Chemically modified adeno-associated virus

Abstract

The present invention relates to chemically modified adeno-associated viruses (AAV) and their use in gene therapy.

Description

[Technical field]

[0001] The present invention relates to chemically modified adeno-associated viruses (AAV) and their use in gene therapy. [Background technology]

[0002] Gene therapy was originally developed to correct defective genes underlying inherited diseases. It is now increasingly used to treat a wide range of acquired diseases, including cancer.

[0003] Gene therapy is based on the therapeutic delivery of nucleic acids to the patient's cell nucleus. The nucleic acid can then be inserted into the genome of the targeted cell or remain episomal. The delivery of therapeutic nucleic acids to the subject's target cells can be performed by various methods, including the use of synthetic and viral vectors. Among the many available viral vectors (e.g., retroviruses, lentiviruses, adenoviruses, and the like), recombinant adeno-associated viruses (AAV) have gained popularity as a versatile vector for gene therapy, especially for in vivo applications. The main advantages of recombinant AAV (rAAV) are their broad tropism, their high transduction efficiency, their persistent episomal expression, and their high safety profile, especially because wild-type AAV has not been associated with any human disease.

[0004] Human clinical trials with rAAV have demonstrated durable expression at therapeutic levels when targeting tissues such as the retina, liver, or motor neurons. Several clinical trials using rAAV as a gene vector are underway for a wide range of disorders. The FDA and EMA have recently approved Voretigene neparvovec (Luxturna®), an adeno-associated virus vector serotype 2 (AAV2) capsid containing cDNA encoding the human retinal pigment epithelium 65 kDa protein (hRPE65) for the treatment of vision loss due to inherited retinal dystrophies caused by confirmed biallelic RPE65 mutations. As a further example, Zolgensma® (onasemnogene abeparvovec-xioi) has just been approved by the FDA for the treatment of pediatric patients under the age of 2 with spinal muscular atrophy (SMA). Zolgensma® is an AAV9 vector capable of delivering a functional, non-mutated copy of the defective gene in SMA, the SMN1 gene, in motor neurons.

[0005] Despite these successes, certain clinical trials have shown some limitations of these vectors in the treatment of certain diseases. Their first limitation is their immunogenicity. Due to their non-integrating nature, systemic gene therapy with AAV vectors, especially in pediatric patients, can be limited by tissue proliferation resulting in dilution of the vector over time. However, re-administration of the vector can be hindered by persistent neutralizing antibodies (Nab) induced after the first administration of the viral vector. Moreover, it has been further shown that pre-existing humoral immunity against certain AAV serotypes, especially serotype 2 AAV, is widespread in humans. Anti-AAV neutralizing antibodies (NAb) can completely block transduction in target tissues, resulting in lack of efficiency, especially when the vector is administered directly into the bloodstream. As a result, subjects seropositive for AAV-Nab are generally excluded from gene therapy trials.

[0006] A further limitation of AAVs is their broad tropism, which can result in transgene expression in tissues other than the one in which transgene expression is desired.

[0007] AAV as a gene vector may also suffer from a reduced therapeutic index. Sometimes, high doses of AAV are required to achieve effective transduction. For example, AAV2 vectors can efficiently target the liver, but transgene expression may be limited to a very small amount of transfected hepatocytes due to intracellular proteasome-mediated degradation of the vector, so that high doses of AAV-2 may be required to achieve the required therapeutic effect. Such high doses not only impose a burden on vector production, but also increase the risk of immune responses, among which the induction of Nabs.

[0008] Several strategies have been proposed to overcome the shortcomings of AAV, especially of serotype 2 AAV (AAV2), in gene therapy, certain of which are based on the modification of the capsid protein of the vector.

[0009] In this regard, it has been shown that mutations in surface-exposed tyrosine residues on AAV2 allow it to avoid phosphorylation and subsequent ubiquitination, thereby avoiding proteasome-mediated degradation. Indeed, the lack of capsid ubiquitination is believed to improve intracellular transport of viral vectors to the nucleus, resulting in increased transduction efficiency in hepatocytes both in vitro and in vivo (Zhong et al., PNAS, 2008, 105, 7827-7832; Markusic et al., Molecular Therapy, 2010, 18, 2048-2056). Similar results were observed for AAV8 in gene transfer efficiency in retinal cells (Petrs-Silva, 2009, Molecular Therapy, 17:463-471).

[0010] Chemical modification of viral capsids has also been proposed to introduce ligands into the capsid or mask certain exposed amino acids to alter the antigenicity, tropism, or transduction efficiency of AAV. As a first strategy, some studies have explored the genetic incorporation of unnatural amino acids with modified side chains containing reactive groups such as alkyne, azide, or aminophenyl groups, which allow subsequent selective coupling of ligands by orthogonal reactions. For example, in International Patent Application Publication No. 2015 / 062516, unnatural amino acids such as azide-containing amino acids are genetically inserted into the capsid prior to the coupling step with the ligand by click reaction to change the tropism of the capsid to target cells.

[0011] An alternative strategy consists in the direct chemical modification of the viral capsid without any prior site-directed mutagenesis of the capsid protein.

[0012] Therein, Lee et al. (Biotechnol Bioeng 2005, 92:24-34) investigate the possibility of conjugating the AAV surface with activated polyethylene glycol chains to protect the vector from neutralizing antibodies. Horowitz et al. (Bioconj Chem, 2011, 22(4):529-532) describe a chemical approach to selectively mask arginine residues on the viral capsid based on glycosylation with methylglyoxal. The resulting chemically modified AAV retained the ability to infect neurons in the mouse brain and redirected from the liver to skeletal and cardiac muscles after systemic infection in mice. Furthermore, these glycosylated AAV showed antigenic changes that may allow them to escape antibody neutralization. Finally, WO 2017 / 212019 proposes a method for chemically modifying the AAV capsid by covalently coupling ligands bearing isothiocyanate groups that react with amino groups present in amino acid residues such as lysine or arginine. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] International Patent Application Publication No. 2015 / 062516 [Patent Document 2] International Patent Application Publication No. 2017 / 212019 [Patent Document 3] International Patent Application Publication No. 2014144229 [Patent Document 4] EP2292779 [Patent Document 5] EP1310571 [Non-patent literature]

[0014] [Non-Patent Document 1] Zhong et al., PNAS, 2008, 105, 7827-7832 [Non-Patent Document 2] Markusic et al., Molecular Therapy, 2010, 18, 2048-2056 [Non-Patent Document 3] Petrs-Silva, 2009, Molecular Therapy, 17:463-471. [Non-Patent Document 4] Lee et al., Biotechnol Bioeng 2005, 92:24-34 [Non-Patent Document 5] Horowitz et al., Bioconj Chem, 2011, 22(4):529-532 [Non-Patent Document 6] Naso et al., Biodrugs, 2017, 31:317-334 [Non-Patent Document 7] Koerber et al., Molecular Therapy (2008), 16(10), pp. 1703-1709 [Non-Patent Document 8] Deverman et al., Nat Biotechnol (2016), 34(2), 204-209 [Non-Patent Document 9] Buening et al., Curr Opin Pharmacol (2015), 24, 94-104 [Non-Patent Document 10] Chemical Science, 2020, 11, pp. 1122-1131 Summary of the Invention [Problem to be solved by the invention]

[0015] However, there remains a need for new methods that allow for modulation of the properties of AAV when used as a gene delivery vector in gene therapy. [Means for solving the problem]

[0016] The present invention relates to an adeno-associated virus (AAV) having at least one chemically modified tyrosine residue in a capsid, the chemically modified tyrosine residue being represented by the following formula (I):

[0017] [ka]

[0018] (In the formula, - X1 is

[0019] [ka]

[0020] is selected from the group consisting of - Ar is an optionally substituted aryl or heteroaryl moiety. It relates to the adeno-associated virus.

[0021] X1 is preferably in the ortho position to the phenol group.

[0022] In some embodiments, at least one chemically modified tyrosine residue has the following formula (Ia):

[0023] [ka]

[0024] (In the formula, X1 and Ar are as defined above, - spacer is a group for linking the "Ar" group to the functional moiety "M" and preferably contains up to 1000 carbon atoms, preferably in the form of a chemical chain which may optionally contain heteroatoms and / or cyclic moieties; - n is 0 or 1, - M is a functional moiety comprising a group selected from a steric shielding agent, a labeling agent, a cell type specific ligand, a drug moiety, and combinations thereof. It is.

[0025] In some particular embodiments, X1 is of formula (a) and / or "Ar" is selected from substituted or unsubstituted phenyl, pyridyl, naphthyl, and anthracenyl.

[0026] In some other embodiments, at least one chemically modified tyrosine in the capsid of the AAV has the following formula (1c):

[0027] [ka]

[0028] (In the formula, - X2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -NH(C=S)- or -(C=S)-NH-, preferably -(C=O)-NH- or -(C=O)-O-, - X2 is in the para-, meta- or ortho-position of the phenylene group, preferably in the para-position; - spacer, n, and M are as defined above. It is.

[0029] A "spacer", when present, is an optionally substituted, saturated or unsaturated, linear or branched C-C 40 "M" may be selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyldiamines, and combinations thereof, and / or "M" may comprise or consist of a mono- or polysaccharide, a hormone, e.g., a steroid hormone, a peptide such as RGD peptide, muscle targeting peptide (MTP), or Angiopep-2, a protein or fragment thereof, a membrane receptor or fragment thereof, an aptamer, an antibody including heavy chain antibodies and fragments thereof such as Fab, Fab', and VHH, an ScFv, a spiegelmer, a peptide aptamer, a vitamin, and a drug such as a CB1 and / or CB2 ligand.

[0030] In some other embodiments, the “spacer” (if present) is a linear or branched C2-C 20 alkyl chains of 1 to 20 monomers, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymer having from 2 to 20 monomers, and / or "M" comprises or consists of a cell type specific ligand derived from a protein selected from transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF, a mono- or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, cross-linked GalNac, or mannose-6-phosphate, an MTP selected from SEQ ID NO:1 to SEQ ID NO:7, and a vitamin such as folic acid.

[0031] As an example, M is a cell type specific ligand for specifically targeting hepatocytes and has the formula (III):

[0032] [ka]

[0033] and / or the "spacer" is a polyethylene glycol chain containing from 2 to 10 monomers.

[0034] As a further example, the AAV can be represented by formula (II):

[0035] [ka]

[0036] The capsid comprises at least one chemically modified tyrosine in said capsid,

[0037] In some other embodiments, the AAV of the invention further comprises at least one additional chemically modified amino acid residue in the capsid that is different from a tyrosine residue, said amino acid residue preferably having the following formula (V):

[0038] [ka]

[0039] (In the formula, - N * is the nitrogen of the amino group of an amino acid residue, e.g., a lysine or arginine residue, - Ar, spacer, n, and M have the same definitions as Ar, spacer, n, and M as defined in formula (II) above. The amino group is chemically modified with a group.

[0040] The AAV of the present invention can be of any type. For example, the AAV can be a recombinant AAV, preferably selected from AAV with a wild-type capsid, naturally occurring serotype AAV, variant AAV, pseudotype AAV, AAV with a hybrid or mutant capsid, and self-complementary AAV.

[0041] The present invention also relates to a method for chemically modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, comprising a step of incubating said AAV with a chemical reagent having a reactive group selected from an aryldiazonium salt and a 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) moiety, under conditions conducive to reacting said reactive group with a tyrosine residue present in the capsid of the AAV to form a covalent bond.

[0042] In some embodiments, the methods of the invention comprise providing an AAV having formula (VId):

[0043] [ka]

[0044] to produce a compound of formula (Ic):

[0045] [ka]

[0046] (In the formula, - X2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -C=S-NH or NH-C=S-, preferably -(C=O)-NH- or -(C=O)-O-, - X2 is in the para-, meta- or ortho-position of the phenyl group, preferably in the para-position; - the spacer is a hydrocarbon group containing from 2 to 400 carbon atoms, - n is 0 or 1, - M is a chemical moiety, including a steric shielding agent, a labeling agent, a cell type specific ligand, or a drug moiety. The method includes obtaining at least one chemically modified tyrosine residue in the capsid of the antibody.

