR sequences.

[0361] 78. The method according to any one of embodiments 74-77, wherein the ratio of vectors encoding the nucleic acid molecules according to (b) and (c) is from about 1 : 1 to about 1 :20, from 1 : 1 to 1 : 10 or from 1 : 1 to 1 :5, preferably (i) from 1 : 1 to 1 :4 or (ii) 1 :2, 1 :3 or 1 :4.

[0362] 79. The method of any one of embodiments 70-78, wherein the vector is selected from a plasmid, shuttle vector, phagemide, cosmid, expression vector, retroviral vector, lentiviral vector, adenoviral vector, MVA vector, and AAV vector, preferably the vector is a plasmid.

[0363] 80. The method of any one of embodiments 70-79, wherein the AAV capsid protein is selected from the group consisting of AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAVrhlO capsid protein or variants thereof or a combination thereof, preferably wherein AAV capsid protein is AAV2 capsid protein or AAV9 capsid protein.

[0364] 81. The method of any one of embodiments 70-80, wherein the SMBD is selected from the group of an antibody fragment, a VHH, an immunoglobulin single variable domain, scFv, Fab, Fab', F(ab')2, Fv and a synthetic or natural binding domain for a cell surface molecule.

[0365] 82. The method of any one of embodiments 70-81 , wherein the SMBD is a VHH that specifically binds DPP6. The method of any one of embodiments 70-82, wherein the SMBD is a VHH that specifically binds to CD38, CD73, PD-L1, or P2X7. The method according to any one of embodiments 070-83, wherein the SMBD is a VHH that specifically binds CD38, preferably, wherein the CD38-specific VHH is selected from the group consisting of MU1053, MU375 and MU551. The method according to any one of embodiments 70-84, wherein the SMBD is a VHH specific that specifically binds CD73, preferably, wherein the CD73-specific VHH is SB 116. The method according to any one of embodiments 70-85, wherein the SMBD is a VHH specific that specifically binds PD-L1, preferably, wherein the PD-L1 specific VHH is B3. The method of any one of embodiments 70-86, wherein the SMBD is inserted in the GH2 / 3 loop of the VP capsid protein. The method of any one of embodiments 70-86, wherein the SMBD is inserted in the GH12 / 13 loop of the VP capsid protein. The method of any one of embodiments 70-86, wherein the SMBD is inserted in the GH12 / 13 loop and in the GH2 / 3 loop of the VP capsid protein. The method according to any one of embodiments 70-86, wherein the AAV serotype is AAV9 and wherein the SMBD is inserted at any position in the region of aa 249 to 254 or aa 249 to 255 of AAV9 VP3 as set out in SEQ ID NO: 27, preferably wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 250 to 253 or amino acids 250 to 252 are modified and / or deleted. The method according to any one of embodiments 70-86, wherein the AAV serotype is AAV2 and wherein the SMBD is inserted at any position in the region of aa 250 to 258 or aa 250 to 257 of AAV2 VP3 as set out in SEQ ID NO: 6, wherein 0-9 amino acids of this region are modified and / or deleted, more preferably, wherein the amino acids 251 to 257 are modified and / or deleted. The method according to any one of embodiments 70-86, wherein the AAV serotype is AAV5 and wherein the SMBD is inserted at any position in the region of aa 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15; preferably wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted, more preferably wherein the amino acids 251 to 253 are modified and / or deleted. 93. The method according to any one of embodiments 70-92, wherein the VP3 capsid protein is a AAV capsid fusion protein according to any one of embodiments 1 to 30.

[0366] 94. The method of any one of embodiments 70-93, wherein the method further comprises a step of transfecting into the host cell a transgene-expression plasmid.

[0367] 95. The method of any one of embodiments 70-94, wherein the host cell is selected from hamster BHK21 cells, HEK cells, HEK293 cells and derivatives thereof.

[0368] 96. The method of embodiment 94 or 95, wherein the ratio of transgene-expression plasmid and / or the helper-plasmid to Rep / Cap-plasmid is in the range from 1 :2 to 1 :20, preferably from 1 :2 to 1 : 10 and more preferably from 1 :2 to 1 :5.

[0369] 97. The method of any one of embodiments 70-96, wherein the method further comprises the step of incubating the transfected host cells under incubation conditions suitable for producing AAV vector particles.

