Chemically modified adeno-associated viruses

Abstract

The present invention relates to a chemically modified adeno-associated virus (AAV), a method for preparing the same, and its use in gene therapy. The chemically modified AAV is preferably prepared by incubating the AAV with a chemical reagent having an N-substituted luminol moiety under conditions conducive to reaction of the chemical reagent with tyrosine residues exposed on the surface of the AAV capsid, preferably by electrochemical methods.

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. Now, gene therapy is increasingly being used to treat a wide range of acquired diseases, such as cancer.

[0003] Gene therapy is based on the therapeutic delivery of nucleic acids into the nucleus of a patient's cells. The nucleic acid can then be inserted into the genome of the targeted cell or can remain episomal. Delivery of therapeutic nucleic acids to target cells of a subject can be carried out 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 (AAVs) have gained popularity as versatile vectors for gene therapy, particularly in vivo applications. The main advantages of recombinant AAVs (rAAVs) are their broad tropism, their high transduction efficiency, their persistent episomal expression, and their high safety profile, especially because wild-type AAVs have not been associated with any human diseases.

[0004] Human clinical trials using 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 approved Voretigene neparvovec, an adeno-associated viral vector serotype 2 (AAV2) capsid containing cDNA encoding the human retinal pigment epithelial 65 kDa protein (hRPE65), for the treatment of vision loss due to inherited retinal dystrophy caused by confirmed biallelic RPE65 mutations. As a further example, Zolgensma® (onasemnogene abeparvovec-xioi) has 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 gene defective in SMA, the SMN1 gene, in motor neurons.

[0005] Despite these successes, certain clinical trials have demonstrated several limitations of these vectors in the treatment of certain diseases. The primary limitation is their immunogenicity. Due to their non-integrating nature, systemic gene therapy using AAV vectors, especially in pediatric patients, can be limited by tissue proliferation, which leads to vector dilution over time. However, re-administration of the vector can be hindered by persistent neutralizing anti-AAV antibodies (NAb) induced after the initial administration of the viral vector. Furthermore, it has been shown that pre-existing humoral immunity to certain AAV serotypes, particularly serotype 2 AAV, is widespread in humans. Anti-AAV neutralizing antibodies (NAb) can completely block transduction in target tissues, resulting in a lack of efficacy, 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 tissue in which transgene expression is desired.

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

[0008] Several strategies have been proposed to overcome the drawbacks of AAV, especially serotype 2 AAV (AAV2), in gene therapy. Some of these strategies are based on AAV surface remodeling, i.e., modification of AAV capsid proteins.

[0009] The first option is to genetically modify the viral capsid. For example, it has been shown that mutations in surface-exposed tyrosine residues on AAV2 can prevent phosphorylation and subsequent ubiquitination, thereby avoiding proteasome-mediated degradation (Zhong et al., PNAS, 2008, 105, 7827-7832; Markusic et al., Molecular Therapy, 2010, 18, 2048-2056).

[0010] Another option is to introduce chemical modifications into the viral capsid to introduce ligands into the capsid or to mask certain exposed amino acids in order to alter the antigenicity, tropism, or transduction efficiency of AAV. For this purpose, it has been proposed to genetically incorporate unnatural amino acids with modified side chains (e.g., as in International Patent Application Publication No. 2015 / 062516). For example, unnatural amino acids containing azide groups are genetically inserted into the capsid prior to the coupling step with a ligand by a click reaction to alter its tropism for 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] In doing so, WO 2017 / 212019 proposes a method for chemically modifying AAV capsids by covalently coupling ligands bearing isothiocyanate groups that react with amino groups present in amino acid residues such as lysine or arginine.

[0013] WO 2021 / 005210 describes a method for chemically modifying tyrosine residues present in capsids by reaction with aryldiazonium-bearing ligands.

[0014] However, there remains a need for new methods that allow for modulation of the properties of AAV gene delivery vectors in gene therapy. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Patent Application Publication No. 2017 / 212019 [Patent Document 2] International Patent Application Publication No. 2021 / 005210 [Patent Document 3] International Patent Application Publication No. 2014144229 [Patent Document 4] EP2292779 [Patent Document 5] EP1310571 [Non-patent literature]

[0016] [Non-Patent Document 1] Zhong et al., PNAS, 2008, 105, 7827-7832 [Non-patent document 2] Markusic et al., Molecular Therapy, 2010, 18, pp. 2048-2056 [Non-patent document 3] Naso et al., Biodrugs, 2017, 31:317-334 [Non-patent document 4] Koerber et al., Molecular Therapy (2008), 16(10), pp. 1703-1709 [Non-patent document 5] Deverman et al., Nat Biotechnol (2016), 34(2), pp. 204–209 [Non-patent document 6] Buening et al., Curr Opin Pharmacol (2015), 24, 94-104 [Non-Patent Document 7] Koniev, O., Wagner, A., Chem. Soc. Rev., 44, p. 5495 (2015) [Non-patent document 8] Kolb et al., Angew. Chem. Int. Ed. 2001, 40, 2004-2021 [Non-Patent Document 9] Rudolf et al., Current opinion in Chemical Biology, 2013, 17:110-117 [Non-Patent Document 10] S. Depienne et al., Chem. Sci., 2021, 12, pp. 15374-15381 [Non-Patent Document 11] Horowitz et al., Bioconj Chem, 2011, 22(4):529-532 [Non-Patent Document 12] C.-C. Lin et al., Org. Lett., 2007, 9, pp. 2131-2134 Summary of the Invention

[0017] The present invention provides an adeno-associated virus (AAV) having at least one chemically modified tyrosine residue in the capsid, wherein the chemically modified tyrosine residue is represented by formula (I):

[0018] [ka]

[0019] (In the formula, -R A is -(Y) n -M, C1-C6 alkyl, optionally substituted C6-C 14 aryl, or optionally substituted (C6-C 14 aryl)-(C1-C3 alkyl), - Each R B are independently of the formula -(Y) n -M group, hydrogen or halogen, C1-C6 alkyl, C6-C 14 Aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C 10 is a substituent selected from the group consisting of alkoxyalkyl, or C2-C6 alkoxycarbonyloxy, However, R B Groups and R A at least one group of the formula -(Y) n -M group, k is 1 or 2; - n is 0 or 1 Y is a spacer, and - M is a functional moiety This is about AAV.

[0020] In some embodiments, R A is C1-C3 alkyl, phenyl, or benzyl, preferably methyl or benzyl, more preferably methyl, and / or one or two R B But the formula -(Y) n -M group, and other R B is hydrogen. In additional embodiments, R A is C1-C3 alkyl, phenyl, or benzyl, preferably methyl or benzyl, and one R B Ga-(Y) n -M and the remaining R B is H.

[0021] In some other embodiments, R A is -(Y) n -M and R B The group is H.

[0022] In certain embodiments, the at least one chemically modified tyrosine residue is one in which Y is a chemical chain group containing 2 to 500 carbon atoms and is selected from the group consisting of polymers, including homopolymers, copolymers, and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains, which may optionally be interrupted by one or several heteroatoms and / or by one or several cyclic or heterocyclic moieties, which may optionally have heteroatoms such as S, O, and NH at at least one of their termini, and which may optionally be substituted by one or several substituents, and combinations thereof.

[0023] In particular, Y is one or more heteroatoms selected from -O-, -S-, -N(R)- (wherein R is H or C1-C3 alkyl), -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and -NH-CS-; and / or - C5-C such as cycloalkyl, cycloalkenyl, or aromatic groups 20 a carbocyclic moiety; and / or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyl or heteroaryl having 5 to 20 ring atoms; may be optionally interrupted by - optionally having at least one terminal thereof a heteroatom group selected from -O-, -S-, -N(R)- (R is H or C1-C3 alkyl), -ON(R)- (R is H or C1-C3 alkyl), -N(C1-C3 alkoxy)-, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and NH-CS-; It can be a saturated or unsaturated hydrocarbon having from 2 to 100 carbon atoms.

[0024] In certain embodiments, the AAV is a functional moiety wherein M comprises a group selected from a click chemistry group, a steric shielding agent, a labeling agent, a targeting agent such as a cell type-specific ligand, a drug moiety, an oligonucleotide, and combinations thereof.

[0025] For example, M is - a cell targeting agent selected from click chemistry groups, preferably 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 small chemical molecules such as drugs, e.g. CB1 and / or CB2 ligands; or - a cell type-specific ligand derived from a click chemistry group, a protein selected from transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF, a monosaccharide or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, crosslinked GalNac, or mannose-6-phosphate, sialic acid and its derivatives (e.g., Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5Ac), an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7, and a vitamin such as folic acid. It may comprise or consist of:

[0026] In some additional embodiments, the AAV of the present invention further comprises at least one additional chemically modified amino acid residue in the capsid selected from cysteine, arginine, or lysine. The AAV of the present invention can be of any type. For example, the AAV is a recombinant AAV, preferably selected from AAV with a wild-type capsid, a naturally occurring serotype AAV, a variant AAV, a pseudotype AAV, an AAV with a hybrid or mutant capsid, and a self-complementary AAV.

[0027] In another aspect, the present invention 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 an N-substituted luminol moiety under conditions conducive to reacting said chemical reagent with the tyrosine residue present in the capsid of the AAV to form a covalent bond, preferably carried out by electrochemical methods.

[0028] In certain embodiments, the methods of the invention comprise providing the AAV of formula (X):

[0029] [ka]

[0030] (In the formula, -R A is -(Y) n -M, C1-C6 alkyl, optionally substituted C6-C 14 aryl, or optionally substituted (C6-C 14 aryl)-(C1-C3 alkyl), - Each R B is the formula -(Y) n -M group, hydrogen or halogen, C1-C6 alkyl, C6-C 14 Aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C 10 independently selected from substituents selected from alkoxyalkyl, and C2-C6 alkoxycarbonyloxy; However, R B Groups and R A At least one group in the formula is -(Y) n -M, - n is 0 or 1, Y is a spacer, and - M is a functional moiety in the presence of a potential difference that allows electroactivation of said chemical reagent of formula (X) to an oxidized form capable of reacting with tyrosine residues, resulting in a compound of formula (I):

[0031] [ka]

[0032] (In the formula, -R A and R B is as defined in formula (X), - k is 1 or 2 The method includes obtaining at least one chemically modified tyrosine residue in the AAV capsid.

[0033] In certain embodiments, the methods of the present invention are carried out in an electrochemical system having three electrodes, including a working electrode, a counter electrode, and a reference electrode, by applying a constant potential difference between the working electrode and the reference electrode, the potential difference preferably falling within a range defined as the oxidation potential of the chemical reagent ±200 mV.

[0034] In some embodiments, (i) R A is C1-C6 alkyl, and (ii) at least one R B is the formula -(Y) n -M and other R B is H.

[0035] In other embodiments, the methods of the invention further comprise a step of click reaction, whereby a functional moiety, preferably selected from a ligand and a label, is covalently attached to the AAV capsid, whereby the functional moiety is preferably selected from a ligand and a label. By way of example, the click reaction can be strain-promoted alkyne-azide cycloaddition (SPAAC), meaning that M can be an azide group and the functional moiety comprises a strained alkyne, or vice versa.

[0036] The present invention also relates to an AAV obtainable by the method of the present invention. In a further aspect, the present invention also relates to a pharmaceutical composition comprising an AAV as defined above and at least one pharmaceutically acceptable excipient.

[0037] In a further aspect, the present invention relates to the use of an AAV or pharmaceutical composition of the present invention as a diagnostic agent in vivo or as a drug ex vivo or in vivo, preferably in gene therapy. The present invention also relates to the use of an AAV as defined herein as an in vitro research tool, for example as an in vitro gene transfection agent or as an imaging agent.

[0038] The present invention further relates to the use of an AAV as defined herein in the manufacture of a diagnostic agent or a medicament, in particular for ex vivo or in vivo gene therapy.

[0039] The present invention also provides a compound of formula (X), as defined herein, as an agent for chemically modifying the capsid of AAV by electrochemical bioconjugation:

[0040] [ka]

[0041] The present invention relates to the use of the compound of the present invention. [Brief explanation of the drawings]

[0042] [Figure 1] Figure 1 shows the experimental setup and electrochemical cell assembled for AAV electrobioconjugation. The anode, cathode, and reference electrodes are attached to the electrode holder (A) and immersed in a low-binding vial filled with AAV and luminol solution (B), which is inserted into a 5 mL glass vial to form (ABC). Alligator clips connect the electrical connections from (A) to a potentiostat. The latter is USB-controlled by a computer (software EC-Lab). [Figure 2A] FIG. 1 shows that cyclic voltammetry of N-methylluminol GalNAc derivative 12 (LumGalNAc) at scan rates of 25 mV / s, 50 mV / s, 75 mV / s, and 100 mV / s clearly revealed a diffusion-controlled coherent electrochemical process. [Figure 2B] FIG. 1 shows multicyclic voltammetry (n=6) of LumGalNAc at 100 mV / s outlining the clean, reversible oxidation process. [Figure 3A] Top: Dot blot analysis of viral vectors (2x1010vg) using anti-capsid A20 antibody to detect assembled capsids. Bottom: Dot blot analysis of viral vectors (2x1010vg) using labeled soybean agglutinin (GalNAc-binding lectin) to detect surface-conjugated LumGalNAc. [Figure 3B] Figure 1 shows Western blot analysis of denatured viral vectors (2 x 1010 vg) using (left) polyclonal anti-VP antibodies to detect the three constituent viral proteins (VP1 / VP2 / VP3 1:1:10) or (right) labeled concanavalin A (Man-binding lectin) to detect surface-conjugated LumMan. [Figure 4](Left) Percentage of GFP+ cells quantified by flow cytometry after 48 hours of incubation of electroconjugated glycosylated viral vectors AAV2-LumMan and AAV2-LumGalNAc (carrying a GFP reporter gene) in HEK293 cells at an MOI of 5 × 10. Results from three independent electroconjugation experiments and transduction assays. (Right) Comparison of transduction efficiencies of AAV2 and AAV2-LumGalNAc (1 min) in HEK293 and HuH-7 (expressing the GalNAc receptor) at an MOI of 1 × 10. Percentage of GFP+ cells quantified by flow cytometry after 48 hours of incubation (n = 3). [Figure 5A] FIG. 1 shows some examples of "M" moieties according to the present invention (non-exhaustive list, for illustrative purposes only). [Figure 5B] FIG. 1 shows some examples of "M" moieties according to the present invention (non-exhaustive list, for illustrative purposes only). [Figure 6] FIG. 1 shows dot blot analysis of viral vector (2×10 vg) coupling with N-methylluminol azide derivative (LumN3), using anti-capsid A20 antibody to detect assembled capsids, and DBCO-fluorescein to detect surface-conjugated N3, and mass spectrometry to determine the number of azide derivatives by VP3 molecules. [Figure 7] Figure 1 shows Western blot and silver staining analysis of denatured viral vectors (2 x 1010 vg) using labeled anti-fluorescein antibody, streptavidin, anti-CD62L and anti-CD45 nanobody antibodies to verify the SPAAC reaction and to detect constituent viral proteins. DETAILED DESCRIPTION OF THE INVENTION

[0043] Surface-exposed tyrosine residues have been proposed to play a central role in immunogenicity and in the proteasomal degradation of AAV in cells, and therefore have been identified as residues that can be targeted to improve AAV properties.

[0044] In WO 2021 / 005210, the applicants describe the chemical modification of surface-exposed tyrosine residues by chemical coupling with ligands bearing aryldiazonium functional groups that react specifically with the aromatic ring of the tyrosine side chain.

[0045] The applicants have now developed a new strategy for chemically modifying surface-exposed tyrosine residues in AAV, which differs from that disclosed in the previous application WO2021 / 005210, as it relies on the use of different reactive entities and can be carried out by electrochemical bioconjugation at low potential.

[0046] The electrochemical bioconjugation developed by the applicants is based on the use of specific reagents, i.e., N-substituted luminol derivatives, preferably N-methyl-luminol (NMeLum) derivatives, which are electrochemically activated (i.e., electrooxidized) in situ and then react specifically with aromatic rings present in the side chains of tyrosine residues in the AAV capsid.

[0047] To the best of Applicants' knowledge, electrochemical bioconjugation has not been described or suggested in the prior art for externally modifying the surface of a virus, particularly for chemically modifying tyrosine residues in AAV capsid proteins. Indeed, to the best of our knowledge, electrochemical bioconjugation has mostly been used to chemically modify isolated proteins (see, e.g., Depienne et al., Chem. Sci., 2021, 12, pp. 15374-15381), but has never been used with proteins present in complex systems containing genetic elements, such as viruses.

[0048] Applicants surprisingly demonstrated that subjecting AAV2 particles to an electrochemical potential for 1 hour did not alter viral capsid integrity, nor the viral infectivity and transduction capacities, i.e., both viral proteins and genomes were unchanged by exposure to the electrochemical potential.

[0049] Furthermore, the present applicants have identified NMeLum as a coupling moiety of interest in the context of AAV surface remodeling. NMeLum-containing ligands can be electroactivated at low potentials, regardless of the substituents present on the NMeLum aromatic ring. Notably, the potential used to oxidize NMeLum is low enough to avoid side reactions with most chemical groups other than phenolic residues, meaning that a variety of ligands can be coupled onto AAV capsids via the NMeLum moiety. Furthermore, without intending to be bound by any theory, the present applicants believe that electroactivation of the NMeLum moiety at low potentials allows the formation of stable nitrogen-centered radicals that can specifically react with tyrosine residues in the AAV capsid through radical coupling, while simultaneously avoiding the formation of highly reactive by-products and side reactions with other amino acid residues (e.g., lysine, arginine) present in the AAV capsid.

