Modified adeno-associated virus (AAV) particles for gene therapy

Abstract

The present invention relates to improved adeno-associated virus (AAV) particles for gene delivery and gene therapy. Adeno-associated virus (AAV) particles containing modified capsids are provided. The present invention further relates to methods for producing the improved AAV particles of the present invention by removing native binding sites in the adeno-associated virus (AAV) capsid and introducing ligand-binding sites into the capsid to provide AAV that transduces only specific cells of interest. Additional aspects of the present invention relate to modified AAV particles for use in treating disease, and methods for treating disease, comprising administering the modified AAV particles to a subject in need thereof. Yet a further aspect of the present invention relates to AAV particles of the present invention for cell transfection, for example as a gene delivery tool in basic research.

Description

[Technical Field]

[0001] The present invention relates to improved adeno-associated virus (AAV) particles for gene delivery and gene therapy. Adeno-associated virus (AAV) particles containing modified capsids are provided. The present invention further relates to methods for producing the improved AAV particles of the present invention by removing native binding sites in the adeno-associated virus (AAV) capsid and introducing ligand-binding sites into the capsid to provide AAV that transduces only specific cells of interest. Additional aspects of the present invention relate to modified AAV particles for use in treating disease, and methods for treating disease, comprising administering the modified AAV particles to a subject in need thereof. Yet a further aspect of the present invention relates to AAV particles of the present invention for cell transfection, for example, as a gene delivery tool in basic research. [Background technology]

[0002] Background of the Invention Introducing molecules carrying genetic information into cells is a useful tool in modern medicine and basic research. Preferred methods include the use of gene delivery vehicles derived from viruses, including adenoviruses, retroviruses, vaccinia viruses, and adeno-associated viruses. Among these, adeno-associated viruses (AAVs) are the preferred virus for gene therapy. Wild-type AAVs are relatively small DNA viruses that integrate stably and site-specifically into the genome of infected cells. Importantly, no human diseases have been associated with AAV infection to date. Therefore, adeno-associated viruses (AAVs) have been chosen as gene therapy vectors due to their lack of pathogenicity. Interestingly, over 100 clinical trials using AAV-based vectors are ongoing, and importantly, the first AAV gene therapy, Voretigene neparvovec, for the treatment of inherited retinal diseases was recently approved by the FDA.

[0003] Adeno-associated viruses are members of the Dependovirus genus of the Parvoviridae family, which contain a non-enveloped icosahedral capsid. The AAV genome is approximately 4.7 kilobases long and consists of linear, single-stranded deoxyribonucleic acid (ssDNA), which can be either positive- or negative-sense. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs): rep and cap. The rep frame consists of four overlapping genes encoding the nonstructural replication (Rep) proteins required for the AAV life cycle. The cap frame contains overlapping nucleotide sequences of the structural VP capsid proteins: VP1, VP2, and VP3, which interact together to form a capsid with icosahedral symmetry. The terminal 145 nt are self-complementary and organize to allow the formation of an energetically stable intramolecular duplex that forms a T-shaped hairpin. These hairpin structures serve as origins for viral DNA replication and as primers for the cellular DNA polymerase complex. After AAV infection in mammalian cells, the rep genes (i.e., Rep78 and Rep52) are expressed from the P5 and P19 promoters, respectively, and both Rep proteins function in the replication of the viral genome. Splicing events in the rep ORF result in the expression of four Rep proteins (i.e., Rep78, Rep68, Rep52, and Rep40).

[0004] Generally, viruses can enter cells in various ways, either through direct membrane fusion / permeabilization at the plasma membrane or through endocytosis followed by similar cell membrane breaching in early endosomes. The most common form of viral internalization is via clathrin-mediated endocytosis. This process can be divided into several steps, including: (1) nucleation of clathrin-coated pits, (2) cargo capture in the coated pits, (3) induction of curvature and membrane invagination; and (4) vesicle scission and uncoating. The vesicles deliver their viral contents to early endosomes. Interestingly, the entire process of endocytosis occurs in vivo within a matter of seconds. Examples of viruses internalized by clathrin-mediated endocytosis are dsDNA viruses such as Adenoviridae, adenovirus type 2, adenovirus type 5, adenovirus type 8, adenovirus type 37, canine adenovirus type 2 (CAV-2), adeno-associated virus 2, and adeno-associated virus 5 (Dependvirus).

[0005] Viruses can also be internalized via caveolae, specialized lipid rafts that form 50-70 nm flask-shaped invaginations of the plasma membrane. Caveolin forms the structural backbone of caveolae. Internalization through caveolae is not a constitutive process but occurs only upon cellular stimulation. Viruses that bind to their host cell receptors and are internalized are delivered to early endosomes. Caveolae are a low-volume but highly regulated pathway. Furthermore, some viruses are internalized using several routes that do not use clathrin or caveolin coats and, in some cases, are hijacked by bacteria and viruses to reach host cells. These routes can be further defined by their dependence on various molecules, such as cholesterol, DNM2 / dynamin 2, or small GTPases or tyrosine kinases.

[0006] AAV vectors are being explored as vehicles for targeted gene therapy because they stably and site-specifically integrate into the genome of infected cells and are non-pathogenic.

[0007] Kern et al. (Identification of a Heparin-Binding Motif on Adeno-Associated Vims Type 2 Capsids, Journal of Virology Sep 2003, 77 (20) 11072-11081) disclose that infection of cells by adeno-associated virus (AAV) type 2 (AAV-2) is mediated by binding to heparan sulfate proteoglycans and can be competed for by heparin. Mutational analysis of the AAV-2 capsid protein showed that a group of basic amino acids (arginines 484, 487, 585, and 588, and lysine 532) contributes to heparin binding to HeLa cells. These amino acids are arranged in three clusters in the tripartite spike region of the AAV-2 capsid. Tissue distribution of recombinant AAV-2 mutated at R484 and R585 in mice showed that liver infection was significantly reduced compared with infection with wild-type recombinant AAV, whereas heart infection persisted. They suggested that heparin binding may affect AAV-2 infectivity but is not essential.

[0008] US 5,756,283 (Patent Document 1) discloses a recombinant AAV vector containing a selected transgene under the control of a regulatory sequence, in which the infected cells are contacted with an agent that facilitates conversion of the single-stranded recombinant virus to its double-stranded form, thereby increasing the transduction efficiency of the recombinant AAV into the target cells.

[0009] EP 1664314 B1 (Patent Document 2) describes recombinant AAV vectors carrying capsid protein modifications that result in reduced or eliminated heparin-binding function, based on the identification of capsid protein domains and corresponding amino acid residues involved in heparin binding.