[0047] The present invention also relates to an AAV obtainable or obtained by the method of the present invention. A further object of the present invention is a pharmaceutical composition comprising an AAV as defined above and at least one pharma- ceutically acceptable excipient. Said AAV or said pharmaceutical composition can be used as a diagnostic or drug, preferably in gene therapy. [Brief description of the drawings]

[0048] [Figure 1] Figure 1 shows AAV capsid protein integrity measured by SDS-PAGE and silver staining. 1012vg of AAV2-Luc-GFP and AAV2-GFP vectors were added to a solution of 2, 3, or 4 (3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. 1010vg of each condition was analyzed by SDS-PAGE and silver staining. VP1, VP2, and VP3 are the three proteins that make up the AAV capsid. Capsid protein molecular weights are shown to the right of the image according to the protein ladder. AAV2+2: AAV2 incubated with compound 2 (invention), AAV2+3: AAV2 incubated with compound 3 (comparison), AAV2+4: AAV2 incubated with compound 4 (comparison). [Diagram 2] Figure 1 shows dot blot analysis. 1012vg of AAV2-Luc-GFP or AAV2-GFP vectors were added to a solution of compounds 2 and 4 (3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. 109vg of each condition were analyzed by dot blot using the A20 antibody, which recognizes assembled capsids (A), or FITC-soybean lectin, which recognizes GalNAc sugars (B). [Diagram 3]Figure 1 shows Western blots. 1012vg of AAV2-Luc-GFP or AAV2-GFP vectors were added to a solution of compound 2, 3, or 4 (3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. 1012vg samples were analyzed by Western blot using a polyclonal antibody against capsid proteins to detect VP proteins (A) or using FITC-soybean lectin that recognizes GalNAc sugars (B-C). VP1, VP2, and VP3 are the three proteins that make up the AAV capsid. Capsid protein molecular weights are shown to the right of the image according to a protein ladder. B and C are images from the same blot at short (B) and long (C) exposure times during color development. [Figure 4] Western blots (A-B) and AAV capsid protein integrity measured by SDS-PAGE and silver staining (C). 1012vg of AAV2-Luc-GFP or AAV2-GFP vectors were added to a solution of compound 2 or 4 (3E5 or 3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. 1012vg samples were analyzed by Western blot using polyclonal antibodies against capsid proteins to detect VP proteins (A) or using FITC-soybean lectin that recognizes GalNAc sugars (B), and 1010vg of each condition was analyzed by SDS-PAGE and silver staining (C). VP1, VP2, and VP3 are the three proteins that make up the AAV capsid. Capsid protein molecular weights are shown to the right of the image according to the protein ladder. AAV2+2: AAV2 incubated with compound 2 (invention), AAV2+4: AAV2 incubated with compound 4 (comparison). [Diagram 5]Figure 1 shows dot blot analysis. 1012vg of AAV2-Luc-GFP or AAV2-GFP vectors were added to a solution of compounds 2, 9, and 10 (3E5) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. 109vg of each condition were analyzed by dot blot using the A20 antibody, which recognizes assembled capsids (A), or using FITC-soybean lectin or FITC-concanavalin A lectin, which recognizes GalNAc and mannose sugars (B). [Figure 6] Figure 1 shows dot blot analysis (A-B) and AAV capsid protein integrity measured by SDS-PAGE and silver staining (C). 1012vg of AAV2-Luc-GFP or AAV2-GFP vector was added to a solution of compounds 4 and 6 (3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. Each condition of 109vg was analyzed by dot blot using A20 antibody that recognizes assembled capsids (A) or FITC-soybean lectin that recognizes GalNAc sugars (B), and each condition of 1010vg was analyzed by SDS-PAGE and silver staining (C). VP1, VP2, and VP3 are the three proteins that make up the AAV capsid. Capsid protein molecular weights are shown to the right of the image according to the protein ladder (C). AAV2+4: AAV2 incubated with compound 4 (comparison), AAV2+6: AAV2 incubated with compound 6 (invention). [Figure 7]Figure 1 shows dot blot analysis (A-B) and AAV capsid protein integrity measured by SDS-PAGE and silver staining (C). 1012vg of AAV2-Luc-GFP or AAV2-GFP vectors were added to a solution of compounds 7 and 8 (3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. 109vg of each condition were analyzed by dot blot using A20 antibody that recognizes assembled capsids (A) or FITC-Concanavalin A lectin that recognizes mannose sugars (B), and 1010vg of each condition were analyzed by SDS-PAGE and silver staining (C). VP1, VP2, and VP3 are the three proteins that make up the AAV capsid. Capsid protein molecular weights are shown to the right of the image according to the protein ladder (C). AAV2+7: AAV2 incubated with compound 7 (invention). [Figure 8] Figure 1 shows dot blot analysis (A-B) and AAV capsid protein integrity measured by SDS-PAGE and silver staining (C). 1012vg of AAV8-GFP vector was added to a solution of compounds 2 and 4 (3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. Each condition of 109vg was analyzed by dot blot using ADK8 antibody that recognizes assembled capsid (A) or FITC-soybean lectin that recognizes GalNAc sugars (B), and each condition of 1010vg was analyzed by SDS-PAGE and silver staining (C). VP1, VP2, and VP3 are the three proteins that make up the AAV capsid. Capsid protein molecular weights are shown to the right of the image according to the protein ladder (C). AAV2+2: AAV2 incubated with compound 2 (invention), AAV2+4: AAV2 incubated with compound 4 (comparison). [Figure 9]Figure 1 shows dot blot analysis. 1012vg of AAV2-Luc-GFP or AAV2-GFP vectors were added to a solution of compounds 11 and 12 (3E6 equivalents) in TBS buffer (pH 9.3) and incubated at room temperature for 4 hours. 109vg of each condition were analyzed by dot blot using the A20 antibody, which recognizes assembled capsids (A), or using FITC-soybean lectin, which recognizes GalNAc sugars (B). [Figure 10] FIG. 1 shows the infectivity of AAV2-GFP and AAV2+2:AAV2 chemically modified with compound 2 (invention) (3E5 and 3E6 equivalents) in HEK cells by FACS analysis. [Figure 11] FIG. 1 shows the infectivity of AAV2-GFP and AAV2+2:AAV2 chemically modified with compound 2 (invention) (3E5 and 3E6 equivalents) in HuH7 cells by FACS analysis. [Figure 12A] FIG. 2 shows examples of "M" moieties according to the present invention. [Figure 12B] FIG. 2 shows examples of "M" moieties according to the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] To the best of our knowledge, the methods described in the prior art for chemically modifying AAV capsid target amino acid residues with amino groups, such as arginine and lysine. However, there are other amino acid residues of interest for chemical modification, such as surface-exposed tyrosine residues. In fact, surface-exposed tyrosine may play a central role in the immunogenicity of AAV in cells, as well as in proteasomal degradation. The present inventors have devised a new method that allows chemical modification of tyrosine residues present on the surface of AAV capsid. This method relies on the specific reaction of an aryldiazonium group or a PTAD group with the phenyl group in the tyrosine residue, resulting in covalent coupling.

[0050] The present inventors have prepared several PTAD and diazonium ligands bearing sugar moieties for chemically modifying the capsid of AAV vectors at tyrosine residues.

[0051] As proof of concept, we showed that N-acetylgalactosamine and mannose moieties could be covalently immobilized on the surface of AAV capsids by incubating AAV particles with N-acetylgalactosamine or mannose-bearing aryldiazoniums (compounds 2, 6, 7, and 12) in aqueous buffer at basic pH, as demonstrated by dot blot analysis with soybean and concanavalin A lectin detection.

[0052] The inventors further showed that covalent coupling did not alter the capsid protein subunits VP1, VP2, and VP3 of AAV2 and AAV8, as also demonstrated by immunostaining with A20 and ADK8 antibodies, respectively, suggesting that AAV integrity was maintained. Of note, the inventors further showed that AAV2 vectors chemically modified with diazonium compound 2 remained infectious in non-hepatic cell line HEK cells, but showed a transduction efficiency that was less significant than that observed for non-chemically modified vectors (Figure 10). On the contrary, in hepatic cell line HuH7 (expressing low levels of ASPGR), AAV2 vectors chemically modified with diazonium compound 2 showed increased expression of the transgene, evidenced by higher fluorescence intensity (Figure 11). Thus, chemical modification of capsid tyrosine residues with sugar-containing ligands may result in retargeting of AAVs in vivo and alteration of their tropism, thereby reducing off-target effects.

[0053] Chemically modified AAV of the present invention Thus, the present invention relates to an adeno-associated virus (AAV) having at least one chemically modified tyrosine residue in the capsid.

[0054] As used herein, adeno-associated virus (AAV) refers to a small, non-enveloped virus of the dependoparvovirus family, with a single-stranded linear DNA genome of approximately 5 kb in length. Wild-type AAV has two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The 5' and 3' ORFs code for replication and capsid proteins, respectively. The ITRs contain 145 nucleotides and serve as the AAV genome replication origin and packaging signal. In recombinant AAV, the viral ORFs are replaced by exogenous gene expression cassettes, while replication and capsid proteins are provided in trans.

[0055] Thus, in the context of the present invention, recombinant AAV refers to an AAV in which an exogenous nucleic acid sequence has been introduced into the viral genome. The exogenous nucleic acid sequence may be of any type and is selected taking into account the intended use of the AAV. By way of example, the nucleic acid may comprise any RNA or DNA sequence.

[0056] In a preferred embodiment, the AAV of the present invention is a recombinant AAV.Typically, the recombinant AAV will be used as a gene vector for in vivo or in vitro applications, which means that the AAV of the present invention is a recombinant AAV vector.For a review on AAV as a vector in gene therapy, see Naso et al., Biodrugs, 2017, 31:317-334, the contents of which are incorporated herein by reference.

[0057] By way of example only, a recombinant AAV for use as a vector in gene therapy may contain an exogenous gene expression cassette that replaces the viral ORF and is placed between two ITRs. The cassette may contain a promoter, a gene of interest, and a terminator. The promoter and the gene of interest are selected depending on the tissue / organ to be targeted and the condition to be treated. As another example, a recombinant AAV for use in gene therapy may contain a DNA template for homologous recombination in cells. Such a recombinant AAV may be used in combination with a gene editing tool to promote homologous recombination in targeted cells in vivo, in vitro, or ex vivo. The gene editing tool may be of any type, including but not limited to CRISPR / Cas9, zinc finger nucleases, meganucleases, as well as RNA and DNA encoding the proteins.

[0058] In the context of the present invention, the term "AAV" includes all types of AAV, including wild-type AAV and recombinant or variant AAV, including, but not limited to, AAV with mutated or synthetic capsids, such as AAV with hybrid capsids, pseudotyped AAV, as well as self-complementary AAV (scAAV).

[0059] The wild-type AAV capsid is composed of three overlapping capsid proteins called viral protein 1 (VP1), VP2, and VP3. Capsid engineering refers to amino acid modifications of the capsid proteins, for example in their hypervariable loops.

[0060] As used herein, an "AAV having a genetically engineered capsid" or an "AAV having a mutant capsid" refers to an AAV in which one or several amino acid modifications have been introduced into at least one capsid protein (i.e., VP1 and / or VP2 and / or VP3) compared to the wild-type version of said capsid protein.

[0061] As used herein, an "amino acid modification" encompasses one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 20, 30, 40, 50, or 100) amino acid insertions, deletions, or substitutions.

[0062] As used herein, "chemically modified tyrosine residue" means that at least one tyrosine present in the capsid of the virus has been chemically modified by covalent coupling of a chemical entity, typically by covalent coupling of said chemical entity onto the phenyl ring of the tyrosine. The tyrosine is typically a surface-exposed residue present in VP1, VP2, or VP3. A surface-exposed tyrosine means that the tyrosine is accessible for covalent coupling. Such tyrosine residues can be identified by molecular modeling of the capsid protein or by molecular modeling of the entire capsid itself.

[0063] As used herein, "at least one chemically modified tyrosine residue" includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more chemically modified tyrosine residues.

[0064] In some embodiments, the chemically modified AAV of the present invention comprises several chemically modified tyrosine residues in its capsid.

[0065] The chemically modified tyrosine can be present in VP1 and / or VP2 and / or VP3.

[0066] In some embodiments, at least 0.1%, such as at least 0.5%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or more of the surface-exposed tyrosine residues of the capsid are chemically modified.

[0067] In the context of the present invention, AAV can be either an AAV with a wild-type capsid or an AAV with a mutated and / or synthetic capsid.

[0068] In some embodiments, the AAV of the present invention is a recombinant AAV having a wild-type capsid. In other embodiments, the AAV is a recombinant AAV having a mutant capsid, i.e., having one or more amino acid modifications in at least one capsid protein compared to the corresponding parent capsid protein.

[0069] In a specific embodiment, the AAV is a recombinant AAV having a mutant capsid, wherein the amino acid modifications do not involve any tyrosine residues present in the capsid protein.

[0070] They are AAVs of various serotypes that can be either wild-type or synthetic, all serotypes being contemplated in the context of the present invention.

[0071] A "serotype" is traditionally defined based on the absence of cross-reactivity between antibodies to one virus compared to another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). AAV includes a variety of natural and synthetic (e.g., hybrid, chimeric, or shuffled serotypes) serotypes.

[0072] Such non-limiting serotypes include AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10 (such as -cy10 or -rh10), -11, -rh74, or engineered AAV capsid variants, such as AAV-2i8, AAV2G9, -LK3, -DJ, and -Anc80. In the context of the present invention, synthetic serotypes also include pseudotyped AAV, i.e., AAV resulting from a mixture of capsids and genomes from different viral serotypes, such as AAV2 / 5, AAV2 / 7, and AAV2 / 8, as well as AAV with hybrid capsids from multiple different serotypes, such as AAV-DJ, which contains hybrid capsids from eight serotypes.

[0073] Synthetic serotypes also encompass certain variants in which new glycan binding sites have been introduced into the AAV capsid, as described in particular in International Patent Application Publication No. WO2014144229 (which in particular discloses the AAV2G9 serotype). Other AAV serotypes include those disclosed in EP2292779 and EP1310571. In addition, other AAV serotypes include those obtained by shuffling as described in Koerber et al. (Molecular Therapy (2008), 16(10), pp. 1703-1709), by peptide insertion (e.g., Deverman et al., Nat Biotechnol (2016), 34(2), pp. 204-209), or by rational capsid design (reviewed in Buening et al., Curr Opin Pharmacol (2015), 24, pp. 94-104).

[0074] In some embodiments, the AAV is from a naturally occurring serotype, preferably selected from the group consisting of AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9, and AAVrhlO, more preferably AAV-2. By way of example, the AAV of the present invention may be a recombinant AAV vector of serotype 2.

[0075] AAV can target a wide variety of cells, tissues, and organs. Examples of cells targeted by AAV include, but are not limited to, liver cells; cells of the retina, i.e., photoreceptors, retinal pigment epithelium (RPE); muscle cells, i.e., myoblasts, satellite cells; cells of the central nervous system (CNS), i.e., neurons, glia; cells of the heart; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells, lymphocytes, blood cells such as hematopoietic cells, including hematopoietic stem cells, induced pluripotent stem cells (iPS), and the like. Examples of tissues and organs that can be targeted by AAV include liver, muscle, cardiac muscle, smooth muscle, brain, bone, connective tissue, heart, kidney, lung, lymph node, mammary gland, myelin, prostate, testis, thymus, thyroid, trachea, and the like. Preferred cell types are liver cells, retinal cells, muscle cells, cells of the CNS, cells of the PNS, and hematopoietic cells. Preferred tissues and organs are liver, muscle, heart, eye, and brain.

[0076] The tropism of AAVs can vary depending on their serotypes. For example, AAV-2 may be used to transduce the central nervous system (CNS), kidney, and photoreceptor cells, while AAV-8 is effective in transducing the CNS, heart, liver, photoreceptor cells, retinal pigment epithelium (RPE), and skeletal muscle.