[0050] As a proof of concept, we prepared N-substituted luminol derivatives bearing sugar moieties (i.e., N-acetylgalactosamine (GalNAc) or mannose (Man) sugars) to chemically modify the capsid of AAV vectors on naturally occurring tyrosine residues. We demonstrated that such sugar moieties could be efficiently and covalently coupled to the surface of serotype 2 AAV particles by incubating virus particles with the N-substituted luminol derivatives in a biocompatible buffer (e.g., dPBS, pH 7.4) under a potential low enough to oxidize the N-substituted luminol derivatives to viable radicals (e.g., 750 mV vs. Ag / AgCl as a reference electrode). Effective coupling was obtained for both derivatives after a short time (typically within a few minutes, even within 1 minute), as evidenced by dot blot analysis with soybean agglutinin (SBA lectin-GalNAc-binding protein) and concanavalin A lectin (mannose-binding protein) detection (Figure 3A). Notably, AAV2 capsids incubated with triethylene glycol-derivatized GalNAc (without the N-substituted luminol moiety) could not be detected by SBA, excluding noncovalent ligand adsorption of NMeLum derivatives on the capsid surface during electrochemical bioconjugation.

[0051] We further demonstrated that electrochemical coupling with NMeLum derivatives did not alter the capsid protein subunits of AAV2, as evidenced by dot blot analysis with anti-capsid A20 antibody staining (Figure 3A). On the other hand, denaturation of AAV2 capsids followed by SDS-PAGE separation of VP1, VP2, and VP3 demonstrated a mass shift for each VP protein, confirming that tyrosine residues in the VP proteins were coupled with NMeLum derivatives (Example 3B).

[0052] Applicants further demonstrated that the infection and transduction efficiency of AAV2 was not compromised by electrochemical coupling: electrochemically modified AAV2 vectors carrying a GFP reporter gene exhibited transduction efficiencies in the hepatocyte cell line HuH7 and HEK cells that approached those of non-chemically modified vectors, as evidenced by similar levels of fluorescence using flow cytometry (Example 4 and Figure 4).

[0053] Applicants also decorated the surface of AAV2 with various entities (also called nanobody-VHHs, fluorescein, biotin) in a two-step method involving electrochemical bioconjugation of AAV2 with an azide derivative of N-methylluminol, followed by strain-promoted alkyne-azide cycloaddition (SPAAC) reaction with DBCO-ligands. As shown in Example 6 and illustrated in Figures 6 and 7, each ligand was efficiently coupled onto the viral capsid protein without capsid denaturation.

[0054] In summary, we have demonstrated that N-substituted luminol derivatives can be used to modify surface-exposed tyrosine residues in AAV capsids with specificity and efficacy, without compromising viral structural integrity and transduction efficiency. This method allows for the decoration of AAV capsids with a wide variety of ligands, including sugar moieties, biotin, fluorescent labels, and proteins (e.g., nanobodies).

[0055] The present applicants have developed an electrochemical bioconjugation process that exhibits many advantages, including high reaction rate and conversion, high chemoselectivity toward tyrosine residues, few by-products, and performance under biocompatible conditions. Furthermore, the electrochemical bioconjugation of the present invention avoids the use of numerous chemical entities (e.g., oxidants, catalysts, and / or scavengers), and upon completion of the reaction, unreacted ligands can be easily removed by standard methods (e.g., dialysis). Additionally, the electrodes used in electrochemical bioconjugation do not produce waste and can be reused several times, allowing the process to be easily implemented from laboratory to industrial process scale.

[0056] Thus, the present invention relates to an adeno-associated virus (AAV) having at least one chemically modified tyrosine residue, preferably at least one electrochemically modified tyrosine residue, in its capsid. The chemically modified tyrosine residue results from the reaction of a tyrosine in the capsid with a functional moiety having an N-substituted luminol group. Preferably, the tyrosine residue to be chemically modified is a naturally occurring residue in the capsid, i.e., the tyrosine has not been introduced by mutagenesis.

[0057] Thus, chemically modified adeno-associated viruses (AAVs) contain the following moieties:

[0058] [ka]

[0059] (In the formula, R A is -(Y) n -M (as further defined below) or a hydrocarbon substituent, typically C1-C6 alkyl, optionally substituted C6-C 14 aryl, or optionally substituted (C6-C 14 aryl)-(C1-C3 alkyl), preferably methyl or benzyl, more preferably methyl The AAV capsid comprises a functional moiety, e.g., a ligand, covalently linked to an aromatic ring present in the side chain of a tyrosine residue in the capsid via the formula:

[0060] The present invention also relates to a method for preparing a chemically modified AAV, comprising the step of contacting AAV with a functional moiety having an N-substituted luminol group under conditions that allow the functional moiety having the N-substituted luminol group to react with the aromatic ring of a tyrosine residue present in AAV, thereby covalently linking the functional moiety to AAV. The N-substituted luminol is preferably N-(C1-C6 alkyl) luminol, N-(C6-C 14 aryl(optionally substituted))luminol, or N—[(C6-C 14 aryl)-(C1-C3 alkyl)(optionally substituted)] luminol, most preferably N-methylluminol or N-benzylluminol, more preferably N-methylluminol.

[0061] In a preferred embodiment, such a method is carried out by electrochemical means, i.e., by exposing the reaction medium to a potential difference sufficient to electroactivate (i.e., oxidize) the N-substituted luminol group and promote its reaction with the tyrosine residue.

[0062] The present invention also relates to the use of the resulting chemically modified AAV, preferably electrochemically modified AAV, particularly in gene therapy.

[0063] The invention is described in more detail below.

[0064] - General definition As used herein, the expression "about X," where X is a physical value (eg, voltage), corresponds to X±5%.

[0065] As used in this disclosure, the term "C x ~C y" (where x and y are integers) means that the corresponding hydrocarbon chain contains from x to y carbon atoms. For example, when the term C1-C6 is used, it means that the corresponding hydrocarbon chain can contain from 1 to 6 carbon atoms, particularly 1, 2, 3, 4, 5, or 6 carbon atoms. For example, when the term C2-C5 is used, it means that the corresponding hydrocarbon chain can contain from 2 to 5 carbon atoms, particularly 2, 3, 4, or 5 carbon atoms.

[0066] As used herein, the term "alkyl" refers to a saturated, linear or branched aliphatic group. A preferred alkyl is a "C1-C6 alkyl," which refers to an alkyl having 1 to 6 carbon atoms. Examples of alkyl (or C1-C6 alkyl) include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, and hexyl.

[0067] As used herein, the term "alkene" or "alkenyl" refers to an unsaturated, linear or branched aliphatic group having at least one carbon-carbon double bond. A preferred alkene is a "C2-C6 alkene," which refers to an alkene having 2 to 6 carbon atoms. Examples of alkenes (or C2-C6 alkenes) include, for example, ethenyl, propenyl, butenyl, pentenyl, or hexenyl, and preferably ethenyl (-CH=CH2).

[0068] As used herein, the term "alkyne" or "alkynyl" refers to an unsaturated, linear or branched aliphatic group having at least one carbon-carbon triple bond. A preferred alkyne is a "C2-C6 alkyne," which refers to an alkyne having 2 to 6 carbon atoms. Examples of alkynes (or C2-C6 alkynes) include, for example, ethynyl, propynyl, butynyl, pentynyl, or hexynyl, and preferably ethynyl (-C≡CH).

[0069] As used herein, the term "alkoxy" refers to an alkyl, as defined herein, attached to the rest of the molecule via an ether bond (-O-). In other words, alkoxy can be written as "-O-alkyl." Preferred alkoxy is a C1-C6 alkoxy having 1 to 6 carbon atoms. Examples of alkoxy (or C1-C6 alkoxy) include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy, and hexyloxy.

[0070] As used herein, the term "alkylthio" refers to an alkyl, as defined herein, bonded to the rest of the molecule via a thioether bond (-S-). In other words, alkylthio can be written as "-S-alkyl." Preferred alkylthio is a C1-C6 alkylthio having 1 to 6 carbon atoms. Examples of alkylthio (or C1-C6 alkylthio) include, for example, methylthio, ethylthio, propylthio, isopropylthio, butylthio, pentylthio, and hexylthio.

[0071] As used herein, the term "alkylamino" refers to an alkyl, as defined herein, bonded to the rest of the molecule via an amino bond (-NH-). In other words, alkylamino can be written as "-NH-alkyl." Preferred alkylamino is a C1-C6 alkylamino having 1 to 6 carbon atoms. Examples of alkylamino (or C1-C6 alkylamino) include, for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, pentylamino, and hexylamino.

[0072] As used herein, the term "carbocycle" (or "carbocyclic group") refers to a saturated or unsaturated, aliphatic or aromatic, monocyclic, bicyclic, or tricyclic hydrocarbon group. A carbocyclic group may be, inter alia, cycloalkyl, cycloalkenyl, or aryl.

[0073] As used herein, the term "cycloalkyl" refers to a saturated monocyclic, bicyclic, or tricyclic aliphatic group. It also includes fused, bridged, or spirocyclic cycloalkyl groups. The term "C3-C6 cycloalkyl" refers to a cycloalkyl having 3 to 6 carbon atoms. Examples of cycloalkyl (or C3-C6 cycloalkyl) include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The term "cycloalkyl" can also refer to bridged carbocycles, such as bicyclo[2,2,1]heptanyl, bicyclo[2,2,2]octanyl, or adamantyl.

[0074] As used herein, the term "cycloalkenyl" refers to an unsaturated monocyclic, bicyclic, or tricyclic aliphatic group containing at least one carbon-carbon double bond. It also includes fused, bridged, or spirocyclic cycloalkenyl groups. The term "C3-C6 cycloalkenyl" refers to a cycloalkenyl having 3 to 6 carbon atoms. Examples of cycloalkenyl (or C3-C6 cycloalkenyl) include, but are not limited to, cyclopentenyl and cyclohexenyl.

[0075] As used herein, the term "heterocycle" refers to a saturated or unsaturated, aliphatic or aromatic, monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) group containing at least one heteroatom, such as a nitrogen, oxygen, or sulfur atom. If bicyclic or tricyclic, the rings may be fused, bridged, or have a spiro configuration. Advantageously, the heterocycle contains between 3 and 20 ring atoms, e.g., between 3 and 6 ring atoms, and at least one of the ring atoms is a heteroatom, such as a nitrogen, oxygen, or sulfur atom. In some embodiments, a "heterocycle" is a heterocycloalkyl, heterocycloalkenyl, or heteroaryl. In some embodiments, a heterocycle is a heterocycloalkyl, heterocycloalkenyl, or heteroaryl fused to one or more carbocyclic or heterocyclic moieties (e.g., a heteroaryl fused to a cycloalkyl).

[0076] As used herein, the term "heterocycloalkyl" corresponds to a cycloalkyl group as defined above, wherein at least one carbon atom is replaced with a heteroatom, such as a nitrogen, oxygen, or sulfur atom.

[0077] As used herein, the term "heterocycloalkenyl" corresponds to a cycloalkenyl group, as defined above, in which at least one carbon atom is replaced with a heteroatom, such as a nitrogen, oxygen, or sulfur atom.

[0078] Examples of heterocycles that are heterocycloalkyl or heterocycloalkenyl include aziridinyl, azepanyl, diazepanyl, dioxolanyl, benzo[1,3]dioxolyl, azetidinyl, oxetanyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4-dioxanyl, imidazolidinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, pyrimidinyl, oxozolinyl , oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiooxetanyl, thiopyranyl, thiomorpholinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, dihydrofuranyl, dihydrothiopyranyl, dihydrothiophenyl, dihydropiperidinyl, tetrahydropiperidinyl, tetrahydrothiopyranyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothiophenyl.

[0079] As used herein, the term "aryl" refers to an aromatic ring system, preferably having 6 to 14 atoms, with at least one ring having a conjugated pi-electron system, and optionally substituted. "Aryl" can 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.

[0080] "Heteroaryl" refers to a heteroaryl group. "Heteroaryl" refers to a chemical group, preferably having 5 to 14 ring atoms, where 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. Examples of heterocycles that are heteroaryl groups include triazolyl, furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinazolinyl, and quinolinyl.

[0081] Examples of bicyclic heteroaryl groups include, but are not limited to, 1H-indazolyl, benzo[1,2,3]thiadiazolyl, benzo[1,2,5]thiadiazolyl, benzothiophenyl, imidazo[1,2-a]pyridyl, quinolinyl, indolyl, and isoquinolinyl groups.

[0082] As used herein, (C6-C 14 Aryl)-(C1-C3 alkyl) is at least one (preferably only one) C6-C alkyl group as defined herein. 14 It refers to C1-C3 alkyl as defined herein, substituted by aryl. Preferred (C6-C 14 Aryl)-(C1-C3 alkyl) is phenylmethyl (i.e., benzyl).

[0083] As used herein, the term "alkanoyl" refers to an alkyl, as defined herein, attached to the remainder of the molecule through an oxo group (-C(O)-). In other words, alkanoyl can be written as "-C(O)-alkyl." Preferred alkanoyl is a C1-C6 alkanoyl, having an alkyl chain of 1 to 6 carbon atoms. Examples of alkanoyl (or C1-C6 alkanoyl) include, for example, methanoyl, ethanoyl, propanoyl, isopropanoyl, butanoyl, pentanoyl, and hexanoyl.

[0084] As used herein, the term "acylamino" refers to a group of the formula RC(O)-NH-, where R is C1-C6 alkyl, C3-C 12 The term "acylamino" refers to a group of acylamino, which is a hydrocarbon group such as cycloalkyl, aryl, etc. Preferred acylamino is C1-C6 acylamino, which has a hydrocarbon chain of 1 to 6 carbon atoms.

[0085] As used herein, the term "ester" or "carboxy ester" refers to a -C(O)OR' or R'C(O)O- group, where R' is C1-C6 alkyl, C3-C 12 The term "ester" refers to any hydrocarbon group, such as cycloalkyl, cycloalkyl, or aryl. Preferred esters are C1-C6 esters, having a hydrocarbon chain of 1 to 6 carbon atoms.

[0086] As used herein, "alkoxycarbonyloxy" refers to the group R"-C(O)-O-, where R" is alkoxy.

[0087] As used herein, the term "halogen" includes chlorine, fluorine, iodine, bromine, preferably chlorine or fluorine.

[0088] As used herein, the term "aminoalkyl" refers to an alkyl, as defined above, substituted with one or more (preferably one) amino (-NH2) groups.

[0089] As used herein, the term "alkylaminoalkyl" refers to an alkyl, as defined above, substituted by one or more (preferably one) alkylamino groups, as defined above.

[0090] As used herein, the term "hydroxyalkyl" refers to an alkyl, as defined above, substituted with one or more (preferably one) hydroxy (-OH) groups.

[0091] As used herein, the term "alkoxyalkyl" refers to an alkyl, as defined above, substituted with one or more alkoxy, as defined above.

[0092] As used herein, the term "thioalkyl" refers to an alkyl, as defined above, substituted by one or more (preferably one) --SH groups.

[0093] As used herein, the term "haloalkyl" refers to an alkyl, as defined above, substituted with one or more halogen atoms.

[0094] "Substituted" or "optionally substituted" includes groups substituted with one or several substituents, typically 1, 2, 3, 4, 5, or 6 substituents. Exemplary substituents include C1-C6 alkyl, aryl, C3-C6 cycloalkyl, C3-C6 cycloalkenyl, C2-C6 heterocycle, 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, OH, -NO2, -SO3H, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C6 alkylamino, -C1 ... 10They may be independently selected from alkoxyalkyl, C2-C6 alkoxycarbonyloxy, -CN, -CF3, and C2-C6 alkoxyalkyl.

[0095] Preferred substituents are halogen, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0096] As used herein, N-substituted luminols have the following formula:

[0097] [ka]

[0098] (In the formula, R A is a hydrocarbon substituent, as further defined below, or -(Y) n -M).

[0099] As used herein, N-(C1-C6 alkyl)luminol refers to a compound of formula (B) where R A As used herein, N-methylluminol refers to a compound of formula (B) where R A refers to the compound (wherein methyl is methyl).

[0100] As used herein, N—(C6-C 14 Aryl(optionally substituted) luminols are compounds of formula (B) where R A is an optionally substituted C6 to C 14 It refers to compounds in which the aryl group is aryl.

[0101] As used herein, N—[(C6-C 14 aryl)-(C1-C3 alkyl)(optionally substituted)] luminol is a compound of formula (B) A may be optionally substituted (C6 to C 14 It refers to a compound of the formula (C1-C3 alkyl)-(aryl).

[0102] As used herein, N-benzylluminol is a compound represented by formula (B) where R A is benzyl).

[0103] N-methylluminol is R A N-methylluminol can also be called "2,3-dihydro-2-methyl-1,4-phthalazinedione" and has the following CAS number: 18393-54-9.

[0104] As used herein, "N-substituted luminol derivative" refers to an N-substituted luminol as defined above having one or several substituents on the benzene ring of the phthalazinedione group.

[0105] The phrase "optionally substituted" may be interchanged with the phrase "substituted or unsubstituted" throughout this application.

[0106] - Chemically modified AAV of the present invention According to a first aspect, the present invention refers to an adeno-associated virus (AAV) having at least one chemically modified tyrosine residue in the capsid. More precisely, the chemically modified AAV of the present invention has the formula (A):

[0107] [ka]

[0108] (In the formula, R A is -(Y) n -M, C1-C6 alkyl, optionally substituted C6-C 14 aryl, or optionally substituted (C6-C 14 aryl)-(C1-C3 alkyl) and at least one chemical moiety of

[0109] C6~C 14Aryl or (C6-C 14 The one or more substituents present on the (aryl)-(C1-C3 alkyl) may be of any type. Preferably, said substituents are selected from the group consisting of halogen, —NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0110] Preferably, R A is C1-C3 alkyl, optionally substituted phenyl, or optionally substituted benzyl. More preferably, R A is methyl, phenyl, or benzyl. Even more preferably, R A is methyl, where the "-N" is linked to the aromatic ring of a tyrosine residue in the AAV capsid.