[0010] WO 2017 / 143100 A1 (Patent Document 3) describes methods for modifying AAV capsid polypeptides to exhibit enhanced neutralization profiles, increased transduction and / or tropism in human liver tissue or hepatocytes compared to the non-mutated parent capsid polypeptide.

[0011] The safety and efficacy of human gene therapy remain a major topic of debate. Problems with current vectors include unintended transduction of certain tissues and adverse immune responses. Current approaches using AAV particles for gene delivery suffer from a lack of efficient transduction; that is, to be effective, very high titers of AAV vectors are usually required. Another limitation of current approaches is that many AAV vectors are ineffective at transducing certain cell types, and AAV vectors can have off-target effects due to transduction of inappropriate cell types.

[0012] In view of the above limitations, the underlying objective of the present invention is to provide improved adeno-associated virus (AAV) particles for gene delivery. This is achieved by engineering an adeno-associated virus (AAV) capsid that can transduce specific cells of interest and / or be delivered at a lower titer and still be effective. The above problems are solved by modifying the viral capsid to accept ligand binding and attaching a ligand of interest to the capsid. Optionally, the native binding site in the AAV capsid can be removed before modifying the virus to accept the ligand. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] US 5,756,283 [Patent Document 2] EP 1664314 B1 [Patent Document 3] WO 2017 / 143100 A1 [Non-patent literature]

[0014] [Non-Patent Document 1] Identification of a Heparin-Binding Motif on Adeno-Associated Vims Type 2 Capsids, Journal of Virology Sep 2003, 77 (20) 11072-11081 Summary of the Invention

[0015] According to a first aspect of the present invention, the above object is achieved by providing an adeno-associated virus (AAV) particle comprising a modified capsid, wherein the modified capsid comprises at least one modification selected from removal of a native binding site in the capsid and introduction of a ligand binding site into the capsid.

[0016] The terms "adeno-associated viral vector," "AAV vector," "adeno-associated virus," "AAV virus," "AAV virion," "AAV viral particle," and "AAV particle" are used interchangeably herein and refer to a viral particle composed of at least one AAV capsid protein and a recombinant viral genome enclosed in the capsid. An AAV particle containing a recombinant viral genome having a heterologous polynucleotide to be delivered to a mammalian cell, flanked by AAV inverted terminal repeats, and a transcriptional regulatory region including a promoter, is typically referred to as an "AAV vector particle" or "AAV vector."

[0017] In a preferred embodiment, the adeno-associated virus (AAV) particle of the present invention is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. To date, the most commonly used gene transfer systems are derivatives of viruses, such as adeno-associated virus type 2 (AAV-2). AAV-2 and / or AAV-9 are preferred.

[0018] Currently, over 100 AAV serotypes have been identified, which differ in the ability of their capsid proteins to bind to specific cell surface receptors and can transduce different cell types. AAV2 was the first serotype cloned into a bacterial plasmid and has since been used as a comparison for identifying other serotypes. Twelve serotypes (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12) have been thoroughly tested for their ability to transduce specific cell types, and distinctions have been made between capsid protein motifs that bind to specific cell surface receptors for cell attachment. In the context of the present invention, AAV particles selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12 are preferred. However, it should be understood that any other AAV particles can be used in the context of the present invention.

[0019] The AAV capsid is composed of three overlapping capsid proteins (VP1, VP2, VP3) that contain a unique VP1 N-terminus, a VP1 / VP2 consensus region, and a region common to VP1, VP2, and VP3.

[0020] More preferred is the adeno-associated virus (AAV) particle of the present invention, in which at least one protein in the capsid of the AAV particle is modified, preferably at least one protein is VP1, VP2, and / or VP3. Alternatively, two of the proteins VP1, VP2, and / or VP3 in the capsid are modified, or all three of the proteins VP1, VP2, and VP3 in the capsid are modified. Preferably, at least one portion, for example, one amino acid, of at least one of the proteins to be modified in the capsid is modified. However, it is also possible to modify multiple portions, for example, multiple amino acids, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of portions or amino acids, of the proteins VP1, VP2, and VP3 in the capsid. Preferably, at least one of arginines 484, 487, 585, and 588, and lysine 532 of VP1, and / or the analogous arginines of VP2 or VP3, is removed by substituting them with a different amino acid, such as alanine.

[0021] It is further preferred that the capsid modification comprises both the removal of at least one native binding site and the introduction of at least one ligand-binding site. Alternatively, at least one native binding site of the AAV may be unchanged, i.e., not removed, but at least one ligand-binding site is introduced.

[0022] If the native binding site in the capsid is present and not removed, and at least one additional ligand binding site is introduced into the capsid by modification, the AAV particles of the present invention have a higher infectivity rate at a lower titer of viral particles used. Conversely, if the native binding site in the capsid is removed before introducing at least one additional ligand binding site into the capsid by modification, the tropism of the AAV particles of the present invention is altered.

[0023] Preferred are adeno-associated viruses (AAV) of the invention, wherein the native binding site is a binding site that allows binding to heparan sulfate proteoglycans, preferably removed by substituting at least one of arginine 585 or arginine 588 of VP1 and / or the analogous arginines of VP2 or VP3 with a different amino acid, such as alanine.

[0024] More preferably, the adeno-associated virus (AAV) of the present invention has a ligand-binding site that allows covalent binding of a ligand. More preferably, the ligand-binding site comprises a benzylguanine group, a benzylcytosine group, a chloroalkane group, a dibenzocyclooctyne group, an azide group, a phosphine, or a combination thereof. Alternatively, any haloalkane group can be used. More preferably, the benzylguanine or other group is bound to an available lysine residue. The introduced ligand-binding site is preferably bound to the ε-amino group or primary amine of the available lysine residue.

[0025] Further preferred are adeno-associated virus (AAV) particles of the present invention further comprising a ligand, in particular a HaloTag™, a SNAP-tag™, or a CLIP-tag™, or a phosphine or an azide or a dibenzocyclooctyne group, bound to the benzylguanine group, the benzylcytosine group, the chloroalkane group, the dibenzocyclooctyne group, the azide group, and / or the phosphine. The present invention preferably utilizes tags that can bind to their specific ligands with high affinity, such as SNAP-tag, CLIP-tag, Halo Tag, Lumio Tag, and others.

[0026] Benzylguanine, benzylcytosine, and chloroalkanes are recognized by "suicide" enzymes such as SNAP. In the context of the present invention, benzylguanine or benzylcytosine derivatives can also be used. Benzylguanine or benzylcytosine derivatives are understood to mean modified benzylguanine or benzylcytosine groups that are still recognized by suicide enzymes.