[0077] AAV can be produced by any method known in the art, for example, by transient transfection in a cell line of interest, for example, HEK293 cells, as described in the Examples section. In this regard, reference can be made to Naso et al., Biodrugs, 2017, 31:317-334, which provides an overview on AAV as a vector in gene therapy and describes traditional methods for producing AAV on an industrial scale.

[0078] The AAV of the present invention may have other amino acids of the capsid that have been chemically modified. By way of example, the AAV may comprise one or several amino groups of the capsid that have been modified by the method disclosed in International Patent Application Publication No. 2017 / 212019, i.e., by reacting said amino groups in the capsid with a ligand that has an isothiocyanate reactive group. Alternatively or additionally, the AAV of the present invention may have one or several arginine residues of the capsid that have been modified by glycosylation, e.g., by reaction with methylglyoxal as described in Horowitz (supra).

[0079] Typically, at least one chemically modified tyrosine residue in the capsid has the following formula (I):

[0080] [ka]

[0081] (In the formula, - X1 is

[0082] [ka]

[0083] is selected from the group consisting of

[0084] - Ar is an optionally substituted arylene or heteroarylene moiety. It is.

[0085] In a preferred embodiment, the AAV of the present invention has at least one chemically modified tyrosine residue in its capsid, wherein the at least one chemically modified tyrosine residue has the following formula (Ia):

[0086] [ka]

[0087] (In the formula, - X1 is

[0088] [ka]

[0089] is selected from the group consisting of

[0090] - n is 1 or 0 ("n is 1" means that the group "spacer" is present; "n is 0" means that the spacer is absent), Ar is an optionally substituted aryl or heteroaryl moiety; - Spacer is a chemical group that connects "Ar" to "M", - M is a functional moiety, the nature of which depends on the functional modification of the AAV that is desired to be performed by carrying out the chemical modification. It is.

[0091] - X1 X1 is of formula (a) or (b) as above. Preferably, X1 is of formula (a).

[0092] X1 can be in the ortho, meta, or para position, preferably the para position.

[0093] - Ar group As mentioned above, Ar is a substituted or unsubstituted aryl or heteroaryl moiety.

[0094] As used herein, "aryl" refers to an aromatic ring system, preferably having 6-14 atoms, with at least one ring having a conjugated pi-electron system, which may be optionally substituted. "Aryl" may contain more than one aromatic ring, such as a fused ring system, or an aryl group substituted with another aryl group. Aryl includes, but is not limited to, phenyl, anthracenyl, naphthyl, indenyl, and divalent biphenyl.

[0095] "Heteroaryl" refers to a heteroaryl group. "Heteroaryl group" refers to a chemical group, preferably having 5 to 14 ring atoms, in which 1 to 4 heteroatoms are ring atoms in an aromatic ring, and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and selenium. Suitable heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinazolinyl, and quinolinyl.

[0096] Examples of bicyclic heteroaryl groups include, but are not limited to, 1H-indazolyl, benzo[l,2,3]thiadiazolyl, benzo[l,2,5]thiadiazolyl, benzothiophenyl, imidazo[l,2-a]pyridyl, quinolinyl, indolyl, and isoquinolinyl groups.

[0097] "Substituted" or "optionally substituted" includes groups substituted with one or several substituents, typically 1, 2, 3, 4, 5, or 6 substituents. By way of example, the substituents can be C1-C6 alkyl, aryl groups, C3-C6 cycloalkyl, C2-C6 heterocycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C1-C6 aminoalkyl-, C1-C6 alkylaminoalkyl-, -N3, -NH2, -F, -I, -Br, -Cl, -CN, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, C1-C6 aminoalkyl, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C2-C6 alkoxy, C1-C6 alkylamino, C1-C6 alkoxy ..., C1-C6 alkoxy, C1-C6 alkylaminoalkyl-, -N3, -NH2, -F, -I, -Br, -Cl, -CN, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, C1-C6 aminoalkyl, C1-C6 hydroxyalkyl, C1-C 10 It may be independently selected from alkoxyalkyl, C2-C6 alkoxycarbonyloxy, -CN, -CF3, and C2-C6 alkoxyalkyl.

[0098] Preferred substituents are halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0099] The phrase "optionally substituted" may be replaced throughout this application by the phrase "substituted or unsubstituted."

[0100] In some embodiments, "Ar" is selected from aryl and heteroaryl containing from 5 to 14 ring atoms, e.g., from 6 to 10 ring atoms, where the aryl or heteroaryl may be optionally substituted. By way of example, the aryl or heteroaryl group may contain 1, 2, or 3 substituents independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0101] In some particular embodiments, "Ar" is selected from the group consisting of substituted or unsubstituted phenyl, pyridyl, naphthyl, and anthracenyl. The aromatic compound may contain from one to three substituents, preferably independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0102] - Spacer group A "spacer" is any chemical group and therefore can be present or absent.

[0103] When present, a "spacer" is used to link the "Ar" group with the functional moiety "M". The "spacer" can be any chemical chain, which may contain heteroatoms as well as cyclic moieties such as cycloalkyl or aromatic groups. The "spacer" can contain up to 1000 and even more carbon atoms. The length and chemical nature of the spacer can be optimized depending on the "M" moiety intended to be coupled to the tyrosine residue and the biological effect sought. Indeed, in addition to its linking function, the "spacer" can be used to refine the properties of the functional moiety "M". By way of example, the "spacer" can reduce the steric hindrance of "M" to the capsid, improve the accessibility of "M" for binding with the biological entity of interest, improve the binding of "M" with the entity of interest, and / or increase the solubility of "M".

[0104] In some embodiments, a "spacer" is a chemical chain group comprising from 2 to 1000 carbon atoms, preferably from 2 to 500 carbon atoms, from 2 to 300 carbon atoms, e.g., from 2 to 100 carbon atoms, from 2 to 40 carbon atoms, from 4 to 30 carbon atoms, or from 4 to 20 carbon atoms.

[0105] Typically, the "spacer" is selected from the group consisting of polymers, including homopolymers, copolymers, and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains, optionally interrupted by one or several heteroatoms (e.g., S, O, Se, P, or NH), optionally having heteroatoms such as S, O, and NH at at least one of its termini, and optionally substituted by one or several substituents such as hydroxyl, halogen, C1-C3 alkoxy, -CN, -CF3, or C1-C3 alkyl, and combinations thereof.

[0106] As used herein, a "combination" means that it may include several (e.g., 2, 3, 4, 5, or 6) hydrocarbon, oligomeric, or polymeric chains linked by any suitable group, such as, for example, -O-, -S-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, NH-CS-, phosphodiester, or phosphorothioate groups.

[0107] In some embodiments, the spacer may be selected from the group consisting of polyethers such as polyethylene glycol (PEG) and polypropylene glycol, polyvinyl alcohol (PVA), polyesters such as polylactic acid, polyacrylates, polymethacrylates, polysilicones, polycaprolactones and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), D,L-lactic acid-co-glycolic acid copolymers (PLGA), polymers of alkyldiamines, unsaturated or saturated branched or unbranched hydrocarbon chains, optionally having heteroatoms such as O, NH, and S at at least one terminus, and combinations thereof.

[0108] As used herein, alkyldiamine refers to NH2-(CH2) r -NH2, where r is an integer from 2 to 20, for example, an integer from 2 to 10, such as 2, 3, 4, and 5. Polymers of alkyl diamines (also known as polyamines) are represented by the formula NH2-[(CH2) r -NH] t -H, where r is as defined above and t is an integer of at least 2, e.g., at least 3, 4, 5, 10 or more. Polymers of alkyldiamines of interest are, by way of example, spermidine and spermine.

[0109] By way of example, the spacer comprises at least one polyethylene glycol moiety comprising from 2 to 40 monomers, e.g., from 2 to 10, or from 2 to 6 monomers. By way of example only, the spacer may comprise from 2 to 10 triethylene glycol blocks linked together by a linker. As another example, the spacer may comprise at least one polyethylene glycol moiety comprising from 2 to 40 monomers, e.g., from 2 to 10, or from 2 to 6 monomers. 12 Alternatively, the spacer may be a hydrophilic triethylene glycol ethylamine derivative. Alternatively, the spacer may be a saturated or unsaturated C2-C 40 Hydrocarbon chains, especially C 10 ~C 20 C2-C alkyl chain or C6 alkyl chain 10 It may be an alkyl chain, which may have a group such as NH, S, or O at at least one end.

[0110] By way of further example, the spacer may be selected from spermidine, putrescine, spermine, and combinations thereof.

[0111] In certain embodiments, the spacer group is a linear or branched C-C 20 The polymers are selected from the group consisting of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diamino alkyl polymers, and combinations thereof. Preferably, the polyethylene glycol, polypropylene glycol, PLGA, pHPMA, and alkyl diamine polymers contain from 2 to 40 monomers, preferably from 2 to 10 or from 10 to 20 monomers.

[0112] By way of example, the group "spacer" may contain one or several (eg, 2, 3, 4, or 5) triethylene glycol blocks.

[0113] - "M" part The functional moiety "M" can be of any type. "M" is typically selected depending on the biological effect that is desired when the AAV capsid is chemically modified.

[0114] By way of example, "M" may include a moiety selected from a targeting agent, a steric shielding agent, a labeling agent, or a drug. "M" may also be a (nano)particle, including magnetic (nano)particles and quantum dots. By way of example, M may be an iron, dye, silicon, gold, or carbon (nano)particle.

[0115] In some embodiments, "M" comprises or consists of a labeling agent, for example a fluorescent dye such as fluorescein, rhodamine, boron-dipyrromethene (Bodipy) dyes, and alexa fluor, or a radionuclide.

[0116] In other embodiments, "M" comprises or consists of a steric shielding agent, e.g., an agent capable of masking certain epitopes of the capsid, thereby avoiding binding of neutralizing antibodies. By way of example, "M" can be polyethylene glycol (PEG), pHPMA, or a polysaccharide.

[0117] In certain embodiments, "M" comprises or consists of a steric shielding agent capable of masking tyrosine residues, thereby avoiding proteasomal degradation of AAV in cellulo.

[0118] In some embodiments, M comprises or consists of a cell type specific ligand, i.e., a ligand that allows targeting of a particular type of cell. Such a ligand may allow modifying the tropism of the AAV, i.e., its ability to selectively infect and / or transduce a given cell line, tissue, or organ.

[0119] By way of example, "M" may be a ligand that specifically binds to a biological entity (e.g., a membrane receptor) of the membrane of the targeted cell. The ligand may be, by way of example, a mono- or polysaccharide, a hormone, e.g., a steroid hormone, a peptide such as RGD peptide, angiopep-2, a muscle targeting peptide, a protein or a fragment thereof, a membrane receptor or a fragment thereof, CB1 and CB2 ligands, an aptamer, an antibody including heavy chain antibodies and fragments thereof such as Fab, Fab', and VHH, an ScFv, a spiegelmer, a peptide aptamer, a small chemical molecule known to bind to the targeted biological entity, and the like.

[0120] In some embodiments, "M" comprises or consists of cell type specific ligands derived from proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF.

[0121] In some embodiments, "M" comprises or consists of a cell type specific ligand derived from a monosaccharide or polysaccharide, for example comprising one or more galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine (GalNac), and cross-linked GalNac. The monosaccharide or polysaccharide may be natural or synthetic.

[0122] In another embodiment, "M" comprises or consists of a cell type specific ligand derived from a vitamin such as folic acid.

[0123] According to one embodiment, the cell type specific ligand included in "M" may be derived from or may be in muscle targeting peptide (MTP). The ligand may be ASSLNIA (SEQ ID NO:1); WDANGKT (SEQ ID NO:2); GETRAPL (SEQ ID NO:3); CGHHPVYAC (SEQ ID NO:4); HAIYPRH (SEQ ID NO:5); CYCLICCQLFPLFRC (SEQ ID NO:6), or the sequence of SEQ ID NO:7 as shown below:

[0124] [ka]

[0125] where X is an aminohexanoic acid residue and B is a beta-alanine residue. The amino acid sequence may be selected from the group consisting of:

[0126] As used herein, the cyclic CQLFPLFRC of SEQ ID NO:6 is

[0127] [ka]

[0128] Refers to...

[0129] In certain embodiments, "M" is a cancer cell targeting peptide, including peptides such as RGD, including cyclic RGD.

[0130] When "M" comprises a peptide moiety, such as a muscle targeting peptide (MTP), the peptide moiety may comprise a chemical modification at its M-terminus or C-terminus. By way of example, the N-terminus of the peptide moiety may be acylated or coupled to a moiety such as -C(=O)-(PEG moiety)-NH2.

[0131] In another embodiment, "M" comprises or consists of a cell type specific ligand derived from a small molecule or hormone, such as naproxen, ibuprofen, cholesterol, progesterone, or estradiol.

[0132] In further embodiments, "M" is a CB1 and / or CB2 ligand, such as:

[0133] [ka]

[0134] It comprises or consists of:

[0135] Galactose-derived ligands recognized by the asialoglycoprotein receptor (ASPGPr) can be used to specifically target hepatocytes. Thus, in some embodiments, "M" is a ligand for specifically targeting hepatocytes and has the following formula (IIIa), (IIIb), or (IIIc):

[0136] [ka]

[0137] [ka]

[0138] Contains at least one portion of.

[0139] In some other embodiments, "M" is a ligand for targeting muscle cells, particularly skeletal muscle cells, and contains at least one of the following mannose-6-phosphate moieties:

[0140] [ka]

[0141] Includes.

[0142] In some other embodiments, "M" is a ligand for a photoreceptor or neuronal cell and comprises at least one mannose moiety of the following formula (IIIf):

[0143] [ka]

[0144] Includes.

