Adeno-associated virus conjugation

Abstract

The present invention relates to an adeno-associated virus (AAV) VP1, VP2 or VP3 capsid protein characterized in that a sortase recognition sequence (wherein n and m are in the range of 0 to 25, and X is any natural amino acid) is inserted in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region and the VR-VIII region.

Description

[Technical field]

[0001] The present invention relates generally to the field of gene therapy. In particular, the present invention relates to techniques for conjugating heterologous molecules (e.g., targeting molecules) to AAV capsids in a controlled manner. [Background technology]

[0002] Gene therapy (GT) is a therapeutic modality that involves modifying cell physiology by the addition of a transgene, thereby treating or preventing disease. This innovative technology uses viral vectors to correct or replace defective genes that cause disease. Gene therapy holds great promise for the treatment of rarer, more complex and devastating diseases for which no curative treatment is currently available.

[0003] Adeno-associated virus (AAV) is a small, non-pathogenic virus that is currently the primary gene delivery vehicle in gene therapy. It has broad tissue tropism, is not associated with disease phenotypes, and is not highly immunogenic. Furthermore, AAV has the potential to provide long-lasting therapeutic effects after a single administration.

[0004] Recombinant AAV (rAAV) is an engineered version of the virus in which viral genes have been placed in trans and replaced with an approximately 4.5 kb transgene. Although rAAV-based GT products have generally been shown to be safe and effective, they have several drawbacks. For example, the processes underlying transduction, i.e., cell entry at the target tissue, uncoating and expression of the therapeutic payload of the viral particles within the target cells, are inefficient and therefore require large vector doses to achieve a therapeutic effect. After systemic administration, this can result in accumulation in the liver and immune responses against the therapeutic vector. Importantly, high doses of rAAV have been associated with severe adverse events, such as hepatotoxicity and thrombotic microangiopathy, which have led to the death of several patients in AAV GT trials. Furthermore, depending on the AAV serotype, 40-70% of the human population harbor AAV neutralizing antibodies. These antibodies contribute to the observed immune responses, reducing efficacy, preventing rechallenge of GT products, and leading to seropositivity, which is an exclusion criterion in many GT trials. Next to these biological problems, production of large quantities of highly purified rAAV product is expensive and production capacity is limited.

[0005] One of the most promising approaches to solving these problems is the design of novel capsids that exhibit improved immune evasion and transduction properties. By narrowing the broad tropism of naturally occurring AAV vectors to specific tissues or cell types through capsid engineering, rAAV products become more potent and allow treatment at lower doses. This reduces safety risks and production costs. However, despite significant efforts in capsid engineering (e.g., directed evolution capsid design platforms), no breakthrough innovations have been validated in the clinic. This is due in part to the broad tropism of naturally found AAV serotypes used as starting materials for directed evolution library generation, the difficulties in producing novel rAAV serotypes, and the need to screen capsid libraries in rodents and non-human primates (NHPs), the latter being a barrier to human translation.

[0006] A promising way to engineer capsids with increased specificity for target tissues and lower off-target transduction without affecting manufacturability is to conjugate AAV particles with specific (re)targeting groups at a post-production stage. Several groups have genetically incorporated targeting groups into capsid proteins or conjugated them with adapter molecules that allow covalent or non-covalent attachment after the AAV particles are purified. These rAAV conjugates have higher transduction efficiency in specific tissues and lower off-target effects, for example in the liver. However, the required modifications of the capsid often affect the production yield. In addition, due to the less modular and complex, often multi-component genetic or chemical design set-up, these systems are hardly compatible with current commercial manufacturing practices. More recently, capsid proteins have been genetically fused to nanobodies, resulting in improved targeting. However, the targeting efficiency was highly dependent on the specific AAV nanobody construct. This can be traced back to the unknown effects of nanobody fusions on the unpredictable folding and quaternary structure formation of modified AAV virions, as well as on the supramolecular dynamics caused by direct genetic incorporation of nanobodies. Genetic insertion of large proteinaceous moieties also negatively impacts the efficiency of affinity purification using commercially available resins. Summary of the Invention [Problem to be solved by the invention]

[0007] In summary, conjugating AAV with targeting groups is a promising strategy to improve the transduction profile of rAAV particles for gene therapy, but current methods lack the design and / or performance qualities required for a commercial gene therapy product and have high manufacturing hurdles. Better approaches for AAV conjugation are desperately needed to be clinically and commercially successful. [Means for solving the problem]

[0008] Through extensive experimentation, the inventors have found that a sortase recognition sequence can be introduced internally into the capsid sequence of an AAV particle to allow conjugation of a targeting molecule with the AAV capsid, producing conjugated AAV particles that can target tissues and cells more efficiently and specifically. Surprisingly, in contrast to the general consensus in the art of molecular biology and biotechnology, where sortase recognition sequences are typically used at the C-terminus, the inventors have found that the insertion of a sortase recognition sequence into the VP1-VP2 transition region, the VR-I region, the VR-IV region, and the VR-VIII region maintains the structural integrity of the region after being subjected to a sortase reaction. The use of an internal sortase recognition sequence is particularly valuable for generating new "generations" of tailored AAV particles with improved properties (e.g., more efficient and specific targeting of tissues and cells of interest). In addition, the molecular design considerations made by the inventors allow for increased modularity of AAV production methods and platforms while maintaining similar production efficiency when compared to the appropriate parent AAV serotype.

[0009] Thus, one aspect of the present invention provides an adeno-associated virus (AAV) capsid protein characterized in that a sortase recognition sequence is inserted into one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and the VR-VIII region. In certain embodiments, the sortase recognition sequence has a general sequence motif selected from the group consisting of: Xn-LPXTG-Xm [SEQ ID NO: 10], Xn-NPXTG-Xm [SEQ ID NO: 40], Xn-LPXTA-Xm [SEQ ID NO: 41], Xn-LAXTG-Xm [SEQ ID NO: 42], Xn-LPXAG-Xm [SEQ ID NO: 48], Xn-LPXLG-Xm [SEQ ID NO: 49], Xn-APXTG-Xm [SEQ ID NO: 50], Xn-LPXSG-Xm [SEQ ID NO: 51], Xn-FPXTG-Xm [SEQ ID NO: 52], Xn-XPKTG-Xm, [SEQ ID NO: 53] and Xn-LPEXG-Xm [SEQ ID NO: 54], where n and m range from 0 to 25 and X is any naturally occurring amino acid independently selected for Xn and Xm. Preferably, the (AAV) VP1, VP2, or VP3 capsid protein has inserted in one or more of the VR-I, VR-IV, and VR-VIII regions the sortase recognition sequence Xn-LPXTG-Xm [SEQ ID NO: 10], where n and m range from 0 to 25, and X is any naturally occurring amino acid. Optionally, n and m range from 0 to 20. Preferably, X is glutamic acid (E) or glutamine (Q).

[0010] In certain embodiments, the VP1-VP2 transition region is defined by SEQ ID NO: 1 [PVKTAP] in AAV2 or the corresponding amino acid sequence in another AAV serotype, the VR-I region is defined by SEQ ID NO: 2 [SSQSGASN] in AAV2 or the corresponding amino acid sequence in another AAV serotype, the VR-IV region is defined by SEQ ID NO: 3 [SRTNTPSGTTTQSRLQFSQAGASDIRDQS] in AAV2 or the corresponding amino acid sequence in another AAV serotype, and the VR-VIII region is defined by SEQ ID NO: 4 [QYGSVSTNLQRGNRQAATADVNTQGV] in AAV2 or the corresponding amino acid sequence in another AAV serotype.

[0011] In certain embodiments, the insertion in the VR-IV region is in a fragment having SEQ ID NO:5 [TPSGTTTQS] in AAV2 or a corresponding amino acid sequence in another AAV serotype, and / or the insertion in the VR-VIII region is in a fragment having SEQ ID NO:6 [LQRGNRQAA] in AAV2, or a corresponding amino acid sequence in another AAV serotype.

[0012] In certain embodiments, in a region into which the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted, one or more amino acids in the region are deleted and / or one or more amino acids in the region are substituted.

[0013] Optionally, the AAV is AAV2 or AAV9.

[0014] Optionally, n is between 15 and 20, or between 10 and 15, or between 5 and 10, or between 0 and 5, or is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. Preferably, n is 5 to 20, preferably 5 to 15, more preferably 10 to 15.

[0015] Optionally, m is between 15 and 20, or between 10 and 15, or between 5 and 10, or between 0 and 5, or is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. Preferably, m is between 5 and 20, preferably between 5 and 15, more preferably between 10 and 15.

[0016] In certain embodiments, the linker sequence Xn or Xm consists of at least 80% glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala). Preferably, the linker sequence Xm or Xn consists of amino acids selected from glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala).

[0017] In certain embodiments, X in LPXTG is aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn) or glutamine (Gln), preferably glutamine (Gln). In certain embodiments, one or more lysine (K) of the AAV capsid protein is mutated to glycine (Gly), serine (Ser), or alanine (Ala).

[0018] A further related aspect of the invention is directed to a nucleic acid encoding a capsid protein according to any one of the embodiments described herein.

[0019] An even further related aspect of the invention is directed to an expression vector comprising the nucleic acid of the preceding aspect.

[0020] Further related aspects of the invention relate to AAV particles. In particular, the AAV particles comprise AAV capsid proteins having a sortase recognition sequence inserted in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region and the VR-VIII region. Both AAV particles before and after the sortase conjugation reaction (i.e., conjugated AAV particles) are contemplated. Thus, a further aspect of the invention is directed to AAV particles comprising an AAV capsid protein according to any one of the above embodiments. Furthermore, a still further related aspect of the invention is directed to conjugated AAV particles comprising AAV capsid proteins characterized by a residual sortase recognition sequence (i.e., a modified sortase recognition sequence as present after the conjugation reaction) in the VP1-VP2 transition region, the VR-I region, the VR-IV region and / or the VR-VIII region, wherein the residual sortase recognition sequence is operably linked to a heterologous conjugate molecule.

[0021] In certain embodiments, the conjugated AAV particle is operably linked (i.e., fused) to a heterologous conjugate molecule via a sortase recognition sequence, preferably the conjugate molecule is characterized by the presence of a terminal triglycine amino acid sequence. Optionally, the heterologous conjugate molecule is a small molecule, carbohydrate, lipid, or polypeptide.

[0022] In certain embodiments, the conjugated AAV particle is operably linked (i.e., fused) to a targeting moiety via a sortase recognition sequence. Optionally, the targeting moiety is a ligand for a cell receptor or a protein that binds to a cell surface protein. Optionally, the protein that binds to a cell surface protein is an antibody or a nanobody. Optionally, the antibody or nanobody specifically binds to HER2.

[0023] A still further aspect of the present invention is directed to the use of a conjugated AAV particle according to any one of the embodiments described herein as a medicament.

[0024] Related aspects of the above embodiments of the invention are directed to methods of producing conjugated AAV particles as described herein. In certain embodiments, the methods of producing conjugated AAV particles include: - contacting the cell with one or more nucleic acids encoding AAV capsid proteins, wherein one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, or the VR-VIII region comprises a sortase recognition sequence; - causing the cell to assemble a plurality of AAV capsid proteins into AAV particles and recovering the AAV particles from the cell; and - contacting the AAV particles with a sortase and a heterologous conjugate molecule Includes.

[0025] The above and further aspects and preferred embodiments of the present invention are set out in the following section and in the appended claims, the subject matter of which is specifically incorporated into this specification. [Brief description of the drawings]