[0027] The tag molecule can be any molecule or biomolecule that can specifically bind to another molecule. Examples can include SNAP-tag, CLIP-tag, Lumio-tag, or Halo-tag. For example, the affinity tag can be SNAP-tag, which is a mutant of alkylguanine-DNA alkyltransferase. Importantly, one of the substrates of SNAP-tag is benzylguanine. Commercially available products useful for the present invention include, for example, HaloTag from Promega, LumioTag from Life Technologies, and SNAP / CLIP Tag from NEB.

[0028] Those skilled in the art are well aware of other methods for binding ligands to capsids, such as anti-tag antibodies, streptavidin-biotin, or chemical crosslinking. Any known method can be used in the context of the present invention. For example, unnatural amino acids can be incorporated into both the capsid and the ligand. Then, the ligand can be covalently linked to the capsid using a crosslinker, such as a bioorthogonal crosslinker. In another example, a phosphine or dibenzocyclooctyne group can be incorporated into the capsid, and an azide can be incorporated into the ligand, or vice versa. Then, the ligand can be covalently linked to the capsid by Staudinger reaction or strain-promoted click reaction.

[0029] Any kind of ligand can be bound to said ligand binding site.Preferably, the ligand to be bound is selected from protein ligand such as growth factor or cytokine; toxin subunit such as cholera toxin B subunit; lectin such as isolectin B4 or wheat germ agglutinin; adhesion factor such as lactadherin; antibody such as anti-CD-34 antibody; peptide such as deltorphin opioid receptor ligand; and gene editing nuclease such as Cas9.

[0030] The nucleic acid molecule packaged in the adeno-associated virus (AAV) particle of the present invention can be any type of nucleic acid molecule. Preferably, the nucleic acid molecule is a nucleic acid molecule encoding an intracellular antibody (e.g., for neutralizing a specific protein in the cell), a nucleic acid molecule encoding a peptide toxin (e.g., for blocking an ion channel in a pain pathway), a nucleic acid molecule encoding an optogenetic actuator (e.g., for turning on or off neuronal activity using light), a nucleic acid molecule encoding a pharmacogenetic tool (e.g., for turning on or off neuronal signaling using a chemical ligand without interfering pharmacological effects), a nucleic acid molecule encoding a CRISPR-based editor for precise gene editing, a nucleic acid molecule encoding a CRISPR-epigenetic tool for regulating gene expression, and / or a nucleic acid molecule encoding a suicide gene for inducing cell death.

[0031] Preferably, when the ligand is a gene-editing nuclease such as Cas9, the adeno-associated virus (AAV) particles of the invention further carry as cargo a nucleic acid molecule such as a gRNA and / or specific DNA to be inserted into the host genome.

[0032] Those skilled in the art are aware of other gene editing nucleases other than Cas9, such as Cpfl, TALEN, ZFN or homing endonucleases.In addition, it can be convenient to use DNA guided Argonaute interference system (DAIS) to operate.Basically, the Argonaute (Ago) protein is heterologously expressed from a polynucleotide introduced into the cell in the presence of at least one exogenous oligonucleotide (DNA guide) that provides the Ago protein with the specificity of cleavage at a preselected locus.TALEN and Cas9 systems are described in WO 2013 / 176915 and WO 2014 / 191128, respectively. Zinc finger nucleases (ZFNs) were first described by Kim, YG; Cha, J.; Chandrasegaran, S. ("Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain" (1996). Proc Natl Acad Sci USA 93 (3): 1156-60). Cpf1 is a Class 2 CRISPR-Cas system described by Zhang et al. (Cpf1 is a single RNA-guided endonuclease of a Class 2 CRIPR-Cas System. (2015). Cell;163:759-771). The Argonaute (AGO) gene family was first described in Guo S, Kemphues KJ. (Par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser / Thr kinase that is asymmetrically distributed. (1995). Cell;81(4):611-20).

[0033] Another aspect of the present invention then relates to AAV particles according to the invention for use in the treatment of diseases.

[0034] Any kind of disease can be treated or prevented by the AAV particles for use according to the present invention. Preferably, the disease to be treated or prevented by the AAV particles for use according to the present invention is a disease that can be treated by gene therapy, for example, cancer, inherited monogenic diseases such as hereditary retinal diseases, hereditary skin diseases such as Olmsted syndrome or familial primary localized cutaneous amyloidosis, infectious diseases, adrenoleukodystrophy, α-1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten disease, Becker muscular dystrophy, β-thalassemia, Canavan disease, chronic granulomatous disease, Crigler-Najjar syndrome, Cystic fibrosis, Duchenne muscular dystrophy, Fabry disease, familial adenomatous polyposis, familial hypercholesterolemia, familial lecithin-cholesterol acyltransferase deficiency, Fanconi anemia, galactosialidosis, Gaucher disease, gyrate atrophy, hemophilia A, hemophilia B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's chorea, junctional epidermolysis bullosa, late-onset childhood neuronal ceroid lipofuscinosis, leukocyte adhesion deficiency, limb-girdle muscular dystrophy, and lipoprotein lipase deficiency , metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, ornithine transcarbamylase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, recessive dystrophic epidermolysis bullosa, Sanfilippo A (mucopolysaccharidosis type IIIA), Sanfilippo B (mucopolysaccharidosis type IIIB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, Tay-Sachs disease, Wiskott-Aldrich syndrome, von Gierke disease (glycogenosis type Ia), X-linked tubular myopathy, anemia of end-stage renal failure, angina pectoris (stable, unstable, refractory), coronary artery stenosis, critical limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcer, adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria, pediatric respiratory disease, respiratory syncytial virus, tetanus, tuberculosis, gynecological cancer, breast cancer, ovarian cancer, cervical cancer, vulvar cancer, nervous system cancer, glioblastoma, leptomeningeal carcinomatosis, glioma, astrocytoma, neuroblastoma, retinoblastoma,Digestive system cancer, colon cancer, colorectal cancer, liver metastasis, post-hepatitis liver cancer, pancreatic cancer, gallbladder cancer, hepatocellular carcinoma, genitourinary cancer, prostate cancer, kidney cancer, bladder cancer, anogenital neoplasms, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal cancer, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell carcinoma / non-small cell carcinoma, mesothelioma, blood cancer, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell carcinoma, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis, carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonal neuropathy, late-onset childhood These conditions include neuronal ceroid lipofuscinosis, multiple sclerosis, myasthenia gravis, pain, Parkinson's disease, peripheral neuropathy, spinal muscular atrophy type 2, color vision disorders, age-related macular degeneration, choroideremia, diabetic macular edema, glaucoma, Leber's congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, punctate corneal opacities, X-linked retinoschisis, arthritis (rheumatic, inflammatory, degenerative), degenerative joint disease, severe inflammatory rectal disease, ulcerative colitis, chronic kidney disease, diabetic ulcers / foot ulcers, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft-versus-host disease / transplant patients, oral mucositis, parotid salivary gland hypofunction, systemic sclerosis (scleoderma), type 1 diabetes, and / or wound healing.