[0145] In some embodiments, "M" is multivalent, meaning that it comprises at least two (e.g., 2, 3, 4, 5, or 6) ligand moieties of interest, such as cell type specific ligands as described above. By way of example, M may comprise a multifunctional linker having several (e.g., at least 2, 3, 4, 5, or 6) cell type specific ligands. The cell type specific ligands may be the same or different. By way of example, "M" may be represented by the following formula (IV):

[0146] [ka]

[0147] (wherein n is an integer from 1 to 100, preferably from 1 to 20). may include a portion of

[0148] As another example of a multivalent ligand, "M" may comprise a moiety of formula (IV) in which the GalNac group is replaced by mannose, mannose-6-phosphate, cross-linked GalNac, CB1 and / or CB2 ligands or peptides.

[0149] In certain embodiments, "M" may include both a labeling moiety, such as a fluorescent label or a radionuclide, and a cell type specific ligand. By way of example only, M may be:

[0150] [ka]

[0151] That is, it can be the muscle targeting peptide of SEQ ID NO:1 linked to K-FITC.

[0152] Other examples of chemical moieties that can be used as the "M" moiety are provided in Figures 12A and 12B.

[0153] As mentioned above, a preferred chemically modified AAV is an AAV that contains at least one chemically modified tyrosine of formula (Ia) or (I), where X1 is formula (a).

[0154] In certain embodiments, X1 is in the ortho position of the phenyl group of tyrosine. Thus, the present invention provides a compound of formula (Ib):

[0155] [ka]

[0156] (wherein "Ar", "spacer", n, and "M" are as defined above for formula (I). The present invention relates to an AAV particle comprising at least one chemically modified tyrosine present in the capsid, wherein the at least one chemically modified tyrosine is

[0157] In some embodiments of the invention, the chemically modified tyrosine present in the capsid has formula (Ib) and has the following characteristics: - "Ar" is selected from the group consisting of substituted or unsubstituted phenyl, pyridyl, naphthyl, and anthracenyl, "Ar" may contain from 1 to 3 substituents, preferably independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl, and / or - "Spacer", if present, is an optionally substituted saturated or unsaturated, linear or branched C-C 40 selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of a cell type targeting ligand, preferably selected from mono- or polysaccharides, hormones, e.g. steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTP) or angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, ScFv, spiegelmers, peptide aptamers, vitamins and drugs, small chemical molecules such as CB1 and / or CB2 ligands. The compound may be further characterized by one or more of the following:

[0158] In another embodiment, the chemically modified tyrosine present in the capsid has formula (Ib) and has the following characteristics: "Ar" is selected from optionally substituted phenyl or pyridyl; and / or - "Spacer" (if present) is linear or branched C2-C 40 , preferably C2 to C 20 selected from the group consisting of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF, muscle targeting peptides as described above, and cell type specific ligands derived from mono- or polysaccharides, e.g., containing one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine, or cross-linked GalNac, CB1 and / or CB2 ligands, and vitamins such as folic acid. The compound may be further characterized by one or more of the following:

[0159] In certain embodiments, "Ar" is an optionally substituted phenyl. Typically, the phenyl may be substituted by one or several substituents selected from halogen such as Cl, Br, I, or F, C1-C6 alkyl, C1-C6 hydroxyalkyl, C1-C6 alkoxy, C1-C6 haloalkyl, -CN, -NO2, and the like.

[0160] In some embodiments, "Ar" is an unsubstituted phenyl, said phenyl being linked to a spacer group (when n is 1) or to an M group (when n is 0) by a linker X2.

[0161] Therefore, a further object of the present invention is an AAV comprising at least one chemically modified tyrosine, said chemically modified tyrosine having the following formula (Ic):

[0162] [ka]

[0163] (In the formula, - X2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH and -OC=OO-, O, NH, NH(C=S)-, -(C=S)-NH-, preferably -(C=O)-NH- or -(C=O)-O-, - "spacer", n and M are as defined above; - X2 can be in the para, meta or ortho position of the phenyl group, preferably in the para position. It is.

[0164] In some embodiments of the invention, the chemically modified tyrosine present in the capsid has formula (Ic) and has the following characteristics: - "Spacer", if present, is an optionally substituted saturated or unsaturated, linear or branched C-C 40selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of a cell type targeting ligand, preferably selected from mono- or polysaccharides, hormones, e.g. steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTP) or angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, ScFv, spiegelmers, peptide aptamers, vitamins and drugs, small chemical molecules such as CB1 and / or CB2 ligands. The compound may be further characterized by one or more of the following:

[0165] In another embodiment, the chemically modified tyrosine present in the capsid has formula (Ic) and the following characteristics: - "Spacer" (if present) is linear or branched C2-C 40 , preferably C2 to C 20 selected from the group consisting of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF, muscle targeting peptides as described above, and cell type specific ligands derived from mono- or polysaccharides, e.g., containing one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine, or cross-linked GalNac, CB1 and / or CB2 ligands, and vitamins such as folic acid. The compound may be further characterized by one or more of the following:

[0166] In some embodiments, X2 is -C(=O)NH- and is in the para position. Thus, the present invention also relates to an AAV comprising at least one chemically modified tyrosine present in the capsid, wherein the chemically modified tyrosine has the following formula (Id):

[0167] [ka]

[0168] (wherein n, M, and spacer are as defined above). , regarding AAVs.

[0169] In some embodiments, n is 1.

[0170] In some further or alternative embodiments, the chemically modified tyrosine present in the capsid has formula (Id) and has the following characteristics: - "Spacer", if present, is an optionally substituted saturated or unsaturated, linear or branched C-C 40 selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of a cell type targeting ligand, preferably selected from mono- or polysaccharides, hormones, e.g. steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTP) or angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, ScFv, spiegelmers, peptide aptamers, vitamins and drugs, small chemical molecules such as CB1 and / or CB2 ligands. The compound may be further characterized by one or more of the following:

[0171] In another embodiment, the chemically modified tyrosine present in the capsid has formula (Id) and has the following characteristics: - "Spacer" (if present) is linear or branched C2-C 40 , preferably C2 to C 20 selected from the group consisting of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF, muscle targeting peptides as described above, and cell type specific ligands derived from mono- or polysaccharides, e.g., containing one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine, or cross-linked GalNac, CB1 and / or CB2 ligands, and vitamins such as folic acid. The compound may be further characterized by one or more of the following:

[0172] In some other embodiments, the chemically modified tyrosine present in the capsid is of formula (Ia), where X1 is of formula (b), preferably in the ortho position, and has the following features: - "Spacer", if present, is an optionally substituted saturated or unsaturated, linear or branched C-C 40 selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of a cell type targeting ligand, preferably selected from mono- or polysaccharides, hormones, e.g. steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTP) or angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, ScFv, spiegelmers, peptide aptamers, vitamins and drugs, small chemical molecules such as CB1 and / or CB2 ligands. The compound may be further characterized by one or more of the following:

[0173] In another embodiment, the chemically modified tyrosine present in the capsid is of formula (Ia), where X1 is of formula (b), preferably in the ortho position, and has the following features: - "Spacer" (if present) is linear or branched C2-C 40 , preferably C2 to C 20 selected from the group consisting of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers preferably comprising from 2 to 40 monomers; and / or - "M" comprises or consists of proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF, muscle targeting peptides as described above, and cell type specific ligands derived from mono- or polysaccharides, e.g., containing one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine, or cross-linked GalNac, CB1 and / or CB2 ligands, and vitamins such as folic acid. The compound may be further characterized by one or more of the following:

[0174] By way of example only, the AAV of the invention may have the following formula (IIa), (IIb), (IIc), or (IId):

[0175] [ka]

[0176] The capsid may comprise at least one chemically modified tyrosine.

[0177] The AAV of the present invention has the following formula (IIe), (IIf), or (IIg):

[0178] [ka]

[0179] The capsid may comprise at least one chemically modified tyrosine.

[0180] In all of the above embodiments, particularly those in which at least one chemically modified protein is of formula (I), (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), and (IIg), the AAV is preferably a recombinant AAV, more preferably a recombinant AAV vector.

[0181] As mentioned above, the AAV may have a "naturally occurring" capsid or a genetically modified capsid, i.e., a capsid that contains one or several mutations in at least one capsid protein, i.e., VP1, VP2, and / or VP3.

[0182] In some further or alternative embodiments, the AAV may be a serotype selected from AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9, and AAVrhlO, more preferably AAV-2.

[0183] Alternatively, the AAV is a synthetic serotype.

[0184] In some further embodiments, the AAV of the invention may have at least one additional chemically modified amino acid residue in the capsid that is different from a tyrosine residue, e.g., an arginine or lysine residue. In some embodiments, the amino acid residue has an amino group in its side chain and has the following formula (V):

[0185] [ka]

[0186] (In the formula, - N * is the nitrogen of the amino group of the side chain of an amino acid residue, e.g., a lysine residue, - Ar, spacer, n, and M are as defined in formulae (Ia), (Ib), (Ic), and (Id). It will be appreciated that Ar, spacer, n, and M in formula (V) can be the same or different from those present in at least one chemically modified tyrosine, as described above.

[0187] Modifications at the amino group can be introduced as described in International Patent Application Publication No. 2017212019, the contents of which are incorporated herein by reference.

[0188] Chemical modification of the capsid of an AAV may alter one or several biological functionalities and / or properties. Depending on the nature of "M" covalently attached to the surface of the chemically modified AAV, said chemically modified AAV may have one or several altered biological properties, such as the following biological properties, compared to the same but non-chemically modified AAV: - altered tropism, e.g., increased selectivity of the AAV for a particular organ, tissue, or cell (either administered in vivo or by transducing tissues or cells in culture), or shifted selectivity of the AAV from one tissue / organ / cell to another; and / or - altered immune reactivity of the AAV, e.g., decreased immunogenicity and / or decreased affinity for neutralizing antibodies of the AAV, and / or the AAV elicits an altered humoral response when administered in vivo, e.g., failure to generate neutralizing antibodies directed against the AAV - increased infection efficiency of AAV particles, and / or - Increased transduction efficiency of AAV into specific cells, tissues, or organs - Reduced cytotoxicity when transducing cells in culture - Inducing targeted killing of cancer cells - Visualization / monitoring of AAV particles during in vivo administration or in vitro modification of cells with these AAV particles. - Theragnostic applications; e.g. combining therapeutic and diagnostic agents may have:

[0189] In some embodiments, the chemically modified AAV of the present invention may have a higher transduction efficiency, which may be due to increased intracellular trafficking to the nucleus, decreased proteasomal degradation, more efficient intranuclear decapsidation, more rapid vector genome stabilization, and / or decreased interaction with neutralizing antibodies, and / or decreased antibody-mediated clearance of AAV in vivo, compared to non-chemically modified AAV. In some other embodiments, the AAV may have a higher infection efficiency and / or increased selectivity for a given cell, tissue, or organ, either in vivo or in vitro, compared to non-chemically modified AAV.

[0190] In some other embodiments, when AAV is used as a drug, e.g., as a gene vector, such altered properties may result in an improved therapeutic index of the AAV, which may result in a reduction in the dose that needs to be administered to a patient to achieve the desired therapeutic effect and / or reduced toxicity of the AAV.

[0191] In certain embodiments, the chemically modified AAV of the present invention exhibits preferential tropism for an organ or cell selected from the liver, heart, brain, joints, retina, and skeletal muscle. In alternative or additional embodiments, the chemically modified AAV of the present invention exhibits preferential tropism for a cultured cell selected from, but not limited to, hepatocytes, cardiomyocytes, muscle cells, neurons, motor neurons, retinal pigment cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC), or induced pluripotent stem cells (iPS).

[0192] Methods for preparing chemically modified AAV of the present invention The present invention also relates to a method for chemically modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, comprising a step of incubating said AAV with a chemical reagent carrying a reactive group selected from an aryldiazonium salt and a 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) moiety, under conditions conducive to reacting said reactive group with a tyrosine residue present in the capsid of the AAV to form a covalent bond.

[0193] In a specific embodiment, the method of the invention is for obtaining a chemically modified AAV comprising at least one chemically modified tyrosine residue in the capsid, wherein the chemically modified tyrosine residue has the formula (Ia):

[0194] [ka]

[0195] and wherein the AAV is treated with a chemical reagent selected from:

[0196] [ka]

[0197] under conditions conducive to reacting the chemical reagent with tyrosine residues present in the capsid of the AAV to form covalent bonds.

[0198] Naturally, Ar, spacer and n are as defined above for formula (Ia), - when X1 is of formula (a), the chemical reagent is of formula (VIa), and when X1 is of formula (b), the chemical reagent is of formula (VIb).

[0199] Typically, AAV particles are incubated with the chemical reagent under conditions suitable to promote the formation of a covalent bond between the phenyl group of the tyrosine residue and the chemical reagent without compromising the structural integrity of the AAV.

[0200] The incubation may be carried out in an aqueous buffer having a pH of from 5 to 11, preferably from 7 to 10, for example from 8.5 to 10.5 or from 9 to 10.

[0201] The buffer may be selected from TRIS buffer, sodium carbonate-sodium bicarbonate buffer, phosphate buffer, such as PBS or Dulbecco's phosphate buffered saline (dPBS).

[0202] When the chemical reagent is of formula (VIa), the pH of the aqueous buffer may be basic, e.g., above 7, such as from 8.5 to 10, e.g., from 9 to 10, e.g., from 9.0 to 9.6. Such a basic pH may allow for increased reactivity and / or specificity of the aryl diazonium salt reagent towards the phenyl groups of tyrosine residues. By way of example, a suitable buffer for carrying out incubation of AAV with the aryl diazonium reagent is TRIS buffer.

[0203] The incubation may last from a few minutes to a few hours, for example from 5 minutes to 6 hours, for example from 3 to 4 hours. Typically, the incubation is terminated when a sufficient yield of coupling is achieved.

[0204] The incubation temperature is typically from 10° C. to 50° C. Preferably, the incubation is carried out at room temperature, ie from 18° C. to 30° C., for example at about 20° C.

[0205] The incubation may be carried out with stirring.

[0206] The molar ratio of chemical reagent to AAV particles ranged from 1:10 to 1:10 8 For example, 1:10 5 From 1:10 7 Up to, for example, 1:10 6 ~5:10 6 It could be.