[0026] [Figure 1] (A) Schematic gene buildup of wild-type AAV (wtAAV) and recombinant AAV (rAAV). (B) Schematic of sortase A machinery. [Diagram 2] (A) Schematic diagram of the VP1, VP2, VP3 proteins, and the VP1-VP2, VR-I, VR-IV, and VR-VIII regions. (B) General representation of the internal LPQTG motif adjacent to the linker sequence. [Diagram 3] The LPQTG motif was inserted into AAV2-HB0 VR-IV (between AA 453 and 454) without a linker or flanked by one or two GGSGS [SEQ ID NO: 47] repeats on either side of the motif A band, and VIII-1 and VIII-2 showed bands consistent with the molar mass of the Nb (~19 kDa) conjugated to the N-terminal fragment of VP3, demonstrating the conjugation reaction. Srt-A: sortase A only, Nb: nanobody only. [Figure 4] AAV2, VR-I, 0-1-2 L. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] located at position 265 and flanked on either side by 0, 1 or 2 GGSGS [SEQ ID NO: 47] linkers. Arrows: conjugation products. [Diagram 5] AAV2, VR-IV, 0-1-3-4-5 L. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] located at position 453 and flanked on either side by 0, 1, 3, 4 or 5 GGSGS [SEQ ID NO: 47] linkers. Arrows: conjugation products. [Figure 6] AAV2, VR-VIII 0-1-2-3-4-5L. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] located at position 587 and flanked on either side by 0, 1, 2, 3, 4 or 5 GGSGS [SEQ ID NO: 47] linkers. Arrows: conjugation products. [Figure 7] AAV9, VR-IV 0-1-2-3L. AAV9-A with a LPQTG tag [SEQ ID NO: 43] located at position 455 and flanked on either side by 0, 1, 2 or 3 GGSGS [SEQ ID NO: 47] linkers. Arrows: splice products. [Figure 8] AAV9, VR-VIII 0-1-3L. AAV9-A with a LPQTG tag [SEQ ID NO: 43] located at position 589 and flanked on either side by 0, 1 or 3 GGSGS [SEQ ID NO: 47] linkers. Arrows: splice products. [Figure 9] Comparison of five different constructs: AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] at position 265 and flanked on both sides by two GGSGS [SEQ ID NO: 47] linkers, AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] at position 453 and flanked on both sides by three GGSGS [SEQ ID NO: 47] linkers, AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] at position 587 and flanked on both sides by three GGSGS linkers, AAV9-A with a LPQTG tag [SEQ ID NO: 43] at position 455 and flanked on both sides by three GGSGS [SEQ ID NO: 47] linkers, and AAV9-A with a LPQTG tag [SEQ ID NO: 43] at position 589 and flanked on both sides by three GGSGS [SEQ ID NO: 47] linkers. Arrows: spliced ​​product. [Figure 10] GGG-biotin. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] located at position 587 and flanked on both sides by one GGSGS [SEQ ID NO: 47] linker. Arrow: conjugation product. [Figure 11] HER2 vs. GFP. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] at position 453 and flanked by three GGSGS [SEQ ID NO: 47] linkers, AAV9-A with a LPQTG tag [SEQ ID NO: 43] at position 589 and flanked by three GGSGS [SEQ ID NO: 47] linkers. Arrows: ligation product. [Figure 12]LPQTG vs. LPETG in AAV2_VR-VIII and AAV9_VR-IV. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] located at position 587 and flanked on both sides by three GGSGS [SEQ ID NO: 47] linkers, AAV2-HB0 with a LPETG tag [SEQ ID NO: 44] located at position 587 and flanked on both sides by three GGSGS [SEQ ID NO: 47] linkers, AAV9-A with a LPQTG tag [SEQ ID NO: 43] located at position 455 and flanked on both sides by three GGSGS [SEQ ID NO: 47] linkers, and AAV9-A with a LPETG tag [SEQ ID NO: 44] located at position 455 and flanked on both sides by three GGSGS [SEQ ID NO: 43] linkers. Arrows: splicing product. [Figure 13-1] In vitro targeting. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] at position 587 and flanked on either side by three GGSGS [SEQ ID NO: 47] linkers. (A) % transduced cells, (B) % transduced cells (enlarged from (A)). [Figure 13-2] In vitro targeting. AAV2-HB0 with a LPQTG tag [SEQ ID NO: 43] at position 587 and flanked on either side by three GGSGS [SEQ ID NO: 47] linkers. (C) Flow cytometry results. [Figure 14] Comparison of transduction efficiency of different AAV vectors conjugated with anti-HER2 nanobodies versus non-conjugated vectors. Transduction efficiency is measured by GFP fluorescence. [Figure 15] Comparison of production yields of different constructs. (A) Mean viral genome ("Vg") yield (left Y-axis) and % intact virus particles (right Y-axis; black dots) in lysates of producer cell cultures. (B) Mean viral genome ("Vg") yield (left Y-axis; black dots) and % intact virus particles (right Y-axis) in supernatants of producer cell cultures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0028] The terms "comprising," "comprises," and "comprised of," as used herein, are synonymous with "including," "includes," or "containing," "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. These terms also encompass "consisting of" and "consisting essentially of," which have well-established meanings in patent language. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within each range, as well as the recited endpoints. This applies to numerical ranges whether the numerical range is introduced by the phrase "from or to," or "between," or otherwise.

[0029] The terms "about" or "approximately" as used herein, when referring to a measurable value, e.g., a parameter, amount, duration, etc., are meant to encompass variation of the specified value and variation from the specified value, e.g., variation of the specified value and of no more than + / - 10%, preferably no more than + / - 5%, more preferably no more than + / - 1%, and even more preferably no more than + / - 0.1%, insofar as such variations are appropriate to practice in the disclosed invention. It should be understood that the value to which the modifier "about" or "approximately" refers is itself specifically and preferably disclosed.

[0030] The term "one or more" or "at least one," e.g., one or more elements or at least one element of a group of elements, is itself clear, but by way of further illustration, the term includes reference to, among other things, any one of the elements, or any two or more of the elements, e.g., any three or more, four or more, five or more, six or more, seven or more, etc., of the elements, up to and including all of the elements. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0031] The discussion of the background of the invention herein is included to explain the context of the invention and should not be construed as an admission that any of the material mentioned was published, publicly known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0032] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited herein are incorporated by reference in their entirety. In particular, the teachings or passages of such documents specifically mentioned in this specification are incorporated by reference.

[0033] Unless otherwise specified, all terms (including technical and scientific terms) used in the disclosure of the present invention have the meaning as commonly understood by those skilled in the art to which the present invention belongs. By way of further guidance, definitions of terms are included to better appreciate the teachings of the present invention. When a particular term is defined in relation to a particular aspect of the present invention or a particular embodiment of the present invention, such connotation or meaning is meant to apply throughout the specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise specified. For example, an embodiment directed to a product can also be applied to the corresponding features of the method and application.

[0034] In the following text, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s), unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0035] Throughout this specification, references to "one embodiment" or "embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, although some embodiments described herein include some features and not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the present invention and form different embodiments, as would be understood by one of ordinary skill in the art. For example, the appended claims encompass alternative combinations of the claimed embodiments, as would be understood by one of ordinary skill in the art.

[0036] Unless otherwise indicated, all methods, steps, techniques and operations that are not specifically described in detail can be and are carried out in a manner known per se, as will be clear to those skilled in the art.See, for example, standard handbooks and the general background art referred to herein and further references cited therein (e.g., Sambrook et al., Molecular cloning: a laboratory manual, ISBN 0879693096, 1989 and the corresponding latest edition of the 4th edition, Cold Spring Harbor Laboratory Press, 2012).

[0037] In general, the amino acid sequences and their numbering in the present invention refer to the AAV2 VP1 capsid sequence shown in SEQ ID NO: 7. The same numbering is maintained for the shorter VP2 and VP3 proteins. It is further noted that the insertion of sortase recognition sequences, the optional insertion of linker sequences, and the deletion of amino acids in the capsid protein backbone result in polypeptides of different lengths. And for these polypeptides, the amino acids in the capsid protein backbone remain as defined by SEQ ID NO: 7. The sequence similarity between the AAV2 capsid protein and those of other AAV serotypes allows the identification of corresponding amino acids with reference to SEQ ID NO: 7 in sequence alignments. Exemplarily, reference to the corresponding AAV9 positions is also made throughout this description.

[0038] The present invention is equally applicable to AAV capsid sequences that differ from SEQ ID NO: 7, 8, or 9 (e.g., naturally occurring serotype variants and artificial modifications to alter tropism). Such variants can also occur within the VR regions, as in the case of, for example, the R585A, R588A mutants. Thus, by way of example, the AAV2 positions disclosed herein are also intended to cover the corresponding amino acid positions in other AAV serotype variants, both naturally occurring and artificially generated.

[0039] The term "protein" as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi-synthetically, or synthetically produced proteins. The term also encompasses proteins bearing one or more co-expression or post-expression modifications of the polypeptide chain, such as, but not limited to, glycosylation, acetylation, guanidinylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, and the like. The term further encompasses protein variants or mutants bearing amino acid sequence alterations (e.g., amino acid deletions, additions, and / or substitutions) relative to the corresponding native protein. The term contemplates both full-length proteins and portions or fragments of proteins (e.g., naturally occurring protein portions resulting from processing of such full-length proteins).

[0040] The term "polypeptide" as used throughout this specification generally encompasses a polymeric chain of amino acid residues linked by peptide bonds. Thus, the terms "protein" and "polypeptide" may be used interchangeably herein to refer to a protein, particularly when such a protein is composed of only a single polypeptide chain. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semisynthetically, or synthetically produced polypeptides. The term contemplates both full-length polypeptides and portions or fragments of polypeptides (e.g., naturally occurring polypeptide portions resulting from processing of such full-length polypeptides).

[0041] The term "peptide" as used throughout this specification refers to a short chain of amino acid residues linked by peptide bonds, preferably comprising 50 or fewer amino acids, such as 45 or fewer amino acids, preferably 40 or fewer amino acids, such as 35 or fewer amino acids, more preferably 30 or fewer amino acids, such as 25 or fewer, 20 or fewer, 15 or fewer, or 10 or fewer amino acids. There is no strict maximum length for a peptide to still be considered a peptide. The term peptide may encompass naturally, recombinantly, semi-synthetically, or synthetically produced peptides, such as those discussed for polypeptides above.

[0042] The term "amino acid" encompasses naturally occurring amino acids, naturally encoded or proteinogenic amino acids, non-naturally encoded amino acids, non-naturally occurring amino acids, amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, all of which are D- and L-stereoisomers, provided that their structures allow for such stereoisomeric forms. Amino acids are referred to herein by either their name, their commonly known three letter code, or the one-letter code recommended by the IUPAC-IUB Biochemical Nomenclature Commission. A "naturally encoded amino acid" refers to an amino acid that is one of the 20 common amino acids or pyrrolysine, pyrroline-carboxy-lysine, or selenocysteine. The 20 common amino acids are alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr). Amino acid analogs in which one or more individual atoms are replaced with either a different atom, an isotope of the same atom, or a different functional group are also included.

[0043] "Encode" should be interpreted according to common interpretation in the art, and thus indicates that a nucleic acid sequence, or a portion thereof, corresponds to a particular amino acid sequence, e.g., the amino acid sequence of one or more desired proteins or polypeptides, or to another nucleic acid sequence in a template-transcription product (e.g., RNA or RNA analog) relationship according to the genetic code of the organism in question. Although numerous references are made throughout this specification to modifications in amino acid positions and / or amino acid sequences, it will be apparent that in embodiments in which the AAV capsid protein is translated from a nucleic acid sequence, such modifications are introduced into the encoding nucleic acid sequence.

[0044] The term "nucleic acid" as used throughout the present specification typically refers to a polymer (preferably a linear polymer) of any length essentially composed of nucleoside units. Nucleoside units generally include a heterocyclic base and a sugar group. Heterocyclic bases may include, among others, purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) that are widely present in naturally occurring nucleic acids, other naturally occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated) non-natural or derivatized bases. Nucleic acids can be double-stranded, partially double-stranded, or single-stranded. When single-stranded, nucleic acids can be sense or antisense strands. In addition, nucleic acids can be circular or linear.

[0045] It is understood that a person skilled in the art can evaluate sequence identity between sequences. Methods and tools for verifying sequence identity between different sequences of amino acids or nucleic acids are well known. Such tools include (Protein)BLAST, ClustalW2, SIM alignment tool, TranslatorX, and T-COFFEE. The percentage of identity between two sequences may show slight differences depending on the choice and parameters of the algorithm. The term "sequence identity" as used herein refers to the relationship between sequences at the nucleotide (or amino acid) level. The expression "% identical" is determined by comparing optimally aligned sequences, e.g., two or more, over a comparison window, and the portion of the sequence within the comparison window may include insertions and / or deletions compared to the reference sequence for optimal alignment of the sequences. The reference sequence does not include insertions or deletions. Select a reference window, then determine the number of nucleotides (or amino acids) that are identical between the sequences within the window, and calculate "% identity" by dividing the number of identical nucleotides (or amino acids) by the number of nucleotides (or amino acids) within the window and multiplying by 100. Unless otherwise stated, sequence identity is calculated over the entire length of the reference sequence. An example procedure for determining percent identity between a particular amino acid sequence and the amino acid sequence of a query polypeptide involves aligning two amino acid sequences using the Blast 2 sequence (Bl2seq) algorithm available as a web application or a stand-alone executable program (BLAST version 2.2.31+) at the NCBI website (www.ncbi.nlm.nih.gov) using appropriate algorithm parameters. Examples of suitable algorithm parameters include: matrix=Blosum62, cost to open a gap=11, cost to extend a gap=1, expectation=10.0, word size=3). Those skilled in the art will readily understand that any sequence in the sequence database or shown herein may be a precursor of a peptide, polypeptide, protein, or nucleic acid, and may include a portion processed from a mature molecule.

[0046] "Adeno-associated virus", commonly abbreviated as "AAV", refers to a non-pathogenic parvovirus composed of a 4.7 kb single-stranded DNA genome within a non-enveloped icosahedral capsid. Both the full-length term and its abbreviations may be used to refer to the virus itself or its derivatives. The AAV genome contains three AAV promoters (i.e., p5, p19, and p40; the names refer to their relative map positions) that are responsible for the expression of two open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19) couple with differential splicing of a single AAV intron to drive the production of four Rep proteins (Rep 78, Rep 68, Rep 52, and Rep 40) from the rep gene. The different Rep proteins have distinct enzymatic properties that are involved in various aspects of viral replication. The Cap gene is expressed from the p40 promoter and encodes three capsid proteins, viral protein 1 (VP1), viral protein 2 (VP2), and viral protein 3 (VP3), by alternative splicing and non-consensus translation start sites. VP1, VP2, and VP3 are involved in AAV encapsidation (i.e., VP1, VP2, and VP3 are AAV capsid proteins). Thus, VP1, VP2, and VP3 have overlapping sequences, and VP3 is completely contained within the sequence of VP2, which in turn is contained within VP1. The regions with the highest structural variation (VR) are annotated in the art as VR-I to VR-IX. The present invention is directed to sequence manipulations within the VR-I, VR-IV, and VR-VIII regions. In addition, the present invention encompasses sequence manipulations within the VP1-VP2 transition region (i.e., the region that marks the end of the N-terminal VP1 portion unique to each of the VP proteins and the N-terminus of the VP2 protein sequence). A single consensus polyadenylation site is located at map position 95 of the AAV genome. In addition, an open reading frame present as an alternative reading frame within the cap gene localizes AAV capsid proteins to the nucleolus and produces the assembly activating protein (AAP), a viral protein that functions in the capsid assembly process.The AAV genome contains inverted terminal repeats at both ends.