[0035] As used herein, the term "preventing and / or inhibiting" is intended to include treating an existing disease. Treating, preventing, and / or inhibiting are intended to include, for example, treating, slowing, or alleviating disease progression, alleviating the symptoms of a disease or condition, or curing a disease or condition. An "effective amount" is an amount of AAV particles of the invention that alleviates the symptoms seen in the disease being treated, such as any of the diseases listed above. Alleviating is intended to include, for example, preventing, treating, alleviating the symptoms, or curing the disease or condition. The present invention also includes methods for treating a subject at risk for the development and / or progression of a disease, wherein a therapeutically effective amount of the AAV particles of the invention is administered to the patient. Being at risk for a disease can be due, for example, to phenotypic symptoms that predispose to the disease. As used herein, the terms "prevention" or "preventing" when used in reference to a subject refer to arresting, hindering, and / or delaying the occurrence or development of a disease, particularly symptoms associated with the disease.

[0036] Yet another aspect of the invention relates to the AAV particles described above for use in the treatment of disease, wherein the AAV is administered to a subject in liquid, dry, or semi-solid form, such as in the form of tablets, coated tablets, effervescent tablets, capsules, powders, granules, dragees, troches, pills, ampoules, drops, suppositories, emulsions, ointments, gels, tinctures, pastes, creams, compresses, mouthwashes, plant saps, nasal preparations, inhalation mixtures, aerosols, mouthwashes, mouth sprays, nasal sprays, or room sprays.

[0037] Another aspect of the present invention then relates to a method for producing improved adeno-associated virus (AAV) particles, comprising the step of introducing at least one modification into the capsid of the AAV, preferably wherein the modification comprises introducing at least one ligand binding site into the capsid, optionally wherein a native binding site in the capsid is removed, e.g., previously removed.

[0038] As described above, if the native binding site in the capsid is present and not removed, and at least one additional ligand binding site is introduced into the capsid by modification, the adeno-associated virus (AAV) particles of the present invention have a higher infectivity rate than a lower titer of virus particles used. Conversely, if the native binding site in the capsid is removed before introducing at least one additional ligand binding site into the capsid by modification, the tropism of the adeno-associated virus (AAV) particles of the present invention is altered.

[0039] The adeno-associated virus (AAV) particles produced by the above methods are preferably selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and mixtures thereof.

[0040] Any of the proteins of the adeno-associated virus (AAV) particles to be produced by the above method can be modified. Preferably, in the above method, at least one of the proteins VP1, VP2, or VP3 in the capsid is modified. Alternatively, two of the proteins VP1, VP2, and / or VP3 in the capsid are modified, or all three of the proteins VP1, VP2, and VP3 in the capsid are modified. Preferably, at least one portion, for example, one amino acid, of at least one of the proteins to be modified in the capsid is modified. However, it is also possible to modify multiple portions, for example, multiple amino acids, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any other number of portions or amino acids, of the proteins VP1, VP2, and VP3 in the capsid. Preferably, at least one of arginines 484, 487, 585, and 588, and lysine 532 of VP1, and / or the analogous arginines of VP2 or VP3, is removed by substituting them with a different amino acid, such as alanine.

[0041] Proteins such as VP1, VP2, or VP3 can be chemically modified by reacting specific amino acids. Examples of such modifications are well known in the art and are summarized, for example, in R. Lundblad, Chemical Reagents for Protein Modification, 3rd edition, CRC Press, 2005, which is incorporated herein by reference. Chemical modifications of amino acids include, but are not limited to, acylation, amidination, pyridoxylation of lysine, reductive alkylation, trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS), amide modification of carboxyl groups, and sulfhydryl modification of cysteine ​​to cysteic acid by performic acid oxidation, formation of mercury derivatives, formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and carbamoylation with cyanic acid at alkaline pH. In this regard, those skilled in the art are referred to Chapter 15 of Current Protocols in Protein Science, Eds. Coligan et al. (John Wiley & Sons NY 1995-2000) for a more extensive methodology regarding the chemical modification of proteins.

[0042] Preferably, the above method for generating improved adeno-associated virus (AAV) particles comprises both removal of native binding sites and introduction of ligand-binding sites. Alternatively, the native binding sites of the AAV may remain unchanged, i.e., not removed, but at least one ligand-binding site is introduced.

[0043] In a preferred embodiment, the native binding sites are removed by the above-described method for generating improved adeno-associated virus (AAV) particles, wherein the native binding sites are native binding sites that allow binding to heparan sulfate proteoglycans, preferably by substituting at least one of arginines 585 and 588 of VP1 and / or the analogous arginines of VP2 or VP3 with a different amino acid, such as alanine.

[0044] The introduced ligand binding site allows for covalent attachment of a ligand and is preferably selected from benzylguanine groups attached to available lysine residues, more preferably by reacting the capsid with benzylguanine N-hydroxysuccinimide (BG-NHS) and / or benzylcytosine N-hydroxysuccinimide (BC-NHS).

[0045] The present invention preferably utilizes tags capable of binding to their specific ligands with high affinity, such as SNAP-tag, CLIP-tag, Halo-tag, Lumio-tag, and others. The tag molecule introduced in the above method can be any molecule or biomolecule capable of specifically binding to an additional molecule. Examples include SNAP-tag, CLIP-tag, Lumio-tag, or Halo-tag. For example, the affinity tag can be SNAP-tag, a mutant of alkylguanine-DNA alkyltransferase. Importantly, one of the substrates of SNAP-tag is benzylguanine. Commercially available products useful in the present invention include, for example, HaloTag from Promega, LumioTag from Life Technologies, and SNAP / CLIP Tag from NEB. The introduced ligand binding site is preferably linked to the ε-amino group or primary amine of the available lysine residue.

[0046] Therefore, further preferred is the above method for producing improved adeno-associated virus (AAV) particles, further comprising the step of attaching a ligand, in particular HaloTag™, SNAP-tag™, or CLIP-tag™, to the benzylguanine and / or benzylcytosine groups.

[0047] The ligand to be bound can be any type of ligand, but is preferably selected from protein ligands such as growth factors or cytokines; toxin subunits such as cholera toxin B subunit; lectins such as isolectin B4 or wheat germ agglutinin; adhesion factors such as lactadherin; antibodies such as anti-CD-34 antibodies; peptides such as deltorphin opioid receptor ligands; and gene editing nucleases such as Cas9.