[0207] The medium may contain one or several additional compounds. By way of example, if the chemical reagent is of formula (VIc), the medium may contain an oxidizing agent or a system that generates an oxidizing agent, such as H2O2.

[0208] The counter anion present in the aryl diazonium salt reagent can be of any type, and is preferably TsO - , BF4 - , Cl - , AcO - , PF6 - , TfO - , or CF3CO2 - It is.

[0209] In certain embodiments, the resulting AAV comprises at least one chemically modified tyrosine of formula (Ib). Thus, the chemical reagent has the following formula (VIa):

[0210] [ka]

[0211] (wherein Ar, spacer, n, and M are as defined in formula (Ib). It is.

[0212] In some other embodiments, the resulting AAV comprises at least one chemically modified tyrosine of formula (Ic). Thus, the chemical reagent has the following formula (VId):

[0213] [ka]

[0214] It is.

[0215] In the formula, X2, the spacer, n, and M are as defined in formula (Ic).

[0216] In another embodiment, the resulting AAV comprises at least one chemically modified tyrosine of formula (Id). Thus, the chemical reagent has the following formula (VIe):

[0217] [ka]

[0218] It is.

[0219] wherein the spacer, n, and M are as defined in formula (Id).

[0220] The method of the invention may comprise one or several additional steps before or after the incubation step as described above.

[0221] By way of example, a method of the invention may include a step of providing or producing AAV particles, which may be chemically modified.

[0222] The present invention may also include the step of providing or preparing a chemical reagent.

[0223] The chemical reagents can be produced by synthetic routes. For example, the chemical reagent of formula (VIIa) can be prepared from the nitro derivative by reducing -NO2 to -NH2, for example by hydrogenation using Pd / C as catalyst, followed by the formation of an aryldiazonium group from the aniline derivative, for example by reaction with tBuONO. By way of example only, reference can be made to the synthesis of the compound of formula II described in the Examples section.

[0224] The method of the present invention may also include, after the incubation step, - destroying any unreacted chemical reagents at the end of the incubation step; and / or - removing unreacted reagents, for example by dialysis or tangential flow filtration, and / or - harvesting the chemically modified AAV particles; and / or - purifying the chemically modified AAV particles; and / or - recovering the chemically modified AAV particles; and / or - formulating and / or packaging the chemically modified AAV The method may include one or several additional steps.

[0225] The method of the present invention may further comprise the step of chemically modifying an amino acid residue other than the tyrosine residue of the AAV capsid. By way of example, the additional chemically modified amino acid residue may have an amino group in its side chain.

[0226] In certain embodiments, the methods of the invention comprise administering to the patient an AAV having the following formula (VII):

[0227] [ka]

[0228] Such a step may include incubating a compound of formula (I) with a chemical reagent of formula (I) under conditions conducive to promoting reaction of said chemical reagent with an amino group of an amino acid residue, such as a lysine residue or an arginine residue, present in the capsid of AAV to form a covalent bond. Such a step may include incubating a compound of formula (I) with a chemical reagent of formula (I)

[0229] [ka]

[0230] (In the formula, - N * is the nitrogen of the amino group of an amino acid residue, e.g., a lysine residue, - Ar, spacer, n, and M are as defined in formulae (Ia), (Ib), (Ic), and (Id). It is possible to chemically modify the amino groups present in the amino acid residues of the capsid according to the formula (V). It goes without saying that Ar, spacer, n, and M in formula (V) can be the same or different from those present in at least one chemically modified tyrosine as described above.

[0231] Typically, such steps may be carried out in an aqueous buffer such as TRIS buffer, at a pH of 8 to 10, e.g., a pH of about 9.3, and at a temperature of 10° C. to 50° C., e.g., at room temperature. Details regarding the performance of such steps can be found in International Patent Application Publication No. WO 2017 / 212019, the contents of which are incorporated herein by reference.

[0232] This step can be carried out before, simultaneously with, or after the step of chemically modifying at least one tyrosine residue in the AAV capsid as described above.

[0233] The present invention also relates to a chemically modified AAV obtainable or obtainable by the method of the invention as described above.

[0234] In a further aspect, the present invention also relates to a method for modifying one or several biological properties of AAV, more precisely of a recombinant AAV intended to be used as a gene vector in gene therapy. Indeed, depending on the nature of the "M" part, a method for chemically modifying the capsid of AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of AAV as described above, consists in: - modifying the tropism, e.g., increasing the selectivity of the AAV for a particular organ, tissue, or cell (either administered in vivo or transducing a tissue or cell in culture), or shifting the selectivity of the AAV from one tissue / organ / cell to another; and / or - altering the immune reactivity of the AAV, e.g. reducing the immunogenicity of the AAV and / or reducing the affinity for neutralizing antibodies, and / or eliciting an altered humoral response when administered in vivo, e.g. not generating neutralizing antibodies directed against the AAV; and / or - increasing the infection efficiency of AAV particles; and / or - Reduce off-target effects, i.e. transducing cells that are not necessary to provide the benefit of the drug and may even be harmful - Increase the transduction efficiency of AAV into specific cells, tissues, or organs - Reduces cytotoxicity when transducing cells in culture - Induce targeted killing of cancer cells - Allows visualization / monitoring of AAV particles during in vivo administration or in vitro modification of cells with these AAV particles - Combining therapeutic and diagnostic agents in AAV This may make it possible.

[0235] Uses of the AAVs of the Invention The chemically modified AAV of the present invention may be used as a research tool or as a pharmaceutical agent, e.g., as a vector for delivery of therapeutic nucleic acids such as DNA or RNA, and / or as a diagnostic tool, e.g., as an imaging agent, or a combination of both, including theragnostic uses.

[0236] In some embodiments, the chemically modified AAV of the present invention is used to deliver nucleic acid to a cell and is thus a recombinant AAV.

[0237] The recombinant AAV can be administered to cells in vivo, ex vivo, or in vitro. The cells can be from any mammal, including humans, primates, cows, mice, sheep, goats, pigs, rats, and the like. The cells can be of any type, including hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigment cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPS).

[0238] The recombinant AAV of the present invention can be used to deliver a therapeutic nucleic acid of interest in a subject. Thus, the present invention relates to a method for delivering a therapeutic nucleic acid of interest in a subject in need, comprising administering a chemically modified AAV of the present invention to a subject in need. The recombinant AAV of the present invention can be delivered to a subject by any suitable route. Suitable administration routes include, but are not limited to, inhalation, topical, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal), conjunctival (e.g., intraretinal, subretinal), mucosal (e.g., buccal, nasal), intra-articular, intravitreal, intracranial, intravascular (e.g., intravenous), intraventricular, intracisternal, intraperitoneal, and intralymphatic routes. Typically, the administration route is selected depending on the tissue / organ to be targeted, i.e., depending on the tissue / organ for which transduction is sought.

[0239] The dose of AAV administered to a subject is typically determined by a skilled artisan, taking into account the specific characteristics of the subject, the therapeutic effect desired, and the tissue / organ to be targeted. A single dose or several doses of AAV may be required to achieve the desired therapeutic effect. The AAV of the present invention is typically administered in the form of a pharmaceutical composition, i.e., in a mixture with one or several pharmaceutical excipients.

[0240] The condition that can be treated by administering AAV can be of any type, including genetic disorders as well as acquired disorders.Genetic disorders of interest include inherited muscle disorders such as Duchenne muscular dystrophy, leukodystrophy, spinal muscular atrophy (SMA), hemophilia, sickle cell disease, and inherited retinal dystrophy.Chemically modified AAV can also be used to treat disorders such as cancer, arthritis, osteoarthritis, congenital and acquired heart disease, Parkinson's disease, Alzheimer's disease, and infectious diseases such as Hepatitis C.

[0241] Another object of the present invention is a pharmaceutical composition comprising the chemically modified AAV of the present invention and at least one pharma- ceutically acceptable excipient, which may be selected from well-known excipients such as carriers, preservatives, antioxidants, surfactants, buffers, stabilizers, and the like.

[0242] The present invention further relates to an in vivo or ex vivo method for delivering a nucleic acid of interest in a cell, comprising contacting the cell with the chemically modified AAV of the present invention. The cell may be derived from a patient. After transduction, the cell may be transplanted into a patient in need thereof. The cell may be, for example, a hematopoietic stem cell. The nucleic acid of interest may be of any type and is selected depending on the effect desired.

[0243] As an example, the AAV may contain an exogenous gene expression cassette. The cassette may include a promoter, a gene of interest, and a terminator. As another example, the AAV of the present invention may contain a DNA template for homologous recombination in a cell. Such recombinant AAV may be used in combination with a gene editing tool to promote homologous recombination in a targeted cell. The gene editing tool may be of any type, including but not limited to CRISPR / Cas9, zinc finger nucleases, meganucleases, as well as RNA and DNA encoding the protein.

[0244] The present invention also relates to a host cell transfected with the chemically modified AAV of the present invention, said host cell may be of any type.

[0245] By way of example, the host cell may be a hepatocyte, a cardiomyocyte, a muscle cell, a neuron, a motor neuron, a retinal pigment cell, a photoreceptor, a chondrocyte, a hematopoietic stem cell (HSC), or an induced pluripotent stem cell (iPS).

[0246] Further aspects and advantages of the present invention are disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application. EXAMPLES

[0247] The following compounds were prepared:

[0248] [ka]

[0249] The synthesis of compounds 3, 9, and 10 and the coupling procedures at the amino groups of the capsid of AAV have been previously published in Chemical Science, 2020, 11, pp. 1122-1131. The synthesis of the other compounds is described below.

[0250] 1. Synthesis of Compound 2 (Invention)

[0251] [ka]

[0252] Ligand 2 is prepared as follows:

[0253] [ka]

[0254] Compound A (1 eq., 1.12 mmol) was solubilized in DCM, and DMAP (5 eq., 5.6 mmol), 4-nitrobenzoyl chloride (3 eq., 3.3 mmol) were added. The mixture was stirred at room temperature for 4 h. The organic solution was washed with HCl 1M, water, and NaHCO3, and dried over MgSO4. After concentration, the orange oil was purified by flash chromatography (SiO2, AcOEt / MeOH 85 / 15). A pale yellow solid of compound B was obtained. Yield: 51.7%. NMR 1 H (300 MHz, MeOD): δ (ppm) 8.34 (d, J= 8.98 Hz, 2H), 8.06 (d, J= 9 Hz, 2H), 5.32 (d, J= 3.13 Hz, 1H), 5.06 (dd, J= 3.3 Hz, J= 11 HZ, 1H), 4.61 (d. 13C (75 MHz, MeOD): δ (ppm) 173.66, 172.08, 172.05, 171.75, 168.22, 151.04, 141.44, 129.85 x 2C, 124.66 x 2C, 102.87, 72.18, 71.84, 71.60, 71.39, 71.35, 70.39, 70.21, 68.18, 62.73, 51.56, 41.17, 22.92, 20.55 x 2C, 20.51. HRMS: TOF (ESI+) m / z C 27 H 37 N3O 14 Na (M+Na) + Calculated value 650.2173, measured value 650.2172.

[0255] [ka]

[0256] Compound B (1 eq, 0.6 mmol) was solubilized in a mixture 1:1 H2O / MeOH and Amberlist basic resin was added. The mixture was stirred for 2 h until disappearance of starting material. The resin was filtered and the filtrate was concentrated under vacuum to give the corresponding product C as a white solid. Yield: quantitative. NMR 1 H (400 MHz, MeOD): δ (ppm) 8.33 (d, J= 8.8 Hz, 2H), 8.05 (d, J= 9.2 Hz, 2H), 4.42 (d, J= 8.4 Hz, 1H), 3.93 (m, 2H), 3.82-3.46 (m, 18H), 1.96 (s, 3H) NMR 13 C (100 MHz, MeOD): δ (ppm) 174.12, 168.29, 151.08, 141.44, 129.78, 124.66, 103.12, 76.79, 73.50, 71.62, 71.54, 71.40, 70.41, 69.76, 69. 67, 62.57, 54.29, 41.19, 23.08. HRMS: TOF (ESI+) m / z C 21 H31 N3O 11 Na (M+Na) + Calculated value 524.1856, measured value 524.1852.

[0257] [ka]

[0258] Compound C (1 equiv., 0.6 mmol) was solubilized in MeOH (15 mL) and palladium on carbon (10% w / w) was added. The mixture was placed under an H2 atmosphere and stirred at room temperature for 5 h. The mixture was filtered through celite and the filtrate was concentrated in vacuo to give a white solid corresponding to compound D. Yield: 82.3%. NMR 1 H (400 MHz, MeOD): δ (ppm) 7.60 (d, J= 8.8 Hz, 2H), 6.67 (d, J= 8.8 Hz, 2H), 4.41 (d, J= 8.4 Hz, 1H), 3.76-3.46 (m, 20H), 1.96 (s, 3H) NMR 13 C (100 MHz, MeOD): δ (ppm) 174.16, 170.47, 153.15, 129.97, 123.20, 114.76, 103.12, 76.72, 73.49, 71.57, 71.34, 70.79, 69.74, 69.70, 62.56, 54.29, 40.68, 23.09. HRMS: TOF (ESI+) m / z C 21 H 33 N3O9Na (M+Na) + Calculated value 494.2114, measured value 494.2108.

[0259] Diazonium salt derivative 2 was obtained by adding HBF4 (1.1 equivalents) and tBuONO (1.1 equivalents) in ultrapure water to amino derivative D. After 5 min of vortexing, the formation of diazonium salt was confirmed by using a solution of 2-hydroxynaphthol. A mixture of one drop of each solution shows a red coloration confirming the formation of diazonium salt.