[0047] The term "AAV" as used herein should be understood to include all subtypes and / or serotypes (naturally occurring and recombinant forms; rAAV), unless required or expressly indicated otherwise. Thus, the term "AAV" encompasses AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, or any combination thereof. Those skilled in the art will understand that the above indication refers to a subject that can be infected by the AAV. For example, primate AAV refers to an AAV that can infect primates. The genomic sequences of various AAV serotypes, their native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits have been described in the art. Such sequences can be found in the literature or in public repositories such as GenBank, UniProt, etc. The term "AAV" equally encompasses mosaic AAV and chimeric AAV (including, for example, AAV obtained by directed evolution) that contain custom-designed AAV capsid proteins that do not exist in nature. It has been described in the art that genomic diversity between different serotypes is most concentrated in the hypervariable region (HVR) of the viral capsid, which affects the tissue tropism of AAV. It is known to those skilled in the art that the tissue tropism of AAV vectors depends on other factors (i.e., parameters) such as cell surface receptors, cellular uptake, intracellular processing of AAV, nuclear delivery of the AAV genome, uncoating of AAV, and second strand DNA conversion. The preferred AAV serotypes in the context of the present invention are AAV2 and AAV9, but it is envisioned that any embodiment described herein is equally applicable to any AAV serotype, including mosaic AAV and chimeric AAV.

[0048] The term "tropism" as used herein refers to the preferred targeting of a particular host species or a particular cell type within a host species by a virus (in this context, by AAV). For a particular virus, the tropism of the virus represents the relative preference of the virus. If the viruses prefer the same characteristics (e.g., the second virus is also more successful in infecting the same cells (i.e., the same cell type)) even if the absolute transduction efficiency is not similar, the virus can be considered to have a similar (or the same) tropism when compared to another virus. The second virus may be more efficient than the first virus in infecting all given cell types tested, but if the relative preferences are similar (or the same), the second virus is generally considered in the art to have a similar (or the same) tropism as the first virus.

[0049] An "AAV (virus) particle," "AAV virion," or "AAV (vector) particle" refers to a viral particle composed of at least one AAV capsid protein and a single-stranded nucleic acid strand encapsidated. In certain embodiments, an AAV particle contains a heterologous nucleic acid sequence (i.e., a nucleic acid other than a wild-type AAV genome, such as a transgene, to be delivered to a eukaryotic cell, such as, but not limited to, a mammalian cell or an insect cell). In general, the related term "AAV capsid" refers to the outer surface (i.e., capsid) of an AAV particle. An AAV capsid contains 60 copies (total) of the three VPs predicted to be present in the capsid in a VP1:VP2:VP3 ratio of 1:1:10. Thus, throughout this specification, reference to "AAV capsid proteins" encompasses each of the VP1, VP2, and VP3 proteins, or any selection thereof, where indicated. In instances throughout the specification where the term "conjugated AAV particles" is used, this should be understood to refer to AAV particles that have been subjected to a sortase conjugation reaction.

[0050] "Sortase", used interchangeably with "sortase enzyme", refers to a family of enzymes that play a role in the formation of bacterial cell walls in nature by covalently attaching certain surface proteins to peptidoglycan. In its broadest interpretation, a sortase can be defined as an enzyme that recognizes a stretch of amino acids, cleaves site-specifically within that stretch of amino acids, and finally attaches a substrate moiety immediately C-terminal to the cleavage site via a peptide bond with the N-terminal residue of an acceptor moiety. In other words, a sortase enzyme recognizes a sortase recognition motif in a substrate protein and performs a transpeptidation reaction. In a first reaction step, the sortase cleaves the peptide bond in the sortase recognition motif, forming an acyl intermediate with the cleaved sortase recognition motif. In a second reaction step, the sortase binds to an acceptor moiety that bears a sortase acceptor motif (typically at least one glycine or a stretch of glycines) and transfers the acyl intermediate. This reaction results in the formation of a new peptide bond between the substrate protein and the acceptor moiety. Alternative terms used interchangeably herein and in the art to refer to a "sortase recognition motif" include "sortase recognition sequence" and "sortase recognition tag."

[0051] Different sortases have been described in the art. Non-limiting examples include sortase enzymes naturally occurring in Gram-positive enzymes. Preferred sortase enzymes include sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F. More specific sortase subfamilies have been described and are also encompassed by the term "sortase". It is well within the capabilities of the skilled artisan to assign an identified sortase to the correct class and / or subfamily based on its sequence or functional characteristics (e.g. transpeptidation activity). It is clear that when general reference is made to "sortase" throughout this specification, all naturally occurring and artificially engineered sortase types and subtypes are envisaged, unless otherwise indicated.

[0052] The AAV capsid protein as described herein is intended to accommodate the physical association between the AAV capsid protein and a heterologous conjugate (molecule). The term "heterologous", which is used interchangeably in the art with the term "exogenous", indicates that a certain moiety (in the context of the present invention, a conjugate molecule) does not occur in the natural, unmodified version of AAV. Thus, in accordance with the nomenclature generally accepted in the fields of molecular biology and biotechnology, "heterologous conjugate" and "heterologous conjugate molecule" refer to a molecule present in a host organism that does not naturally contain or express the molecule.

[0053] The inventors have unexpectedly discovered that a sortase recognition sequence can be introduced into a specific internal location in the capsid sequence of an AAV particle to allow conjugation of a targeting molecule with the AAV capsid (to produce a conjugated AAV particle) for more efficient and specific tissue and cell targeting while maintaining similar production efficiency when compared to the appropriate parental AAV serotype. This finding is noteworthy for a number of reasons.

[0054] -There is a general teaching in the art that for biotechnological applications, sortase recognition sequences must be located at, or at least close to, the C-terminus of a protein to be functional. The notion that an internally located sortase recognition sequence in an AAV capsid protein would be functional and accessible is unexpected.

[0055] - Remarkably, even after the sortase reaction, the structural and functional integrity of the (now split) capsids forming the (joined) AAV capsid is maintained. Even upon introduction of an extended linker sequence, the AAV capsid proteins remained susceptible to assembly by the sortase enzyme. Moreover, it was surprisingly found that the insertion of a sortase recognition sequence in combination with a relatively long linker sequence (over the length of 34 amino acids, indicated in the art as the maximum insertion length) in a surface-exposed region does not negatively affect the structural or functional properties of the viral capsid proteins. It could not be expected that the -VP1-VP2 transition region, the VR-I region, the VR-IV region, and the VR-VIII region would be accessible to such an extent that they could bring both the sortase and the acceptor moieties into close proximity to complete the sortase reaction.

[0056] Unexpectedly, our findings can be easily reproduced (or extrapolated) to different AAV serotypes: the sequence diversity of AAV capsid proteins between serotypes does not create a barrier to inserting and using internal sortase recognition sequences.

[0057] Thus, the present invention provides adeno-associated virus (AAV) capsid proteins characterized in that a sortase recognition sequence is inserted into their protein sequence, in particular into the VP1, VP2 or VP3 capsid proteins. More specifically, the present invention relates to adeno-associated virus (AAV) capsid proteins characterized in that a sortase recognition sequence is inserted into one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region and the VR-VIII region. Optionally, the AAV capsid protein is VP1 and the sortase recognition sequence is inserted into VP1-VP2, VR-I, VR-IV, VR-VIII, or any combination thereof. Preferably, the AAV capsid protein is VP1 and the sortase recognition sequence is inserted into VR-I, VR-IV, VR-VIII, or any combination thereof. Alternatively, the AAV capsid protein is VP2 and the sortase recognition sequence is inserted into VR-I, VR-IV, VR-VIII, or any combination thereof. Alternatively, the AAV capsid protein is VP3 and the sortase recognition sequence is inserted into VR-I, VR-IV, VR-VIII, or any combination thereof. Considering that the sequence of VP3 is completely encompassed by the sequence of VP2, which in turn is completely encompassed by the sequence of VP1, the AAV capsid protein may be a protein comprising the sequence of the VP3 protein as defined herein. Alternatively or additionally, the AAV capsid protein may be a protein comprising the sequence of the VP2 protein as defined herein. Still alternatively or additionally, the AAV capsid protein may be a protein comprising the sequence of the VP3 protein as described herein. It is clear that in envisaged embodiments, the VP1, VP2, or VP3 sequence differs from the canonical sequence of these proteins due to the presence of one or more sortase recognition motifs. Thus, the AAV capsid proteins that are the subject of the present invention do not share 100% sequence identity with the naturally occurring VP1, VP2, or VP3 proteins or their coding sequences.Thus, in certain embodiments, the AAV capsid protein comprises an amino acid sequence that is at least about 80% identical, preferably at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 98% identical to the amino acid sequence of a naturally occurring (i.e., wild-type) AAV capsid protein, and the capsid protein comprises at least one sortase recognition sequence. In preferred embodiments, the AAV capsid protein comprises an amino acid sequence that is at least about 80% identical, preferably at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 98% identical to the amino acid sequence of a naturally occurring (i.e., wild-type) AAV2 or AAV9, preferably AAV2 capsid protein.

[0058] In a preferred embodiment, the sortase recognition sequence is operably linked to an N-terminal linker (i.e., the linker sequence N-terminal to the sortase recognition motif). In an alternative preferred embodiment, the sortase recognition sequence is operably linked to a C-terminal linker (i.e., the linker sequence C-terminal to the sortase recognition motif). In a more preferred embodiment, the sortase recognition sequence is adjacent to an N-terminal linker sequence and a C-terminal linker sequence. Optionally, the linker sequence is a GGGGS [SEQ ID NO: 39] sequence or any multiple thereof, or a GGSGS [SEQ ID NO: 47] sequence or any multiple thereof. Optionally, the linker sequence comprises a GGGGS [SEQ ID NO: 39] sequence, a GGSGS [SEQ ID NO: 47] sequence, or any combination thereof.

[0059] It is clear that for any sortase recognition sequence and optional linker sequence described herein, each of these sequences must be operably linked to the N-terminal and C-terminal portions of the AAV capsid protein. The term "operably linked" is well known to those skilled in the art of molecular biology and refers to an arrangement of elements in which the components so described are configured to perform their usual functions. Thus, a sortase recognition sequence operably linked to an internal location within an AAV capsid protein sequence does not interfere with translation of that AAV capsid protein. Control sequences need not be contiguous with the coding sequence, so long as they function to direct expression of that coding sequence.

[0060] Optionally, the sortase recognition sequence is a sequence that corresponds to a sortase recognition sequence of a naturally occurring sortase enzyme (such as, but not limited to, a naturally occurring sortase enzyme in a Gram-positive bacterium). Preferably, the sortase recognition sequence is a sequence that corresponds to the universal sortase recognition sequence of a sortase selected from the group consisting of sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F. Preferably, the sortase recognition sequence is a sequence that corresponds to the universal sortase recognition sequence of sortase A.

[0061] In certain embodiments, the AAV capsid protein comprises two or more sortase recognition sequences for a sortase selected from the group consisting of sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F. In embodiments in which multiple sortase recognition sequences are inserted, the sortase recognition sequences are preferably inserted at different positions in the AAV capsid protein. The sortase recognition sequences in such embodiments may be the same sortase recognition sequence or any combination of sortase recognition sequences for a sortase selected from the group consisting of sortase A, sortase B, sortase C, sortase D, sortase E, and sortase F. Preferably, the AAV capsid protein comprises at least a sortase A recognition sequence.

[0062] Optionally, the sortase recognition sequence is a sequence having a general sequence motif selected from the group consisting of: LPXTG [SEQ ID NO:10], NPXTG [SEQ ID NO:40], LPXTA [SEQ ID NO:41], LAXTG [SEQ ID NO:42], LPXAG [SEQ ID NO:48], LPXLG [SEQ ID NO:49], APXTG [SEQ ID NO:50], LPXSG [SEQ ID NO:51], FPXTG [SEQ ID NO:52], XPKTG [SEQ ID NO:53], and LPEXG [SEQ ID NO:54], where X is any amino acid. Preferably, the sortase recognition sequence is a sequence having the general sequence motif LPXTG [SEQ ID NO:10], where X is any amino acid and X is defined independently for Xn and Xm. In still further preferred embodiments, the sortase recognition sequence is LPQTG [SEQ ID NO: 43], LPETG [SEQ ID NO: 44], LPNTG [SEQ ID NO: 45], LPDTG [SEQ ID NO: 46], NPQTN [SEQ ID NO: 55], QVPTG [SEQ ID NO: 56], LPNTA [SEQ ID NO: 57], LPLTG [SEQ ID NO: 58], APKTG [SEQ ID NO: 59], DPKTG [SEQ ID NO: 60], SPKTG [SEQ ID NO: 61], APATG [SEQ ID NO: 62], LAETG [SEQ ID NO: 63], LPEAG [SEQ ID NO: 64], LPECG [SEQ ID NO: 65], LPESG [SEQ ID NO: 66], or LMVGG [SEQ ID NO: 67]. In a most preferred embodiment, the sortase recognition sequence is LPQTG [SEQ ID NO: 43] or LPETG [SEQ ID NO: 44]. An alternative suitable way of describing the sortase recognition sequences described herein is in the format "Xn-sortase recognition sequence-Xm". This format therefore indicates the optional presence of a linker sequence, where "X" denotes any amino acid, and "n" and "m" are integers indicating the length of the linker sequence (i.e., the amount of amino acids). Clearly, X can be defined independently of one another for Xn and Xm. Thus, in certain embodiments, the sortase recognition sequence is immediately preceded by the sequence Xn and immediately followed by the sequence Xm.Thus, the sortase recognition sequences described herein may alternatively be represented by Xn-LPXTG-Xm [SEQ ID NO: 10], Xn-NPXTG-Xm [SEQ ID NO: 40], Xn-LPXTA-Xm [SEQ ID NO: 40], Xn-LAXTG-Xm [SEQ ID NO: 42], Xn-LPXAG-Xm [SEQ ID NO: 48], Xn-LPXLG-Xm [SEQ ID NO: 49], Xn-APXTG-Xm [SEQ ID NO: 50], Xn-LPXSG-Xm [SEQ ID NO: 51], Xn-FPXTG-Xm [SEQ ID NO: 52], Xn-XPKTG-Xm, [SEQ ID NO: 53], and Xn-LPEXG-Xm, [SEQ ID NO: 54] (wherein X is any amino acid and n and m are in the range of 0 to 25). Preferably, the sortase recognition sequence is a sequence having the general sequence motif Xn-LPXTG-Xm [SEQ ID NO: 10], where X is any amino acid and n and m are in the range of 0 to 25. More preferably, the sortase recognition sequence is Xn-LPQTG-Xm [SEQ ID NO: 43], Xn-LPETG-Xm [SEQ ID NO: 44], Xn-LPNTG-Xm [SEQ ID NO: 45], Xn-LPDTG-Xm [SEQ ID NO: 46], Xn-NPQTN-Xm [SEQ ID NO: 55], Xn-QVPTG-Xm [SEQ ID NO: 56], Xn-LPNTA-Xm [SEQ ID NO: 57], Xn-LPLTG-Xm [SEQ ID NO: 58], Xn- APKTG-Xm [SEQ ID NO: 59], Xn-DPKTG-Xm [SEQ ID NO: 60], Xn-SPKTG-Xm [SEQ ID NO: 61], Xn-APATG-Xm [SEQ ID NO: 62], Xn-LAETG-Xm [SEQ ID NO: 63], Xn-LPEAG-Xm [SEQ ID NO: 64], Xn-LPECG-Xm [SEQ ID NO: 65], Xn-LPESG-Xm [SEQ ID NO: 66], or Xn-LMVGG-Xm [SEQ ID NO: 67]. Most preferably, the sortase recognition sequence is Xn-LPQTG-Xm [SEQ ID NO: 43] or Xn-LPETG-Xm [SEQ ID NO: 44].