[0048] A further aspect of the present invention then relates to a method for treating a disease that can be treated by gene therapy, comprising the step of administering AAV particles according to the present invention to a subject in need thereof.

[0049] In the context of the present invention, the term "subject", when used in certain embodiments, preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human. The term "patient" preferably refers to a mammal, e.g., a human patient, such as a mouse, rat, guinea pig, rabbit, horse, cattle, cow, cat, dog, monkey, or preferably a human, for whom diagnosis, prognosis, or treatment is desired. A subject of the present invention may be at risk of contracting a disease, such as a bacterial infection, a viral infection, a fungal infection, or a parasitic infection. A more detailed description of medical indications relevant in the context of the present invention is provided elsewhere herein.

[0050] The cells and / or subjects treated with the AAV particles of the present invention are preferably of mammalian origin, such as human origin.Nevertheless, the present invention can be advantageously used in veterinary medicine, cell culture procedures, or even plant cell diseases, depending on the similarity of the cell entry mechanism.Preferably, the cells treated are mammalian cells, prokaryotic cells, or plant cells.Most preferably, the cells treated are human cells.

[0051] Yet another aspect of the present invention relates to the above-described method for treating a disease, comprising administering AAV particles according to the present invention to a subject in need thereof, wherein the AAV particles are administered to the subject in liquid, dry, or semi-solid form, such as tablets, coated tablets, effervescent tablets, capsules, powders, granules, dragees, lozenges, pills, ampoules, drops, suppositories, emulsions, ointments, gels, tinctures, pastes, creams, compresses, mouthwashes, plant sap, nasal preparations, inhalation mixtures, aerosols, mouthwashes, mouth sprays, nasal sprays, or room sprays.

[0052] The diseases treated by the above-described methods for treating a disease, comprising the step of administering AAV particles to a subject, are preferably cancer, inherited monogenic diseases such as inherited retinal diseases, inherited skin diseases such as Olmsted syndrome or familial primary localized cutaneous amyloidosis, infectious diseases, adrenoleukodystrophy, alpha-1 antitrypsin deficiency, aromatic L-amino acid deficiency, Batten disease, Becker muscular dystrophy, beta thalassemia, Canavan disease, chronic granulomatous disease, Crigler-Najjar syndrome, cystic fibrosis, Duchenne muscular dystrophy, Fabry's disease, and the like. disease, familial adenomatous polyposis, familial hypercholesterolemia, familial lecithin-cholesterol acyltransferase deficiency, Fanconi anemia, galactosialidosis, Gaucher disease, gyrate atrophy, hemophilia A and B, Hurler syndrome (mucopolysaccharidosis type I), Hunter syndrome (mucopolysaccharidosis type II), Huntington's chorea, junctional epidermolysis bullosa, late-onset childhood neuronal ceroid lipofuscinosis, leukocyte adhesion deficiency, limb-girdle muscular dystrophy, lipoprotein lipase deficiency, metachromatic leukodystrophy, Sly syndrome (mucopolysaccharidosis type VII), Netherton syndrome, Oncogene Lunitine transcarbamylase deficiency, Pompe disease, purine nucleoside phosphorylase deficiency, recessive dystrophic epidermolysis bullosa, Sanfilippo A (mucopolysaccharidosis type IIIA), Sanfilippo B (mucopolysaccharidosis type IIIB), sickle cell disease, severe combined immunodeficiency, spinal muscular atrophy, Tay-Sachs disease, Wiskott-Aldrich syndrome, von Gierke disease (glycogenosis type Ia), X-linked myotubular myopathy, anemia of end-stage renal failure, angina pectoris (stable, unstable, refractory), coronary artery stenosis, critical limb ischemia, heart failure, intermittent claudication, myocardial ischemia, peripheral vascular disease, pulmonary hypertension, venous ulcer , adenovirus infection, cytomegalovirus infection, Epstein-Barr virus infection, hepatitis B infection, hepatitis C infection, HIV / AIDS, influenza, Japanese encephalitis, malaria, pediatric respiratory diseases, respiratory syncytial virus, tetanus, tuberculosis, gynecological cancer, breast cancer, ovarian cancer, cervical cancer, vulvar cancer, nervous system cancer, glioblastoma, leptomeningeal carcinomatosis, glioma, astrocytoma, neuroblastoma, retinoblastoma, digestive cancer, colon cancer, colorectal cancer, liver metastasis, post-hepatitis liver cancer, pancreatic cancer, gallbladder cancer, hepatocellular carcinoma, genitourinary cancer, prostate cancer, kidney cancer, bladder cancer,Anogenital neoplasms, skin cancer, melanoma (malignant / metastatic), head and neck cancer, nasopharyngeal cancer, squamous cell carcinoma, esophageal cancer, lung cancer, adenocarcinoma, small cell carcinoma / non-small cell carcinoma, mesothelioma, blood cancer, leukemia, lymphoma, multiple myeloma, sarcoma, germ cell carcinoma, Li-Fraumeni syndrome, thyroid cancer, Alzheimer's disease, amyotrophic lateral sclerosis, carpal tunnel syndrome, chronic traumatic brain injury, cubital tunnel syndrome, diabetic neuropathy, epilepsy, giant axonal neuropathy, late-onset childhood neuronal ceroid lipofuscinosis, multiple sclerosis, myasthenia gravis, pain, Parkinson's disease, peripheral neuropathy, spinal muscular atrophy type 2 The disease is selected from the group consisting of color vision deficiency, age-related macular degeneration, choroideremia, diabetic macular edema, glaucoma, Leber's congenital amaurosis, macular telangiectasia type 2, retinitis pigmentosa, punctate corneal opacities, X-linked retinoschisis, arthritis (rheumatic, inflammatory, degenerative), degenerative joint disease, severe inflammatory rectal disease, ulcerative colitis, chronic kidney disease, diabetic ulcers / foot ulcers, detrusor overactivity, erectile dysfunction, fractures, hearing loss, hereditary inclusion body myopathy, graft-versus-host disease / transplant patients, oral mucositis, parotid salivary gland hypofunction, systemic sclerosis, type 1 diabetes mellitus, and wound healing, or a combination thereof.

[0053] Further preferred is a method for treating a disease comprising administering AAV particles according to the present invention to a subject in need thereof, wherein the AAV particles are administered to the subject or cells in the form of a pharmaceutical composition, e.g., in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer. Preferably, the composition is administered to the subject in the form of a spray, coating, foam, lotion, gel, mouthwash, oral formulation, or injection. The composition can be administered to the subject systemically, orally, or by any other clinically / medically acceptable method.