[0111] 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 approximately 5 kb long. Wild-type AAV has two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The 5' and 3' ORFs encode 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.

[0112] Therefore, in the context of the present invention, a recombinant AAV refers to an AAV in which an exogenous nucleic acid sequence, such as a transgene sequence, has been introduced into the viral genome. The exogenous nucleic acid sequence can be of any type and is selected taking into account the intended use of the AAV. For example, the nucleic acid can include any RNA or DNA sequence.

[0113] 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 of 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.

[0114] By way of example only, a recombinant AAV for use as a vector in gene therapy can contain an exogenous gene expression cassette that replaces the viral ORF and is placed between two ITRs. The cassette can include a promoter, a gene of interest, and a terminator. The promoter and gene of interest are selected depending on the target tissue / organ and the condition to be treated. As another example, a recombinant AAV for use in gene therapy can contain a DNA template for homologous recombination in cells. Such a recombinant AAV can 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 can be of any type, including, but not limited to, CRISPR / Cas9, zinc finger nucleases, meganucleases, as well as RNA and DNA encoding the proteins.

[0115] 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 mutant or synthetic capsids, such as AAV with hybrid capsids, pseudotyped AAV, as well as self-complementary AAV (scAAV).

[0116] The wild-type AAV capsid is composed of three overlapping capsid proteins called viral protein 1 (VP1), VP2, and VP3. More precisely, the wild-type AAV capsid is composed of a total of 60 copies of the viral protein subunits VP1, VP2, and VP3 in a ratio of 1:1:10.

[0117] Capsid engineering refers to amino acid modifications of said capsid proteins, for example in their hypervariable loops.

[0118] As used herein, "AAV with a genetically engineered capsid" or "AAV with 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.

[0119] As used herein, an "amino acid modification" includes the insertion, deletion, or substitution of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 20, 30, 40, 50, or 100) amino acids.

[0120] In some embodiments, the AAV has a genetically mutated capsid, where the mutation does not involve a naturally occurring tyrosine residue or does not result in the insertion of a tyrosine residue. In other words, the AAV of the present invention may have a wild-type capsid, or may have a mutant capsid in which the naturally occurring tyrosine residue is preserved. In certain embodiments, the AAV of the present invention is selected from wild-type AAV and recombinant or variant AAV having a wild-type AAV capsid.

[0121] 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. In a specific embodiment, the AAV is a recombinant AAV having a mutant capsid, wherein the amino acid modifications are not related to any tyrosine residues naturally present in the wild-type capsid protein.

[0122] As used herein, "chemically modified tyrosine residue" means that at least one tyrosine present in the viral capsid has been chemically modified by covalent coupling with a chemical entity, typically by covalent coupling of the chemical entity to the phenyl ring of the tyrosine. The tyrosine is typically a surface-exposed residue present in VP1, VP2, or VP3. By way of example, there are approximately 360 tyrosine residues believed to be exposed in the AAV2 capsid. 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 the entire capsid itself. 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, 150, 200, 250, or more chemically modified tyrosine residues.

[0123] In some embodiments, the chemically modified AAV of the present invention comprises several chemically modified tyrosine residues in its capsid. The chemically modified tyrosines may be present on VP1, VP2, and / or VP3. In some embodiments, at least 0.1%, e.g., 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.

[0124] 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.

[0125] A "serotype" is traditionally defined based on the lack 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.

[0126] 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, e.g., 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 the mixing of capsids and genomes from different viral serotypes, e.g., AAV2 / 5, AAV2 / 7, and AAV2 / 8, as well as AAV with hybrid capsids from multiple different serotypes, e.g., AAV-DJ, which contains hybrid capsids from eight serotypes.

[0127] 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. 2014144229 (which specifically discloses the AAV2G9 serotype). Other AAV serotypes include those disclosed in EP 2292779 and EP 1310571. 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).

[0128] In some embodiments, the AAV is a naturally occurring serotype, preferably selected from the group consisting of AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9, and AAVrhlO, and more preferably AAV-2. By way of example, the AAV of the present invention can be of the AAV-2 or AAV-9 serotype.

[0129] 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; retinal cells (i.e., photoreceptors, retinal pigment epithelium (RPE); muscle cells (i.e., myoblasts, satellite cells); central nervous system (CNS) cells (i.e., neurons, glia); cardiac cells; peripheral nervous system (PNS) cells; 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, CNS cells, PNS cells, and hematopoietic cells. Preferred tissues and organs are liver, muscle, heart, eye, and brain.

[0130] The tropism of AAVs can vary depending on their serotype: 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.

[0131] AAV can be produced by any method known in the art, for example, by transient transfection in a cell line of interest, such as HEK293 cells as described in the Examples section. In this regard, see Naso et al., Biodrugs, 2017, 31:317-334, which provides a review on AAV as a vector in gene therapy and describes traditional methods for producing AAV on an industrial scale.

[0132] The AAV of the present invention may have other amino acids of the capsid that have been chemically modified. For example, the AAV may contain 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 the amino groups in the capsid with a ligand having an isothiocyanate-reactive group. Alternatively or additionally, the AAV of the present invention may contain one or several arginine residues of the capsid that have been modified by glycation, for example, by reaction with methylglyoxal as described in Horowitz et al. (Bioconj Chem, 2011, 22(4):529-532). Alternatively or additionally, the AAV of the present invention may contain one or several cysteine ​​residues of the capsid that have been modified by reacting the cysteine ​​residue with a chemical reagent having a reactive group selected from maleimide, vinylsulfonamide, and 3-(carboxy derivative)acrylamide.

[0133] In some embodiments, the present invention provides an adeno-associated virus (AAV) having at least one chemically modified tyrosine residue in the capsid, wherein the chemically modified tyrosine residue has formula (I):

[0134] [ka]

[0135] (In the formula, -R A is -(Y) n -M, C1-C6 alkyl, optionally substituted C6-C 14 aryl, or optionally substituted (C6-C 14 aryl)-(C1-C3 alkyl), - Each R B is the formula -(Y) n -M group, hydrogen or halogen, C1-C6 alkyl, C6-C 14Aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 thioalkyl, C1-C6 alkylthio, C2-C 10 independently selected from the group consisting of substituents selected from the group consisting of alkoxyalkyl, and C2-C6 alkoxycarbonyloxy; However, R A and R B At least one of the groups is of the formula -(Y) n -M, k is 1 or 2; - n is 0 or 1 Y is a spacer, and - M is a functional moiety is.

[0136] Preferably, R A is C1-C3 alkyl, optionally substituted phenyl, or optionally substituted benzyl. More preferably, R A is methyl, phenyl, or benzyl. Even more preferably, R A is methyl.

[0137] In the formulas described in this application (e.g., formula (I)), the following moieties:

[0138] [ka]

[0139] (In the formula,

[0140] [ka]

[0141] represents the bond where the tyrosine is attached to the rest of the protein) represents a tyrosine within a capsid protein (i.e., VP1, VP2, or VP3).

[0142] An N-substituted luminol moiety can be added ortho to the phenolic group in the tyrosine residue.

[0143] In the above formula (I), "k is 1" means the following formula (II):

[0144] [ka]

[0145] (In the formula, R A and R B is as defined herein) is attached to a tyrosine residue.

[0146] Typically, when k is 1, at least one chemically modified tyrosine residue in the capsid has formula (Ia):

[0147] [ka]

[0148] (In the formula, R A and R B are as defined herein) It can be expressed as:

[0149] "k is 2" means that two moieties of formula (II) as defined above are attached to tyrosine residues. Typically, when k is 2, at least one chemically modified tyrosine residue in the capsid has formula (Ib):

[0150] [ka]

[0151] (In the formula, R A and R B are as defined herein) In such formula (Ib), each R in the moiety of formula (II) A and R B may be the same or different from those of the other moieties of formula (II), and are preferably the same. In other words, the two moieties of formula (II) bound to the tyrosine residue in formula (Ib) may be different or the same, and are preferably the same.

[0152] It goes without saying that the chemically modified AAV of the present invention may have one or several chemically modified tyrosine residues of formula (Ia) and one or several chemically modified tyrosine residues of formula (Ib).

[0153] In some embodiments, the tyrosine residues of formula (Ia) represent, on average, at least 50%, e.g., at least 60%, 70%, 80%, 90%, or 95%, of the total number of chemically modified tyrosine residues found in a population of chemically modified AAVs of the invention.

[0154] -R A In the above formula (I), R A is C1-C6 alkyl, optionally substituted C6-C 14 aryl, or optionally substituted (C6-C 14 In some embodiments, R A teeth: - -(Y) n -M (as defined below); - C1-C6 alkyl; - Halogen, C1-C6 alkyl, C6-C 14Aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C 10 Alkoxyalkyl, C2-C6 alkoxycarbonyloxy, and (C6-C 14 C6-C alkyl) optionally substituted with one or more (preferably one) groups selected from 14 aryl; or - Halogen, C1-C6 alkyl, C6-C 14 Aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C 10 Alkoxyalkyl, C2-C6 alkoxycarbonyloxy, and (C6-C 14 (C6-C aryl)-(C1-C3 alkyl), optionally substituted with one or more (preferably one) groups selected from 14 Aryl)-(C1-C3 alkyl) is.

[0155] Preferably, the substituents are selected from the group consisting of halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0156] Preferably, R A is C1-C3 alkyl (e.g., methyl), phenyl, or benzyl. More preferably, R A is methyl or benzyl. Even more preferably, R A is methyl.

[0157] -R B In the above formula (I), each R B are independently of the formula -(Y) n -M (wherein n and M are as defined above), hydrogen, or halogen, C1-C6 alkyl, C6-C 14 Aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C 10 is a substituent selected from alkoxyalkyl and C2-C6 alkoxycarbonyloxy, However, R A and R B At least one of the groups is -(Y) n The condition is that it is -M.

[0158] In some embodiments, R A is selected from methyl, phenyl, or benzyl, preferably methyl. In such embodiments, at least one R B is -(Y) n -M.

[0159] In some other embodiments, R A Ha-(Y) n -M. Preferably, R B Ha-(Y) n -M is not. For example, R A Ha-(Y) n -M and all R B is H.

[0160] In a preferred embodiment, the chemically modified tyrosine residue of formula (I) comprises one or two (preferably one) R B is the formula -(Y) n -M group.

[0161] More preferably, the chemically modified tyrosine residue of formula (I) has one or two (preferably one) R B is the formula -(Y) n -M group and other R B is hydrogen.

[0162] -(Y) n -M can be any position on the aromatic ring (i.e., any R B Preferably, the moiety of formula (II) as defined above can be of formula (II-a) or (II-b):

[0163] [ka]

[0164] (In the formula, each R B are independently hydrogen, halogen, C1-C6 alkyl, C6-C 14 Aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C 10 is a substituent selected from alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, and preferably, each R B is hydrogen) is.

[0165] R A is as defined above, preferably -(Y) n -Not M.

[0166] "n is 1" means that the spacer Y is present. "n is 0" means that the spacer Y is absent.

[0167] In a preferred embodiment, the chemically modified tyrosine residue of formula (I) is: -R A is methyl or benzyl; and - one or two (preferably one) R B is the formula -(Y) n -M group, and other R B is hydrogen It is something.

[0168] In a preferred embodiment, the moiety of formula (II) as defined above has formula (II-c) or (II-d):

[0169] [ka]

[0170] (In the formula, R A is C1-C3 alkyl, optionally substituted phenyl, or optionally substituted C1-C3 alkyl-phenyl, preferably methyl or benzyl) is.

[0171] Spacer Y Y is a spacer that links the N-substituted luminol moiety and the functional moiety M. Y can be present (when n is 1) or absent (when n is 0). When Y is absent, the N-substituted luminol and M are directly linked to each other.

[0172] Y can be any chemical chain (e.g., a hydrocarbon chain) that can contain heteroatoms as well as cyclic moieties, such as cycloalkyl, cycloalkenyl, aromatic groups, or heterocyclic moieties such as heterocycloalkyl or heteroaryl. Y can contain up to 1000 carbon atoms and even more. The length and chemical nature of the spacer can be optimized depending on the functional moiety "M" intended to be coupled to the tyrosine residue and the desired biological effect. Indeed, in addition to its linking function, Y can be used to refine the properties of the functional moiety "M". For example, Y 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 to the entity of interest, and / or increase the solubility of M.

[0173] In some embodiments, Y is a chemical chain group containing 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.

[0174] Typically, Y is a polymer, including homopolymers, copolymers, and block polymers, peptides, oligosaccharides, and the like, optionally interrupted by one or several heteroatom groups (e.g., S, O, Se, P, -C(O)-, -NHC(O)-, -OC(O)-, -N(R)- (wherein R is H or C1-C3 alkyl)) and / or by one or several cyclic or heterocyclic moieties, and / or by one or several heteroatom groups (e.g., S, O, -C(O)-, -NHC(O)-, -OC(O)-, -N(R)- (wherein R is H or C1-C3 alkyl)). and a saturated or unsaturated hydrocarbon chain, which may optionally have (O)-, -N(R)- (wherein R is H or C1-C3 alkyl), -ON(R)- (wherein R is H or C1-C3 alkyl), or -N(C1-C3 alkoxy)- at at least one of its termini, and / or may be optionally substituted with one or several substituents (e.g., hydroxyl, halogen, C1-C3 alkoxy, -CN, -CF3, or C1-C3 alkyl), and combinations thereof.

[0175] As used herein, "combination" means that the spacer group Y can comprise any suitable group, such as, for example, -O-, -S-, -N(R)- (wherein R is H or C1-C3 alkyl), -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, NH-CS-, a phosphate group, or a phosphodiester or phosphorothioate group, as well as several (e.g., 2, 3, 4, 5, or 6) hydrocarbon chains, oligomeric chains, or polymeric chains linked (or connected) by a cyclic or heterocyclic group.

[0176] Typically, the group used to link several hydrocarbon, oligomeric, or polymeric chains together (also called a connector) results from the reaction used to connect these different chains together. By way of example, the connector can be -NHC(O)- in the case of amide coupling reactions, "N" in the case of reductive amination, or a triazole derivative in the case of click chemistry, which involves the reaction of an azide with an alkyne group.

[0177] In some embodiments, Y may be selected from the group consisting of polyethers such as polyethylene glycol (PEG) and polypropylene glycol, polyesters such as polyvinyl alcohol (PVA), polylactic acid, polyacrylates, polymethacrylates, polysilicones, polycaprolactones, and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), poly(D,L-lactic-co-glycolic acid) (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.

[0178] As used herein, alkyldiamines are 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 alkyldiamines (also known as polyamines) have 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.

[0179] By way of example, Y can include 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, Y can include from 2 to 10 triethylene glycol blocks linked together by a linker. As another example, Y can include C 12 Alternatively, Y may be a saturated or unsaturated C2-C 40 Hydrocarbon chains, especially C 10 ~C 20 C2 to C alkyl chains, such as C6 alkyl chains 10It may be an alkyl chain, which may have at least one terminus group such as NH, S, or O. As an example, Y may be putrescine.

[0180] In certain embodiments, Y is an optionally substituted saturated or unsaturated linear or branched C-C 40 In certain embodiments, Y is selected from the group consisting of a hydrocarbon chain, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof. 20 The polymers are selected from the group consisting of alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diaminoalkyl polymers, and combinations thereof. Preferably, the polyethylene glycol, polypropylene glycol, PLGA, pHPMA, and alkyldiamine polymers contain from 2 to 40 monomers, preferably from 2 to 10 or from 10 to 20 monomers.

[0181] By way of example, Y may include one or several (eg, two, three, four, or five) triethylene glycol blocks.

[0182] In some embodiments, Y is one or more heteroatoms selected from -O-, -S-, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -N(R)- (wherein R is H or C1-C3 alkyl), -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and -NH-CS-; and / or - C5-C such as cycloalkyl, cycloalkenyl, or aromatic groups 20 a carbocyclic moiety; and / or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyl or heteroaryl having 5 to 20 ring atoms; may be arbitrarily interrupted by; and hydrocarbon chains (for example, alkyl chains) having 2 to 100 carbon atoms, 2 to 40 carbon atoms, 2 to 30 carbon atoms, or 2 to 20 carbon atoms, which may optionally have a heteroatom group selected from -O-, -S-, -N(R)- (R is H or C1-C3 alkyl), -ON(R)- (R is H or C1-C3 alkyl), -N(C1-C3 alkoxy), -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, or NH-CS- at at least one terminus.

[0183] In some embodiments, Y is one or more heteroatoms selected from —O—, —S—, —N(R)— (wherein R is H or C1-C3 alkyl), —C(O)—, —NHC(O)—, and —OC(O)—; and / or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyl or heteroaryl having 5 to 20 ring atoms; may be arbitrarily interrupted by; and a hydrocarbon chain (for example, an alkyl chain) having 2 to 100 carbon atoms, 2 to 40 carbon atoms, 2 to 30 carbon atoms, or 2 to 20 carbon atoms, which may optionally have a heteroatom group selected from -O-, -S-, -N(R)- (R is H or C1-C3 alkyl), -ON(R)- (R is H or C1-C3 alkyl), -N(C1-C3 alkoxy), -C(O)-, -NHC(O)-, and -OC(O)- at at least one terminus.

[0184] The heterocycle (or "heterocyclic moiety") may be a triazolyl or a member of the following group:

[0185] [ka]

[0186] The ring may be a triazolyl fused to another ring such as one selected from:

[0187] When the above heterocyclic moieties (i), (ii), (iii), (iv), (v), (vi), (vii), and (viii) are part of the spacer Y, the symbol

[0188] [ka]

[0189] represents the bond connecting such heterocyclic moiety to the remainder of spacer Y. Such heterocyclic moiety may be present in spacer Y if its synthetic route includes a step in which an azide reacts with a strained alkyne (e.g., DBCO).