[0063] As noted above, in certain embodiments, the sortase recognition sequence is preceded and / or followed by a linker sequence (i.e. embodiments in which the sortase recognition sequence is preceded or followed by an Xn and / or Xm sequence, where n and / or m are at least 1, and where the X in Xn and Xm can be defined independently of each other). Thus, the invention contemplates embodiments in which n and / or m are 0, but equally contemplates embodiments in which n and / or m are at least 1. Thus, in certain embodiments, n and / or m are an integer between 0 and 25, such as at least 2, at least 3, at least 4 or at least 5, preferably between 5 and 20, such as between 10 and 15.

[0064] Optionally, n and / or m are an integer between 15 and 20, or between 15 and 20. Optionally, n and / or m are an integer between 10 and 15, or between 10 and 15. Optionally, n and / or m are an integer between 5 and 10, or between 0 and 5. Optionally, n and / or m are 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0. Further contemplated are embodiments in which n is 0 and m is an integer between 0 and 25, and preferably between 0 and 20. Also contemplated are embodiments in which m is 0 and n is an integer between 0 and 25, and preferably between 0 and 20.

[0065] The exact sequence of the linker sequences Xn and Xm is not particularly limiting to the present invention. However, preferred Xn and Xm sequences (or their characteristics) are described in the following paragraphs. Preferably, the linker sequences Xn, Xm, or both Xn and Xm consist of at least 65%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% glycine, serine, threonine, and alanine. More preferably, the linker sequences Xn, Xm, or both Xn and Xm consist of glycine, serine, threonine, and alanine. Alternatively, the linker sequences Xn, Xm, or both Xn and Xm consist of at least 65%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% glycine and serine. In further alternative embodiments, the linker sequences Xn, Xm, or both Xn and Xm consist of glycine and serine. In certain embodiments, the linker sequence is GGSGS [SEQ ID NO: 47].

[0066] Each of the regions described herein into which a sortase recognition sequence may be inserted has been described many times throughout the art, and therefore, identifying these regions is well within the capabilities of one of ordinary skill in the art. Optionally, the VP1-VP2 transition region is defined by SEQ ID NO: 1 [PVKTAP]. Optionally, the VR-I region is defined by SEQ ID NO: 2 [SSQSGASN]. Optionally, the VR-IV region is defined by SEQ ID NO: 3 [SRTNTPSGTTTQSRLQFSQAGASDIRDQS]. Optionally, the VR-VIII region is defined by SEQ ID NO: 4 [QYGSVSTNLQRGNRQAATADVNTQGV]. In a preferred embodiment, the VR-I region is defined by SEQ ID NO: 2 [SSQSGASN], the VR-IV region is defined by SEQ ID NO: 3 [SRTNTPSGTTTQSRLQFSQAGASDIRDQS] and the VR-VIII region is defined by SEQ ID NO: 4 [QYGSVSTNLQRGNRQAATADVNTQGV]. In a further preferred embodiment, the VP1-VP2 transition region is defined by SEQ ID NO: 1 [PVKTAP], the VR-I region is defined by SEQ ID NO: 2 [SSQSGASN], the VR-IV region is defined by SEQ ID NO: 3 [SRTNTPSGTTTQSRLQFSQAGASDIRDQS] and the VR-VIII region is defined by SEQ ID NO: 4 [QYGSVSTNLQRGNRQAATADVNTQGV]. It should be understood that SEQ ID NO:1-4 correspond to sequences without a sortase recognition motif inserted, i.e., sequences that the inventors have found to be amenable to one or more sortase recognition motifs, optionally flanked by one or more linkers. Corresponding amino acid sequences in AAV serotypes other than AAV2 are likewise envisioned.

[0067] In embodiments in which a sortase recognition sequence is inserted in the VR-IV region, the sortase recognition sequence may preferably be inserted in a fragment having SEQ ID NO:5 [TPSGTTTQS] and / or the insertion in the VR-VIII region is in a fragment having SEQ ID NO:6 [LQRGNRQAA]. In such embodiments, the sortase recognition sequence may be inserted after the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th amino acid of SEQ ID NO:5. In alternative such embodiments, the sortase recognition sequence may be inserted after the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th amino acid of SEQ ID NO:6. In further alternative embodiments, a first sortase recognition sequence may be inserted after the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th amino acid of SEQ ID NO:5, and a second sortase recognition sequence may be inserted after the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, or 9th amino acid of SEQ ID NO:6.

[0068] A sortase recognition sequence as described herein may be inserted into the VP1-VP2 transition region, VR-I region, VR-IV region, and / or VR-VIII region by inserting a sortase recognition sequence into the genomic sequence of the region. The insertion may be an insertion without removal of canonical amino acids in the region. In such an embodiment, the sortase recognition sequence is introduced in addition to the original sequence. Alternatively, a sortase recognition sequence as described herein may be inserted into the VP1-VP2 transition region, VR-I region, VR-IV region, and / or VR-VIII region by insertions and deletions (resulting in substitutions or replacements) in the genomic sequence of the region.

[0069] The deletion or substitution may correspond to any number of amino acids of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and / or the VR-VIII region, as long as the structural integrity of said regions (and consequently the AAV capsid protein) is maintained. In some embodiments, the functional integrity of the AAV proteins may be affected in that the insertion site disrupts certain functionalities (e.g., the HSPG binding site of VR-VIII in AAV2), but these are not critical for further use of the junction protein. However, in further embodiments, both the structural and functional integrity of the regions (and consequently the AAV capsid protein) are maintained.

[0070] Without wishing to be bound by theory, the inventors note that surface exposed lysines may optionally reduce the reaction efficiency of sortase-mediated transpeptidation by allowing the generation of impermissible by-products. Thus, in an embodiment of the invention, one or more surface exposed lysines are mutated, for example, to glycine, serine or alanine. The choice of which lysines to target is based on their distance from the loop where an insertion site, such as an LPXTG tag, is added.

[0071] One of skill in the art can identify amino acids that correspond to those disclosed herein for AAV serotypes other than AAV2. Generally, lysine mutations adjacent to the region where the sortase recognition sequence is inserted are envisaged.

[0072] Exemplarily, the lysines 30 Å from the VR-I region of AAV2 correspond to K258, K321, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K692, and K706; the lysines 30 Å from the VR-IV region of AAV2 correspond to K258, K309, K313, K321, K490, K5 lysines 30 Å from the VR-VIII region of AAV2 correspond to K309, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K688, K692, and K706; lysines 30 Å from the VR-VIII region of AAV2 correspond to K309, K490, K507, K527, K532, K544, K549, K556, K620, K640, K688, and K706.

[0073] Illustratively, lysines within 15 Å of the VR-I region of AAV2 correspond to K258, K507, K527, K549, and K706; lysines within 15 Å of the VR-IV region of AAV2 correspond to K258, K490, K507, K532, K544, K549, K556, and K665; and lysines within 15 Å of the VR-VIII region correspond to K490, K507, K527, and K532.

[0074] Illustratively, the lysine 5 Å from the VR-IV region of AAV2 is K549, and the lysine 5 Å from the VR-VIII region of AAV2 is K507.

[0075] Optionally, the sortase recognition sequence is inserted into the VR-I region and one or more of the lysines at positions K258, K321, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K692, K706 in AAV2 or the corresponding amino acids in another serotype are mutated. In a preferred embodiment, the sortase recognition sequence is inserted into the VR-1 region and one or more of the lysines at positions K258, K507, K527, K549 and K706 in AAV2 or the corresponding amino acids in another serotype are mutated. Optionally, a sortase A recognition sequence is inserted into the VR-I region and mutates one or more of the lysines at positions K258, K321, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K692, K706 in AAV2 or the corresponding amino acid in another serotype. Optionally, the sortase A recognition sequence is Xn-LPXTG-Xm [SEQ ID NO: 10].

[0076] Optionally, in embodiments contemplated herein, a sortase recognition sequence is inserted into the VR-IV region and one or more of the lysines at positions K258, K309, K313, K321, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K688, K692, and K706 in AAV2 or the corresponding amino acids in another serotype are mutated. In a preferred embodiment, a sortase recognition sequence is inserted into the VR-IV and one or more of the lysines at positions K258, K490, K507, K532, K544, K549, K556, and K665 in AAV2 or the corresponding amino acids in another serotype are mutated. Optionally, a sortase A recognition sequence is inserted into the VR-IV region, and one or more of the lysines at positions K258, K309, K313, K321, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K688, K692 and K706 in AAV2 or the corresponding amino acid in another serotype are mutated. Optionally, the sortase A recognition sequence is Xn-LPXTG-Xm [SEQ ID NO: 10]. In a further preferred embodiment, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into the VR-IV region, and the lysine at position K549 in AAV2 or the corresponding amino acid in another serotype is mutated, preferably to glycine or alanine.

[0077] Optionally, a sortase recognition sequence is inserted into the VR-VIII region and one or more of the lysines at positions K309, K490, K507, K527, K532, K544, K549, K556, K620, K640, K688, and K706 in AAV2, or the corresponding amino acids in another serotype, are mutated. In a preferred embodiment, a sortase recognition sequence is inserted into the VR-VIII region and one or more of the lysines at positions K490, K507, K527, and K532 in AAV2, or the corresponding amino acids in another serotype, are mutated. Optionally, a sortase A recognition sequence is inserted into the VR-VIII region and mutates one or more of the lysines at positions K309, K490, K507, K527, K532, K544, K549, K556, K620, K640, K688, and K706 in AAV2, or the corresponding amino acids in another serotype. Optionally, the sortase A recognition sequence is Xn-LPXTG-Xm [SEQ ID NO: 10].

[0078] In a further preferred embodiment, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into the VR-VIII region and the lysine at position K507, or the corresponding amino acid in another serotype, is mutated, preferably to glycine or alanine.

[0079] Further aspects of the present invention are directed to nucleic acids (i.e., nucleic acid sequences) encoding any of the AAV capsid proteins described herein, and their use in methods for producing AAV particles. Thus, nucleic acid sequences encoding any of the AAV capsid proteins described herein with a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and the VR-VIII region are encompassed. The nucleic acid may be DNA, RNA, variants, or any combination of DNA and RNA. Methods for constructing the nucleic acid constructs of the present disclosure are well known. The nucleic acid may be a nucleic acid further comprising a promoter sequence. The term "promoter" as defined herein is a region of DNA that initiates transcription of a particular gene, thus allowing the gene to be transcribed. The promoter is recognized by RNA polymerase, which then initiates transcription. Thus, a promoter includes a DNA sequence that is either directly bound by RNA polymerase or is involved in the recruitment of RNA polymerase. A promoter sequence may also contain an "enhancer region," which is one or more regions of DNA that can bind proteins (i.e., trans-acting factors) to enhance the transcription level of genes in a gene cluster. Enhancers are typically at the 5' end of the coding region, but may be distant from the promoter sequence, for example, in an intron region of the gene or 3' to the coding region of the gene. A promoter may be located close to the start codon of the gene, typically upstream (5') of the gene, in a preferred embodiment on the same strand. Promoters may vary in size, preferably being about 100 to 1000 nucleotides in length.