[0054] A further aspect of the present invention then relates to a pharmaceutical composition comprising the AAV particles according to the present invention together with at least one pharmaceutically acceptable carrier and / or diluent, i.e., in combination with a pharmaceutically acceptable additive, carrier, diluent, solvent, filter, lubricant, excipient, binder, or stabilizer. Preferably, the composition is administered to the subject in the form of a spray, coating, foam, lotion, gel, mouthwash, oral formulation, or injection. The composition can be administered to the subject systemically, orally, or by any other clinically / medically acceptable method.

[0055] A further aspect of the present invention then relates to a kit comprising: a) AAV particles as disclosed and / or for use according to the invention, or pharmaceutical compositions comprising AAV particles as disclosed according to the invention; b) written instructions for applying the AAV particles or the pharmaceutical composition to the target; and Optionally, a container holding the AAV particles or composition and written instructions for use.

[0056] Another aspect of the present invention relates to the use of the above kit for preventing, treating and / or inhibiting a viral infection in a subject in need of said treatment.

[0057] Yet another aspect of the present invention relates to AAV particles according to the present invention for use in cell transfection, for example as a gene delivery tool in research. The use may also be for cosmetic purposes, and the present invention includes methods for cosmetic treatment similar to the medical treatments disclosed herein. For this purpose, administering AAV particles according to the present invention to a subject or cell can also be achieved in the form of a cosmetic composition, for example, in combination with cosmetically safe and acceptable additives, carriers, diluents, solvents, filters, lubricants, excipients, binders, or stabilizers. Preferably, the composition is administered to the subject in the form of a spray, coating, foam, lotion, gel, mouthwash, oral formulation, or injection. The composition can be administered to the subject systemically, orally, or by any other clinically / cosmetically acceptable method.

[0058] Those skilled in the art are aware of methods using AAV-derived vectors for gene transfer in vitro and in vivo, such as those described in WO 93 / 09239, US4797368, US5139941, and EP 488 528.

[0059] An additional aspect of the present invention relates to a kit comprising: a) AAV particles for transfection of cells; b) written instructions for using the AAV particles for transfection of cells; and Optionally, a container holding the AAV particles and written instructions.

[0060] Preferred features of each aspect of the present invention apply mutatis mutandis to each of the other aspects. Prior art documents referred to herein are incorporated to the fullest extent permitted by law. Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

[0061] [The present invention 1001] An adeno-associated virus (AAV) particle comprising a modified capsid, wherein the modified capsid comprises at least one modification selected from the removal of a native binding site in the capsid and the introduction of a ligand binding site into the capsid. [The present invention 1002] 1001. The AAV particle of the present invention, wherein the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12. [The present invention 1003] The AAV particle of the present invention 1001 or 1002, wherein at least one protein in the capsid is modified, and preferably, said at least one protein is VP1, VP2, and / or VP3. [The present invention 1004] The AAV particle of any of claims 1001 to 1003, wherein the modification of the capsid comprises removal of at least one natural binding site and introduction of at least one ligand binding site. [The present invention 1005] Any of the AAV particles of the present inventions 1001 to 1004, wherein the native binding sites that allow binding to heparan sulfate proteoglycans are preferably removed by substituting at least one of arginine 585 and arginine 588 of VP1 and / or the analogous arginines of VP2 or VP3 with a different amino acid, such as alanine. [The present invention 1006] Any of the AAV particles of the present inventions 1001 to 1005, wherein the ligand binding site is a ligand binding site suitable for covalent binding of a ligand, and is preferably selected from a benzylguanine group, a benzylcytosine group, a chloroalkane group, an azide group, a dibenzocyclooctyne group, a phosphine, or a combination thereof. [The present invention 1007] The AAV particle of the present invention 1006 further comprising a ligand bound to the benzylguanine group, the benzylcytosine group, the chloroalkane group, the azide group, the dibenzocyclooctyne group, and / or the phosphine, in particular HaloTag™, SNAP-tag™, or CLIP-tag™, an azide, a dibenzocyclooctyne group, or a phosphine. [The present invention 1008] The AAV particle of the present invention 1006 or 1007, wherein the ligand is selected from a protein ligand, a toxin subunit, a lectin, an adhesion factor, an antibody, a peptide, and a gene-editing nuclease. [The present invention 1009] A method for producing improved adeno-associated virus (AAV) particles, the method comprising introducing at least one modification into the capsid of the AAV, wherein a native binding site in the capsid is removed and / or at least one ligand binding site is introduced into the capsid. [The present invention 1010] The method of the present invention 1009, wherein the native binding site is a native binding site that allows binding to heparan sulfate proteoglycans, preferably removed by substituting at least one of arginine 585 and arginine 588 of VP1 and / or the analogous arginines of VP2 or VP3 with a different amino acid, such as alanine. [The present invention 1011] 10. The method of claim 1009 or 1010, wherein the ligand binding site is a ligand binding site suitable for covalent attachment of a ligand, and is preferably selected from a benzylguanine group, a benzylcytosine group, a chloroalkane group, an azide group, a dibenzocyclooctyne group, a phosphine, or a combination thereof. [The present invention 1012] A pharmaceutical composition comprising any of the AAV particles of the present inventions 1001 to 1008, together with at least one pharmaceutically acceptable carrier and / or diluent. [The present invention 1013] An AAV particle of any of claims 1001 to 1008, or a pharmaceutical composition of claim 1012, for use in the treatment of a disease, preferably a disease that can be treated by gene therapy, such as cancer, a hereditary monogenic disease, a hereditary skin disease, an infectious disease, type I diabetes, and wound healing. [The present invention 1014] A method for treating a disease that can be treated by gene therapy, comprising the step of administering any of the AAV particles of the present inventions 1001 to 1008 or the pharmaceutical composition of the present invention 1012 to a subject in need thereof, wherein the disease is preferably a disease that can be treated by gene therapy, such as cancer, a hereditary monogenic disease, a hereditary skin disease, an infectious disease, type I diabetes, and wound healing. [The present invention 1015] Use of any of the AAV particles of the present invention 1001 to 1008 for transfection of cells, for example as a gene delivery tool. [The present invention 1016] Use of the AAV particles of the present invention for cosmetic purposes. The present invention is further illustrated in the following examples and figures, which are provided for illustrative purposes only and are not intended to limit the invention in any way, for purposes of the present invention, all cited documents are incorporated herein by reference in their entirety. [Brief explanation of the drawings]