[0190] In some embodiments, Y represents a group represented by formula (III): Y1-Y2-Y3(III) (In the formula, - each of Y1 and Y3 is a polymer, including homopolymers, copolymers and block polymers, a peptide, an oligosaccharide, optionally interrupted by one or several heteroatoms (e.g., O, N, S) and / or by a group selected from -C(O)-, -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC(=O)-O-, -NH(C=S)- or -(C=S)-NH-; independently selected from the group consisting of saturated or unsaturated, branched or straight hydrocarbon chains, and combinations thereof, which may optionally have a heteroatom group (e.g., -O-, -S-, -N(R)- (wherein R is H or C1-C3 alkyl, -ON(R)- (wherein R is H or C1-C3 alkyl), -N(C1-C3 alkoxy)-, -C(O)-, -NHC(O)-, and -OC(O)-) at at least one terminus, and which may optionally be substituted with one or several substituents; and - Y2 is a cyclic or heterocyclic moiety having 5 to 20 ring atoms, such as a heterocycloalkyl or heteroaryl having 5 to 20 ring atoms. It is a spacer.

[0191] In certain embodiments, each of Y1 and Y3 is selected from the group consisting of polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diaminoalkyl polymers, linear or branched C2-C 20 an alkyl chain (which may be optionally interrupted by one or more heteroatoms (e.g., O, N, S) and / or by a group selected from -C(O)-, -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC(=O)-O-, -NH(C=S)-, or -(C=S)-NH-, and which may optionally have a heteroatom group (e.g., -O-, -S-, -N(R)- (wherein R is H or C1-C3 alkyl), -ON(R)- (wherein R is H or C1-C3 alkyl), -N(C1-C3 alkoxy)-, -C(O)-, -NHC(O)-, and -OC(O)-) at at least one terminus), and combinations thereof.

[0192] In certain embodiments, Y2 is triazolyl or selected from among heterocyclic moieties (i), (ii), (iii), (iv), (v), (vi), (vii), and (viii) above.

[0193] In some embodiments, Y has, at one of its termini (typically the terminus linked to M), a heteroatom selected from -ON(R)- (wherein R is H or C1-C3 alkyl) and -N(C1-C3 alkoxy)-, preferably a heteroatom selected from -ON(Me)-, -O-NH-, and -N(OMe)-.

[0194] For example, Y may have the formula:

[0195] [ka]

[0196] where q is an integer from 2 to 10 and R is H or methyl.

[0197] functional moiety M The functional moiety "M" can be of any type. "M" is typically selected depending on the biological effect desired when the AAV capsid is chemically modified. Alternatively, M can be a reactive group, such as a click chemistry reactive group, selected to enable subsequent coupling. M can also be a ligand that enables specific, non-covalent coupling of another entity to the surface of the AAV, such as biotin / strept(avidin), a cationic binding group (e.g., nitrilotriacetic acid (NTA) for binding to a His tag), a protein binding tag, or a ligand / anti-ligand pair (e.g., an antibody / antigen, such as biotin / anti-biotin antibody and digoxigenin / anti-digoxigenin antibody, or a ligand / receptor).

[0198] M can also be a labeling moiety.

[0199] Thus, "M" may comprise a moiety selected from a chemically reactive group such as a click chemistry reactive group, a targeting agent, a steric shielding agent, a labeling agent, an oligonucleotide, 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.

[0200] 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.

[0201] In other embodiments, "M" comprises or consists of a steric shielding agent, e.g., a substance that can mask 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.

[0202] 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.

[0203] In another embodiment, "M" can be an oligonucleotide such as a messenger RNA (mRNA) or an antisense oligonucleotide such as a small interfering RNA (siRNA), shRNA, snoRNA, and meroduplex (mdRNA).

[0204] In some embodiments, M comprises or consists of a targeting agent, i.e., a ligand that allows targeting a specific organ, tissue, cell, or protein of interest, such as a cell surface protein, receptor, or oligosaccharide, e.g., a cell surface protein present on the surface of a specific cell line or tumor cell.

[0205] By way of example, the targeting agent may be a cell type specific ligand, ie, a ligand that allows for targeting of a particular type of cell.

[0206] Such ligands may make it possible to modify the tropism of AAV, ie, its ability to selectively infect and / or transduce a given cell line, tissue, or organ.

[0207] By way of example, "M" can be a ligand that specifically binds to a biological entity (e.g., a membrane receptor) in the membrane of a targeted cell. The ligand can be of any type, for example, a peptide, a protein, an oligosaccharide, or a small chemical entity. By way of example, M can be a monosaccharide or polysaccharide, a hormone, e.g., a steroid hormone, a peptide such as RGD peptide, angiopep-2, a muscle-targeting peptide, or the like, a membrane receptor or a fragment thereof, a CB1 and CB2 ligand, an aptamer, an antibody including a heavy chain antibody and fragments thereof such as Fab, Fab', and VHH (also called nanobody), an ScFv, a spiegelmer, a peptide aptamer, a small chemical molecule known to bind to the targeted biological entity, and the like.

[0208] In certain embodiments, M is a full-length antibody or an antibody comprising an antigen-binding domain derived from an antibody.

[0209] As used herein, the term "antibody" refers to an immunoglobulin or a fragment or derivative thereof and encompasses any polypeptide containing an antigen-binding domain, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific (e.g., bispecific), humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. The term "antibody" also includes antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind their target, such as Fab, F(ab'), Fv, scFv, Fd, dAb, and other antibody fragments (e.g., VHH derived from single-chain antibodies). Typically, such fragments contain an antigen-binding domain. The term "antigen-binding domain" or "antigen-binding fragment" refers to a portion of an antibody that contains amino acids responsible for specific binding between the antibody and the antigen. If the antigen is large, the antigen-binding domain may only bind to a portion of the antigen. The portion of an antigen molecule that is responsible for specific interaction with an antigen-binding domain is called an "epitope" or "antigenic determinant." The antigen-binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). However, it does not necessarily comprise both (see, e.g., antigen-binding fragments of single-chain antibodies and VHH fragments). Typically, an antigen-binding fragment or domain contains at least a portion of the variable regions (heavy and light chains) of an antibody sufficient to form an antigen-binding site (e.g., one or more CDRs, generally all CDRs) and thus retains the binding specificity and / or activity of the antibody.

[0210] As used herein, a "full-length antibody" (also referred to herein as an immunoglobulin or Ig) refers to a protein having the structure constituting the natural biological form of an antibody, including variable and constant regions. The term "full-length antibody" encompasses both monoclonal and polyclonal full-length antibodies, including wild-type full-length antibodies, chimeric full-length antibodies, and humanized full-length antibodies, but this list is not exhaustive. In most mammals, including humans and mice, full-length antibodies are generally tetrameric in structure. The tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (typically having a molecular weight of about 25 kDa) and one "heavy chain" (typically having a molecular weight of about 50-70 kDa). In human immunoglobulins, light chains are classified as kappa light chains and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. Thus, "isotype" as used herein refers to any of the immunoglobulin classes defined by the chemical and antigenic properties of their constant regions. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

[0211] 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.

[0212] In some other embodiments, "M" comprises or consists of a cell-type specific ligand derived from a monosaccharide or polysaccharide, including, for example, one or more galactoses, mannose-6-phosphate, N-acetylgalactosamine (GalNAc) and cross-linked GalNAc, and sialic acid and its derivatives (e.g., Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5Ac). The monosaccharide or polysaccharide can be natural or synthetic.

[0213] In another embodiment, "M" comprises or consists of a cell type specific ligand derived from a vitamin such as folic acid.

[0214] According to one embodiment, the cell type specific ligand included in "M" can be derived from or within a muscle targeting peptide (MTP), such as 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), cyclic CQLFPLFRC (SEQ ID NO: 6), or the following: The amino acid sequence may comprise an amino acid sequence selected from the group consisting of the sequence of SEQ ID NO: 7 as set forth in RXRRXRRXRFQILYRXRXRXRX (SEQ ID NO: 7), where X is either an aminohexanoic acid residue or a beta-alanine residue as set forth in the sequence listing.

[0215] As used herein, cyclic CQLFPLFRC of SEQ ID NO: 6 means:

[0216] [ka]

[0217] Refers to...

[0218] In certain embodiments, "M" is a cancer cell targeting peptide, including peptides such as RGD, including cyclic RGD.

[0219] In some other embodiments, M is a cell-type targeting ligand selected from antibodies and fragments thereof.

[0220] When "M" includes a peptide moiety such as a muscle-targeting peptide (MTP), the peptide moiety can include chemical modifications at its M-terminus or C-terminus. For example, the N-terminus of the peptide moiety can be acylated or coupled to a moiety such as -C(=O)-(PEG moiety)-NH2.

[0221] 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.

[0222] In additional embodiments, "M" is a CB1 and / or CB2 ligand, such as:

[0223] [ka]

[0224] Comprises or consists of.

[0225] 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 (Va), (Vb), or (Vc):

[0226] [ka]

[0227] It includes at least one part of.

[0228] In some other embodiments, "M" is a ligand for targeting muscle cells, particularly skeletal muscle cells, and comprises at least one of the following mannose-6-phosphate moieties:

[0229] [ka]

[0230] Includes:

[0231] 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 (Vf):

[0232] [ka]

[0233] Includes:

[0234] In some other embodiments, "M" is a ligand for a Siglec protein (sialic acid-binding immunoglobulin-like lectin). In some embodiments, M is a sialic acid moiety or a derivative thereof. As used herein, "sialic acid moieties and derivatives thereof" includes moieties that contain one or more N-acylated neuraminic acid units and, optionally, one or more other saccharide units, such as galactose moieties.

[0235] More specifically, M can be a sialic acid moiety or a derivative thereof, said moiety having the formula (Vg):

[0236] [ka]

[0237] wherein R5 is alkyl, aryl, heteroaryl, haloalkyl (e.g., —CH2-Hal (wherein Hal is halogen)), —OR6, —NR7R8, —SR9, —CH2OR 10, -CH2NR 11 R 12 , or -CH2SR 13 (In the formula, R6, R 7、 R8, R9, R 10 , R 11、 R 12 , and R 13 are each independently selected from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group. It comprises or consists of at least one part of

[0238] In certain embodiments, R5 is alkyl, -OR6, -CH2OR 10、 -CH2-Hal, wherein Hal, R6, R 10 is as defined herein.

[0239] In more particular embodiments, R5 is methyl, -CH2OH, or -CH2-F.

[0240] In certain embodiments, M is selected from Neu5Ac, Neu5Acα2-6Gal, and Neu5Acα2-8Neu5Ac moieties.

[0241] As used herein, a Neu5Ac moiety refers to an N-acetylneuraminic acid moiety. Neu5Ac has the following formula (Vh):

[0242] [ka]

[0243] It can be expressed as:

[0244] As used herein, a Neu5Acα2-6Gal moiety refers to a moiety consisting of an N-acetylneuraminic acid unit and a galactose unit linked by an α2-6 bond. The Neu5Acα2-6Gal moiety has the following formula (Vi):

[0245] [ka]

[0246] It can be expressed as:

[0247] As used herein, a Neu5Acα2-8Neu5Ac moiety refers to a moiety consisting of two N-acetylneuraminic acid units linked by an α2-8 bond. The Neu5Acα2-8Neu5Ac moiety has the following formula (Vj):

[0248] [ka]

[0249] It can be expressed as:

[0250] In some embodiments, "M" is multivalent, meaning that it includes at least two (e.g., two, three, four, five, or six) ligand moieties of interest, such as cell-type-specific ligands as described above. By way of example, M can include a multifunctional linker bearing several (e.g., at least two, three, four, five, or six) cell-type-specific ligands. The cell-type-specific ligands can be the same or different.

[0251] As an example, "M" may be represented by the following formula (VI):

[0252] [ka]

[0253] (where n is an integer from 1 to 100, preferably from 1 to 20). may include a portion of

[0254] As another example of a multivalent ligand, "M" can comprise a moiety of formula (VI) in which the GalNAc group is replaced by mannose, 6-mannose phosphate, cross-linked GalNAc, sialic acid or a derivative thereof (e.g., as shown above, e.g., Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5AcNeu5Ac), CB1 and / or CB2 ligands or peptides.

[0255] In some embodiments, M is an oligosaccharide selected from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, galactans, and mannans.

[0256] In certain embodiments, "M" may include both a labeling moiety, such as a fluorescent label or radionuclide, and a cell type-specific ligand. By way of example only, M may be:

[0257] [ka]

[0258] That is, it may be the muscle targeting peptide of SEQ ID NO: 1 linked to K-FITC.

[0259] Other examples of chemical moieties that can be used as the "M" moiety are provided in Figures 5A and 5B.

[0260] In certain embodiments, M comprises or consists of a cell targeting agent, preferably selected from mono- or polysaccharides, hormones, e.g., steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTPs), 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 small chemical molecules such as drugs, e.g., CB1 and / or CB2 ligands.

[0261] In another embodiment, 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, sialic acid and its derivatives (e.g., Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5Ac), an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7, and a vitamin such as folic acid.

[0262] In certain embodiments, M is a chemically reactive group, more preferably a biocompatible chemically reactive group. As used herein, M can enable covalent interaction between the AAV and the entity of interest without significantly altering the functionality of the AAV (and thus in a biocompatible manner). In other words, the functional moiety can include a chemically reactive group that can facilitate the formation of a covalent bond with the entity of interest. For example, the functional moiety can include a chemically reactive group suitable for forming a covalent bond by click chemistry or a bioconjugation reaction. Bioconjugation reactions involve reactions between amino acids such as lysine, cysteine, or tyrosine and a reactive group, as detailed in Koniev, O., Wagner, A., Chem. Soc. Rev., 44, 5495 (2015).

[0263] Preferably, M is a click chemistry reactive group, hereinafter also referred to as a "click chemistry group".

[0264] As used herein, "click chemistry group" refers to any reactive chemical group that can participate in a click chemistry reaction. Preferably, M is not a thiol (-SH).

[0265] The term "click reaction" or "click chemistry" was introduced by Sharpless in 2001. "Click chemistry" generally refers to a chemical reaction characterized by high yield, high functional group selectivity, is easy to perform, and generates harmless by-products. "Click reactions" can typically be performed with high efficiency in complex media. Click reactions are typically used to create a covalent heteroatom linkage (CXC) between two entities of interest. For general reviews of click chemistry, see Kolb et al., Angew. Chem. Int. Ed. 2001, 40, 2004-2021, and Rudolf et al., Current opinion in Chemical Biology, 2013, 17:110-117.

[0266] Examples of click chemistry reactions include, but are not limited to, Staudinger ligation, azido-ene or azide-alkyne click chemistry, carbonyl condensation, sydnone-alkyne cycloaddition, tetrazole-ene reaction, nitrile oxide-ene click chemistry, nitrile imine-ene click chemistry, inverse electron demand Diels-Alder ligation, isonitrile-tetrazine click chemistry, Suzuki-Miyaura coupling, or His tag. Preferably, the click chemistry reaction is not a thiol-ene reaction or a thiol-maleimide reaction.

[0267] By way of example, M may comprise or consist of an azide (-N3), a phosphine such as a triarylphosphine, an aldehyde, a ketone, a hydrazide, an oxyamine, a nitrile oxide, an oxime, a hydroximoyl chloride, a chlorooxime, a nitrile imine, a hydrazone, a hydrazonoyl chloride, a chlorohydrazone, a tetrazine, a tetrazole, an isonitrile, an aryl halide, an arylboronic acid, an oligohistidine, a nickel complex, or a nickel ligand.

[0268] In a preferred embodiment, M is N3.

[0269] In some embodiments of the present invention: - spacer Y, if present, one or more heteroatoms selected from —O—, —S—, —NH—, —C(O)—, —NHC(O)—, and —OC(O)—; and / or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyl or heteroaryl having 5 to 20 ring atoms, preferably triazolyl, optionally fused to dibenzazepinyl; may be arbitrarily interrupted by; and optionally having a heteroatom group selected from —O—, —S—, —NH—, —C(O)—, —NHC(O)—, and —OC(O)— at at least one of its termini; a hydrocarbon chain (e.g., an alkyl chain) having from 2 to 100 carbon atoms, from 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms; M comprises or consists of a click chemistry group, preferably a cell targeting agent selected from a mono- or polysaccharide, a hormone, e.g. a steroid hormone, a peptide such as an RGD peptide, a muscle targeting peptide (MTP) or angiopep-2, a membrane receptor or a fragment thereof, an aptamer, an antibody including a heavy chain antibody and fragments thereof such as Fab, Fab' and VHH, an ScFv, a spiegelmer, a peptide aptamer, a vitamin, and a small chemical molecule such as a drug, e.g. a CB1 and / or CB2 ligand.

[0270] Preferably, Y is of formula (III) as defined above.

[0271] In some other embodiments: - spacer Y, if present, one or more heteroatoms selected from —O—, —S—, —NH—, —C(O)—, —NHC(O)—, and —OC(O)—; and / or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyl or heteroaryl having 5 to 20 ring atoms, preferably triazolyl, optionally fused to dibenzazepinyl; may be arbitrarily interrupted by; and optionally having a heteroatom group selected from —O—, —S—, —NH—, —C(O)—, —NHC(O)—, and —OC(O)— at at least one of its termini; a hydrocarbon chain (e.g., an alkyl chain) having from 2 to 100 carbon atoms, from 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms; and - M comprises or consists of a click chemistry group, 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, sialic acid and its derivatives (e.g., Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5Ac), an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7, and a cell type-specific ligand derived from a vitamin such as folic acid.

[0272] Preferably, Y is of formula (III) as defined above.

[0273] In some embodiments: - Y has at one of its termini (typically the terminus linked to M) a heteroatom selected from -ON(R)- (wherein R is H or C1-C3 alkyl) and -N(C1-C3 alkoxy)-, preferably a heteroatom selected from -ON(Me)-, -O-NH-, -N(OMe)-; and M is a sugar, a disaccharide, or an oligosaccharide, preferably selected from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, galactans, and mannans.