[0080] The term nucleic acid therefore encompasses (recombinant) nucleic acid vectors and (recombinant) nucleic acid expression vectors, which are further aspects of the present invention. Nucleic acid (expression) vectors are known to the skilled artisan to be suitable for transporting the nucleic acid of the present invention to cells in an environment such as, but not limited to, an organism, tissue or cell culture. Such vectors are illustratively useful for producing an open reading frame encoding the AAV capsid protein that is the subject of the present specification. In such an embodiment, the AAV capsid protein that is the subject of the present specification may be expressed by the nucleic acid in in vitro or in vivo conditions (i.e., the nucleic acid encodes at least the amino acid sequence of the AAV capsid protein). Thus, the nucleic acids described herein may be suitable for producing or aiding in the production of AAV particles and ultimately joint AAV particles, which (optionally joint) AAV particles are described in more detail throughout the present specification. Recombinant expression vector refers to a nucleic acid that encodes a protein, the nucleic acid being capable of expressing the encoded protein, in this context the AAV capsid protein. Examples of such vectors include plasmids, nucleic acid viral vectors, and viral genomes (including both DNA and RNA genomes). Thus, recombinant AAV vectors are contemplated by the present disclosure.

[0081] The term "recombinant AAV vector" is used interchangeably with terms such as "recombinant AAV", "recombinant AAV virus" and "recombinant AAV viral particle" to indicate that the genomic DNA encapsulated in the AAV viral capsid contains heterologous nucleic acid. In the recombinant AAV vector of the present invention, at least the AAV capsid protein is replaced with a heterologous nucleic acid comprising an AAV capsid protein characterized by one or more sortase recognition sequences in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region and / or the VR-VIII region. The coding sequences and elements contained in the vector can be expressed in a suitable host cell by any suitable means for introducing the vector inside the cell. Suitable methods include, by way of example only, but are not limited to, infection, transformation, transduction and transfection. In addition to the therapeutic and / or reporter gene (i.e., the "payload" envisioned to be delivered to the target cell), the vector may contain a number of components (i.e., elements, features) whose function is to regulate expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, introns, Kozak sequences, polyA sequences, selection elements, or origins of replication.

[0082] In certain embodiments, the nucleic acid comprises a sequence encoding an AAV capsid protein having an amino acid sequence that is at least about 80% identical, preferably at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 98% identical to the amino acid sequence of a naturally occurring (i.e., wild-type) AAV capsid protein, wherein the capsid protein comprises at least one sortase recognition sequence. In a preferred embodiment, the nucleic acid comprises a sequence encoding an AAV capsid protein having an amino acid sequence that is at least about 80% identical, preferably at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 98% identical to the amino acid sequence of a naturally occurring (i.e., wild-type) AAV2 or AAV9, preferably AAV2 capsid protein.

[0083] The present invention aims to provide a robust and efficient means for attaching heterologous conjugate molecules to AAV particles (resulting in conjugated AAV particles), and more specifically to AAV capsid proteins. Thus, a still further aspect of the present invention is directed to AAV particles that comprise a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and the VR-VIII region of the AAV capsid protein. Those skilled in the art will appreciate that after performing the sortase conjugation reaction, the sortase recognition sequence is modified, as the C-terminal glycine residue is "cleaved" and the remaining portion of the sortase recognition sequence is linked to a different glycine than the conjugate molecule. Thus, those skilled in the art will appreciate that both AAV particles that serve as starting materials for conjugation (i.e., "input" material) are envisioned, but so are the AAV particles that result from performing the sortase conjugation reaction (i.e., "output" material; conjugated AAV particles). Generally, throughout this disclosure, the sortase recognition sequence portion of an AAV capsid protein following a sortase conjugation reaction is referred to as a "modified sortase recognition sequence," or alternatively, as a "residual sortase recognition sequence." It should thus be understood that the residual sortase recognition sequence referred to herein physically connects, and preferably operably links, the conjugated AAV capsid protein with a heterologous conjugate molecule (such as, but not limited to, the conjugate molecules further described below).

[0084] In other words, the invention therefore provides AAV particles comprising genomically modified AAV capsid proteins, wherein the genomic modification is the presence of a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region and / or the VR-VIII region. Thus, a genomic modification should be considered for any AAV particle in which the AAV capsid protein does not have a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region and / or the VR-VIII region.

[0085] Thus, in certain embodiments, the AAV particle comprises an AAV capsid protein characterized by the presence of a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and / or the VR-VIII region. In alternative embodiments, the conjugated AAV particle comprises an AAV capsid protein characterized by the presence of a residual sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and / or the VR-VIII region operably linked to a heterologous conjugate molecule. Considering the internal location of the sortase recognition sequence and the details of the sortase conjugation reaction, the AAV capsid protein after the conjugation reaction (which may be the VP1, VP2, or VP3 protein) effectively exists in the AAV capsid as two separate proteins: a first N-terminal protein that contains the VP portion N-terminal of the remaining sortase recognition sequence, the remaining sortase recognition sequence, and the conjugate molecule; and a second C-terminal protein that contains the VP portion C-terminal of the (first) sortase recognition sequence. The sortase recognition sequence is inserted into the AAV capsid protein sequence such that both portions retain structural integrity after the conjugation reaction has taken place.

[0086] With regard to the conjugated AAV particles, the inventors further unexpectedly observed that conjugated AAV particles in which a relatively low portion of the AAV capsid protein is conjugated show a more significant improvement in transduction efficiency compared to conjugated AAV particles in which a relatively high portion of the AAV capsid protein is conjugated. It is clear that the lower amount of conjugated AAV capsid protein in the conjugated AAV particles may be the result of a lower amount of AAV capsid protein containing a sortase recognition sequence or of (optionally intentionally) non-optimal parameters for carrying out the conjugation reaction. Both embodiments are contemplated by the present disclosure.

[0087] Thus, optionally, the ratio of non-conjugated capsid protein (i.e., unmodified capsid protein or non-conjugated capsid protein) to conjugated capsid protein (i.e., modified capsid protein) in the conjugated AAV particles is between about 1 / 59 and about 59 / 1, preferably between about 1 / 20 and about 20 / 1, preferably between about 1 / 15 and about 15 / 1, preferably between about 1 / 10 and about 10 / 1, more preferably between about 1 / 9 and about 9 / 1, more preferably between about 1 / 8 and about 8 / 1, more preferably between about 1 / 7 and about 7 / 1, more preferably between about 1 / 6 and about 6 / 1, more preferably between about 1 / 5 and about 5 / 1. In a preferred embodiment, the ratio of non-joined capsid proteins (i.e., non-modified capsid proteins) to jointed capsid proteins (i.e., modified capsid proteins) in the jointed AAV particles is less than about 1 / 5, preferably less than about 1 / 10, preferably less than about 1 / 15, preferably less than about 1 / 20. In a preferred embodiment, the jointed AAV particles contain at least one jointed capsid protein (i.e., at most 59 non-joined capsid proteins). In a preferred embodiment, the jointed AAV particles contain 1 to 20 jointed capsid proteins, preferably 1 to 15 jointed capsid proteins, preferably 1 to 10 jointed capsid proteins, more preferably 1 to 5 jointed capsid proteins. In a specific embodiment, the jointed AAV particles contain one jointed capsid protein. Preferably, the jointed AAV particles contain at least 5% jointed AAV capsid proteins. However, the inventors have found that the present invention allows efficient joining of capsid proteins, if desired, which is interesting. Thus, in certain embodiments, the joined AAV particles comprise at least 20, preferably at least 30, more preferably at least 40, such as at least 50 joined capsid proteins.

[0088] In general, each AAV particle is assumed to contain at least one VP1 and at least one VP2, theoretically in a ratio of VP1:2:3 or 5:5:50. It will therefore be appreciated that the number of junctional proteins in a particle can be influenced by introducing sortase recognition sequences into one or more regions of the capsid protein.

[0089] Optionally, the VP1, VP2 or VP3 proteins in an AAV particle comprising (optionally joined) a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and the VR-VIII region have identical amino acid sequences (i.e., VP1 capsid proteins with different sequences are not assembled into a single assembled AAV particle, VP2 capsid proteins with different sequences are not assembled into a single assembled AAV particle, and / or VP3 capsid proteins with different sequences are not assembled into a single assembled AAV particle). In such embodiments, the sortase recognition sequences may be different and / or the sortase recognition sequence(s) may be inserted into different regions of the AAV capsid proteins.

[0090] The details of the heterologous conjugate molecule are not particularly limiting for the present invention, provided that they are acceptable for sortase-mediated conjugation molecules. Generally, this involves the presence of an N-terminal triglycine sequence (i.e., GGG). By way of example and not limitation, suitable heterologous conjugate molecules may be small molecules, carbohydrates, lipids, or proteins.

[0091] In a preferred embodiment, the heteroconjugate molecule is a targeting moiety. As used herein, the term "targeting moiety" encompasses any molecule that can preferentially bind to a particular tissue, cell type, and / or organ over other respective tissues, targets, and / or organs. The details of the targeting moiety are not particularly limiting to the invention, and thus examples include, but are not limited to, ligands of cell receptors and proteins that bind to cell surface proteins. Thus, preferred heteroconjugate molecules include antibodies and antibody fragments (e.g., but are not limited to, nanobodies). Optionally, in embodiments where the heteroconjugate molecule is an antibody or antibody fragment (e.g., an antibody), the heteroconjugate molecule specifically binds to human epidermal growth factor receptor 2 (HER2).

[0092] A related aspect of the present invention is directed to the conjugated AAV particles described herein for use as a medicament. Accordingly, a method of treating a subject in need thereof is encompassed by the present invention, the method comprising administering the conjugated AAV particles to the subject. The use of the conjugated AAV particles as described herein for the manufacture of a medicament is also envisioned. Particular medical conditions for which the subject of the present invention may be used include proliferative diseases (i.e., cancer) and tissue-specific diseases (e.g., liver disease). It is clear that the products described herein that are correlated with the conjugated AAV particles (e.g., the AAV capsid proteins described herein, the nucleic acids described herein, and the nucleic acid vectors described herein) may also be used for medical purposes.

[0093] The terms "subject", "patient" and "subject in need" may be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, specifically human and non-human mammals. The term "mammal" or "mammalian subject" refers to any animal classified as such, and thus includes, but is not limited to, humans, livestock, commercial animals, farm animals, zoo animals, sports animals, pets and laboratory animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows; primates, such as apes, monkeys, orangutans and chimpanzees; canines, such as dogs and wolves; felines, such as cats, lions and tigers; equines, such as horses, donkeys and zebras; food animals, such as cows, pigs and sheep; ungulates, such as deer and giraffes; rodents, such as mice, rats, hamsters and guinea pigs. A preferred patient is a human subject. Particularly preferred are human subjects, including both genders and all age categories thereof.

[0094] A related aspect of the invention is directed to a method of producing conjugated AAV particles, comprising expressing in a cell a nucleic acid encoding a capsid protein as described herein, allowing the cell to form AAV particles, recovering the AAV particles, and conjugating them to a molecule of interest. - contacting the cell with one or more nucleic acids encoding AAV capsid proteins, wherein one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, or the VR-VIII region comprises a sortase recognition sequence; - causing the cells to assemble the plurality of AAV capsid proteins into AAV particles, and recovering the AAV particles from the cells; and - contacting the AAV particles with a sortase and a heterologous conjugate molecule Includes.

[0095] Those skilled in the art will understand that between the step of providing one or more nucleic acids encoding multiple AAV capsid proteins to cells and the step of collecting AAV particles, a step of culturing the cells is performed to allow the transcription and translation of AAV capsid proteins and assembly into AAV particles. Optionally, the one or more nucleic acid sequences recited in this method correspond to three nucleic acid sequences: a first nucleic acid encoding rep and cap genes, a second nucleic acid including a transgene region and an ITR region, and a third nucleic acid encoding any helper protein for the assembly of AAV particles. In certain embodiments, a step of lysis of the cells contained in the cell culture is performed before the step of collecting AAV particles. The method of producing AAV particles has been described many times in the art and is therefore known to those skilled in the art.

[0096] In a preferred embodiment, the sortase recognition sequence is a sortase A recognition sequence, more preferably the sequence Xn-LPXTG-Xm [SEQ ID NO: 10].

[0097] In a preferred embodiment, the AAV capsid protein contains a sortase recognition sequence in one or more of the VR-I, VR-IV, or VR-VIII regions.

[0098] Optionally, prior to contacting the AAV particles with the sortase and the heterologous conjugate molecule, the method further comprises the additional step of enriching, purifying, and / or isolating the assembled AAV particles.Alternatively and / or in addition to the preceding embodiments, prior to contacting the AAV particles with the sortase and the heterologous conjugate molecule, the method further comprises the step of depleting or removing AAV capsid proteins that are not part of the assembled AAV particles.

[0099] In certain embodiments, in the conjugation step of the methods contemplated herein, the AAV capsid proteins, sortase, and heterologous conjugate molecules are provided in a ratio of 1:1:1. In alternative embodiments, an excess of sortase and heterologous conjugate molecules are provided such that a molecular ratio of 1:>1:>1 is maintained. In yet further embodiments, an excess of heterologous conjugate molecules are provided such that a ratio of 1:1:>1 is maintained, preferably a ratio of 1:1:>5, and more preferably a ratio of 1:1>10. In a preferred embodiment, the AAV capsid proteins, sortase, and heterologous conjugate molecules are provided in a ratio of about 1:0.1:1.

[0100] Optionally, after contacting the AAV particles with the sortase and the heterologous conjugate molecule, the method comprises the further step of enriching, purifying, and / or isolating assembled joint AAV particles comprising at least one joint AAV capsid protein. In a further embodiment, the method comprises the further step of enriching, purifying, and / or isolating assembled joint AAV particles containing at least 10%, preferably at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, or even 100% of the joint AAV capsid proteins.