[0370] 98. The method of embodiment 97, wherein the incubation conditions comprise a temperature in the range of 25 to 40°C, preferably in the range of 30 to 40 °C, more preferably in the range of 32 to 40°C, even more preferably in the range of 35 to 39°C, most preferably in the range of 36 to 38 °C.

[0371] 99. The method of embodiment 97 or 98, wherein the incubation conditions comprise an atmosphere having a CO2-content of 5% - 10%, preferably 6% to 9%, more preferably 7% to 9%.

[0372] 100. The method of any one of embodiments 97-99, wherein the incubation conditions comprise an atmosphere having a CO2-content of 5% - 10%, preferably 6% to 9%, more preferably 7% to 9%.

[0373] 101. The method of any one of embodiments 97-100, wherein the incubation conditions comprise an incubation period in the range of 60 to 80 hours, preferably 65 to 75 hours, more preferably 68 to 74 hours, even more preferably 70-72 hours.

[0374] 102. The method of any one of embodiments 70-101, wherein the method further comprises the step of harvesting the recombinant AAV vector particles.

[0375] 103. The method of embodiment 102, wherein harvesting comprises lysing the host cells under lysing conditions to obtain a lysate.

[0376] 104. The method of embodiment 103, lysing comprises incubating the cells und lysing-incubating conditions. 105. The method of any one of embodiments 102 to 104, wherein harvesting comprises separating the recombinant AAV capsid from the lysate.

[0377] REFERENCES

[0378] Adachi et al. (2014) Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing. Nat Commun, 5:3075.

[0379] Bartlett et al. (1999) Targeted adeno-associated virus vector transduction of nonpermisive cells mediated by a bispecific F(ab’gamma)2 antibody. Nat Biotechnol, 17: 181-6.

[0380] Boucas et al. (2009) Engineering andeno-associated virus serotype 2-based targeting vectors using a new insertion site position 453- and single point mutations. J Gene Med 11 : 1103-13

[0381] Els-Conrath et al. (2001) Camel single domain antibodies as modular building units in bispecific and bivalent antibody constructs. J Biol Chem, 276: 7346-50.

[0382] Girod et al. (2002) The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity. J Gen Virol. 83(Pt5):973-8.

[0383] Judd et al. (2012) Random Insertion of mCherry Into VP3 Domain of Adeno-associated Virus Yields Fluorescent Capsids With no Loss of Infectivity. Mol Ther Nucleic Acids 1 : e54.

[0384] Munch et al. (2013) Displaying high-affinity ligands on adeno-associated viral vectors enables tumor cell-specific and safe gene transfer. Mol Ther 21 : 109-18

[0385] Nagy et al. (2003). Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

[0386] Warrington et al. (2004) Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J Virol. 78(12):6595-609.

[0387] Wistuba et al. (1995) Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes comprising Rep and Cap proteins. J Virol. 1995 Sep;69(9):5311-9.

Claims

CLAIMS1. An adeno-associated virus (AAV) capsid fusion protein comprising a VP3 AAV capsid protein and a surface marker binding domain (SMBD), wherein the SMBD is inserted in the GH2-GH3 loop of the VP3 AAV capsid protein.

2. An AAV vector particle comprising(a) a VP AAV capsid protein with an inserted surface marker binding domain (SMBD); and(b) a wild-type VP AAV capsid protein that does not comprise an inserted SMBD; wherein the VP AAV capsid protein is VP1, VP2 and / or VP3.

3. The AAV vector particle of claim 2, wherein the VP AAV capsid protein is VP3.

4. The AAV capsid fusion protein of claim 1, or the AAV vector particle of claim 3, wherein the VP3 AAV capsid protein with the inserted SMBD comprises in the following order:(i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein;(ii) a second sequence segment which comprises a first peptide linker;(iii) a third sequence segment which comprises the SMBD;(iv) a fourth sequence segment which comprises a second peptide linker; and(v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein.

5. The AAV capsid fusion protein of claim 1 or 4, or the AAV vector particle of claim 3 or 4, wherein the VP3 AAV capsid protein is selected from the group consisting of AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAVrhlO capsid protein or variants thereof or a combination thereof.

6. The AAV capsid fusion protein of claim 1, 4 or 5, or the AAV vector particle according to any one of claims 2-5, wherein the SMBD is selected from the group consisting of an antibody fragment, a VHH, an immunoglobulin single variable domain, scFv, Fab, Fab', F(ab')2, Fv and a synthetic or natural binding domain for a cell surface molecule.