[0274] In such embodiments, Y is: - at one of its ends (typically the end linked to M), has a heteroatom selected from -ON(R)- (wherein R is H or C1-C3 alkyl) and -N(C1-C3 alkoxy)-, preferably a heteroatom selected from -ON(Me)-, -O-NH-, and -N(OMe)-; - optionally having at the other one of its termini a heteroatom selected from -O-, -S-, -NH-, -C(O)-, -NHC(O)-, and -OC(O)-, preferably -O-; - optionally interrupted by one or more heteroatomic groups selected from -O-, -S-, -NH-, -C(O)-, -NHC(O)- and -OC(O)-, preferably -NHC(O)-, It can be a hydrocarbon chain (eg, an alkyl chain) having from 2 to 100 carbon atoms, from 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms.

[0275] For illustrative purposes only, -(Y) n -M can be any one of the following formulas:

[0276] [ka]

[0277] where p is an integer from 2 to 10, R is H or methyl, and Ms is a monosaccharide (e.g., mannose or GalNAc), a disaccharide moiety, or an oligosaccharide preferably selected from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, galactans, and mannans.

[0278] In all the above embodiments, the AAV is preferably a recombinant AAV, more preferably a recombinant AAV vector.

[0279] As mentioned above, AAV may have a "naturally occurring" capsid or a genetically modified capsid, i.e., a capsid containing one or several mutations in at least one capsid protein, i.e., VP1, VP2, and / or VP3.

[0280] In some further or alternative embodiments, the AAV can be a serotype selected from AAV1, AAV4, AAV6, AAV7, AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9, and AAVrhlO, preferably AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9, and AAVrhlO, more preferably AAV-2 or AAV-9. Alternatively, the AAV is a synthetic serotype.

[0281] In some further embodiments, the AAV of the present invention may have at least one additional chemically modified amino acid residue in the capsid that is different from a tyrosine residue, for example, an arginine or lysine residue, in its side chain, having a structure of formula (VIII):

[0282] [ka]

[0283] (In the formula, N* is the nitrogen of the amino group of the side chain of an amino acid residue, such as a lysine or arginine residue, and - Y' has the same definition as Y, n' is 0 or 1, and M' has the same definition as M) has a modified amino group.

[0284] It is understood that Y', n', and M' can be the same as or different from those present in at least one chemically modified tyrosine as described above.

[0285] Modifications on the amino group can be introduced as described in International Patent Application Publication No. WO 2017212019, the contents of which are incorporated herein by reference.

[0286] In some embodiments, the amino acid residue has formula (IX):

[0287] [ka]

[0288] (In the formula, - X" is

[0289] [ka]

[0290] (In the formula, Z is -O-, -S-, or -N(R4)-; R1, R2, R3, and R4 are each independently selected from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, which groups may be optionally substituted; w is 0 or 1, - R0 is hydrogen, halogen, alkyl, aryl, heteroaryl, or alkoxy, and said groups may be optionally substituted. is selected from the group consisting of - Y" has the same definition as Y, n" is 0 or 1, and M" has the same definition as M) is a modified cysteine ​​residue of

[0291] It is understood that Y", n", and M" can be the same or different from those present in at least one chemically modified tyrosine as described above.

[0292] Chemical modification of the AAV capsid can alter one or several biological functions and / or properties. Depending on the nature of "M" covalently attached to the surface of the chemically modified AAV, the chemically modified AAV may have one or several altered biological properties, such as the following biological properties, compared to the same but unmodified AAV: - altered tropism, e.g., increased selectivity of the AAV for a particular organ, tissue, or cell (either administered in vivo or 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 cell death in cancer cells - Visualization / monitoring of AAV particles during in vivo administration or in vitro cell modification using these AAV particles. - Theragnostic applications; e.g., combining therapeutic and diagnostic agents may have:

[0293] In some embodiments, the chemically modified AAV of the present invention may have higher transduction efficiency, which may be due to increased intracellular transport to the nucleus, decreased proteasomal degradation, more efficient intranuclear decapsidation, more rapid vector genome stabilization, and / or decreased interaction with neutralizing antibodies and / or reduced antibody-mediated clearance of AAV in vivo, compared to non-chemically modified AAV. In some other embodiments, the AAV may have 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.

[0294] In some other embodiments, when AAV is used as a drug, e.g., as a gene vector, such modified 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.

[0295] In certain embodiments, the chemically modified AAV of the present invention exhibits preferential tropism for an organ or cell selected from the group consisting of 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, myocytes, neurons, motor neurons, retinal pigment cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs).

[0296] Methods for preparing chemically modified AAV of the present invention The present invention also relates to a method for modifying the capsid of an AAV with a chemical reagent having an N-substituted luminol moiety, and as mentioned above, such a chemical reagent can be a functional moiety "M" as described above linked to an N-substituted luminol moiety either directly or via a spacer Y.

[0297] More specifically, the object of the present invention is 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 the step of incubating said AAV with a chemical reagent having an N-substituted luminol moiety under conditions conducive to reacting said chemical reagent with the tyrosine residue present in the capsid of the AAV to form a covalent bond, said N-substituted luminol moiety being selected from the group consisting of N-(C1-C6 alkyl) luminol, N-(C6-C 14 aryl(optionally substituted))luminol, or N—[(C6-C 14the luminol moiety is an N-methyl or N-benzyl luminol moiety, and more preferably an N-methyl luminol moiety.

[0298] The coupling conditions typically allow for in situ activation of the N-substituted luminol moiety to an oxidized entity capable of reacting with the phenyl group of the tyrosine residue. Preferably, the coupling conditions allow for the formation of a nitrogen-centered radical in the N-substituted luminol moiety. Such oxidation can be carried out by any method known to those skilled in the art for activating N-substituted luminol, preferably to a single radical, for example, by an enzymatic system such as horseradish peroxidase / H2O2 or lactase / O2, or by electrochemical methods.

[0299] As explained above, the applicants have shown that chemical reagents containing N-substituted luminols have an oxidation potential of about 0.6 V versus a saturated calomel electrode (SCE), regardless of the substituents present on the nitrogen atom or aromatic ring of the N-substituted luminol, and that the resulting oxidized entities formed at this potential are reactive with the phenyl group of tyrosine.

[0300] Moreover, applicants have shown that subjecting AAV to such low potentials does not compromise its structural and functional integrity.

[0301] Finally, we have successfully modified tyrosine residues in AAV capsids specifically and efficiently by incubating AAV particles with a chemical reagent bearing an N-substituted luminol moiety under a constant low potential difference for a short period of time.

[0302] Therefore, the chemically modified AAV of the present invention is preferably prepared by electrochemical methods.

[0303] As used herein, "electrochemical methods" refers to a branch of chemical methods in which a reaction between entities of interest is induced by subjecting the entities to an electrical potential difference.

[0304] In certain embodiments, the method of the present invention refers to a method for chemically modifying the capsid of an AAV with a chemical reagent having an N-substituted luminol moiety by electrochemical method, i.e., by contacting the AAV with the chemical reagent under a potential difference that allows the chemical reagent to be electrically activated. Typically, the potential difference satisfies that it does not significantly impair the integrity of the AAV particle.

[0305] As used herein, "electroactivation of a chemical reagent of the present invention" refers to the oxidation, using an electrical potential difference, of a chemical reagent of the present invention to an oxidized form capable of reacting with a phenyl group present in a tyrosine residue.

[0306] In a more specific embodiment, the present invention provides an adeno-associated virus (AAV) having formula (X):

[0307] [ka]

[0308] (In the formula, R A and R B is as above) and a chemical reagent of formula (X) under electrochemical conditions conducive to reacting the chemical reagent with a tyrosine residue present in the capsid of the AAV to form a double bond.

[0309] Preferred chemical reagents of formula (X) are:

[0310] [ka]

[0311] especially,

[0312] [ka]

[0313] (In the formula, R A is C1-C3 alkyl, optionally substituted phenyl, or optionally substituted phenyl-(C1-C3 alkyl), preferably methyl or benzyl, and Y, n, and M are as defined above. is.

[0314] More particularly, such a reagent of formula (X) is incubated with AAV to obtain at least one chemically modified tyrosine residue in the capsid of formula (I) as defined above.

[0315] The electrochemical conditions (also referred to herein as potential conditions) are selected to allow for oxidation of the chemical reagent of formula (X) without compromising the integrity of the AAV.

[0316] As used herein, "potential conditions" refers to a potential difference relative to a reference electrode that allows for the oxidation of a chemical reagent, preferably to a radical.

[0317] This potential difference is close to the oxidation potential (determined relative to the reference electrode) of the chemical reagent. Typically, the potential difference to be applied is generally within the range of the oxidation potential (OP) of the chemical reagent minus 200 mV to the OP plus 500 mV, preferably minus 150 mV to the OP plus 400 mV.

[0318] In certain embodiments, the potential difference to be applied is equal to or substantially close to the oxidation potential (OP) of the chemical reagent, for example, equal to the oxidation potential of the chemical reagent ±200 mV or ±150 mV, more preferably ±100 mV, such as ±50 mV or ±25 mV.

[0319] The potential difference to be applied to oxidize the chemical reagent in the method of the present invention will vary depending, inter alia, on the standard potential of the reference electrode used to carry out the method.

[0320] For a given chemical reagent, the oxidation potential can be determined by standard procedures well known to those skilled in the art, such as cyclic voltammetry, see the methods described in the Examples section (see Example 2).

[0321] Typically, the AAV and chemical reagents are exposed to a potential difference (or equivalently, a "voltage"). Any suitable electrochemical device can be used to apply the voltage. In some embodiments, the voltage is applied using an electrochemical device comprising a three-electrode system, i.e., a working electrode, an auxiliary electrode, and a reference electrode. The working electrode refers to the electrode where the reaction of interest occurs. Depending on whether the reaction at the electrode is reduction or oxidation, the working electrode is called the cathode or anode, respectively. In the context of the present invention, the working electrode is the anode. In the context of the present invention, the auxiliary electrode (also called the counter electrode) is the cathode. The electrochemical device may further comprise a vial, electrical connection means, and means for controlling the potential difference between the reference electrode and the working electrode, typically a potentiostat.

[0322] For example, on a laboratory scale, AAV and chemical reagents are mixed in a suitable buffer, and the resulting mixture is placed in a suitable vial (e.g., a plastic or glass vial) into which three electrodes are inserted. The three electrodes are connected to a potentiostat by suitable electrical connections. The auxiliary electrode can be isolated, for example, by using a glass frit, to avoid the formation of by-products. The potentiostat is used to maintain the potential difference between the reference electrode and the working electrode at a constant value that selectively allows the oxidation of the chemical reagent.

[0323] Anode, cathode, and reference electrodes that can be used in electrochemical processes are well known to those skilled in the art.

[0324] Examples of materials from which the anode can be made include, but are not limited to, carbon (e.g., glassy carbon or graphite), lead bronze, tungsten, niobium, copper, magnesium, titanium, zinc, stainless steel, platinum, gold, silver, aluminum, boron-doped diamond, tin, nickel, cobalt, preferably carbon anodes (e.g., glassy carbon or graphite).

[0325] Examples of materials from which the cathode can be made include, but are not limited to, nickel, platinum, silver, lead bronze, tungsten, niobium, copper, magnesium, titanium, zinc, stainless steel, gold, aluminum, boron-doped diamond, tin, nickel, and cobalt, with a platinum cathode being preferred.

[0326] Examples of reference electrodes include, but are not limited to, a standard hydrogen electrode (SHE), a normal hydrogen electrode (NHE), a reversible hydrogen electrode (RHE), a saturated calomel electrode (SCE), a copper-copper(II) sulfate electrode (CSE), a silver chloride electrode, a palladium-hydrogen electrode, a dynamic hydrogen electrode (DHE), and a mercury-mercury sulfate electrode (MSE), preferably a silver chloride electrode.

[0327] Reference electrode potentials are readily available in reference handbooks. Examples of reference potentials defined for SHE are provided herein as follows (a non-exhaustive list): - Standard Hydrogen Electrode (SHE): E=0.000V - Normal Hydrogen Electrode (NHE): E≒0.000V, - Reversible Hydrogen Electrode (RHE): E=0.000V, - Saturated calomel electrode (SCE): E=+0.241V, saturated, - Copper-copper(II) sulfate electrode (CSE): E=+0.314V, - Silver chloride electrode: E=+0.197V, in saturated KCl - Silver chloride electrode: E=+0.210V, in 3.0mol KCl / kg - Silver chloride electrode: E=+0.22249V in 3.0mol KCl / L.

[0328] Preferably, the electrochemical device used to apply the voltage comprises: - a silver chloride electrode as a reference electrode, - a platinum electrode as the cathode, and - Carbon (e.g., graphite) electrode as the anode Includes:

[0329] The shape and form of the electrode are not particularly limited. For example, a carbon electrode may be in the form of a mesh, layer, or crucible. Examples of electrode shapes include, but are not limited to, a plate, a wire, or a flat rod. The size of the electrode can be adjusted by those skilled in the art depending on the scale of the process, particularly the volume of the incubation medium. The surface of the electrode in contact with the incubation medium can also be adjusted by those skilled in the art. Preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total surface of each electrode is in contact with the incubation medium.

[0330] The voltage can be set and controlled by any suitable device, typically a potentiostat connected to the electrodes (ie, cathode, anode, reference electrode) of the electrochemical device.

[0331] The voltage is set to a value that allows activation (e.g., oxidation) of the N-substituted luminol moiety of the chemical reagent. The voltage to be applied can be readily determined by one skilled in the art by cyclic voltammetry in the incubation medium of interest (i.e., herein, the buffer of interest), particularly through determination of the oxidation potential of the chemical reagent relative to a reference electrode.

[0332] As mentioned above, the potential difference to be applied during incubation is generally within the range of the oxidation potential (OP) of the chemical reagent minus 200 mV to the OP plus 500 mV, preferably minus 150 mV to the OP plus 400 mV.

[0333] In certain embodiments, the potential difference to be applied is equal to or substantially close to the oxidation potential (OP) of the chemical reagent, for example, equal to the oxidation potential of the chemical reagent ±150 mV, more preferably ±100 mV, such as ±50 mV or ±25 mV.

[0334] In some embodiments, incubation is carried out at a voltage equal to the oxidation potential of such chemical reagent ±150 mV, preferably ±100 mV or ±50 mV, more preferably ±40 mV or ±30 mV, for example ±20 mV or ±10 mV.

[0335] Typically, the oxidation potential of the chemical reagents used in the present invention is about 0.75 V vs. Ag / AgCl in saturated KCl. The incubation can be carried out at a voltage selected from values ​​within the range of +0.55 V to 1.25 V vs. Ag / AgCl in saturated KCl, preferably between +0.60 V and 1.0 V vs. Ag / AgCl in saturated KCl, more preferably about +0.70 V to +0.85 V vs. Ag / AgCl in saturated KCl, e.g., 0.70 V to 0.80 V, e.g., about 0.75 V vs. Ag / AgCl in saturated KCl, where the reference potential for Ag / AgCl in saturated KCl is +0.197 V.

[0336] As another example, incubation may be carried out at a voltage between +0.55V and +0.65V vs. SCE, preferably between +0.57V and +0.63V vs. SCE, more preferably at about +0.6V vs. SCE, where the reference potential of SCE is +0.241V.

[0337] 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 7.0 to 8.0, for example about 7.4. The concentration of the buffering agent is at least 30 mM, preferably at least 50 mM and up to 1 M.

[0338] The buffer may be selected from suitable biocompatible buffers, such as TRIS buffer, sodium carbonate-sodium bicarbonate buffer, phosphate buffers such as PBS or Dulbecco's phosphate buffered saline (dPBS), or Good's buffer.

[0339] The incubation time during which the potential difference is applied can vary depending on several parameters, such as (i) the volume of the buffer and the dimensions of the vial, (ii) the surface of the electrode in contact with the incubation medium, (iii) the amount of chemical reagent and the amount of AAV to be chemically modified, (iv) the chemical reagent, (v) the solubility of the chemical reagent, (vi) the voltage, and (vii) the stirring speed.

[0340] Incubation can last from a fraction of a second to several hours, for example, from 5 seconds to 120 minutes, for example, from 10 seconds to 60 minutes, for example, from 15 seconds to 30 minutes, for example, from 30 seconds to 10 minutes, for example, from 5 seconds to 20 seconds, from 20 seconds to 40 seconds, from 40 seconds to 60 seconds, from 1 minute to 2 minutes, from 2 minutes to 3 minutes, from 3 minutes to 5 minutes, from 5 minutes to 7 minutes, or from 7 minutes to 10 minutes. In some embodiments, effective coupling is obtained within 5 minutes, for example, within 1 minute, of incubation under the potential difference.

[0341] The incubation temperature is typically from 10° C. to 40° C. Preferably, the incubation is carried out at room temperature, ie, from 18° C. to 30° C., for example, at about 20° C.

[0342] In some embodiments, the reaction is carried out under stirring, preferably under orbital stirring. In some embodiments, the reaction is carried out without stirring.

[0343] AAV titers are typically between 1E11 vg / mL and 1E14 vg / mL, for example, 1E12 vg / ml.

[0344] The concentration of the chemical reagent can be from 0.01 mM to 50 mM, for example, from 0.1 mM to 20 mM, such as from 0.1 mM to 10.0 mM or from 0.5 mM to 5.0 mM.

[0345] The ratio of chemical reagent concentration to AAV particles can be from 3E2 to 3E7.

[0346] In another specific embodiment, the method of the present invention relates to a method for chemically modifying the capsid of an AAV with a chemical reagent having an N-substituted luminol moiety using an enzyme system, i.e., by incubating the AAV and the chemical reagent in the presence of an enzyme system that allows activation of the chemical reagent. Preferably, the chemical reagent is a compound of formula (X), as described above.

[0347] The enzyme system consists in particular of a combination of an enzyme and an oxidizing agent, for example a peroxidase (eg horseradish peroxidase) with H2O2, or lactase with O2.