[0101] Optionally, the AAV capsid protein comprises a plurality of different sortase recognition sequences, and the AAV particle is contacted with the different sortases and junction molecules in a sequential manner. The plurality of different sortase recognition sequences may be provided in a single AAV capsid protein, or may be provided by using a collection of AAV capsid proteins, each of which comprises a different sortase recognition sequence (optionally at different locations in the AAV capsid protein). Preferably, an AAV capsid protein is provided that comprises a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, or the VR-VIII region. In certain embodiments, a combination of a naturally occurring AAV capsid protein and an AAV capsid protein that comprises a sortase recognition sequence in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, or the VR-VIII region is provided.

[0102] It is clear that any embodiment described herein with respect to such AAV capsid proteins is equally applicable to such (isolated) AAV capsid proteins, but also to AAV capsid proteins encoded by nucleic acids or nucleic acid vectors, and to (assembled) AAV particles, and vice versa.

[0103] In addition to the embodiments described herein above, the present invention further relates to an adeno-associated virus (AAV) VP1, VP2 or VP3 capsid protein characterized in that a sortase recognition sequence Xn-LPXTG-Xm [SEQ ID NO:10] (wherein n and m are in the range of 0 to 20, and X is any naturally occurring amino acid) is inserted in one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region and the VR-VIII region. In certain embodiments, an adeno-associated virus (AAV) VP1, VP2 or VP3 capsid protein is characterized by the insertion of a sortase recognition sequence, Xn-LPXTG-Xm [SEQ ID NO:10], into one or more of the VP1-VP2 transition region, the VR-I region, the VR-IV region, and the VR-VIII region, wherein the VP1-VP2 transition region is defined by SEQ ID NO:1 [PVKTAP], the VR-I region is defined by SEQ ID NO:2 [SSQSGASN], the VR-IV region is defined by SEQ ID NO:3 [SRTNTPSGTTTQSRLQFSQAGASDIRDQS], and the VR-VIII region is defined by SEQ ID NO:4 [QYGSVSTNLQRGNRQAATADVNTQGV]. In certain embodiments, the insertion in the VR-IV region is in a fragment having SEQ ID NO: 5 [TPSGTTTQS] and / or in a fragment having SEQ ID NO: 6 [LQRGNRQAA]. In certain embodiments, one or more amino acids are deleted in the region where the sortase recognition site is inserted.

[0104] In certain embodiments of any of the above embodiments, the AAV is AAV2 or AAV9. In further certain embodiments of any of the above embodiments, n is between 15 and 20, between 10 and 15, between 5 and 10, between 0 and 5, or is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In particular, it is envisaged that m is between 15 and 20, between 10 and 15, between 5 and 10, between 0 or 5, or is 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid. In certain embodiments of any of the above embodiments, the linker sequence Xn or Xm comprises at least 80% Gly, Ser, Thr, and Ala. Preferably, the linker sequence Xm or Xn consists of amino acids selected from Gly, Ser, Thr, and Ala. In certain embodiments of any of the above embodiments, X in LPXTG is Asp, Glu, Ans, or Gln, typically Gln. In certain embodiments of any of the above embodiments, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into the VR-1 region and one or more of the lysines at positions K258, K321, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K692, K706 are mutated. In further particular embodiments, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into the VR-1 region and one or more of the lysines at positions K258, K507, K527, K549, and K706 are mutated. In a further particular embodiment, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into the VR-IV region and one or more of the lysines at positions K258, K309, K313, K321, K490, K507, K527, K532, K544, K549, K556, K620, K640, K649, K665, K688, K692, and K706 are mutated. In a further particular embodiment, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into the VR-IV region and one or more of the lysines at positions K258, K490, K507, K532, K544, K549, K556, and K665 are mutated.In a further particular embodiment, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into VR-IV and the lysine at position K549 is mutated. In a further particular embodiment, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into VR-VIII and one or more of the lysines at positions K309, K490, K507, K527, K532, K544, K549, K556, K620, K640, K688, and K706 are mutated. In a further particular embodiment, the sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into VR-VIII and one or more of the lysines at positions K490, K507, K527, and K532 are mutated. In further particular embodiments, a sortase recognition sequence LPXTG [SEQ ID NO: 10] is inserted into VR-VIII and one or more of the lysines at position K507 are mutated to Gly or Ala. In preferred embodiments of the embodiments described herein, the one or more lysines are mutated to Gly, Ser or Ala. The present invention also provides nucleic acids encoding the capsid proteins of any one of the above embodiments herein. The present invention further provides expression vectors comprising these nucleic acids. The present invention further provides AAV particles comprising the above AAV capsid proteins herein. In particular embodiments, the ratio of unmodified AAV protein to modified protein is 1 / 20, 1 / 10 or 1 / 5 to 5 / 1, 10 / 1 or 20 / 1. In particular embodiments, the AAV particles are fused to a conjugate (e.g., a small molecule, carbohydrate, polypeptide) via a sortase recognition site. In further particular embodiments, the AAV particles are fused to a targeting moiety (e.g., a ligand for a cell receptor, or a protein that binds to a cell surface protein) via a sortase recognition site. In further particular embodiments, the protein that binds to a cell surface protein is an antibody or nanobody, e.g., an antibody or nanobody that binds to HER2. The invention further provides uses of the AAV particles described herein, e.g., the AAV particles described herein for use as a medicament.

[0105] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. It is therefore intended to embrace all such alternatives, modifications, and variations within the spirit and broad scope of the appended claims as set forth below. The aspects, descriptions, and embodiments of the present invention disclosed herein are further supported by the following non-limiting examples. EXAMPLES

[0106] Example 1: General design information, considerations, and exploration results Adeno-associated virus (AAV) is a small, non-pathogenic, non-enveloped ssDNA virus. Its genome (4.7 kb) consists of two major open reading frames: the rep gene encodes the four replication proteins; the cap gene encodes the three structural proteins of the AAV capsid (VP1, VP2, and VP3), which are formed through alternative splicing and alternative start codons. In a 1:1:10 ratio, they form a 60-subunit capsid coat. Flanking the rep and cap genes are the inverted terminal repeats (ITRs), which are the only genetic elements required for viral DNA replication and packaging. As a result, the viral rep and cap genes can be provided in trans during the production process, and a transgene of interest of ∼4.5 kb can be inserted between the ITRs, resulting in a recombinant AAV (rAAV) viral vector (Figure 1A).

[0107] Sortase reactions are generally described in numerous examples throughout the art and are therefore known to those skilled in the art. A general overview of the Sortase A reaction is shown in Figure 1B. This results in efficient and controlled antibody-drug coupling with a homogenous and predictable drug-to-antibody ratio. This technique has not previously been used for enzymatic coupling of targeting moieties to AAV vectors.

[0108] The targeting ability of AAV vectors depends on the targeting ability of the ligand. Nanobodies are preferred moieties suitable for this. They combine a small and compact structure with high specificity, high stability and low immunogenicity profile, and sortase A technology is widely used for nanobody conjugation to drugs or other nanobodies. Furthermore, nanobodies can be designed against virtually any cell receptor. The invention is illustrated using the anti-HER 2 (human epidermal growth factor receptor 2) nanobody 2Rs15d, which is used in breast cancer radionuclide therapy.

[0109] The sortase A LPXTG recognition motif [SEQ ID NO: 10] can function as part of an exposed loop in the target protein. Previous literature has shown that peptide insertions of up to 34 amino acids into the variable regions (VR)-IV and VR-VIII of the AAV capsid are well tolerated. The placement of the LPXTG recognition motif [SEQ ID NO: 10] in VR-IV and VR-VIII is shown in (Figures 2A and 2B). Since the LPXTG [SEQ ID NO: 10] binding cleft on the sortase A enzyme is a fairly deep binding pocket, the length and sequence of the LPXTG [SEQ ID NO: 10] flanking linker is taken into consideration.

[0110] In order to weaken the natural affinity of AAV2, AAV2-HB0, whose ability to bind to heparan sulfate proteoglycan receptors has been abolished by mutations (R585A and R588A), is used in the examples of the present invention, however, it is clear that the mutations of this particular AAV2 mutant are not limiting in the context of the present invention, and the findings described herein are readily applicable to other AAV serotypes in general and other AAV2 mutations.

[0111] Sortase A mediated coupling reactions are performed using Ab recognition (e.g. HA or FLAG) and affinity tag (e.g. His) tagged nanobody proteins. Recombinant 2Rs15d nanobody is used in this example.

[0112] Different constructs are tested in experiments to assess ligand binding using sortase A. The LPQTG motif [SEQ ID NO: 43] with an optional linker was inserted as shown in Table 1.

[0113] [Table 1]

[0114] Nanobodies are typically synthesized using the Gly 5 N-terminally tagged with 3xFLAG-His 6 The unconjugated and undesired products are removed using diafiltration.

[0115] AAV2 allows the insertion of LPQTG [SEQ ID NO: 43] into loops IV and VIII with different linker lengths. The LPQTG motif [SEQ ID NO: 43] was inserted into AAV2-HB0 VR-IV (between AA453 and 454) VR-VIII (between AA587 and 588) without a linker or flanked by one or two GGSGS [SEQ ID NO: 47] repeats on either side of the motif. Gly5-nanobodies were then conjugated using sortase A. Western blots were performed and imaged with anti-VP antibodies (detecting VP1, 2 and 3) and anti-FLAG antibodies (detecting nanobodies). As the sortase A reaction cleaves the VP primary structure during conjugation, a 17-18 kDa band was expected and seen in VIII-1 and VIII-2 (Figure 3).

[0116] Example 2: AAV2_VR-I 0-2L Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15) [AAV2-HB0 with an LPQTG tag located at position 265 and flanked on both sides by 0, 1 or 2 GGSGS linkers] were mixed with sortase A and anti-GFP nanobody (SEQ ID NO:11) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 100-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. The mixtures were incubated for 16 hours at 25° C. The reacted samples were then run on an SDS-PAGE gel with an equal amount of vector in sortase reaction buffer without sortase A or nanobody and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:1000 rabbit anti-VP1 / 2 / 3 (Progen #61084) for 16 hours at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-rabbit antibody (Dako P0448) for 1 hour at room temperature and developed using chemiluminescence.

[0117] In parallel, equal amounts of unreacted nanobodies were loaded on another SDS-PAGE gel, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0118] Results and Discussion Anti-VP1 / 2 / 3 stained blots with nanobody-conjugated samples (Figure 4) show a faint band just above the 55 kDa reference band (arrow) that increases in intensity as the linker length increases from 0 to 1 to 2 GGSGS [SEQ ID NO: 47] elements. This apparent molecular weight is consistent with sortase A-mediated backbone cleavage yielding a 53 kDa C-terminal VP fragment.

[0119] Similarly, a faint band was seen just above the 35 kDa marker, which may refer to a nanobody-conjugated VP1 or VP2 fragment, however, the 100-fold excess of nanobody and an unknown 40 kDa species in the nanobody preparation make identification of the fainter band difficult.

[0120] In conclusion, the LPQTG tag [SEQ ID NO: 43] can be inserted into VR-I and conjugation is not very efficient. Conjugation efficiency increases with increasing linker length, but is generally not very efficient at this insertion site.

[0121] Example 3: AAV2_VR-IV 0-5L Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO: 16 to SEQ ID NO: 20) [AAV2-HB0 with an LPQTG tag located at position 453 and flanked on both sides by 0, 1, 3, 4 or 5 GGSGS linkers] were mixed with sortase A and anti-GFP nanobody (SEQ ID NO: 11) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 10-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. An identical reaction mixture was also made but without sortase A or nanobody. The reaction mixture was incubated at 25 °C for 16 h. Samples were then run on an SDS-PAGE gel and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:1000 rabbit anti-VP1 / 2 / 3 (Progen #61084) for 16 hours at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-rabbit antibody (Dako P0448) for 1 hour at room temperature and developed using chemiluminescence.

[0122] In parallel, the reaction mixture was loaded onto another SDS-PAGE gel, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0123] Results and Discussion A distinct band of 37-39 kDa is clearly visible in anti-VP1 / 2 / 3 stained blots with nanobody-conjugated samples (Figure 5). The intensity increases from 0 to 1-3 linker repeats and appears to stabilize as the linker length increases to 4 and 5 linker repeats. This apparent molecular weight is consistent with sortase A-mediated backbone cleavage yielding C-terminal VP fragments of 32-35 kDa, considering that most fragments migrate slightly higher than the expected molecular weight.

[0124] Similarly, a band is seen around the 55 kDa marker, which represents the nanobody-conjugated N-terminal VP fragment, which is not seen in the absence of linker fragment and is barely visible when one linker repeat is seen, but is clearly seen with 3, 4, or 5 linker repeats, with no obvious difference in intensity between 3, 4, or 5 repeats.

[0125] In conclusion, the LPQTG tag [SEQ ID NO: 43] can be inserted into VR-IV in AAV2 with good conjugation efficiency, which increases with increasing linker length and appears to plateau at three GGSGS linker repeats on each side of the LPQTG tag.

[0126] Example 4: AAV2_VR-VIII 0-5L Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO:21 to SEQ ID NO:26) [AAV2-HB0 with an LPQTG tag located at position 587 and flanked on both sides by 0, 1, 2, 3, 4 or 5 GGSGS linkers] were mixed with sortase A and anti-GFP nanobody (SEQ ID NO:11) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 10-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. An identical reaction mixture was also made but without sortase A or nanobody. The reaction mixture was incubated at 25 °C for 16 h. Samples were then run on an SDS-PAGE gel and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:1000 rabbit anti-VP1 / 2 / 3 (Progen #61084) for 16 hours at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-rabbit antibody (Dako P0448) for 1 hour at room temperature and developed using chemiluminescence.