7. The AAV vector particle according to any one of claims 3-6, wherein the SMBD is inserted in the GH2-GH3 loop and / or the GH12-13 of the VP3 AAV9 capsid protein.

8. The AAV capsid fusion protein of any one of claims 1, and 4-6, or the AAV vector particle according to any one of claims 3-7, wherein the AAV serotype is AAV9 and wherein the SMBD is inserted at any position in the region of aa 249 to 255 of AAV9 VP3 as set out in SEQ ID NO: 27; preferably, wherein 0, 1, 2, 3, 4, 5, or 6 amino acids of this region are modified and / or deleted, more preferably wherein amino acids 250 to 252 are modified and / or deleted.

9. The AAV capsid fusion protein of any one of claims 1, and 4-6, or the AAV vector particle according to any one of claims 3-7, wherein the AAV serotype is AAV2 and wherein the SMBD is inserted at any position in the region of aa 250 to 257 of AAV2 VP3 as set out in SEQ ID NO: 6, preferably wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted, more preferably, wherein the amino acids 251 to 257 are modified and / or deleted.

10. The AAV capsid fusion protein of any one of claims 1, and 4-6, or the AAV vector particle according to any one of claims 3-7, wherein the AAV serotype is AAV5 and wherein the SMBD is inserted at any position in the region of aa 250 to 254 of AAV5 VP3 as set out in SEQ ID NO: 15; preferably wherein 0, 1, 2, 3, 4, 5, 6 or 7 amino acids of this region are modified and / or deleted; more preferably wherein amino acids 251 to 253 are modified and / or deleted.

11. The AAV vector particle according to any one of claims 3-10, wherein the SMBD is inserted in the GH2-GH3 loop and / or the GH12-13 of the VP3 AAV2 capsid protein.

12. The AAV capsid fusion protein of claim 1, 4-6, or 8-10, or the AAV vector particle according to any one of claims 2-8, wherein the SMBD is a VHH that specifically binds to DPP6, and the AAV serotype is AAV9.

13. The AAV vector particle according to any one of claims 3-12, wherein the SMBD is inserted in the GH2-GH3 loop and / or the GH12-13 of the VP3 AAV5 capsid protein.

14. The AAV vector particle according to any one of claims 2-13, wherein the VP AAV capsid protein comprises the AAV capsid fusion protein of claim 1, 4-6, 8-10, or 12.

15. The AAV vector particle according to any one of claims 2-14, wherein the VP1 and / or VP2 capsid proteins do not comprise an SMBD insert.

16. A nucleotide sequence encoding the AAV capsid fusion protein according to any one of claims 1, 4-6, 8-10, or 12.

17. A vector comprising the nucleotide sequence according to claim 16.

18. Use of the AAV capsid fusion protein according to any one of claims 1, 4-6, 8-10, or 12, the AAV vector particle according to any one of claims 2 to 15, the nucleotide sequence according to claim 16, the vector according to claim 17 for introducing a defined nucleic sequence into a target cell.

19. Use of the AAV capsid fusion protein according to any one of claims 1, 4-6, 8-10, or 12, the AAV vector particle according to any one of claims 2 to 15, the nucleotide sequence according to claim 16, the vector according to claim 17 for improving the transduction efficiency of the AAV targeted cells.

20. A method for the preparation of a recombinant AAV, wherein the recombinant AAV comprises an SMBD inserted into a VP capsid protein selected from VP1, VP2 and / or VP3, wherein the method comprises transfecting into a host cell the following vectors:(a) one or more vectors comprising a nucleic acid molecule encoding the AAV helper functions;(b) a vector comprising a nucleic acid molecule encoding a VP1, VP2 and / or VP3 capsid protein with an inserted SMBD; and(c) a vector comprising a nucleic acid molecule encoding a wild-type AAV VP1, VP2 and / or VP3 capsid protein; preferably encoding a wild-type AAV VP1 capsid protein, a wild-type AAV VP2 capsid protein and a wild-type AAV VP3 capsid protein; optionally wherein vector (c) comprises a nucleic acid molecule encoding at least the wildtype VP capsid protein of the VP capsid protein that is encoded by the nucleic acid molecule of vector (b) in the form of a VP capsid protein with an inserted SMBD, and wherein the ratio of vectors comprising the nucleic acid molecules according to (b) and (c) is from about 1 : 1 to about 1 :20, from 1 : 1 to 1 : 10 or from 1 : 1 to 1 :5, preferably from 1 :1 to 1 :4, or wherein the ration of vectors comprising the nucleic acid molecules according to (b) and (c) is 1 :2, 1 :3 or 1 :4.