[0260] 2. Synthesis of compound 4 (comparison)

[0261] [ka]

[0262] Compound A (1 eq., 0.2 mmol) was solubilized in DCM (15 mL) and DMAP (5 eq., 1.1 mmol), benzoyl chloride (3 eq., 0.8 mmol) were added. The mixture was stirred at room temperature for 4 h. The organic solution was washed with HCl 1M, water, and NaHCO3, and dried over MgSO4. After concentration, the yellow oil was purified by flash chromatography (SiO2, AcOEt / MeOH 8 / 2). A white solid of compound E was obtained. Yield: 67%. NMR 1 H (400 MHz, MeOD): δ (ppm) 7.83 (d, J= 8.4 Hz, 2H), 7.48 (m, 3H), 5.31 (d, J= 3.2 Hz, 1H), 5.06 (dd, J= 11.2 Hz, J= 3.2 Hz, 1H), 4.61 (d, NMR 13 C (100 MHz, MeOD): δ (ppm) 173.59, 172.07, 172.06, 171.76, 170.31, 135.72, 132.66, 129.56, 128.34, 102.77, 72.23, 71.84, 71.65, 71.46, 71.36, 70.58, 70.10, 68.23, 62.75, 51.65, 40.88, 22.90, 20.56, 20.54, 20.52. HRMS: TOF (ESI+) m / z C 27 H 39 N2O 12 (M+H) +Calculated value 583.2503, measured value 583.2504.

[0263] [ka]

[0264] Compound E (1 eq, 0.15 mmol) was solubilized in a mixture 1:1 H2O / MeOH and Amberlist basic resin was added. The mixture was stirred for 2 hours until disappearance of starting material. The resin was filtered and the filtrate was concentrated under vacuum to give the corresponding product as a colorless oil. The oil was then lyophilized to give compound 4 as a white solid. Yield: quantitative. NMR 1 H (400 MHz, MeOD): δ (ppm) 7.83 (m, 2H), 7,49 (m, 3H), 4.42 (d, J= 8.4 Hz, 1H), 3.93 (m, 2H), 3.81-3.56 (m, 16H), 3.46 (m, 1H), 1.96 (s, 3H)NMR 13 C (100 MHz, MeOD): δ (ppm) 174.14, 170.37, 135.69, 132.66, 129.57, 128.32, 103.09, 76.77, 73.52, 71.63, 71.58, 71.40, 70.60, 69.73, 69.70, 62.55, 54.32, 40.91, 23.07 HRMS: TOF (ESI+) m / z C 21 H 32 N2O9Na (M+Na) + Calculated value: 479.2006, measured value: 479.1997.

[0265] 3. Synthesis of Compound 5 (Invention)

[0266] [ka]

[0267] To a solution of A (160 mg, 0.246 mmol) in dry DCM (5 mL) was added 6-nitro-2-nicotinoyl chloride (91 mg, 0.492 mmol) and DMAP (90 mL, 0.738 mmol). After stirring at 20° C. for 2 h, the reaction mixture was diluted with DCM and washed with 1M HCl and saturated aqueous NaHCO3. The combined organic phases were washed with brine, dried over MgSO4, and the mixture was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, AcOEt / MeOH: 90 / 10) to give amide F (94 mg, 0.149 mmol) as a white solid. Yield: 61%. 1 H NMR (300 MHz, CDCl3): δ = 1.92 (s, 3H, AcO), 1.97 (s, 3H, AcO), 2.01 (s, 3H, AcO), 2.13 (s, 3H, NHAc), 3.63-3.94 (m, 13H), 4.06-4.21 (m, 3H, H-2, H-6a, H-6b), 4.76 (d, 1H, J 1,2 = 8.6 Hz, H-1), 5.12 (dd, 1H, J 3,2 = 11.3 Hz, J 3,4 = 3.5 Hz, H-3), 5.28 (dd, 1H, J 4,3 = 3.4 Hz, J 4,5 = 0.8 Hz, H-4), 6.29 (d, 1H, J = 9.1 Hz, NHAc), 8.22 (d, 1H, J = 7.9 Hz, Py), 8.37 (d, 1H, J = 8.1, J = 0.9 Hz, Py), 8.55 (d, 1H, J = 7.7, J = 0.9 Hz, Py). 13C NMR (100.6 MHz, CDCl3): δ = 20.6 (2 x CH3, 2 x AcO), 20.7 (CH3, AcO), 20.69 (CH3, AcO), 23.2 (CH3, NHAc), 39.4 (CH2), 50.9 (CH, C-5), 61.5 (CH2, C-6), 66.8 (CH, C-4), 68.7 (CH2), 69.7 (CH2), 70.49 (CH2), 70.51 (CH2) 70.6 (CH), 70.7 (CH, C-3), 71.1 (CH2), 101.9 (CH, C-1), 120.1 (CH, Py), 127.7 (CH, Py), 141.6 (CH, Py), 149.6 (C, HRMS (ESI): m / z C 26 H 37 N4O 14 [M + H] + Calculated value: 629.2306, Measured value: 629.2307

[0268] [ka]

[0269] Compound F (88 mg, 0.139 mmol) was dissolved in anhydrous MeOH (2.8 mL) and sodium methoxide (42 μL of a 1 M solution in MeOH, 0.042 mmol) was added. The mixture was stirred for 2 h, neutralized by adding acidic resin Amberlite IR120(H), filtered and the solvent was evaporated to dryness. The crude product of the reaction was used in the next step without further purification.

[0270] To a solution of the deprotected sugar (0.139 mmol) in MeOH (3 mL) was added Pd / C (24 mg, 20% w). The resulting suspension was stirred under H2 atmosphere (1 atm) for 8 h. Pd / C was removed by filtration through Celite® and the filtrate was evaporated under reduced pressure. The crude product of the reaction was lyophilized to give aniline G (54 mg, 0.114 mmol) as a white solid. Yield: 82% 1 H NMR (300 MHz, CD3OD): δ = 1.97 (s, 3H, NHAc), 1.49 (t, 2H, J = 5.9 Hz), 3.57-3.80 (m, 13H), 3.84 (bd, 1H, J = 3.1 Hz), 4.06 (m, 2H), 4.43 (d, 1H, J 1,2 = 8.5 Hz, H-1), 6.69 (dd, 1H, J = 8.4 Hz, J = 0.8 Hz, Py), 7.30 (dd, 1H, J = 7.3, J = 0.7 Hz, Py), 7.53 (dd, 1H, J = 8.2, J = 7.3 Hz, Py). 13 C NMR (100.6 MHz, CDCl3): δ = 23.1 (CH3, NHAc), 40.2 (CH2), 54.3 (CH, C-5), 62.6 (CH2, C-6), 69.7 (CH), 69.8 (CH2), 70.8 (CH2), 71.40 (CH2), 71.47 (CH2), 71.54 (CH2), 70.6 (CH), 73.4 (CH, C-3), 76.7 (CH), 103.2 (CH, C-1), 112.0 (CH, Py), 113.1 (CH, Py), 139.4 (CH, Py), 148.9 (C, Py), 160.2 (C, Py), 167.5 (C, CO), 174.2 (C, NHAc). HRMS (ESI): m / z C 20 H 32 N4O9[M + Na] + Calculated value: 495.2067, Measured value: 495.2063.

[0271] Diazonium salt derivative 5 was obtained by adding HBF4 (1.1 equivalents) and tBuONO (1.1 equivalents) in ultrapure water to amino derivative G. After vortexing for 5 minutes, the formation of the diazonium salt was confirmed by using a solution of 2-hydroxynaphthol. A mixture of one drop of each solution shows a red coloration, which confirms the formation of the diazonium salt.

[0272] 4. Synthesis of Compound 6 (Invention)

[0273] [ka]

[0274] To a solution of 6-(((benzyloxy)carbonyl)amino)-2-naphthoic acid (210 mg, 0.644 mmol) and HOBt (87 mg, 0.644 mmol) in dry DCM (4.3 mL) was added dropwise DIC (90 mL, 0.738 mmol) at 0° C. After stirring for 15 min, a solution of A (280 mg, 0.429 mmol) containing TEA (88 μL, 0.644 mmol) was added dropwise. After stirring for 2 h at room temperature, the reaction mixture was diluted with DCM and washed with 1 M HCl and saturated aqueous NaHCO3. The combined organic phase was washed with brine, dried over MgSO4, and the mixture was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, AcOEt / MeOH: 95 / 5) to give the amide H (94 mg, 0.120 mmol) as a white solid. Yield: 28%. 1 H NMR (300 MHz, CD3OD): δ = 1.91 (s, 3H, AcO), 1.94 (s, 3H, AcO), 1.97 (s, 3H, AcO), 2.05 (s, 3H, NHAc), 3.53-3.91 (m, 13H), 4.00 (d, 2H, J = 4.0 Hz), 4.07 (dd, 1H, J = 11.3 Hz, J = 8.5 Hz), 4.42 (s, 2H, PhCH2), 4.48 (d, 1H, J 1,2 = 8.5 Hz, H-1), 5.00 (dd, 1H, J 3,2 = 11.3 Hz, J 3,4 = 3.4 Hz, H-3), 5.24 (dd, 1H, J 4,3 = 3.4 Hz, J 4,5 = 0.8 Hz, H-4), 6.73 (d, 1H, J = 2.3 Hz), 7.06 (d, 1H, J = 8.9 Hz, J = 2.3 Hz), 7.19–7.41 (m, 5H), 7.53 (d, 1.7 Hz, J = 8.8). 8.18 (d, 1H, J = 1.8 Hz). 13 C NMR (100.6 MHz, CD3OD): δ = 20.5 (CH3, AcO), 20.6 (2 x CH3, 2 x AcO), 22.9 (CH3, NHAc), 40.8 (CH2), 48.3 (CH2), 51.5 (CH, C-76), CH 2,6,61. C-4), 70.1 (CH2), 70.7 (CH2), 71.3 (CH2), 71.4 (CH2), 71.6 (CH2), 71.7 (CH), 72.2 (CH, C-3), 102.8 (CH, C-1), 104.3 (CH, Ar (CH2), 12,0 126.9 (CH, Ar), 127.3 (C, Ar), 127.9 (C, Ar), 128.0 (CH, Ar), 128.4 (2 X CH, Ar), 128.9 (CH, Ar), 129.5 (2 X CH, Ar), 130.9 (CH71 (C, 8), 1 (C), 170.6 (C, AcO), 171.8 (C, AcO), 172.0 (2 x C, AcO, CO), 173.6 (C, NHAc). HRMS (ESI): m / z C 39 H 48 N3O 14 [M + H]+ Calculated value: 782.3136, Measured value: 782.3142.

[0275] [ka]

[0276] Compound H (86 mg, 0.109 mmol) was dissolved in MeOH (2.2 mL) and basic resin (190 mg) was added. The mixture was stirred for 2 h, filtered and the solvent was evaporated to dryness. The crude product of the reaction was used in the next step without further purification.

[0277] To a solution of the deprotected crude sugar (0.109 mmol) in MeOH (4 mL) was added Pd / C (17 mg, 20% w). The resulting suspension was stirred under H2 atmosphere (1 atm) for 6 h. Pd / C was removed by filtration through Celite® and the filtrate was evaporated under reduced pressure. The crude product of the reaction was lyophilized to give aniline I (51 mg, 0.098 mmol) as a white solid. Yield: 90%. 1 H NMR (300 MHz, CD3OD): δ = 1.95 (s, 3H, NHAc), 3.43 (t, 2H, J = 5.4 Hz), 3.48-3.84 (m, 21H), 3.92 (m, 2H), 4.38 (d, 1H, J 1,2 = 8.5 Hz, H-1), 6.97 (d, 1H, J = 2.2 Hz), 7.05 (dd, 1H, J = 8.7 Hz, J = 2.2 Hz), 7.57 (d, 1H, J = 8.7 Hz), 7.73 (m, 2H), 8.19 (d, 1H, J = 1.8 Hz). 13C NMR (100.6 MHz, CD3OD): δ = 23.1 (CH3, NHAc), 40.9 (CH2), 54.3 (CH, C-2), 62.5 (CH2, C-6), 69.68 (CH, C-4), 69.74 (CH2), 71.4 (CH2), 71.6 (2 X CH2), 73.5 (CH), 76.7 (CH, C-3), 103.1 (CH, C-1), 108.3 (CH, Ar), 120.4 (CH, Ar), 125.1 (CH, Ar), 127.7 (C, Ar), 128.3 (C, Ar), 128.9 (CH, Ar), 131.2 (CH, Ar), 138.4 (C), 149.2 (C), 170.7 (C, NHAc), 174.1.0 (C, CO). HRMS (ESI): m / z C 25 H 35 N3O9[M + Na] + Calculated value: 544.2271, Measured value: 544.2270.

[0278] Diazonium salt derivative 6 was obtained by adding HBF4 (1.1 equivalents) and tBuONO (1.1 equivalents) in ultrapure water to amino derivative I. After 5 min of vortexing, the formation of diazonium salt was confirmed by using a solution of 2-hydroxynaphthol. A mixture of one drop of each solution shows a red coloration confirming the formation of diazonium salt.

[0279] 5. Synthesis of Compound 7 (Invention)

[0280] [ka]

[0281] Compound J (1 eq, 1.38 mmol, 0.900 g) was solubilized in DCM, and DMAP (3 eq, 4.14 mmol, 0.506 g), 4-nitrobenzoyl chloride (3 eq, 4.14 mmol, 0.769 g) were added. The mixture was stirred at room temperature for 4 h. The organic solution was washed with HCl 1M, water, and NaHCO3, and dried over MgSO4. After concentration, the orange oil was purified by flash chromatography (SiO2, AcOEt / MeOH 85 / 15) to give a pale yellow solid K (0.270 g, 0.43 mmol). Yield: 31.1%. 1 H NMR (300 MHz, CDCl3): δ (ppm) 8.27 (m, 2H), 7.99 (m, 2H), 7.19 (br s, 1H), 5.29 (m, 3H, H2, H3, H4), 4.86 (d, 1H, J =1.6 Hz, H-1), 4.25 (m, 1H, H-6), 4.07 (m, 2H), 3.87-3.66 (m, 13H, H-5, H-6, PEG), 2.15 (s, 3H, AcO), 2.09 (s, 3H, AcO), 2.03 (s, 3H, AcO), 1.98 (s, 3H, AcO). 13 C NMR (75 MHz, CDCl3): δ (ppm) 170.86 (C, CO), 170.30 (2 x C, 2 123.83 (2 x C, CH), 97.81 (CH, C-1), 70.78 (CH2), 70.43 (CH2), 70.14 (CH2), 69.81 (CH), 69.68 (CH), 68.71 (CH2), 67.48 (CH2), 66.23 (CH), 62.60 (CH2), 40.25 (CH2), 21.00 (CH3, AcO), 20.88 (CH3, AcO), 20.82 (2 x CH3, 2 x AcO). HRMS: TOF (ESI+) m / z C 27 H 36 N2O15 Calculated value of Na: 651.2013, measured value: 651.2002.