[0127] In parallel, the reaction mixture was loaded onto another SDS-PAGE gel together with a sample containing the same amount of nanobody alone, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0128] Results and Discussion In anti-VP1 / 2 / 3 stained blots with nanobody-conjugated samples, a distinct band of 17-20 kDa is clearly visible (Figure 6). The intensity increases from 0 to 1 to 3 to 4 linker repeats (2 linker repeats are less intense) and appears to be lower again at 5 linker repeats. This apparent molecular weight is consistent with sortase A-mediated backbone cleavage yielding a 17-20 kDa C-terminal VP fragment.

[0129] Similarly, a band is seen around the 70 kDa marker, which migrates slightly higher as in the case of the unreacted VP1 / 2 / 3 band, indicative of the nanobody-conjugated N-terminal VP fragment. This band is not seen in the absence of linker fragment, increases from 1 to 2 to 3 to 4 linker repeats, and is again slightly lower for 5 linker repeats.

[0130] In conclusion, the LPQTG tag [SEQ ID NO: 43] can be inserted into VR-VIII in AAV2 with moderate conjugation efficiency, which increases with increasing linker length and appears to peak at four GGSGS [SEQ ID NO: 47] linker repeats on each side of the LPQTG tag [SEQ ID NO: 43].

[0131] Example 5: AAV9_VR-IV 0-3L Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO:27 to SEQ ID NO:30) [AAV9-A with an LPQTG tag located at position 455 and flanked on both sides by 0, 1, 2 or 3 GGSGS linkers] were mixed with sortase A and anti-GFP nanobody (SEQ ID NO:11) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 10-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. The mixture was incubated for 16 hours at 25°C. The reacted samples were then run on an SDS-PAGE gel with an equal amount of vector in sortase reaction buffer without sortase A or nanobody and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:1000 mouse anti-VP1 / 2 / 3 (Progen #65158) for 16 hours at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 hour at room temperature and developed using chemiluminescence.

[0132] In parallel, equal amounts of unreacted AAV and equal amounts of unreacted nanobody were loaded on a separate SDS-PAGE gel, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0133] Results and Discussion A distinct band at 37 kDa is clearly visible in anti-VP1 / 2 / 3 stained blots with nanobody-conjugated samples (Figure 7). The intensity increases from 0 to 1 linker repeat, with 1, 2 and 3 linker repeats having similar intensity. This apparent molecular weight is consistent with sortase A-mediated backbone cleavage yielding a 33 kDa C-terminal VP fragment, considering that most fragments migrate slightly higher than the expected molecular weight.

[0134] In anti-FLAG gels, a band at 45 kDa is seen, representing the nanobody-conjugated N-terminal VP fragment, which is not seen in the absence of linker fragment but increases in intensity with one, two or three linker repeats.

[0135] In conclusion, the LPQTG tag [SEQ ID NO: 43] can be inserted into the VR-IV in AAV9 with good conjugation efficiency. The conjugation efficiency increases with increasing linker length, reaching its highest value with three GGSGS [SEQ ID NO: 47] linker repeats on each side of the LPQTG tag [SEQ ID NO: 43], and longer linker lengths have not been tested with this construct.

[0136] Example 6: AAV9_VR-VIII 0-1-3L Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33) [AAV9-A with an LPQTG tag located at position 589 and flanked on either side by 0, 1 or 3 GGSGS linkers] were incubated with sortase A and anti-GFP nanobody (SEQ ID NO:11) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 10-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. The mixtures were incubated at 25° C. for 16 hours. The reacted samples, together with samples without sortase A, were then run on an SDS-PAGE gel and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:1000 mouse anti-VP1 / 2 / 3 (Progen#65158) for 16 hours at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) at room temperature for 1 hour and developed using chemiluminescence.

[0137] In parallel, the reaction mixture, together with a sample without sortase A, was loaded and run on another SDS-PAGE gel, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0138] Results and Discussion Anti-VP1 / 2 / 3 stained blots with nanobody-conjugated samples show a faint band at 17 kDa (Figure 8). The band is indistinguishable with one linker repeat, but the intensity increases with one to three linker repeats. This apparent molecular weight is consistent with sortase A-mediated backbone cleavage yielding a 17 kDa C-terminal VP fragment.

[0139] In anti-FLAG gels, a band at 70 kDa is seen, representing the nanobody-conjugated N-terminal VP fragment, which is very faint in the absence of linker fragment but increases in intensity with 1 to 3 linker repeats.

[0140] In conclusion, the LPQTG [SEQ ID NO: 43] tag can be inserted into VR-VIII in AAV9, but the conjugation is inefficient. Conjugation efficiency increases with increasing linker length, reaching its highest value with three GGSGS [SEQ ID NO: 47] linker repeats on each side of the LPQTG tag [SEQ ID NO: 43], and longer linker lengths have not been tested with this construct.

[0141] Example 7: AV2_VR-I 2L, AAV2_VR-IV 3L, AAV2_VR-VIII 3L, AAV9_VR-IV 3L, and AAV2_VR-VIII 3L Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:33) [AAV2-HB0 with a LPQTG tag located at position 265 and flanked by two GGSGS linkers on either side, AAV2-HB0 with a LPQTG tag located at position 453 and flanked by three GGSGS linkers on either side, AAV2-HB0 with a LPQTG tag located at position 587 and flanked by three GGSGS linkers on either side, AAV9-A with a LPQTG tag located at position 455 and flanked by three GGSGS linkers on either side, and AAV9-A with a LPQTG tag located at position 589 and flanked by three GGSGS linkers on either side] were incubated with sortase A and anti-HER2 nanobody (SEQ ID NO:12) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 10-fold higher than that of the AAV viral proteins in the mixture, while the nanobodies were present at a 100-fold higher molar concentration compared to the AAV viral proteins. The mixture was incubated at 25°C for 16 hours. The reacted samples were then run on an SDS-PAGE gel with an equal amount of vector in sortase reaction buffer without sortase A or nanobodies and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:1000 rabbit anti-VP1 / 2 / 3 (Progen#61084) for 16 hours at 4°C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-rabbit antibody (Dako P0448) for 1 hour at room temperature and developed using chemiluminescence.

[0142] In parallel, the reaction mixture was loaded onto another SDS-PAGE gel, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0143] Results and Discussion Anti-VP1 / 2 / 3 stained blots show several bands related to C-terminal VP fragments generated by sortase A (Figure 9). For AAV2_VR-I, the ~58 kDa band is rather weak, indicating inefficient coupling. For AAV2_VR-IV and AAV9_VR-IV, the ~38 kDa band is clearly visible, indicating efficient coupling. For AAV2_VR-VIII and AAV9_VR-VIII, the ~18 kDa band is seen at a higher intensity than AAV2_VR-I, but at a lower intensity than AAV2_VR-IV and AAV9_VR-IV, indicating intermediate coupling.

[0144] The same findings are reflected in the FLAG stained blot: the bands corresponding to the junction AAV2_VR-I and AAV9-VR-VIII are completely obscured by the background from the nanobodies. The band corresponding to the junction AAV2_VR-VIII is very faintly visible. The bands corresponding to AAV2_VR-IV and AAV9_VR-IV are clearly visible.

[0145] In conclusion, insertion into VR-IV shows the highest coupling efficiency, insertion into VR-VIII seems to work, albeit with lower efficiency, and insertion into VR-I is possible, albeit with a very low relative efficiency.

[0146] Example 8: GGG-Biotin Materials and Methods The LPQTG-containing AAV vector (SEQ ID NO: 22 (587_1L_pdAAVe_008)) [AAV2-HB0 with an LPQTG tag located at position 587 and flanked on both sides by one GGSGS linker] was incubated with sortase A and biotin-tagged anti-GGG peptide (GGG-[K(biotin)]-amide, Cambridge Research Biochemicals, crb1000649h) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 100-fold higher than the molar concentration of AAV viral proteins in the mixture, while the GGG-biotin peptide was present at a 250-fold higher molar concentration compared to the AAV viral proteins (sample (1)). Control samples contained (2) "no AAV", (3) no sortase A, (4) no GGG-biotin peptide, (5) GGG-biotin peptide only, (6) GGG-biotin peptide with BSA, and (7) BSA only. In the control reactions, all components were at the same concentrations as in reaction (1), and the mass of BSA in samples (6) and (7) was identical to the mass of viral protein in sample (1). The reaction mixtures were incubated at 25° C. for 16 hours.

[0147] The reaction samples were then run on an SDS-PAGE gel and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:10000 streptavidin-HRP (ThermoScientific, 21130) and developed using chemiluminescence.

[0148] As a reference for the molecular weight of VP on the gel, an AAV-only sample blotted and imaged with anti-VP1 / 2 / 3 antibody was included from a different blot.

[0149] Results and Discussion In streptavidin-HRP blots, sortase A-mediated biotin labeling of VP1, 2 and 3 could be observed in sample (1) and all negative controls showed no sign of conjugation (Figure 10). The molecular weight is lower than that of the unconjugated VP, as expected due to the removal of the 17 kDa C-terminal fragment.

[0150] Example 9: HER2 vs. GFP Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO: 24, SEQ ID NO: 30) [AAV2-HB0 with a LPQTG tag located at position 453 and flanked on both sides by three GGSGS linkers, AAV9-A with a LPQTG tag located at position 589 and flanked on both sides by three GGSGS linkers] were incubated with sortase A and anti-HER2 nanobody (SEQ ID NO: 12), anti-GFP nanobody (SEQ ID NO: 11) or without nanobody and with anti-GFP nanobody (SEQ ID NO: 11) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 10-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. The mixtures were incubated for 16 hours at 25° C. Reaction samples were then run on an SDS-PAGE gel and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:1000 rabbit anti-VP1 / 2 / 3 (Progen #61084) for 16 hours at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-rabbit antibody (Dako P0448) for 1 hour at room temperature and developed using chemiluminescence.

[0151] In parallel, the reaction mixture was loaded onto another SDS-PAGE gel, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0152] Results and Discussion As observed in previous experiments, sortase A-mediated nanobody conjugation of AAV2, which has a LPQTG [SEQ ID NO: 43] insertion at position 587 flanked on each side by three GGSGS [SEQ ID NO: 47] linker repeats, is visible but inefficient (Figure 11). Sortase A-mediated nanobody conjugation of AAV9, which has a LPQTG [SEQ ID NO: 43] insertion at position 455 flanked on each side by three GGSGS [SEQ ID NO: 47] linker repeats, is seen with good conjugation efficiency.

[0153] Most importantly from this experiment, there appears to be no difference in conjugation efficiency between anti-HER2 and anti-GFP nanobodies.

[0154] Example 10: LPQTG vs. LPETG in AAV2_VR-VIII and AAV9_VR-IV Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO:24, SEQ ID NO:34, SEQ ID NO:30, SEQ ID NO:35) [AAV2-HB0 with a LPQTG tag located at position 587 and flanked by three GGSGS linkers on either side, AAV2-HB0 with a LPETG tag located at position 587 and flanked by three GGSGS linkers on either side, AAV9-A with a LPQTG tag located at position 455 and flanked by three GGSGS linkers on either side, and AAV9-A with a LPETG tag located at position 455 and flanked by three GGSGS linkers on either side] were incubated with sortase A and anti-HER2 nanobody (SEQ ID NO:12) in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 5-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 50-fold higher molar concentration compared to the AAV viral proteins. The mixture was incubated for 20 hours at 25°C. The reaction samples were then run on an SDS-PAGE gel and blotted onto a PVDF membrane. After blocking, the membrane was incubated with 1:500 rabbit anti-VP1 / 2 / 3 (Progen#61084) for 16 hours at 4°C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-rabbit antibody (Dako P0448) for 1 hour at room temperature and developed using chemiluminescence.

[0155] In parallel, the reaction mixture was loaded and run on another SDS-PAGE gel, which was blotted onto a PVDF membrane and stained with 1:1000 mouse anti-FLAG antibody (Novus Bio NBP1-97410) for 16 h at 4° C. After washing, the gel was incubated with 1:10000 HRP-conjugated goat anti-mouse antibody (Dako P0447) for 1 h at room temperature and developed using chemiluminescence.

[0156] Results and Discussion As observed in previous experiments, sortase A-mediated nanobody conjugation of AAV2, which has an LPQTG insertion at position 587 flanked on each side by three GGSGS [SEQ ID NO: 47] linker repeats, is visible but inefficient (Figure 12). Sortase A-mediated nanobody conjugation of AAV9, which has an LPQTG [SEQ ID NO: 43] insertion at position 455 flanked on each side by three GGSGS [SEQ ID NO: 47] linker repeats, is seen with good conjugation efficiency.

[0157] From both anti-VP1 / 2 / 3 and anti-FLAG blots, LPETG [SEQ ID NO: 44] appears to have slightly higher conjugation efficiency compared to LPQTG [SEQ ID NO: 43], at least for anti-HER2 nanobody conjugation to AAV9 containing an LPXTG [SEQ ID NO: 10] insertion at position 455 and flanked by three GGSGS [SEQ ID NO: 47] linker repeats on each side.

[0158] Example 11: In vitro targeting Materials and Methods LPQTG-containing AAV vector (SEQ ID NO: 24) [AAV2-HB0 with an LPQTG tag located at position 587 and flanked by three GGSGS linkers on either side] was incubated with sortase A and anti-HER2 nanobody (SEQ ID NO: 12) ("conjugated anti-HER2 Nb"), sortase A and anti-GFP nanobody (SEQ ID NO: 11) ("conjugated anti-GFP Nb"), anti-HER2 nanobody without sortase A (SEQ ID NO: 12) ("unconjugated with Nb"), and neither nanobody nor sortase A ("unconjugated without Nb") in 1× sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5). The molar concentration of sortase A was 10-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. The reactions were incubated at 25°C for 20 hours and then dialyzed against PBS using a Micro Float-A-Lyzer 100 kDa MWCO (Repligen, F235049) to remove sortase A enzyme and unreacted nanobodies. Finally, viral genome titers were determined using ddPCR.