[0062] [Figure 1] A schematic diagram of a SNAP-tagged ligand with a BG-modified virus is shown. [Figure 2] This shows that the ΔHSPG viral particles according to the invention no longer have infectious activity (dark photograph); this construct was tested in sensory neurons of a fluorescent reporter mouse model. The inset shows a phase-contrast microscopy image of the cells. [Figure 3] As in Figure 2, wheat germ agglutinin (WGA) fusions fully restored viral transduction efficiency to 100% when tested in sensory neurons of a fluorescent reporter mouse model (fluorescent cells). [Figure 4] The neurotrophic factors NGF (A), NT3 (B), and BDNF (C) deliver the virus to different neuronal populations. The inset shows a microscopic image of the cells. This construct was tested in sensory neurons of a fluorescent reporter mouse model, as in Figure 2. [Figure 5] This shows that cholera toxin B subunit transported virus retrogradely to neuronal cell bodies when injected into the skin. The inset shows a microscopic image of the cells. This construct was tested in sensory neurons in a fluorescent reporter mouse model, as in Figure 2. [Figure 6] Shown is trigeminal ganglion sensory neuron tissue (A) 3 weeks after injection with virus carrying NGF ligand IV according to the invention, stained with an antibody against TrkA (receptor for NGF) (B). At least 80% overlap can be seen (C). [Figure 7] Shown is staining of a section from Figure 6 with antibodies against NF200 and IB4, which primarily label other neurons (mechanoreceptors (green / gray) and nonpeptidergic nociceptors (blue / dark gray) respectively). Infected cells in red (light gray) are largely distinct from the green and blue cells. [Figure 8]This shows that gene delivery is more efficient when using ligand-conjugated viruses. WGA-modified constructs strongly increased delivery (B). A) Standard AAV9 variant PHP.S; B) WGA-modified PHP.S variant of A). DETAILED DESCRIPTION OF THE INVENTION [Example]

[0063] The goal of the experiments conducted in connection with the present invention was to engineer adeno-associated virus (AAV) capsids so that the virus transduces only cells of interest. This was achieved by removing the natural cell-binding site in the native AAV capsid protein. The engineered virus is then appropriately modified (especially chemically) to accept selective, controlled ligand binding. Such desired ligands are then covalently attached to the virus and tested in vitro in cells and in vivo in mice. While AAV2 is used, these examples can be readily applied to other AAV capsids as well.

[0064] 1. Removal of the native binding site in AAV2 AAV2 binds to heparan sulfate proteoglycans through arginines 585 and 588. These positions were mutated to alanines to create the deletion ΔHSPG.

[0065] Plasmid pTAV2-0 contains the entire AAV-2 genome from pAV-2, including both inverted terminal repeats, cloned into the BamHI site of pBluescript II. Subplasmids containing the appropriate fragments of AAV-2 were generated and used as templates for site-directed mutagenesis reactions. Mutagenesis was performed using the Stratagene (Amsterdam, The Netherlands) QuikChange site-directed mutagenesis kit according to the manufacturer's protocol. For each mutant, two complementary PCR primers were designed to contain the sequence of the substitution, flanked by 15–20 complementary base pairs on each side of the mutation. Mutant plasmids were identified by DNA sequencing. The fragment containing the appropriate mutation was then subcloned into a plasmid backbone containing the remainder of the protein (e.g., pTAV2-0), and the entire fragment was sequenced to check for additional PCR mutations.

[0066] 2. Chemical modification of ΔHSPG to accept ligands Typically, selective attachment of ligands to proteins, e.g., protein labeling, is achieved by incorporating bioorthogonal groups into the protein followed by chemoselective modification. This approach is also referred to as "tag and modify." Various bioorthogonal reactions have been developed, including (1) carbonyl-mediated condensation reactions, (2) azide-mediated "click" reactions, and (3) inverse electron-demand Diels-Alder cycloaddition (DA). INV ) and other cycloaddition reactions, (4) transition metal-catalyzed coupling and decaging reactions, and (5) labeling reactions at cysteine ​​residues.

[0067] Subsequently, benzylguanine (BG) was conjugated to the exposed lysines by reacting the virus with benzylguanine NHS ester (SNAP tag substrate, i.e., BG-NHS). To this end, non-aqueous DMSO was added to the vial containing the dried SNAP tag ligand BG-NHS at room temperature using a needle to the desired final concentration (e.g., 20 mM). The amine-functionalized target protein was diluted to the desired final concentration with solvent (PBS). The two preparations were mixed and incubated at room temperature for 180 minutes, after which unreacted components were removed using a centrifugal 100 Kda MWCO filter unit.

[0068] 3. Covalent attachment of ligands There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag® fusion, and labeling of the fusion with a SNAP-tag substrate of choice. The SNAP-tag is a DNA repair protein, human O 6 SNAP-tag is a small protein based on human β-alkylguanine-DNA-alkyltransferase (hAGT). The SNAP-tag substrate in this case is a guanine leaving group attached to a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate covalently binds to the SNAP-tag.

[0069] The SNAP-tag protein labeling system allows for the specific covalent attachment of virtually any molecule to a protein of interest (see 4. below for a description of this invention).

[0070] Recombinant ligands bearing a C-terminal SNAP tag were produced in E. coli or mammalian cell suspension cultures. For covalent conjugation, the SNAP-tagged ligands were then conjugated to the BG-modified virus by adding a saturating concentration of ligand and incubating overnight at room temperature (see Figure 1). Excess unreacted ligand was removed by passing the reaction through a centrifugal 100 Kda MWCO filter unit.

[0071] For the purposes of the present invention, a mammalian expression plasmid (pSNAP) encoding a SNAP-tag® flanked by restriction sites for cloning a gene of interest is used. f The experiments were performed according to the instructions of the SNAP-Cell® Starter Kit (NEB) containing the following, with modifications for this purpose.

[0072] 4. In vitro and in vivo testing In the context of the present invention, the above strategy was tested with several classes of ligands, namely, protein ligands such as growth factors, cytokines, etc.; toxin subunits such as cholera toxin B subunit; lectins such as isolectin B4 or wheat germ agglutinin; adhesion factors such as lactadherin; antibodies such as anti-CD-34 (a stem cell marker); and peptides such as deltorphin, an opioid receptor ligand.

[0073] First, our ΔHSPG viral particles were shown to no longer have infectious activity when tested in sensory neurons of a fluorescent reporter mouse model (Fig. 2). When tested in sensory neurons of the same fluorescent reporter mouse model, wheat germ agglutinin (WGA, a lectin) fusions completely restored viral transduction efficiency to 100% (Fig. 3).