21. The method of claim 20, wherein the SMBD is:(a) inserted in the VP1 capsid protein, but not in the VP2 and / or VP3 capsid proteins,(b) inserted in the VP2 capsid protein, but not in the VP1 and / or VP3 capsid proteins, or(c) inserted in the VP3 capsid protein, but not in the VP1 and / or VP2 capsid proteins;and wherein preferably vector (c) comprises a nucleic acid molecule encoding a wild-type AAV VP1 capsid protein, a wild-type AAV VP2 capsid protein and a wild-type AAV VP3 capsid protein.

22. The method of claim 20 or 21, wherein the SMBD is inserted into a VP3 capsid protein, and wherein the vector according to (b) comprises in the following order:(i) a first sequence segment which comprises a first part of the VP3 AAV capsid protein;(ii) a second sequence segment which comprises a first peptide linker;(iii) a third sequence segment which comprises the SMBD;(iv) a fourth sequence segment comprising a second peptide linker; and(v) a fifth sequence segment which comprises the second part of the VP3 AAV capsid protein.

23. The method according to any one of claims 20-22, wherein the SMBD is inserted in a VP3 capsid protein, and wherein the SMBD is further inserted in the VP1 and / or VP2 capsid proteins.

24. The method according to any one of claims 20-23, wherein the method comprises further transfecting into the host cell(d) a vector comprising a nucleic acid molecule encoding a transgene expression cassette, optionally flanked with ITR sequences.

25. The method of any one of claims 20-24, wherein the AAV capsid protein is selected from the group consisting of AAV1 capsid protein, AAV2 capsid protein, AAV3 capsid protein, AAV4 capsid protein, AAV5 capsid protein, AAV6 capsid protein, AAV7 capsid protein, AAV8 capsid protein, AAV9 capsid protein, AAV10 capsid protein, AAV11 capsid protein, AAV12 capsid protein, AAVrhlO capsid protein or variants thereof or a combination thereof, preferably wherein AAV capsid protein is AAV2 capsid protein or AAV9 capsid protein.

26. The method of any one of claims 20-25, wherein the SMBD is selected from the group of an antibody fragment, a VHH, an immunoglobulin single variable domain, scFv, Fab, Fab', F(ab')2, Fv and a synthetic or natural binding domain for a cell surface molecule.

27. The method of any one of claims 20-26, wherein the SMBD is inserted (a) in the GH2 / 3 loop of the VP capsid protein, (b) in the GH12 / 13 loop of the VP capsid protein or (c) in the GH12 / 13 loop and in the GH2 / 3 loop of the VP capsid protein.

28. The method according to any one of claims 20-27, wherein the VP3 capsid protein is an AAV capsid fusion protein according to any one of claims 1, 4-6, 8-10, or 12.

29. The method of any one of claims 20-28, wherein the method further comprises a step of transfecting into the host cell a transgene-expression plasmid.

30. The method of claim 29, wherein the ratio of transgene-expression plasmid and / or the helper-plasmid to Rep / Cap-plasmid is in the range from 1 :2 to 1 :20, preferably from 1 :2 to 1 :10 and more preferably from 1 :2 to 1 :5.

31. Use of (i) the AAV capsid fusion protein according to any one of claims 1, 4-6, 8-10, or 12, (ii) the AAV vector particle according to any one of claims 2 to 15, (iii) the nucleotide sequence of claim 16, or (iv) the vector of claim 17 in gene therapy.

32. The (i) AAV capsid fusion protein according to any one of claims 1, 4-6, 8-10, or 12, the (ii) AAV vector particle according to any one of claims 2 to 15, the (iii) nucleotide sequence of claim 16, or (iv) the vector of claim 17 for use in gene therapy.

33. A method of delivering a transgene to a cell of a subject, said method comprising administering to the subject an AAV vector particle according to any one of claims 2 to 15, wherein said AAV vector particle comprises said transgene.

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