[0159] MCF-10A (HER2 negative) and BT-474 (HER2 positive) cells were cultured in MEBM (Lonza) medium supplemented with 10% FBS and RPMI1640 (Gibco) and MEBM (Lonza) supplemented with Glutamax and 10% FBS, respectively. Cells were seeded at 50.000 cells in 90 μl medium and mixed with vector at an MOI of 1E5 vg / cell in 30 μl. This mixture was plated in a 96-well plate and after 3 hours, 80 μl medium was added. After 72 hours, cells were washed with PBS, trypsinized, spun down, and resuspended in 400 μl PBS containing 1:10000 PerCP-Cy5.5-conjugated anti-HER2 antibody (BioLegend 324416). After 30 minutes of incubation at room temperature, the cell suspension was analyzed using an Attune flow cytometer.

[0160] Results and Discussion LPQTG-containing AAV vectors (SEQ ID NO: 24), with or without nanobodies and regardless of conjugation, do not transduce MCF-10A HER2-negative cells (Figures 13A, 13B, and 13C), most likely a result of the HB0 mutation, as unmodified AAV2 efficiently transduces MCF-10A cells (86%) at an MOI of 1E5 (data not shown).

[0161] For BT-474 HER2-positive cells, transduction efficiency is dramatically higher with AAV vectors conjugated with anti-HER2 nanobodies compared to unconjugated or anti-GFP nanobody-conjugated vectors.

[0162] These results clearly show that conjugation of our vector with a targeting nanobody leads to increased transduction efficiency and specificity (Figures 13A, 13B, and 13C).

[0163] Example 12: Comparison of transduction efficiency Materials and Methods LPQTG-containing AAV vectors (SEQ ID NO:24 and SEQ ID NO:30) [AAV2-HB0 with a LPQTG tag located at position 587 and flanked on both sides by three GGSGS linkers, and AAV9-A with a LPQTG tag located at position 455 and flanked on both sides by three GGSGS linkers] were incubated with sortase A and anti-HER2 nanobody (SEQ ID NO:12) ("conjugated anti-HER2 Nb") in 1x sortase reaction buffer (300 mM Tris, 150 mM NaCl, 5 mM CaCl2, pH 7.5), as well as with anti-HER2 nanobody (SEQ ID NO:12) without sortase A ("unconjugated to Nb"). The molar concentration of sortase A was 10-fold higher than the molar concentration of AAV viral proteins in the mixture, while the nanobody was present at a 100-fold higher molar concentration compared to the AAV viral proteins. The reactions were incubated at 25°C for 20 hours and then dialyzed against PBS using a Micro Float-A-Lyzer 100 kDa MWCO (Repligen, F235049) to remove sortase A enzyme and unreacted nanobodies. Finally, viral genome titers were determined using ddPCR.

[0164] BT-474 (HER2 positive) cells were cultured in MEBM (Lonza) medium supplemented with 10% FBS and RPMI1640 (Gibco) and MEBM (Lonza) supplemented with Glutamax and 10% FBS, respectively. Cells were seeded at 50.000 cells in 90 μl medium and mixed with vector at an MOI of 1E5vg / cell in 30 μl or with an equivalent dilution of unreacted nanobody alone. This mixture was seeded in a 96-well plate and after 3 hours, 80 μl medium was added. After 72 hours, cells were washed with PBS, trypsinized, spun down and resuspended in 400 μl PBS containing 1:10000 PerCP-Cy5.5-conjugated anti-HER2 antibody (BioLegend 324416). After 30 minutes of incubation at room temperature, the cell suspension was analyzed using an Attune flow cytometer.

[0165] Results and Discussion Transduction efficiency was 92-fold (AAV2_VR-VIII3L, SEQ ID NO: 24) and 8-fold (AAV9_VR-IV3L, SEQ ID NO: 30) higher for AAV vectors conjugated with anti-HER2 nanobodies compared to non-conjugated vectors (Figure 14).

[0166] These results clearly show that the conjugation of our vector with a targeting nanobody leads to an increase in transduction efficiency.

[0167] Interestingly, this effect is more than 10-fold more pronounced for AAV2_VR-VIII3L, which has a lower conjugation efficiency (~2%) compared to AAV9_VR-IV 3L (~12%), which may be due to the insertion position or the base serotype used.

[0168] Example 13: Comparison of production yields of different constructs Materials and Methods HEK293 cells were seeded at 3.6E+06 viable cells per 10 cm Petri dish in DMEM with 10% FBS and transfected with a transgene plasmid (encoding ITR-flanked CAG-GFP), a plasmid encoding adenoviral helper genes, and a plasmid encoding rep / cap using PEIpro according to the manufacturer's recommendations. AAV2, AAV9, AAV2-VR-VIII-LPQTG-3L [SEQ ID NO: 24], and AAV9-VR-IV-LPQTG-3L [SEQ ID NO: 30] were each transfected with Duplo. The transfected plates were stored at 37° C. and 5% CO2, and the medium was replaced with DMEM with 5% FBS 24 hours after transfection. The supernatant was harvested 72 hours after transfection. The cells were removed using trypsin and stored in a separate Falcon tube. Both tubes were spun at 1000 g for 5 minutes. The medium supernatant was transferred to a new tube and the cell fraction supernatant was discarded.

[0169] The cell pellet was then lysed using three freeze / thaw cycles, treated with DNAse for 1 hour at 37°C, and finally centrifugation was used to remove insoluble debris. Viral genome ("Vg") titers were determined from the supernatant and cellular fractions using ddPCR. Viral particle ("Vp") titers were determined from the supernatant and cellular fractions using ELISA.

[0170] Results and Discussion Although there is some variability in terms of production yield and percentage full, the adhesion production of the mutant vectors is similar to that of the ancestral (AAV2 and AAV9) serotypes (Figures 15A and 15B). In other words, the insertion of the LPQTG site flanked by 15 amino acids on each side does not result in a significant decrease in production yield.

[0171] Sequences referenced in this application AAV2 wild type Cap VP2 start T138 VP3 start M203

[0172] [Table 2]

[0173] [Table 3]

[0174] [Table 4]

[0175] HB 0 Refer to variants R585A and R588A Recognition motif: Xn-LPXTG-Xm LPXTG [SEQ ID NO: 10] X is any natural AA, or X is DEQN, or X is Q. n= 0-20 m= 0-20

[0176] Surface-exposed lysines within 30 Å of the VR-I loop: K258 K321 K490 K507 K527 K532 K544 K549 K556 K620 K640 K649 K665 K692 K706 Surface-exposed lysine within 15 Å of loop VR-I: K258 K507 K527 K549 K706

[0177] Surface-exposed lysines within 30 Å of the VR-IV loop: K258 K309 K313 K321 K490 K507 K527 K532 K544 K549 K556 K620 K640 K649 K665 K688 K692 K706 Surface-exposed lysine within 15 Å of the VR-IV loop: K258 K490 K507 K532 K544 K549 K556 K665 Surface-exposed lysine within 5 angstroms of the VR-IV loop: K549

[0178] Surface-exposed lysine within 30 angstroms of the VR-VIII loop: K309 K490 K507 K527 K532 K544 K549 K556 K620 K640 K688 K706 Surface-exposed lysine within 15 angstroms of the VR-VIII loop: K490 K507 K527 K532 Surface-exposed lysine within 5 angstroms of the VR-VIII loop: K507

[0179] [Table 5]

[0180] [Table 6]

[0181] [Table 7]

[0182] [Table 8]

[0183] [Table 9]

[0184] [Table 10]

[0185] [Table 11]

[0186] [Table 12]

[0187] [Table 13]

[0188] VP1 capsid VR-IV region amino acids 453-454 NTPSGLPQTGTTTQS [SEQ ID NO:36] VP1 capsid VR-VIII region amino acids 587-588 LQRGNGGSGSLPQTGGGSGSRQAAT [SEQ ID NO:37] VP1 capsid VR-VIII region amino acids 587-588 double linker LQRGNGGSGSGGSGSLPQTGGGSGSGGSGSRQAAT [SEQ ID NO:38] Linker sequence 1: GGGGS [SEQ ID NO:39]

[0189] [Table 14]

[0190] [Table 15]

Claims

1. An adeno-associated virus (AAV) capsid protein characterized by having a saltase recognition sequence inserted in one or more of the VP1-VP2 transfer region, VR-I region, VR-IV region, or VR-VIII region.

2. The AAV capsid protein according to claim 1, characterized in that the inserted saltase recognition sequence is a sequence having a general sequence motif selected from the group consisting of Xn-LPXTG-Xm [SEQ ID NO: 10], Xn-NPXTG-Xm [SEQ ID NO: 40], Xn-LPXTA-Xm [SEQ ID NO: 41], Xn-LAXTG-Xm [SEQ ID NO: 42], Xn-LPXAG-Xm [SEQ ID NO: 48], Xn-LPXLG-Xm [SEQ ID NO: 49], Xn-APXTG-Xm [SEQ ID NO: 50], Xn-LPXSG-Xm [SEQ ID NO: 51], Xn-FPXTG-Xm [SEQ ID NO: 52], Xn-XPKTG-Xm [SEQ ID NO: 53], and Xn-LPEXG-Xm [SEQ ID NO: 54] (wherein n and m are in the range of 0 to 25, and X is any natural amino acid).

3. The AAV capsid protein according to claim 1, characterized in that the inserted saltase recognition sequence has a general sequence motif Xn-LPXTG-Xm [SEQ ID NO: 10].

4. The AAV capsid protein according to claim 2, wherein n and / or m are in the range of 0 to 20.

5. The AAV capsid protein according to claim 2, wherein X is glutamine or glutamic acid.

6. The capsid protein according to claim 1, wherein the saltase recognition sequence is inserted into a VP1-VP2 transfer region defined by PVKTAP [SEQ ID NO: 1] in AAV2 or the corresponding amino acid sequence in another AAV serotype, the VR-I region is defined by SSQSGASN [SEQ ID NO: 2] in AAV2 or the corresponding amino acid sequence in another AAV serotype, the VR-IV region is defined by SRTNTPSGTTTQSRLQFSQAGASDIRDQS [SEQ ID NO: 3] in AAV2 or the corresponding amino acid sequence in another AAV serotype, and / or the VR-VIII region is defined by QYGSVSTNLQRGNRQAATADVNTQGV [SEQ ID NO: 4] in AAV2 or the corresponding amino acid sequence in another AAV serotype.

7. The capsid protein according to claim 1, wherein the saltase recognition sequence is inserted into the VR-IV region of a fragment having TPSGTTTQS [SEQ ID NO: 5] in AAV2, and / or the insertion in the VR-VIII region is in a fragment having LQRGNRQAA [SEQ ID NO: 6] in AAV2 or the corresponding amino acid sequence in another AAV serotype.

8. The capsid protein according to claim 1, wherein in the region where the saltase recognition sequence LPXTG [SEQ ID NO: 10] is inserted, one or more amino acids in the said region are deleted or substituted.

9. The capsid protein according to claim 1, wherein the AAV is AAV2 or AAV9.

10. The capsid protein according to claim 2, wherein n is between 5 and 20, preferably between 5 and 15, and more preferably between 10 and 15.

11. The capsid protein according to claim 2, wherein m is between 5 and 20, preferably between 5 and 15, and more preferably between 10 and 15.

12. The capsid protein according to claim 2, wherein the linker sequence Xn or Xm consists of at least 80% glycine, serine, threonine, and alanine.

13. The capsid protein according to claim 2, wherein the linker sequence Xm or Xn consists of an amino acid selected from glycine, serine, threonine, and alanine.

14. The capsid protein according to claim 2, wherein X in LPXTG [SEQ ID NO: 10] is aspartic acid, glutamic acid, asparagine, or glutamine, preferably glutamine in LPXTG [SEQ ID NO: 10].

15. The capsid protein according to claim 1, wherein one or more lysine (K) molecules of the AAV capsid protein are mutated to glycine (Gly), serine (Ser), or alanine (Ala), preferably mutated to glycine (Gly).

16. A nucleic acid encoding a capsid protein according to any one of claims 1 to 15.

17. An expression vector comprising the nucleic acid described in claim 16.

18. AAV particles comprising the AAV capsid protein described in claim 1.

19. A conjugated AAV particle containing an AAV capsid protein, characterized by a residual saltase recognition sequence in the VP1-VP2 transition region, VR-I region, VR-IV region, and / or VR-VIII region, wherein the residual saltase recognition sequence is operably linked to a heterozygous conjugate molecule.

20. The conjugated AAV particle according to claim 19, wherein the ratio of unconjugated capsid AAV protein to conjugated AAV capsid protein is between 1 / 59 and 59 / 1, preferably between 1 / 20 and 20 / 1, preferably between 1 / 10 and 10 / 1, and more preferably between 1 / 5 and 5 / 1.

21. The conjugated AAV particle according to claim 19, operably linked via a residual saltase recognition sequence to a heterozygous conjugate molecule characterized by the presence of a terminal triglycine amino acid sequence.

22. The bonded AAV particle according to claim 19, operably connected to the targeting portion via the remaining saltase recognition sequence.

23. A conjugated AAV particle according to any one of claims 19 to 22, for use as a pharmaceutical.

24. - A step of contacting cells with one or more nucleic acids encoding an AAV capsid protein in which one or more of the VP1-VP2 transfer region, VR-I region, VR-IV region, or VR-VIII region contains a saltase recognition sequence; - A step of having the cells assemble multiple AAV capsid proteins into AAV particles, and recovering the AAV particles from the cells; and - A step of bringing the AAV particles into contact with saltase and heterogeneous compound molecules. A method for producing bonded AAV particles, including

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