[0074] We then tested several factors and found that the neurotrophic factors NGF, NT3, and BDNF (protein ligands) delivered the virus to different specific neuronal populations in a fluorescent reporter mouse model, depending on the factor used (Figure 4). Cholera toxin B subunit (toxin) specifically targeted the virus to neuronal cell bodies in a retrograde manner (i.e., specific to a cellular compartment / part) (Figure 5). In similar studies, lactadherin (an adhesion factor) specifically targeted the virus to macrophages and neurons exposing phosphatidylserine, and deltorphin (a peptide) specifically targeted the virus to neurons expressing μ and δ opioid receptors.

[0075] In the experiment shown in Figure 6, virus carrying NGF ligand IV was injected into the trigeminal ganglion, and then sensory neuron tissue was harvested and analyzed three weeks later. Sectioned tissue was stained with an antibody against TrkA (the receptor for NGF) and very good overlap was found. Because the TrkA antibody is not perfect, an 80% overlap is highly relevant.

[0076] In the experiment shown in Figure 7, sections from Figure 6 were stained with antibodies to NF200 and IB4, which label other neurons (mechanoreceptors and nonpeptidergic nociceptors, respectively). Again, these markers are not perfect, but it can be seen that the green and blue cells are distinct from the red infected cells.

[0077] As a negative control, IL31 receptor knockout mice were infected with IL31 ligand via virus, but no infection occurred.

[0078] In summary, all ligand-tagged viruses successfully and specifically transduce only cells expressing the respective receptor, both when applied to cultured cells in vitro and when injected into mice in vivo, i.e., they can be injected systemically or locally to selectively target different cell populations.

[0079] Additional in vivo studies using ligand-bound AA A) Targeting TrkA+ nociceptors In this example, we targeted TrkA+ nociceptors in the peripheral nervous system with ligand-conjugated AAV. NGF binds to TrkA but does not activate it. R121W The ligand was conjugated to the ΔHSPG-AAV2 vector carrying the tdTomato cargo. Mice were injected subcutaneously, intraneurally, retroorbitally, or intraperitoneally with this construct. After 3 weeks, fluorescence was detected and quantified using a TrkA antibody.

[0080] For retroorbital application, 80% of TrkA+ cells were found to be infected by NGF-AAV. 83% of NGF-AAV-infected cells were TrkA+. It was also found that different administration routes did not significantly differ in their highly effective results.

[0081] B) Targeting IL31RA+ itch receptors In this example, IL31RA was targeted with a ligand-binding AAV. IL31 binds to IL31RA but does not activate IL31RA. K134A The ligand was conjugated to the above-mentioned ΔHSPG-AAV2 carrying the tdTomato cargo. This construct was injected into wild-type and IL31RA knockout mice. Three weeks later, fluorescence was detected using a keratin 14 antibody. Targeted cells were found to be essentially completely positive for K14. Importantly, no fluorescence was detected in IL31RA knockout mice.

[0082] C) Targeting using AAV with isolectin B4 In this example, isolectin B4 (IB4) was conjugated to the above-described ΔHSPG-AAV2 carrying the tdTomato cargo. IB4 can be used as a marker for the vasculature, nonpeptidergic nociceptors, and / or microglia. This construct was injected subcutaneously, intraneurally, or intraspinally. Fluorescence was detected after 3 weeks. Targeted cells were found to be essentially completely positive, regardless of the route of administration.

[0083] D) Targeting with AAV containing wheat germ agglutinin In this example, wheat germ agglutinin (WGA) was conjugated to the above-described ΔHSPG-AAV2 carrying the tdTomato cargo. WGA binds to N-acetylglucosamine and most neuronal membranes and is used as a (transsynaptic) tracer. This construct was injected intravenously into P1 neonatal mice or intracortically into adult mice. Fluorescence was detected after 3 weeks.

[0084] Gene delivery was found to be more efficient when using ligand-conjugated viruses (see Figure 8). When cultured DRG neurons were infected with the AAV9 variant PHP.S (1E+9 VG), the WGA-modified constructs described above showed a strong increase in delivery (see Figure 8B).

Claims

1. 1. An adeno-associated virus (AAV) particle comprising a chemically functionalized capsid protein, the chemically functionalized capsid protein comprises at least one ligand binding site; the ligand binding site is covalently bound to a primary amine of a native lysine residue of the capsid protein and comprises a dibenzocyclooctyne group or an azide group; and An AAV particle, wherein a ligand is covalently bound to the ligand binding site.

2. The AAV particle of claim 1, wherein the ligand binding site comprises a dibenzocyclooctyne group and the ligand comprises an azide group.

3. The AAV particle of claim 1, wherein the ligand binding site comprises an azide group and the ligand comprises a dibenzocyclooctyne group.

4. 2. The AAV particle of claim 1, wherein the ligand is attached to the ligand-binding site via a strain-promoted click reaction.

5. The AAV particle of claim 1, wherein the chemically functionalized capsid protein is selected from one or more of VP1, VP2, and VP3.

6. The AAV particles of claim 1, which have a higher infectivity rate at a lower titer compared to unmodified AAV particles of the same serotype.

7. The AAV particle of claim 1, wherein the chemically functionalized capsid protein is further modified to remove native mammalian cell binding sites.

8. The AAV particle of claim 7, wherein the natural mammalian cell binding site is a heparan sulfate proteoglycan binding site.

9. The AAV particle of claim 1, which has modified tropism compared to unmodified AAV particles of the same serotype.

10. The AAV particle of claim 1, wherein the ligand is selected from a protein ligand, a toxin subunit, a lectin, an adhesion factor, an antibody, a peptide, and an enzyme.

11. 1. A method for producing adeno-associated virus (AAV) particles comprising a chemically functionalized capsid protein, comprising: covalently attaching at least one ligand binding site to a primary amine of a native lysine residue of a capsid protein of the assembled virus particle; and attaching a ligand to the ligand-binding site via a strain-promoted click reaction; The method, wherein the ligand binding site comprises an azide group or a dibenzocyclooctyne group.

12. 12. The method of claim 11, wherein the step of covalently linking at least one ligand binding site to the capsid protein is performed before the step of binding a ligand to the ligand binding site.

13. 12. The method of claim 11, further comprising modifying the capsid protein to remove the native mammalian cell binding site.

14. A pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and / or diluent and AAV particles described in any one of claims 1 to 10.

15. A pharmaceutical composition for treating a patient having a genetic abnormality, comprising at least one pharmaceutically acceptable carrier and / or diluent and an AAV particle described in any one of claims 1 to 10, wherein the genetic abnormality is a disease that can be treated by gene therapy.

16. 16. The pharmaceutical composition of claim 15, wherein the disease is selected from cancer, a hereditary monogenic disease, a hereditary skin disease, an infectious disease, type 1 diabetes, and wound healing.

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