[0348] The pH, buffer, temperature and duration of incubation, as well as the concentrations of chemical reagents and AAV, can be similar to those described above for the electrochemical conditions.

[0349] The method of the present invention may comprise one or several additional steps before or after the incubation step (eg electrochemical or enzymatic incubation) as described above.

[0350] By way of example, the methods of the invention may include a step of providing or producing AAV particles to be chemically modified.

[0351] The method of the present invention may also include the step of providing or preparing a chemical reagent.

[0352] The chemical reagent can be prepared by a synthetic route. For example, the chemical reagent of formula (X) can be prepared from methyl-hydrazine and phthalic anhydride or phthalate, and can be substituted with a functionalizable group. For illustrative purposes only, see the synthesis of compounds 7 or 12 described in the Examples section.

[0353] To prepare chemical reagents of formula (X), where Y has a heteroatom selected from -ON(R)- (where R is H or C1-C3 alkyl) and -N(C1-C3 alkoxy)- at the terminus linked to M, and M is an oligosaccharide, sugar, or disaccharide, the corresponding precursors having terminal functional groups of -O-NH(R) and -NH(C1-C3 alkoxy), respectively, can be contacted with the oligosaccharide.

[0354] When the above electrochemical conditions are used, the method of the present invention may also include the step of determining the oxidation potential of the chemical reagent relative to a reference electrode, such as Ag / AgCl in saturated KCl or a saturated calomel electrode (SCE).

[0355] The method of the present invention may also comprise one or several additional steps after the incubation step, such as: removing unreacted reagents, for example by dialysis, tangential flow filtration, centrifugation through a porous membrane, steric exclusion chromatography, 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.

[0356] The methods of the present invention may further comprise the step of chemically modifying amino acid residues other than tyrosine residues in the AAV capsid.

[0357] By way of example, the additional chemically modified amino acid residue may have an amino group (eg, lysine, arginine) or may be cysteine.

[0358] In certain embodiments, the methods of the invention involve providing an AAV having formula (XI):

[0359] [ka]

[0360] Such a step may include incubating a compound of formula: with a chemical reagent of formula: 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.

[0361] [ka]

[0362] (In the formula, N* is the nitrogen of the amino group of an amino acid residue, such as a lysine or arginine residue, - Y' has the same definition as Y, n' is 0 or 1, and M' has the same definition as M) It is understood that Y', n', and M' may be the same as or different from those present in at least one chemically modified tyrosine as described above.

[0363] Typically, such a process 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., room temperature. Details regarding the implementation of such a process can be found in International Patent Application Publication No. WO 2017 / 212019, the contents of which are incorporated herein by reference.

[0364] This step can be carried out before or after the step of chemically modifying at least one tyrosine residue in the capsid of the AAV as described herein.

[0365] In particular or alternative embodiments, the methods of the present invention comprise reacting a compound of formula (XIIa), (XIIb), or (XIIc):

[0366] [ka]

[0367] (In the formula, Z is -O-, -S-, or -N(R4)-; R1, R2, R3, and R4 are each independently selected from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, which groups may be optionally substituted; w is 0 or 1, R0 is hydrogen, halogen, alkyl, aryl, heteroaryl, or alkoxy, which may be optionally substituted; - Y" has the same definition as Y, n" is 0 or 1, and M" has the same definition as M) The method may include incubating the AAV with the chemical reagent of the present invention under conditions conducive to reacting the chemical reagent with cysteine ​​residues present in the capsid of the AAV to form a covalent bond.

[0368] It is understood that Y", n", and M" can be the same as or different from those present in at least one chemically modified tyrosine residue as described above.

[0369] Such a process makes it possible to chemically modify the cysteine ​​residues of the capsid according to the following formula:

[0370] [ka]

[0371] (In the formula, - X" is

[0372] [ka]

[0373] (In the formula, Z is -O-, -S-, or -N(R4)-; R1, R2, R3, and R4 are each independently selected from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, which groups may be optionally substituted; w is 0 or 1, - R0 is hydrogen, halogen, alkyl, aryl, heteroaryl, or alkoxy, and said groups may be optionally substituted. is selected from the group consisting of - Y" has the same definition as Y, n" is 0 or 1, and M" has the same definition as M)

[0374] This process is - chemically modifying at least one tyrosine residue in the capsid of the AAV as described above, and / or - chemically modifying at least one amino group (e.g., derived from arginine or lysine) in the capsid of the AAV as described above; It can be performed before or after

[0375] Advantageously, the functional group M of the chemical reagent (chemical reagent of formula (X), (Xa), (Xb), or (Xc)) is compatible with the conditions of the incubation step (e.g., electrochemical or enzymatic incubation) described above. In other words, the functional group M in the chemical reagent should not be oxidized under the oxidizing conditions of the incubation step or react with activated N-substituted luminol. Preferably, the functional group M of the chemical reagent does not contain any phenol moiety. By way of example, such a functional group is not the MTP of SEQ ID NO: 1 linked to K-FITC as defined above, nor the MTP of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 7 as defined above, nor the ligand of formula (IVb) as defined above.

[0376] More generally, the group M present in the chemical reagent of formula (X), (Xa), (Xb), or (Xc) may not contain any chemical group with an oxidation potential equal to or lower than the oxidation potential of the N-substituted luminol moiety.

[0377] The introduction of functional groups on the surface of AAVs that have chemical groups incompatible with electrochemical activation as described above can be carried out in two steps: the introduction of chemical moieties on the surface of the AAVs that are capable of undergoing a click chemistry reaction, and then the introduction via click chemistry of the functional moiety of interest that has such incompatible chemical moieties.

[0378] Indeed, when M present in the chemical reagent of formula (X), (Xa), (Xb), or (Xc) is a click chemistry group, the chemically modified AAV can be subjected to a supplementary step aimed at coupling a functional group Z' by reaction with the M group. The implementation of this supplementary step is particularly suitable when Z' is a functional group that is incompatible with the conditions of the above-mentioned incubation step (e.g., electrochemical or enzymatic incubation). The incompatibility of the functional group can be due, for example, to the ability of such a functional group to be oxidized under the oxidizing conditions of the incubation step (i.e., the functional group has an oxidation potential lower than or equal to the oxidation potential of N-substituted luminol) or its ability to react with activated N-substituted luminol.

[0379] Z' may be a functional group containing a phenol moiety or other moieties that can be found in certain fluorophores that are not compatible with electrochemical methods. Examples include xanthene derivatives such as fluorescein and rhodamine, and cyanine 3 / 5 / 7 (Cy3, Cy5, Cy7). Other examples are peptides / polypeptides containing tyrosine residues, such as therapeutic antibodies and therapeutic proteins. For example, Z may be a functional group containing a phenol moiety, such as MTP of SEQ ID NO: 1 linked to K-FITC as defined above, MTP of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 7 as defined above, or a ligand of formula (IVb) as defined above.

[0380] Accordingly, the present invention also provides a method for grafting a functional moiety Z' onto a tyrosine residue in an AAV capsid, said method comprising the steps of: - preparing a chemically modified AAV having at least one chemically modified tyrosine comprising a functional M group, preferably by a method as described above, wherein the functional M group is a click chemistry group; and - coupling the functional group Z' with the group M by a click reaction; The present invention relates to a method, including:

[0381] In such embodiments, M is a click chemistry group that is compatible with the conditions of the incubation step (e.g., electrochemical or enzymatic incubation) described above, i.e., a click chemistry group that cannot be oxidized under the oxidizing conditions of the incubation step (i.e., has an oxidation potential higher than that of N-substituted luminol) or cannot react with activated N-substituted luminol. Preferably, in such embodiments, M is an azide, a phosphine, an aldehyde, a ketone, a hydrazide, an oxyamine, a nitrile oxide, an oxime, a hydrochloride, a chlorooxime, a nitrile imine, a hydrazone, a hydrazonoyl chloride, a chlorohydrazone, a tetrazine, an isonitrile, an aryl halide, an arylboronic acid, an oligohistidine, a nickel complex, or a nickel ligand. More preferably, in such embodiments, M is an azide.

[0382] Typically, the chemically modified AAV of the present invention has the following formula (XIII): Q-(W) r -Z' (XIII) (In the formula, Q is a click chemistry group capable of reacting with M via a click chemistry reaction, - r is 0 or 1, - W is a spacer, and - Z' is a functional group different from the click chemistry group. can react with compounds of the formula:

[0383] Q is an azide (-N3), alkene, or alkyne (especially cyclooctyne (OCT), aryllesscyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa-dibenzocyclooctyne, The compound may comprise or consist of a strained alkyne such as octyne (ODIBO), carboxymethyl monobenzocyclooctyne (COMBO), or benzocyclononyne), a phosphine, an aldehyde, a ketone, a hydrazide, an oxyamine, a nitrile oxide, an oxime, a hydroximoyl chloride, a chlorooxime, a nitrile imine, a hydrazone, a hydrazonoyl chloride, a chlorohydrazone, a tetrazine, an isonitrile, an aryl halide, an arylboronic acid, an oligohistidine, a nickel complex, or a nickel ligand.

[0384] Examples of complementary click chemistry groups and click chemistry reactions include azide-alkyne click chemistry (M = azide and Q = alkyne (e.g., strained endocyclic alkyne)), Staudinger ligation (M = azide and Q = phosphine), carbonyl condensation (M = aldehyde or ketone, and Q = hydrazide or oxyamine), sydnone-alkyne cycloaddition (M = sydnone and Q = alkyne), tetrazole-ene reaction (M = tetrazole and Q = alkene), nitrile oxide-ene click chemistry (M = nitrile oxide or aldehyde, oxime, or hydrazine chloride). Examples of such chemistry include, but are not limited to, nitrile imine-ene click chemistry (M = nitrile imine or aldehyde, hydrazone, hydrazonoyl chloride, or chlorohydrazone, and Q = alkene or alkyne), inverse electron demand Diels-Alder ligation (M = alkene and Q = tetrazine), isonitrile-tetrazine click chemistry (M = isonitrile and Q = tetrazine), Suzuki-Miyaura coupling (M = aryl halide and Q = arylboronic acid).

[0385] In the above-mentioned list of click chemistry groups involved in click chemistry reactions, M and Q can be interchanged, provided that M is a click chemistry group that is compatible with the conditions of the incubation step (e.g., electrochemical or enzymatic incubation) described above. All of the above-mentioned chemical reactions result in a covalent linkage.

[0386] In certain embodiments, M is azide (-N3) and Q is an alkyne (eg, a -C≡CH group, or a strained alkyne such as those mentioned above).

[0387] Preferably, M and Q are not thiol groups (-SH) and the click chemistry reaction is not a thiol-ene reaction or a thiol-maleimide reaction.

[0388] Preferred click reactions are metal-free reactions, i.e., click reactions that do not require the presence of a metal catalyst such as a copper salt.

[0389] In certain embodiments, the click reaction of interest is strain-promoted alkyne-azide cycloaddition (SPAAC), which means that M can be an azide group and Q can be a strained alkyne, as described above.

[0390] W is typically a spacer having the same definition as Y in formula (I).

[0391] Z' is typically a functional group having the same definition as M in (I) as defined herein, except that it is not a click chemistry group. By way of example, Z' can be a targeting agent such as a cell type-specific targeting ligand, preferably selected from 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, an oligonucleotide, a vitamin, and a small chemical molecule such as a drug, e.g., a CB1 and / or CB2 ligand.

[0392] By way of example, Z' can be a cell type-specific ligand derived from proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF, muscle-targeting peptides as described above, as well as mono- or polysaccharides containing, for example, one or several galactoses, mannose, mannose-6-phosphate, N-acetylgalactosamine or cross-linked GalNAc, sialic acid and its derivatives (e.g., Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5Ac), CB1 and / or CB2 ligands, and vitamins such as folic acid.

[0393] As mentioned above, Z' may be a functional group that is incompatible with the conditions of the incubation step (e.g., electrochemical or enzymatic incubation) described above, in particular a functional group that contains a phenol moiety and / or has an oxidation potential lower than or equal to the oxidation potential of N-substituted luminol, such as MTP of SEQ ID NO: 1 linked to K-FITC as defined above, MTP of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 7 as defined above, or a ligand of formula (IVb) as defined above.

[0394] The present invention also relates to a chemically modified AAV obtainable or obtainable by the method of the invention as described above.

[0395] 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" moiety, 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, can involve: - modifying tropism, e.g., increasing the selectivity of the AAV for a particular organ, tissue, or cell (either administered in vivo or transducing tissues or cells 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 causing the AAV to elicit 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 drug's benefit 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 cell killing of cancer cells - Allows visualization / monitoring of AAV particles during in vivo administration or in vitro cell modification using these AAV particles - Combining therapeutic and diagnostic agents in AAV This may make it possible.

[0396] Uses of the AAV of the Invention The chemically modified AAV of the present invention may be used as a research tool or as a pharmaceutical, 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.

[0397] In some embodiments, the chemically modified AAV of the present invention is used to deliver nucleic acids, particularly exogenous nucleic acids such as transgenes, to cells and is therefore a recombinant AAV.

[0398] The recombinant AAV can be administered to cells in vivo, ex vivo, or in vitro. The cells can be derived 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 (iPSCs).

[0399] The recombinant AAV of the present invention can be used to deliver a therapeutic nucleic acid of interest to a subject. Accordingly, the present invention relates to a method for delivering a therapeutic nucleic acid of interest to a subject in need thereof, comprising administering a chemically modified AAV of the present invention to a subject in need thereof. 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 desired.

[0400] 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 desired therapeutic effect, 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., as a mixture with one or several pharmaceutical excipients.

[0401] Conditions 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, leukodystrophies, spinal muscular atrophy (SMA), hemophilia, sickle cell disease, and inherited retinal dystrophies. Chemically modified AAV can also be used to treat disorders such as cancer, arthritis, arthropathy, congenital and acquired heart disease, Parkinson's disease, Alzheimer's disease, and infectious diseases such as hepatitis C.

[0402] Another object of the present invention is a pharmaceutical composition comprising the chemically modified AAV of the present invention and at least one pharmaceutically acceptable excipient, which may be selected from well-known excipients such as carriers, preservatives, antioxidants, surfactants, buffers, stabilizers, and the like.

[0403] 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, for example, be a hematopoietic stem cell. The nucleic acid of interest may be of any type and is selected depending on the desired effect.

[0404] For 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 a recombinant AAV may be used in combination with a gene editing tool to promote homologous recombination in targeted cells. 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.

[0405] The present invention also relates to a host cell transfected with a chemically modified AAV of the present invention, said host cell being of any type.

[0406] 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).

[0407] 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. [Example]

[0408] 1. General theory Organic synthesis: Most chemical reagents and anhydrous solvents were purchased from Sigma Aldrich®, Carbosynth®, Acros Organics®, Alfa Aesar®, or TCI Chemical®. All reagents were stored according to detailed specifications and used without further purification. Reactions requiring anhydrous conditions were carried out under a positive pressure of nitrogen or argon. Routine reaction monitoring was performed using thin-layer chromatography (TLC) on Merck 60 F254 silica gel plates. Color development was carried out under UV light (254 nm) or by immersion in solutions of cerium molybdate, potassium permanganate, sulfuric acid, or vanillin followed by heating. Purification by silica gel chromatography was carried out on 60M silica 0.04–0.063 mm. 1H and 13C NMR spectra were recorded on a Bruker Avance 300 or Bruker Avance 400 spectrometer. NMR spectra were assigned based on the following 1D and 2D experiments: 1H, 13C, DEPT-135, COSY, HSCQ, HMBC, and NOESY. All chemical shifts (δ) are shown in ppm on the x-axis, using residual solvent as the internal standard. Coupling constants (J) are reported in Hz, and peak multiplicities are noted according to the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, dd = double doublet, dt = double triplet, and br = broad signal. The atomic numbers used in NMR assignments differ from those used in compound nomenclature. High-resolution mass spectrometry (HRMS) was recorded on a Xevo GL-XS Qtof spectrometer coupled to a Waters Acquity H-class LC instrument. Ionization sources were used using available methods (ESI+, ESI-, ASAP+, ASAP-). A tolerance of 5 ppm was applied between the calculated and experimental values.

[0409] Electrobioconjugation: Aqueous buffers were obtained from Sigma-Aldrich® or Thermofisher®. Fluorescent cyclooctyne was purchased from Jena Biosciences®. Nanobody cyclooctyne and anti-nanobody antibodies were purchased from NanoTag®. An SP-50 potentiostat was purchased from BioLogic®. Electrosynthesis equipment, including ElectraSyn 2.0, electrodes, and vials, was purchased from IKA®. Chronocoulometry experiments were performed with a three-electrode system connected to the SP-50 potentiostat for voltage control. All data were recorded using EC-Lab software. The three-electrode system typically consisted of a graphite plate as the anode, a platinum plate as the cathode, and Ag / AgCl as the reference (a thin silver rod immersed in saturated aqueous KCl solution and protected from the electrolysis mixture by porous fritted glass). Before each experiment, the electrodes used were thoroughly washed with EtOH, SDS, and distilled water, and the working electrode was repolished on high-grit sandpaper (<1200 grit) to prevent potential passivation.