[0282] [ka]

[0283] Compound K (1 equiv, 0.99 mmol, 625 mg) was solubilized in a mixture 1:1 HO / MeOH and Amberlist basic resin was added. The mixture was stirred for 2 h until disappearance of starting material. The resin was filtered and the filtrate was concentrated under vacuum to give product L as a white solid (475 mg, 1 mmol, quantitative, not dried). 1 H NMR (300 MHz, MeOD): δ (ppm) 8.32 (m, 2H), 8.04 (m, 2H), 4.72 (d,1H, J =1.72 Hz), 3.82 (m, 3H), 3.72-3.54 (m, 16H). 13 C NMR (75 MHz, MeOD): δ (ppm) 168.28 (C, CO), 151.05 (C), 141.43 (C), 129.75 (2 x CH, Ar), 124.62 (2 x CH, Ar), 101.74 (C-1), 74.63, 72.58, 72.13, 71.61, 71.42, 71.36, 70.47, 68.65, 67.68, 62.98, 41.22 (CH2). HRMS: TOF (ESI+) m / z C 19 H 27 N2O 11 Calculated value 459.1615, measured value 459.1611

[0284] [ka]

[0285] To a solution of L (90 mg, 0.197 mmol) in MeOH (3 mL) was added Pd / C (25 mg, 20% w). The resulting suspension was stirred under H2 atmosphere (1 atm) for 4 h. Pd / C was removed by filtration through Celite® and the filtrate was evaporated under reduced pressure. The crude product of the reaction was lyophilized to give the aniline M (69 mg, 0.160 mmol) as a white solid. Yield: 81%. 1 H NMR (400 MHz, CD3OD): δ = 3.52-3.68 (m, 14H), 3.69-3.74 (2H, m), 3.78-3.85 (3H, m), 6.67 (d, 1H, J = 8.8 Hz, Ar), 7.71 (d, 1H, J = 8.8 Hz, Ar). 13 C NMR (100.6 MHz, CDCl3): δ = 40.7 (CH2), 62.9 (CH2, C-6), 68.6 (CH), 70.8 (CH2), 71.28 (CH2), 71.32 (CH2), 71.37(CH2), 71.5 (CH), 72.1 (CH), 72.6 (CH), 74.6 (CH), 101.7 (CH, C-1), 114.8 (2 x CH, Ar), 123.2 (C, Ar), 129.9 (2 x CH, Ar), 153.1 (C, Ar),170.2 (C, amide). HRMS (ESI): m / z C 19 H 30 NO9Na [M + Na] + Calculated value: 453.1849, Measured value: 453.1841.

[0286] Diazonium salt derivative 7 was obtained by adding HBF4 (1.1 equivalents) and tBuONO (1.1 equivalents) in ultrapure water to amino derivative M. After 5 minutes of vortexing, the formation of the diazonium salt was confirmed by using a solution of 2-hydroxynaphthol. A mixture of one drop of each solution shows a red coloration confirming the formation of the diazonium salt.

[0287] 6. Synthesis of Compound 8 (Comparative)

[0288] [ka]

[0289] Compound J (1 eq, 0.3 mmol, 0.230 g) was solubilized in DCM, and DMAP (3 eq, 0.9 mmol, 0.12 g), 4-nitrobenzoyl chloride (3 eq, 0.9 mmol, 0.167 g) were added. The mixture was stirred at room temperature for 4 h. The organic solution was washed with HCl 1M, water, and NaHCO3, and dried over MgSO4. After concentration, the orange oil was purified by flash chromatography (SiO2, AcOEt / MeOH: 85 / 15) to give a pale yellow solid N (70 mg, 0.1 mmol). Yield: 34%. 1 H NMR (400 MHz, CD3OD): δ (ppm) 7.82 (m, 2H), 7.49 (m, 3H), 5.26 (m, 3H, H2, H3, H4), 4.87 (d, J=1.36 Hz, 1H), 4.23 (dd, J=5 Hz, J=12,3 Hz, 1H, H6), 4.10 (m, 2H, H5, H6), 3.82 (m, 1H), 3.71-3.58 (m, 12H), 2.12 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H). 13C NMR (100 MHz, CD3OD): δ (ppm) 172.33 (C, CO), 171.55 (C, CO), 171.49 (C, CO), 171.47 (C, CO), 170.23 (C, CO), 135.69 (C), 132.62 (CH), 129.53 (2 X CH), 128.29 (2 67.30 (CH2), 63.57 (CH2, C-6), 40.93 (CH2), 20.65 (CH3, AcO), 20.62 (CH3, AcO), 20.61 (CH3, AcO), 20.55 (CH3, AcO). HRMS: TOF (ESI+) m / z C 27 H 38 N0 13 Calculated value 584.2343, measured value 584.2344

[0290] [ka]

[0291] Compound N (1 equiv, 0.1 mmol, 70 mg) was solubilized in a mixture 1:1 HO / MeOH and Amberlist basic resin was added. The mixture was stirred for 2 h until disappearance of starting material. The resin was filtered and the filtrate was concentrated under vacuum to give the corresponding product 8 (50 mg, 0.1 mmol, quantitative) as a white solid. 1 H NMR (400 MHz, D2O): δ (ppm) 7.84 (m, 2H), 7.69 (m, 1H), 7.60 (m, 2H), 4.88 (d, J=1.68 Hz, 1H), 3.97 (m, 1H), 3.93-3.63 (m, 18H). 13C NMR (100 MHz, D2O): δ (ppm) 171.11 (C, CO), 133.65 (C), 132.21 (CH, Ar), 128.83 (2 CH, Ar), 127.10 (2 CH, Ar), 99.93 (C-1), 72.74, 70.54, 69.99, 69.66, 69.58, 68.89, 66.76, 66.37, 60.93, 39.57. HRMS: TOF (ESI+) m / z C 19 H 29 Calculated value of NO9Na: 438.1740, measured value: 438.1730.

[0292] 7. Synthesis of Compound 11 (Comparative)

[0293] [ka]

[0294] A mixture of compound O (550 mg, 0.917 mmol) and 20% palladium on carbon (110 mg) in ethyl acetate (9.2 mL) was stirred at room temperature under a hydrogen atmosphere (1 atm) for 2 h. The reaction mixture was filtered through a pad of Celite and the solvent was evaporated in vacuo to give the hydrogenated product as a yellow amorphous solid, which was used without further purification.

[0295] Ethyl carbazate (95 mg, 0.917 mmol) was added to a solution of CDI (149 mg, 0.917 mmol) in dry THF (9.2 mL) and stirred at room temperature under nitrogen for 15 min. Then the crude product (0.917 mmol, dissolved in 4 mL THF) was added dropwise and stirring was continued overnight. The mixture was diluted with AcOEt and washed with 0.5 M aqueous HCl. The organic phase was dried and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, EtOAc-MeOH, 90:10) to give P (311 g, 0.541 mmol) as a white solid. Yield: 59%. 1 H NMR (300 MHz, CDCl3): δ = 1.25 (t, 3H, J = 7.1 Hz, CH3CH2O), 1.88 (s, 3H, AcO), 1.92 (s, 3H, AcO), 2.03 (s, 3H, AcO), 2.09 (s, 3H, NHAc), 3.59-4.10 (m, 16H), 4.16 (d, 2H, J = 7.1 Hz, CH3CH2O), 4.73 (d, 1H, J = 8.6 Hz, H-1), 5.05 (dd, 1H, J 3,2 = 11.2 Hz, J 3,4 = 3.3 Hz, H-3), 5.26 (bd, 1H, J 4,3 = 3.3 Hz, H-4), 6.78 (d, 3H, J = 9.0 Hz, Ph, NH), 7.16 (s, 1H, NH), 7.22 (s, 1H, NH), 7.24 (d, 2H, J = 9.0 Hz, Ph), 7.67 (s, 1H, NH). 13 C NMR (100.6 MHz, CDCl3): δ = 14.4 (CH3, CH3CH2O), 20.7 (3 x CH3, 3 x AcO), 23.0 (CH3, NHAc), 50.4 (CH, C-5), 61.6 (CH2, C-6), 62.3 (CH2, CH3CH2O), 66.7 (CH, C-4), 67.6 (CH2), 68.6 (CH2), 69.9 (CH2), 70.5 (CH, CH2), 70.83 (CH), 70.86 (CH2), 71.2 (CH2), 101.9 (CH, C-1), 114.9 (2 x CH, Ph), 121.7 (2 x CH, Ph), 131.5 (C, Ph), 154.8 (C), 156.4 (C), 157.5 (C), 170.4 (C, AcO), 170.5 (C, AcO), 170.7 (C, AcO), 171.1 (C, NHAc). HRMS (ESI): m / z C 30 H 44 N4O 15 Na [M + Na] +Calculated value: 723.2701, Measured value: 723.2700.

[0296] [ka]

[0297] Compound P (286 mg, 0.497 mmol) was dissolved in dry MeOH (10 mL) and sodium methoxide (149 μL of a 1 M solution in MeOH, 0.149 mmol) was added. The mixture was stirred for 4 h, K2CO3 (137 mg, 0.994 mmol) was added, and the resulting mixture was stirred at 75 °C for 1.5 h (μW, 50 W). The mixture was diluted with 10 mL H2O, neutralized with Amberlite IR120 (H), filtered, and the solvent was evaporated to dryness. The substrate was dissolved in water and subjected to lyophilization to give 11 (226 mg, 0.427 mmol) as a brown solid. Yield: 86%. 1 H NMR (300 MHz, MeOD): δ = 1.97 (s, 3H, NHAc), 3.45 (ddd, 1H, J = 6.5 Hz, J = 5.3 Hz, J = 1.0 Hz, H-5), 3.53 (dd, 1H, J 3,2 = 10.7 Hz, J 3,4 = 3.3 Hz, H-3), 3.62-3.79 (m, 10H), 3.86-3.97 (m, 4H), 4.19 (m, 2H), 4.44 (d, 1H, J 1,2 = 8.4 Hz, H-1), 6.78 (d, 3H, J = 9.0 Hz, Ph, NH), 7.24 (d, 1H, J = 9.1 Hz, Ph), 7.35 (d, 1H, J = 9.1 Hz, Ph). 13C NMR (100.6 MHz, CDCl3): δ = 23.0 (CH3, NHAc), 54.4 (CH, C-5), 62.6 (CH2, C-6), 68.9 (CH2), 69.7 (CH), 70.8 (CH2), 71.6 (CH2), 71.7 (CH2), 71.8 (CH2), 73.6 (CH), 76.7 (CH), 103.1 (CH, C-1), 116.2 (2 x CH, Ph), 125.6 (C, Ph), 129.0 (2 x CH, Ph), 156.1 (2 XC, Urazol), 160.1 (C, Ph), 174.2 (C, HRMS (ESI): m / z C22H31N4O11 [M - H]+ calculated value: 527.1989, measured value: 527.1974.

[0298] 8. Synthesis of Compound 12 (Invention)

[0299] [ka]

[0300] After oxidation of compound 11 in the presence of 1.1 equivalents of hydantoin in acetonitrile, compound 12 was obtained. The formation of the PTAD derivative 12 was confirmed by the red coloration of the solution.

[0301] 3. Preparation of chemically modified AAV - AAV2 and AAV8 production and purification AAV2 vectors were generated from two plasmids: pHelper, PDP2-KANA encoding AAV Rep2-Cap2 and adenovirus helper genes (E2A, VA RNA, and E4), and pVector ss-CAG-Luc-eGFP or pVector ss-CAG-eGFP. AAV8 vectors were generated from two plasmids: pHelper, PDP8-KANA encoding AAV Rep2-Cap8 and adenovirus helper genes (E2A, VA RNA, and E4), and pVector ss-CAG-eGFP. All vectors were generated by transient transfection of HEK293 cells using the calcium phosphate-HeBS method. 48 hours after transfection, vector-producing cells were harvested. For extraction of AAV2 particles, cells were first treated with Triton-1% and benzonase (25 U / mL) at 37°C for 1 hour. This step was omitted for AAV8 vectors, but the subsequent steps were identical. The bulk was centrifuged at 2000 rpm for 20 min and subjected to freeze-thaw cycles to liberate vector particles. Cell debris was removed by centrifugation at 2500 rpm for 15 min. Cell lysates were precipitated with PEG overnight and clarified by centrifugation at 4000 rpm for 1 h. The precipitate was then incubated with benzonase at 37°C for 30 min and collected by centrifugation at 10000g for 10 min at 4°C. Vectors were purified by two-fold cesium chloride (CsCl) gradient ultracentrifugation. The virus suspension was then transferred to a Slide-a-Lyzer cassette (Pierce) with gentle agitation, followed by centrifugation in dPBS (CaCl) for 1 h. ++ and Mg ++ The cells were subjected to four consecutive rounds of dialysis against 100 mM NaCl (containing

[0302] - Coupling and purification Diazonium salt derivatives 2, 5, 6, and 7 were obtained by adding HBF4 (1.1 equivalents) and tBuONO (1.1 equivalents) in ultrapure water to the corresponding amino compounds. After 5 minutes of vortexing, the formation of diazonium salts was confirmed by using a solution of 2-hydroxynaphthol. A drop of each solution mixed together shows a red coloration, confirming the formation of diazonium salts. Then, AAV2-GFP-Luc, AAV2-GFP, or AAV8-GFP (1E12vg, 2.49nmol) was added to a solution of TRIS buffer pH=9.3 containing 2 (invention), 3 (comparison), 4 (comparison), 5 (invention), 6 (invention), 7 (invention), or 8 (comparison) at a molar ratio of 3E5 or 3E6 equivalents, and incubated at 20°C for 4 hours. The vector-containing solution was then dialyzed against dPBS+0.001% Pluronic to remove free molecules not coupled to the AAV capsid.