[0410] Example 1 Synthesis of N-methylluminol derivatives Compound 1: Azide derivative of N-methylluminol

[0411] [ka]

[0412] The azide derivative was prepared in five steps from dimethyl 4-hydroxyphthalate as previously reported (S. Depienne et al., Chem. Sci., 2021, 12, pp. 15374-15381). It is formed as a mixture of two regioisomers in a 60 / 40 ratio. 1 H NMR (400.16 MHz, DMSO-d 6 , 298.15 K): δ H8.15 (d, J=8.8 Hz, 0.4H, H Ar ), 7.91 (d, J=8.8 Hz, 0.6H, H Ar ), 7.62 (d, J=2.7 Hz, 0.6H, H Ar ), 7.46 (dd, J=8.8 Hz, J=2.7 Hz, 0.6H, H Ar ), 7.43 (dd, J=8.8 Hz, J=2.7 Hz, 0.4H, H Ar ), 7.33 (d, J= 2.7 Hz, 0.4H, H Ar ), 4.36 (double t, 2H, OCH2CH2N), 3.72 (double t, 2H, OCH2CH2N), 3.55 + 3.53 (double s, 3H, NCH3). 13 C NMR (100.62 MHz, DMSO-d 6 , 298.15 K): δ C 161.2, 160.8, 157.0, 156.9, 150.2, 149.9, 130.8, 128.7, 126.6, 122.7, 121.8, 121.0, 118.7, 108.3, 106.2, 67.4, 49.4, 37.6, 37.3. HRMS (ASAP - ): m / z C 11 H 10 N5O3[MH] - Calculated value: 260.0783 Measured value: 260.0784.

[0413] Synthesis of mannose and GalNAc derivatives

[0414] [ka]

[0415] compound 3 To a suspension of commercially available tBuOK (793 mg, 7.07 mmol, 1 equiv.) in anhydrous THF (25 mL) was added commercially available diethylene glycol 2 (1.34 mL, 14.13 mmol, 2 equiv.) at 0 °C under a positive nitrogen atmosphere. The mixture was stirred at room temperature for 30 min. Then, an 80% solution of propargyl bromide in toluene (0.61 mL, 7.07 mmol, 1 equiv.) was solubilized in 5 mL of anhydrous THF and added dropwise to the mixture. The reaction was stirred at room temperature for 18 h (completion monitored by TLC; Rf = 0.3 in pure AcOEt), and the mixture was filtered through a pad of Celite washed with THF. The resulting solution was concentrated under reduced pressure, and the residue was purified by silica gel chromatography (2:8 CyHex / AcOEt to 100% AcOEt) to give 3 (735 mg, 72%) as a colorless oil. 1 H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 4.20 (d, J=2.4 Hz, 2H, -CH2C≡CH), 3.75-3.65 (m, 6H, H 鎖 ), 3.60 (m, 2H, H 鎖 ), 2.44 (t, J=2.4 Hz, 1H, -CH2C≡CH); HRMS (ES + ): m / z C7H 12 O3Na [M+Na] + Calculated value 167.0684 Measured value 167.0689

[0416] compound 5 To a solution of commercially available peracetylated mannose 4 (1.7 g, 4.39 mmol, 1 equiv.) in anhydrous MeCN (50 mL, 0.1 M) was added mono-O-propargyl chain 3 (950 mg, 6.59 mmol, 1.5 equiv.) at room temperature under a positive nitrogen atmosphere. The mixture was cooled to 0 °C, and BF3.OEt2 (1.6 mL, 13.18 mmol, 3 equiv.) was added dropwise. The reaction was stirred at room temperature for 48 h (completion monitored by TLC; Rf = 0.3 in 4:6 CyHex / AcOEt) and quenched with a saturated solution of NaHCO3. The aqueous layer was extracted three times with AcOEt, and the combined organic layers were washed once with HO and once with brine, then dried over MgSO4, filtered, and concentrated under reduced pressure. The crude compound obtained was purified by silica gel chromatography (60:40 to 40:60 CyHex / AcOEt) to give 5 (1.28 g, 63%) as a colorless oil. 1 H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 5.36 (dd, J=10.0 Hz, J=3.3 Hz, 1H, H 3 ), 5.32-5.24 (m, 2H, H 4 + H 2 ), 4.87 (d, J=1.6 Hz, 1H, H 1 ), 4.30 (dd, J=12.8 Hz, J=5.3 Hz, 1H, H 6a ), 4.20 (d, J=2.4 Hz, 2H, CH2C≡CH), 4.11-4.04 (m, 2H, H 5 , H 6b ), 3.87-3.77 (m, 1H, H 鎖 ), 3.72-3.63 (m, 7H, H 鎖 ) , 2.44 (t, J=2.4 Hz, 1H, CH2C≡CH), 2.15 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 1.98 (s, 3H, COCH3); 13 C NMR (75.48 MHz, CDCl3, 298.15 K): δ C170.8, 170.2, 170.0, 169.9, 97.9, 79.8, 74.7, 70.7, 70.2, 69.8, 69.3, 69.3, 68.6, 67.5, 66.3, 62.6, 58.6, 21.0, 20.9, 20.8, 20.8; HRMS (ES + ): m / z C 21 H 30 O 12 Na [M+Na] + Calculated value 497.1635 Measured value 497.1630

[0417] compound 6 To a solution of 5 (600 mg, 1.26 mmol, 1.1 equiv.) in dioxane (12 mL) was added the prepared azide derivative 1 (300 mg, 1.15 mmol, 1 equiv.) and sodium ascorbate (273 mg, 1.38 mmol, 1.2 equiv.). Then, a solution of CuSO4.HO (123 mg, 0.69 mmol, 0.6 equiv.) in water (3 mL) was added, and the reaction was heated to 70 °C and stirred while protected from light by aluminum foil. After 45 min, completion was monitored by TLC (in 9:1 DCM / MeOH, Rf = 0.3), and the mixture was cooled to room temperature. Chelex Resin® was added (4 spatulas), and the mixture was allowed to stir for 10 min. The resin was filtered off and washed twice with MeOH. The resulting solution was concentrated under reduced pressure and purified by silica gel chromatography (92:8 DCM / MeOH) to give 6 (750 mg, 89%) as a white solid. 1 H NMR (400.16 MHz, MeOD-d 4 , 298.15 K): δ H 8.17 (d, J=8.8 Hz, 0.6H, H Ar ), 8.12 (s, 1H, 1xC=CH トリアゾール ), 7.94 (d, J=8.8 Hz, 0.4H, H Ar ), 7.67 (d, J=2.5 Hz, 0.4H, H Ar ), 7.42-7.35 (m, 1.6H, H Ar ), 5.28-5.20 (m, 3H, H2 + H 3 + H 4 ), 4.94-4.89 (m, 2H, ArOCH2CH2N), 4.86 (d, J=1.3 Hz, 1H, H 1 ), 4.65 (s, 2H, H 鎖 ); 4.63-4.58 (m, 2H, ArOCH2CH2N), 4.24-4.17 (m, 1H, H 6a ), 4.13-4.05 (m, 2H, H 6b + H 5 ), 3.83-3.77 (m, 1H, H 7 ), 3.69-3.60 (m, 10H, H 7 + NCH3+ 6xH 鎖 ) ; 13 C NMR (100.62 MHz, MeOD-d 4 , 298.15 K): δ C 172.4, 171.6, 171.5, 171.5, 163.3, 162.8, 159.9, 159.8, 152.9, 152.3, 146.3, 131.9, 129.9,127.9, 125.9, 124.1, 123.4, 122.5, 109.5, 107.6, 98.9, 71.6, 71.2, 70.8, 70.8, 70.7, 69.8, 68.4, 68.2, 67.3, 65.1, 63.6, 50.8, 38.9, 38.7, 20.7; HRMS (ES - ): m / z C 32 H 40 N5O 15 [MH] - Calculated value 734.2521 Measured value 734.2522

[0418] Compound 7 (LumMan) To a solution of prepared 6 (100 mg, 0.14 mmol, 1 equiv.) in anhydrous 3:2 MeOH / THF (0.9 mL MeOH / 0.6 mL THF, 0.1 M) was added a 1 M solution of MeONa in anhydrous MeOH (0.27 mL, 0.27 mmol, 2 equiv.), and the reaction was allowed to stir at room temperature under a nitrogen atmosphere. After 30 min, completion was monitored by TLC (85:15 DCM / MeOH, Rf = 0-0.1), and Dowex-50 Acidic Resin® (previously reactivated with concentrated HCl and washed with water and MeOH) was added (four spatulas). The mixture was allowed to stir for 15 min, and the resin was filtered off and washed twice with MeOH. The resulting solution was concentrated under reduced pressure and lyophilized to give 7 (72 mg, 94%) as a white solid that required no further purification. 1 H NMR (400.16 MHz, DMSO-d 6 , 298.15 K): δ H 8.21+8.20 (double s, 1H, C=CH トリアゾール ), 8.11 (d, J=8.7 Hz, 0.4H, H Ar ), 7.88 (d, J=8.7 Hz, 0.6H, H Ar ), 7.61 (d, J=2.5 Hz, 0.6H, H Ar ), 7.45-7.37 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, H Ar ), 7.31 (d, J=2.5 Hz, 0.4H, H Ar ), 4.82 (m, 2H, ArOCH2CH2N), 4.65-4.57 (m, 3H, H 1 + ArOCH2CH2N), 4.53 (s, 2H, OCH2C トリアゾール ), 3.69-3.28 (m, 17H, 6x carb + 8H 鎖 + NCH3) ; 13 C NMR (100.62 MHz, DMSO-d 6 , 298.15 K): δ C161.2, 160.7, 144.0, 130.7, 128.6, 126.6, 124.4, 122.7, 121.8, 121.1, 108.5, 99.99, 73.9, 70.9, 69.7, 69.5, 68.9, 66.9, 66.7, 63.5, 61.2, 48.8, 37.6; HRMS (ES - ): m / z C 24 H 32 N5O 11 [MH] - Calculated value 566.2098 Measured value 566.2100

[0419] compound 9 To a solution of commercially available peracetylated galactosamine 8 (2 g, 5.14 mmol, 1 equiv.) in anhydrous DCM (25 mL, 0.2 M) at 0 °C under a positive nitrogen atmosphere, TMSOTf (3.25 mL, 17.98 mmol, 3 equiv.) was added dropwise. The ice bath was removed, and the reaction was heated to 50 °C and stirred for 5 h. Completion was monitored by TLC (in 98:2 DCM / MeOH, Rf = 0.3), and the reaction was quenched with a saturated solution of NaHCO . The aqueous layer was extracted three times with DCM, and the combined organic layers were washed once with HO and once with brine, then dried over MgSO , filtered, and concentrated under reduced pressure. The resulting crude compound 9 (1.65 g, an orange oily solid) was used in the next step without further purification.

[0420] compound 10 To a solution of freshly prepared crude oxazoline 9 (1.43 g, 4.34 mmol, 1.25 equiv) in anhydrous DCM (30 mL, 0.1 M) was added mono-O-propargyl chain 3 (500 mg, 3.47 mmol, 1 equiv) at room temperature under a positive nitrogen atmosphere. The mixture was cooled to 0 °C, and TMSOTf (0.31 mL, 1.74 mmol, 0.5 equiv) was added. After stirring at room temperature for 24 h, completion was monitored by TLC (Rf = 0.25 in 95:5 DCM / MeOH), and the reaction was quenched with a saturated solution of NaHCO. The aqueous layer was extracted once with DCM, and the combined organic layers were washed once with HO and once with brine, then dried over MgSO, filtered, and concentrated under reduced pressure. The crude compound obtained was purified by silica gel chromatography (95:5 DCM / MeOH) to give 10 (670 mg, 42%) as a yellow oil. 1 H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 6.46 (d, J=9.5 Hz, 1H, NHAc), 5.29 (m, 1H, H 4 ), 4.99 (dd, J=11.1 Hz, J=3.4 Hz, 1H, H 3 ), 4.82 (d, J=8.7 Hz, 1H, H 1 ), 4.34-4.21 (m, 3H, H 2 + -CH2C≡CH), 4.14 (m, 2H, H 6a,b ), 3.92-3.56 (m, 9H, H 5 + 8H 鎖 ), 2.49 (t, J=2.4 Hz, 1H, -CH2C≡CH), 2.14 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 1.97 (s, 3H, COCH3), 1.95 (s, 3H, COCH3); 13 C NMR (75.48 MHz, CDCl3, 298.15 K): δ C170.6, 170.5, 170.5, 170.4, 102.5, 79.3, 75.4, 72.1, 71.1, 70.6, 70.1, 69.2, 68.2, 66.7, 61.7, 58.3, 50.5, 23.17, 20.8, 20.7, 20.7; HRMS (ES + ): m / z C 21 H 31 NO 11 Na [M+Na] + Calculated value: 496.1495 Measured value: 496.1797

[0421] compound 11 To a solution of prepared 10 (585 mg, 1.24 mmol, 1.1 equiv.) in dioxane (9.5 mL, final concentration 0.1 M) was added prepared azide derivative 1 (293 mg, 1.12 mmol, 1 equiv.) and sodium ascorbate (267 mg, 1.35 mmol, 1.2 equiv.). Then, an aqueous solution (2.5 mL) of CuSO4.HO (120 mg, 0.67 mmol, 0.6 equiv.) was added, and the reaction was heated to 70 °C and stirred while protected from light by aluminum foil. After 45 min, completion was monitored by TLC (in 9:1 DCM / MeOH, Rf = 0.35), and the mixture was cooled to room temperature. Chelex Resin® was added (4 spatulas), and the mixture was allowed to stir for 15 min. The resin was filtered off and washed twice with MeOH. The resulting solution was concentrated under reduced pressure and purified by silica gel chromatography (93:7 DCM / MeOH) to afford 11 (680 mg, 83%) as a white solid. 1 H NMR (400.16 MHz, DMSO-d 6 , 298.15 K): δ H 11.57 (br s, 1H, NH), 8.21+8.20 (double s, 1H, C=CH トリアゾール ), 8.10 (d, J=8.7 Hz, 0.4H, H Ar ), 7.87 (d, J=8.7 Hz, 0.6H, H Ar), 7.80 (d, J=9.2 Hz, 1H, NHAc), 7.60 (d, J=2.5 Hz, 0.6H, H Ar ), 7.45-7.36 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, H Ar ), 7.30 (d, J=2.5 Hz, 0.4H, H Ar ), 5.21 (d, J=3.3 Hz, 1H, H 4 ), 4.97 (dd, J=11.2 Hz, J=3.4 Hz, 1H, H 3 ), 4.82 (m, 2H, ArOCH2CH2N), 4.60 (m, 2H, ArOCH2CH2N), 4.57-4.50 (d + s, J=8.7 Hz, 3H, H 1 + OCH2C トリアゾール ), 4.02 (m, 3H, H 5 + H 6a,b ), 3.87 (m, 1H, H 2 ), 3.77 (m, 1H, H 鎖 ), 3.61-3.45 (m, 10H, 7H 鎖 + NCH3), 2.09 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.89 (s, 3H, COCH3), 1.75 (s, 3H, COCH3) ; 13 C NMR (100.62 MHz, DMSO-d 6 , 298.15 K): δ C 169.9, 169.8, 169.6, 169.3, 161.3, 160.7, 144.0, 130.7, 128.6, 126.6, 124.4, 122.8, 121.8, 121.1, 108.6, 100.9, 70.5, 69.9, 69.7, 69.4, 68.9, 68.2, 66.8, 66.7, 63.4, 61.4, 49.4, 48.8, 22.7, 20.5, 20.4, 20.4 ; HRMS (ES + ): m / z C 32 H 43 N6O 14 [M+H]+ Calculated value: 735.2837 Measured value: 735.2832

[0422] Compound 12 (LumGalNAc) To a solution of prepared 11 (200 mg, 0.27 mmol, 1 equiv.) in anhydrous MeOH (3 mL, 0.1 M) was added a 1 M solution of MeONa in anhydrous MeOH (0.4 mL, 0.4 mmol, 1.5 equiv.), and the reaction was allowed to stir at room temperature under a nitrogen atmosphere. After 3 h, completion was monitored by TLC (in 85:15 DCM / MeOH, Rf = 0-0.05), and Dowex-50 Acidic Resin® (previously reactivated with concentrated HCl and washed with water and MeOH) was added (four spatulas). The mixture was allowed to stir for 15 min, and the resin was filtered off and washed twice with MeOH. The resulting solution was concentrated under reduced pressure and lyophilized to give pure 12 (100 mg, 61%) as a white solid that required no further purification. 1 H NMR (400.16 MHz, DMSO-d 6 , 298.15 K): δ H 8.21+8.20 (double s, 1H, C=CH トリアゾール ), 8.11 (d, J=8.7 Hz, 0.4H, H Ar ), 7.88 (d, J=8.7 Hz, 0.6H, H Ar ), 7.61 (d, J=2.5 Hz, 0.6H, H Ar ), 7.57 (d, J=9.0 Hz, 1H, NHAc), 7.45-7.37 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, H Ar ), 7.31 (d, J=2.5 Hz, 0.4H, H Ar ), 4.82 (m, 2H, ArOCH2CH2N), 4.61 (m, 2H, ArOCH2CH2N), 4.52 (s, 2H, OCH2C トリアゾール ), 4.29 (d, J=8.5 Hz, 1H, H 1 ), 3.78 (m, 2H, 2H 鎖 ), 3.70 (m, 1H, H 2), 3.64 (m, 1H, H 4 ), 3.58-3.48 (m, 11H, NCH3+ H 6a,b + 6H 鎖 ), 3.42 (dd, J=10.6 Hz, J=3.3 Hz, 1H, H 3 ), 3.30 (t, J=6.1 Hz, 1H, H 5 ), 1.77 (s, 3H, NHAc); 13 C NMR (100.62 MHz, DMSO-d 6 , 298.15 K): δ C 169.5, 161.1, 160.8, 144.0, 130.7, 128.7, 126.6, 124.4, 122.7, 121.8, 121.1, 108.5, 101.3, 75.3, 71.6, 69.7, 69.6, 68.9, 67.5, 66.7, 63.5, 60.5, 52.0, 48.8, 48.6, 22.9; HRMS (ES + ): m / z C 26 H 37 N6O 11 [M+H] + Calculated value 609.2520 Measured value 609.2523