[0303] For modification of the amino groups of the AAV2 capsid with compounds 9 and 10, the procedure is described in Chemical Science, 2020, 11, pp. 1122-1131.

[0304] For the double modification of tyrosine and amino groups of AAV2-GFP, coupling was performed in a molar ratio of 3E5 equivalents, first with 2 and tyrosine modification, and 5 min later with amino group modification using compound 10, and incubated for 4 hours at 20° C. The solution containing the vector was then dialyzed against dPBS+0.001% Pluronic to remove free molecules not coupled to the AAV capsid.

[0305] For PTAD derivative 12 (invention, 3E6 equivalents) and urazole derivative 11 (comparison, 3E6 equivalents), the compounds were added in small portions to AAV2-GFP in TBS buffer. Five minutes after addition, the solution containing the vector was dialyzed against dPBS+0.001% Pluronic to remove free molecules not coupled to the AAV capsid.

[0306] 4. Characterization of chemically modified AAVs (inventive and comparative) Materials and Methods - Viral genome extraction 3 μL of AAV2, AAV8, or chemically modified AAV2 or AAV8 was treated with 20 units of DNase I (Roche #04716728001) at 37° C. for 45 minutes to remove residual DNA in the vector samples. After DNase I treatment, 20 μL of Proteinase K 20 mg / mL (MACHEREY-NAGEL #740506) was added and incubated at 70° C. for 20 minutes. DNA was then extracted from the purified AAV vectors using extraction columns (NucleoSpin® RNA Virus).

[0307] - Quantitative real-time PCR analysis Quantitative real-time PCR (qPCR) was performed using a StepOnePlus™ Real-Time PCR System Upgrade (Life technologies). All PCRs were performed in a 20 μL final volume PCR containing primers, probe, PCR Master Mix (TaKaRa), and 5 μL of template DNA (plasmid standard or sample DNA). qPCR was performed with an initial denaturation step at 95°C for 20 s, followed by 45 cycles of denaturation at 95°C for 1 s and annealing / extension at 56°C for 20 s. Plasmid standards were prepared by serially diluting seven times (10% of the plasmid) of plasmid pTR-UF-11 (ATCC® MBA-331™) linearized with Sca-I restriction enzyme. 8 ~10 2 The plasmid p53 was generated using a plasmid p53 containing 10 copies of the p53 gene.

[0308] - Western Blotting All vectors were denatured with Laemmli sample buffer at 100°C for 5 min and separated on SDS-PAGE 10% Tris-glycine polyacrylamide gels (Life Technologies). Precision plus Protein All Blue Standards (BioRad) were used as molecular weight size markers. The proteins were transferred to nitrocellulose membranes using transfer buffer (25 mM tris / 192 mM glycine / 0.1 (w / v) SDS / 20% MeOH) in a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) at 150 mA for 1 h, and the membranes were saturated with 5% semi-skimmed milk in PBS-Tween (0.1%) or 1% gelatin, 0.1% Igepal in PBS-Tween (0.01%) for 2 h at room temperature. After saturation, the membranes were probed overnight at 4° C. with antisera against AAV2 and chemically modified AAV2 (polyclonal or B1 monoclonal), as well as with FITC-soybean agglutinin or FITC-concanavalin A. To remove unbound reagents, three washes were performed between each step with PBS-Tween (0.1%) for 15 min at room temperature. Bands were visualized by chemiluminescence using alkaline phosphatase (AP) or horseradish peroxidase (HRP)-conjugated secondary antibodies and captured on X-ray film.

[0309] - Immuno dot blot Before assembling a dot blot manifold (Bio-Rad), undenatured and chemically modified AAV2 or AAV8 were deposited onto nitrocellulose paper that had been briefly soaked in PBS. The nitrocellulose membranes were treated as for Western blotting. To visualize assembled capsids, the membranes were probed with antiserum against AAV2 capsid (A20 antibody) or AAV8 (ADK8 antibody).

[0310] - Infectivity by FACS analysis HEK293 and HUH7 cells were grown in 24-well plates at approximately 2.5 × 10 5 Cells were seeded at a density of 1000 cells / well. Cells were then incubated overnight at 37°C and 5% CO2 to reach 50% confluence. Cells were transduced with AAV2 or chemically modified AAV2 vectors at MOIs of 1E4 and 1E5 in culture medium. All AAV vectors encoded GFP. 48 hours after transduction, the percentage of GFP-positive cells was measured by FACS analysis. Cells were dissociated with trypsin-EDTA (Sigma-Aldrich), fixed with 4% paraformaldehyde, and analyzed on a BD-LSRII flow cytometer (BD Bioscience). All data were processed by FlowJo (V10; Flowjo LLC, Ashland, OR).

[0311] - results The results are shown in Figures 1 to 11.

[0312] To evaluate the purity and integrity of these GalNAc-AAV2 and GalNAc-AAV8, silver staining of the different samples was performed. As shown in Figure 1-A, Figure 4-C, Figure 6-C, and Figure 8-C, and as expected, the ratio VP1:VP2:VP3 was intact after reaction and subsequent dialysis. To evaluate the purity and integrity of Man-AAV2, silver staining of the samples was performed. As shown in Figure 7-C, and as expected, the ratio VP1:VP2:VP3 was intact after reaction and subsequent dialysis. For GalNAc-AAV2, GalNAc-AAV8, and Man-AAV2, the molecular weight of each VP increased with the coupling of ligands 2, 6, and 7.

[0313] The coupling reaction of sugars on the AAV surface was further studied by dot blot analysis using soybean lectin and concanavalin A lectin, which selectively bind GalNAc and mannose residues, respectively. Immunostaining with A20 antibody, which recognizes assembled AAV2 capsids, and ADK8 antibody, which binds assembled AAV8 capsids, respectively, was also performed. Positive dots with A20 and ADK8 antibodies indicated that AAV2 and AAV8 were intact after the coupling procedure with compounds 2, 4, 6, 7, 8, 9, 10, 11, and 12 and subsequent dialysis (Figures 2-A, 5-A, 6-A, 7-A, 8-A, and 9-A).

[0314] Positive signals with soybean lectin were observed only when AAV2 was co-incubated with diazonium compounds 2 and 6 and PTAD derivative 12, and when AAV8 was co-incubated with compound 2, demonstrating efficient click tyrosine reactions in AAV2 and AAV8 capsids (Figures 2B, 6B, 8B, and 9B). Indeed, no detection was observed for compound 4, which lacks the diazonium group, and compound 11, which lacks the PTAD group, proving that compounds 2, 6, and 12 (invention) were covalently linked to AAV2 and AAV8 capsids and not physically adsorbed thereto.

[0315] A positive signal with mannose-binding concanavalin A lectin was observed when AAV2 was co-incubated with compound 7 (invention), revealing an efficient click tyrosine reaction in AAV2 (Figure 7-B). Indeed, no detection was observed for compound 8 (comparison), which lacks the diazonium group, proving that compound 7 was covalently linked to AAV2 and not physically adsorbed thereto.

[0316] For dual modification of the tyrosine and amino groups of the AAV2 capsid, sequential co-incubation with 2 and 10 showed positive dots with concanavalin A and soybean lectin, demonstrating efficient click-tyrosine reaction and amino group modification in AAV2 (Figure 5B).

[0317] Western blot analysis using capsid-specific polyclonal antibodies demonstrated that the VP capsid subunit was intact despite the chemical process for AAV2-Luc and AAV2-GFP (Figures 3A and 4A). Importantly, the bands detected by lectin clearly demonstrated that Compound 2 (invention) was covalently linked to VP for AAV2-Luc and AAV2-GFP (Figures 3B, 3C, and 4B).

[0318] In contrast, capsid subunits derived from AAV2-Luc and AAV2-GFP incubated with 3 and 4 (comparison) at the same ratio did not yield positive bands after incubation with the specific lectins, indicating that no coupling occurs without the diazonium functional group (Figures 3B, 3C, 4B).

[0319] The effect of chemical coupling with compound 2 (invention), which contains a GalNAC moiety, on the infectivity of AAV2-GFP vectors was evaluated.

[0320] Infectivity was assessed by FACS analysis in HEK293 and HuH7 cells. Both the % of cells expressing GFP and the intensity of GFP were measured. As shown in Figures 10 and 11, GalNAC-AAV2-GFP particles are infectious in both cell lines. Chemical modification of these particles has a significant effect on transduction efficiency and off-targeting properties. When using the hepatic cell line HuH7, viral particles chemically modified with compound 2 showed higher levels of transduction than those obtained when using the non-hepatic cell line HEK293.

Claims

1. An adeno-associated virus (AAV) comprising a viral genome into which an exogenous nucleic acid sequence has been introduced, the virus having the ability to infect a cell and having at least one chemically modified tyrosine residue in a capsid, the chemically modified tyrosine residue being represented by the following formula (I): 【Chemistry 1】 (In the formula, -X 1 teeth, 【Chemistry 2】 is selected from the group consisting of - Ar is an optionally substituted aryl or heteroaryl moiety. , the adeno-associated virus.

2. The at least one chemically modified tyrosine residue has the following formula (Ia): 【Chemistry 3】 (In the formula, -X 1 and Ar are as defined in claim 1, - spacer is a group for linking the "Ar" group to the functional moiety "M" and preferably contains up to 1000 carbon atoms, preferably in the form of a chemical chain which may optionally contain heteroatoms and / or cyclic moieties; - n is 0 or 1, - M is a functional moiety including a steric shielding agent, a labeling agent, a cell type specific ligand, or a drug moiety. The AAV of claim 1,

3. X 1 and / or "Ar" is selected from substituted or unsubstituted phenyl, pyridyl, naphthyl, and anthracenyl.

4. At least one chemically modified tyrosine has the following formula (1c): 【Chemistry 4】 (In the formula, -X 2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -NH(C=S)- or -(C=S)-NH-, preferably -(C=O)-NH- or -(C=O)-O-; -X 2 is in the para, meta or ortho position of the phenyl group, preferably in the para position; - spacer, n and M are as defined in claim 2. The AAV of any one of claims 1 to 3,

5. - "Spacer", if present, is an optionally substituted saturated or unsaturated, linear or branched C 2 ~C 40 and / or selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyldiamines, and combinations thereof; - comprising or consisting of a cell type targeting ligand, preferably selected from mono- or polysaccharides, hormones, such as steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTP) or Angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, ScFv, spiegelmers, peptide aptamers, vitamins and drugs such as CB1 and / or CB2 ligands, An AAV described in any one of claims 2 to 4.

6. - The "spacer" (if present) is a linear or branched C 2 ~C 20 , polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers having from 2 to 20 monomers; and / or - "M" comprises or consists of a cell type specific ligand derived from a protein selected from transferrin, epidermal growth factor (EGF) and basic fibroblast growth factor beta FGF, a mono- or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, cross-linked GalNac or mannose-6-phosphate, an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7, and a vitamin such as folic acid, An AAV described in any one of claims 2 to 4.

7. - M is a cell type specific ligand for specifically targeting hepatocytes, having the formula (III): 【Chemistry 5】 and / or - the spacer is a polyethylene glycol chain containing from 2 to 10 monomers, An AAV described in any one of claims 2 to 6.

8. At least one chemically modified tyrosine has the following formula (II): 【Chemistry 6】 The AAV of claim 7.

9. It further has at least one additional chemically modified amino acid residue in the capsid different from a tyrosine residue, said amino acid residue preferably being of the following formula (V): 【Chemistry 7】 (In the formula, - N * is the nitrogen of the amino group of an amino acid residue, e.g., a lysine or arginine residue, - Ar, spacer, n and M have the same definitions as Ar, spacer, n and M of formula (II) defined in claim 2. The AAV of any one of claims 1 to 8, having an amino group chemically modified with a group.

10. The AAV according to any one of claims 1 to 9, which is a recombinant AAV, preferably selected from an AAV having a wild-type capsid, a naturally occurring serotype AAV, a variant AAV, a pseudotype AAV, an AAV having a hybrid or mutant capsid, and a self-complementary AAV.

11. A method for preparing an AAV comprising a viral genome into which an exogenous nucleic acid sequence has been introduced, having at least one chemically modified tyrosine residue in its capsid, and capable of infecting a cell, the method comprising the step of chemically modifying at least one tyrosine residue in the capsid with a chemical reagent having a reactive group selected from aryldiazonium and 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) moieties, under conditions conducive to reacting the reactive group with a tyrosine residue present in the capsid of the AAV to form a covalent bond.

12. The AAV may be represented by the formula (VId): 【Chemistry 8】 to produce a compound of formula (Ic): 【Chemistry 9】 (In the formula, -X 2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -C=S-NH or NH-C=S-, preferably -(C=O)-NH- or -(C=O)-O-, -X 2 is in the para, meta or ortho position of the phenyl group, preferably in the para position; - the spacer is a hydrocarbon group containing from 2 to 400 carbon atoms, - n is 0 or 1, - M is a chemical moiety, including a steric shielding agent, a labeling agent, a cell type specific ligand, or a drug moiety. The method of claim 11, comprising obtaining at least one chemically modified tyrosine residue in the capsid of said virus.

13. A pharmaceutical composition comprising an AAV according to any one of claims 1 to 10 and at least one pharma- ceutically acceptable excipient.

14. A pharmaceutical composition according to claim 13 for use as an in vivo diagnostic agent.

15. 14. The pharmaceutical composition according to claim 13 for use in gene therapy.

16. An in vivo diagnostic agent comprising an AAV described in any one of claims 1 to 10.

17. A gene therapy agent comprising an AAV described in any one of claims 1 to 10.

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