[0423] - Synthesis of LumBiotin

[0424]

change

[0425] Biotin-propargyl derivative was prepared from commercially available biotin-acid as described in the literature (C.-C. Lin et al., Org. Lett., 2007, 9, 2131-2134). To a solution of biotin-propargyl (70 mg, 0.25 mmol, 1.1 equiv.) in dioxane (2 mL) was added luminol-azide derivative (59 mg, 0.22 mmol, 1 equiv.) and sodium ascorbate (54 mg, 0.27 mmol, 1.2 equiv.). An aqueous solution (0.5 mL) of CuSO4.HO (24 mg, 0.13 mmol, 0.6 equiv.) was then added, and the reaction was heated to 70 °C, protected from light by aluminum foil, and stirred. After 45 min, completion was monitored by TLC (Rf=0.2 in 85:15 DCM / MeOH) and the mixture was cooled to room temperature. Chelex Resin® was added (2 spatulas) and the mixture was allowed to stir for 10 min. The resin was filtered off and washed twice with MeOH. The resulting solution was concentrated under reduced pressure and purified by silica gel chromatography (90:10 to 85:15 DCM / MeOH) to give 15 (61 mg, 50%) as a white solid. 1 H NMR (400.16 MHz, DMSO-d 6 , 298.15 K): δ H 8.25 (t, J=5.5 Hz, 1H, NH アミド ), 8.11 (d, J=8.7 Hz, 0.3H, H Ar ), 8.00+7.99 (double s, 1H, C=CH トリアゾール ), 7.88 (d, J=8.7 Hz, 0.7H, H Ar ), 7.56 (d, J=2.5 Hz, 0.7H, H Ar ), 7.44-7.35 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, H Ar ), 7.31 (d, J=2.5 Hz, 0.4H, H Ar ), 6.38 (br s, 1H, NH 尿素 ), 6.33 (br s, 1H, NH 尿素 ), 4.79 (m, 2H, NCH2CH2O ルミノール), 4.58 (m, 2H, NCH2CH2O ルミノール ), 4.28 (d, J=5.5 Hz, 2H, NCH2C トリアゾール , overlaps with m, 1H, H 2 ), 4.11 (m, 1H, H 3 ), 3.54+3.52 (doubled s, 3H, NCH3), 3.08 (m, 1H, H 4 ), 2.80 (dd, J=12.3 Hz, J=5.1 Hz, 1H, H 1a ), 2.57 (dd, J=12.3 Hz, J=2.0 Hz, 1H, H 1b ), 2.09 (t, J=7.5 Hz, 2H, CH2CONH アミド ), 1.65-1.39 (m, 4H, H 鎖 (H 5a + H 5b (including)), 1.36-1.21 (m, 2H, H 鎖 ) ; 13 C NMR (100.62 MHz, DMSO-d 6 , 298.15 K): δ C 172.9, 163.6, 162.0, 161.6, 146.1, 131.7, 129.6, 127.5, 124.2, 123.7, 122.7, 122.0, 109.5, 67.7, 61.9, 60.1, 56.3, 49.68, 35.9, 35.0, 29.1, 28.9, 26.1; HRMS (ES - ): m / z C 24 H 29 N8O5S [MH] - Calculated value 541.1982 Measured value 541.1984

[0426] Example 2 Electrochemical behavior of luminol derivatives The electrochemical behavior of luminol derivatives was measured by cyclic and multicyclic voltammetry at 1 mM in 1:1 MeCN / PB 100 mM pH 7.4. The measurements were recorded at a 2 mm glassy carbon disk cathode, along with a platinum cathode and a saturated calomel or silver chloride (saturated KCl) electrode as a reference (examples in Figure 2). For cyclic voltammetry, scan rates of 25 mV / s, 50 mV / s, 75 mV / s, 100 mV / s, and 250 mV / s were recorded between -0.1 and 1.1 V. For multicyclic voltammetry, six cycles were recorded at 100 mV / s between -0.1 and 1.1 V. Between experiments, the GCE electrode was resanded on high-grit sandpaper to prevent potential passivation. Cyclic voltammetry of the compounds in pure aqueous buffer using a silver chloride (saturated KCl) reference electrode was also performed to obtain their exact oxidation potentials in electrobioconjugation conditions.

[0427] Example 3 AAV2 production and purification and electrochemical bioconjugation AAV2 vectors were generated from two plasmids: (i) pHelper, PDP2-KANA encoding AAV Rep2-Cap2 and adenovirus helper genes (E2A, VA RNA, and E4), and (ii) pVector ss-CAG-eGFP containing the ITRs. All vectors were generated by transient transfection of HEK293 cells using the calcium phosphate-HeBS method. AAV2-transfected cells were harvested 48 hours posttransfection and treated with Triton-1% and benzonase (25 U / mL) for 1 hour at 37°C. The resulting bulk was subjected to freeze-thaw cycles to release vector particles. Cell debris was removed by centrifugation at 2500 rpm for 15 minutes. Cell lysates were precipitated with PEG overnight and clarified by centrifugation at 4000 rpm for 1 hour. The precipitate was then incubated with benzonase for 30 min at 37°C and collected after centrifugation at 10,000 g for 10 min at 4°C. The vector was purified by double cesium chloride (CsCl) gradient ultracentrifugation. The virus suspension was then subjected to four consecutive rounds of dialysis against dPBS (containing Ca2+ and Mg2+) in a 10 kDa MWCO Slide-a-Lyzer cassette (Pierce) with gentle agitation.

[0428] Electrobioconjugation procedure: In a low-binding vial, 100 μL of AAV2-GFP (1E12 vg in dPBS pH 7.4) was added to 900 μL dPBS pH 7.4 containing the appropriate amount of N-methylluminol derivative (LumGalNAc).

[0429] The electrochemical setup was assembled (Figure 1) and 750 mV vs. Ag / AgCl was applied under gentle orbital shaking at room temperature for the duration of the study. After modification, excess unreacted luminol anchor was removed by dialysis against dPBS pH 7.4 (+0.001% poloxamer) in 10 kDa MWCO cassettes for four consecutive rounds.

[0430] Example 4 Characterization of chemically modified AAV bearing GalNAc derivatives of N-methylluminol - Materials and Methods *Viral genome (vg) titration: To determine the titer (vg / mL) of all AAV samples, 3 μL 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®) was added, and the mixture was incubated at 70°C for 20 minutes. DNA was then extracted from the purified AAV vector using an extraction column (NucleoSpin® RNA Virus). Quantitative real-time PCR (qPCR) was performed using a StepOnePlus™ Real-Time PCR System Upgrade (Life Technologies). All PCRs were performed in a final volume of 20 μL containing primers and probes targeting the ITR2 sequence, 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 seconds, followed by 45 cycles of denaturation at 95°C for 1 second and annealing / extension at 56°C for 20 seconds. Plasmid standards were diluted in seven serial dilutions (10 8 ~10 2 The vector was constructed using the plasmid p53 (containing 10 copies of the vector p53).

[0431] *Dot blot, Western blot, and silver staining: For dot blot analysis, the nitrocellulose membrane was briefly soaked in PBS before assembling the dot blot manifold (BioRad) and then transfected with AAV vectors (2 × 10 10 vg). The resulting nitrocellulose membrane was then processed for appropriate characterization (see below: capsid integrity or glycan detection). For silver nitrate or Western blot analysis, all AAV vectors (2 × 10 10(vg) were denatured with Laemmli sample buffer (5 μL) at 100°C for 5 minutes and separated by SDS-PAGE on a 10% Tris-glycine polyacrylamide gel (Life Technologies). Precision Plus Protein All Blue Standards (BioRad) were used as molecular weight size markers. After electrophoresis at 120 V for 200 minutes, the gels were either silver stained (PlusOne Silver Staining Kit, Protein, GE Healthcare®) or transferred to nitrocellulose membranes for Western blotting. Proteins were transferred at 150 mA for 10 minutes in a Trans-Blot SD Semi-Dry Transfer Cell (BioRad®) using 25 mM Tris / 192 mM glycine / 0.1 (w / v) SDS / 20% MeOH buffer. The resulting nitrocellulose membranes were then processed for appropriate characterization (see below: viral capsid protein or glycan detection).

[0432] *Capsid integrity: Membranes were saturated with PBS containing 5% semi-skimmed milk and 0.1% Tween for 2 hours at room temperature. After saturation, the membranes were probed overnight at 4°C with a mouse anti-capsid A20 primary antibody (Kleinschmidt®, diluted 1:20 in milk). The membranes were then washed three times at room temperature for 15 minutes with PBS-0.1% Tween and probed with a secondary antibody, anti-mouse-HRP (Dako®, diluted 1:2000 in milk) for 1 hour and 30 minutes at room temperature. Finally, the membranes were washed three times at room temperature for 15 minutes with PBS-0.1% Tween. Bands were detected by topical treatment with H2O2 / luminol for 1 minute, followed by chemiluminescence visualization on X-ray film.

[0433] *Viral capsid protein detection: Membranes were saturated with PBS containing 5% semi-skimmed milk and 0.1% Tween for 2 hours at room temperature. After saturation, the membranes were probed overnight at 4°C with a rabbit polyclonal anti-AAV capsid protein primary antibody (PROGEN Biotechnik®, diluted 1:2000 in milk solution). The membranes were then washed three times at room temperature for 15 minutes with PBS-0.1% Tween and probed with a secondary antibody, anti-rabbit-HRP (Jackson®, diluted 1:20,000 in milk solution), for 1 hour and 30 minutes at room temperature. Finally, the membranes were washed three times at room temperature for 15 minutes with PBS-0.1% Tween. Band detection was performed by topical treatment with H2O2 / luminol for 1 minute, followed by chemiluminescence visualization on X-ray film.

[0434] *Carbohydrate detection: Membranes were saturated with PBS containing 1% gelatin, 0.1% igepal, and 0.1% Tween for 2 hours at room temperature. After saturation, the membranes were probed overnight at 4°C with soybean agglutinin-fluorescein lectin (Vector Laboratories®, diluted 1:200 in PBS-0.1% Tween) for GalNAc detection or concanavalin A-fluorescein lectin (Vector Laboratories®, diluted 1:200 in PBS-0.1% Tween) for mannose detection. The membranes were then washed three times with PBS-0.1% Tween for 15 minutes at room temperature and probed with the secondary antibody anti-fluorescein-HRP (Abcam®, diluted 1:5000 in PBS-0.1% Tween) for 1 hour and 30 minutes at room temperature. Finally, the membrane was washed three times with PBS-0.1% Tween for 15 min at room temperature, and band detection was performed by topical treatment with H2O2 / luminol for 1 min followed by chemiluminescence visualization on X-ray film.

[0435] - results Glycosylating AAV2 capsids was performed in a single electroconjugation step using luminol derivatives 7 (Man) and 12 (GalNAc) at various concentrations and voltages. The integrity of the assembled viral capsids was preserved for all coupling conditions, as confirmed by dot blot staining with anti-capsid A20 antibody (Figure 3a, top example). Similar dot blot assays using fluorescently labeled GalNAc-binding lectins (soybean agglutinin, SBA) or Man-binding lectins (concanavalin A, Con A) demonstrated efficient electroconjugation of 12 (Figure 3a, bottom example) and 7, respectively, with an observable time-dependent increase in staining intensity. Notably, coupling could be detected after a very short period of conjugation (20 s) in both cases. In contrast, when voltage was applied to a mixture of AAV2 and a GalNAc derivative without a luminol moiety (Ctrl), GalNAc could not be detected by SBA, indicating covalent anchoring of 12 to the capsid during eY-click. All electroconjugated samples were also subjected to capsid denaturation followed by SDS-PAGE separation on a gel. The three constituent envelope proteins (VP1 / VP2 / VP3 1:1:10) were clearly identified by silver nitrate staining and Western blot analysis using anti-VP polyclonal antibodies (Figure 3b, example on the left). A significant mass shift of the VP band was observed overall for eY-click conditions longer than 5 min for both glycans, suggesting an efficient, progressive, and tunable degree of Y-labeling. The labeled lectins SBA and ConA were also used in Western blot staining to detect GalNAc and Man, respectively (example in Figure 3b, right), confirming the covalent conjugation of luminol-glycans on the three constituent proteins of the capsid.

[0436] Example 5 Transduction efficiency of the chemically modified AAV of the present invention - Materials and Methods In vitro transduction: The infectivity of each sample was determined as follows: HEK293 or HuH-7 cells were cultured at 10 in DMEM containing 10% FBS serum and 1% penicillin-streptomycin in 6-well culture plates. 6 The cells were seeded at a density of 1000 cells / well. The cells were then incubated overnight at 37°C with 5% CO2 to reach 50% confluence. The AAV samples were then added at the studied multiplicity of infection (MOI = virus / cell ratio, 10 3 From 10 4 AAV-GFP-infected cells were detected and quantified by fluorescence microscopy and flow cytometry.

[0437] - results The transduction efficiency of electroconjugated glycosylated viral particles carrying a GFP reporter gene was evaluated in the HEK293 cell line. Cells were incubated for 48 hours at various virus / cell ratios, and the percentage of GFP+ cells was measured by flow cytometry. Importantly, a 1-hour voltage control experiment using a GalNAc derivative lacking the luminol moiety resulted in fully preserved AAV infectivity, demonstrating that the applied potential difference did not alter transduction ability. Both the AAV2-LumGalNAc and AAV2-LumMan samples electroconjugated for 20 seconds and 1 minute also showed fully preserved transduction efficiency (Figure 4, left example). Thus, in these examples, a controlled level of glycosylation on the AAV surface was reproducibly achieved only within the 20-second and 1-minute experimental ranges. Additionally, AAV2-LumGalNAc (1 min) showed enhanced transduction capacity compared to AAV2 in HuH-7 cells expressing the GalNAc receptor (example in Figure 4, right), supporting the relevance of viral vector functionalization to promote specific cell line transduction potentially mediated by ligand-receptor interactions.

[0438] Example 6 AAV2 production and purification, electrochemical bioconjugation with LumBiotin and LumN3, and SPAAC reaction The same protocols as described in Example 3 were used for AVV2 production and purification, and electrochemical bioconjugation, except that LumBiotin and LumN3 were used.

[0439] After electrochemical bioconjugation of AAV2 with LumN3, the resulting AAV2-LumN3 was subjected to SPAAC reaction to decorate the AAV2 with nanobody or fluorescein moieties. Briefly, DBCO-fluorescein, DBCO-biotin, DBCO-CD62L nanobody, or DBCO-CD45 nanobody was added to AAV2-LumN3 at concentrations of 10 μM, 15 μM, or 50 μM, and the reaction was carried out at room temperature or 37°C under gentle shaking for 1 hour or 4 hours. After the SPAAC reaction, excess DBCO compound was removed by four consecutive rounds of dialysis against dPBS pH 7.4 (+0.001% poloxamer) in a 10 kDa MWCO cassette.

[0440] - results N3 coating of AAV2 capsids was performed using LumN3 at various concentrations and voltages in a one-step electroconjugation procedure. The integrity of the assembled viral capsids was preserved for all coupling conditions, as confirmed by dot blot staining with anti-capsid A20 antibody (Figure 6). Similar dot blot analysis using DBCO-fluorescein demonstrated efficient electroconjugation of the azide derivatives (Figure 6). Mass spectrometry analysis indicated that an average of four azide derivatives per VP3 protein were coupled onto tyrosine residues (Figure 6).

[0441] After the second SPAAC reaction with DBCO-fluorescein, DBCO-biotin, DBCO-CD62L nanobody, or DBCO-CD45 nanobody, all samples were subjected to capsid denaturation followed by SDS-PAGE separation on a gel. The three component envelope proteins (VP1 / VP2 / VP3 1:1:10) were clearly identified by silver nitrate staining for AAV2 subjected to SPAAC with DBCO-fluorescein, DBCO-CD62L nanobody, and DBCO-CD45 nanobody (Figure 7).

[0442] Labeled anti-fluorescein antibody, streptavidin, anti-CD62L, and anti-CD45 nanobody antibodies were also used in Western blot staining to detect fluorescein, biotin, CD62L nanobody, and CD45 nanobody, respectively (Figure 7), confirming covalent SPAAC conjugation between the azide moiety present on the surface of AAV2 and the DBCO derivative. For the two nanobodies, additional bands corresponding to different molecular weights were observed in Western blots and silver staining, suggesting efficient electrochemical bioconjugation labeling.

Claims

1. An adeno-associated virus (AAV) having at least one chemically modified tyrosine residue in its capsid, wherein the chemically modified tyrosine residue is represented by formula (I): 【Chemistry 1】 (In the formula, -R A is -(Y) n -M, C 1 ~C 6 Alkyl, optionally substituted C 6 ~C 14 aryl, or optionally substituted (C 6 ~C 14 Aryl)-(C 1 ~C 3 alkyl), - Each R B are independently of the formula -(Y) n -M group, hydrogen, or halogen, C 1 ~C 6 Alkyl, C 6 ~C 14 Aryl, C 3 ~C 6 Cycloalkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Alkylamino, C 2 ~C 6 Heterocycle, C 1 ~C 6 Alkanoyl, C 1 ~C 6 Carboxy ester, C 1 ~C 6 Acylamino, -COOH, -CONH 2 , -NO 2 , -SO 3 H, -CN, -CF 3 , C 1 ~C 6 Hydroxyalkyl, C 1 ~C 6 Haloalkyl, C 1 ~C 6 Alkylthio, C 1 ~C 6 Thioalkyl, C 2 ~C 10 Alkoxyalkyl, and C 2 ~C 6 a substituent selected from the group consisting of alkoxycarbonyloxy; However, R A and R B At least one of the groups is -(Y) n -M, k is 1 or 2; - n is 0 or 1 Y is a spacer, and - M is a functional moiety adeno-associated virus (AAV).

2. R A But C 1 ~C 3 2. The AAV of claim 1, which is alkyl, phenyl, or benzyl, preferably methyl or benzyl, more preferably methyl.

3. One or two R's B is the formula -(Y) n -M group, and other R B The AAV of claim 1 or 2, wherein is hydrogen.

4. 4. The AAV of any one of claims 1 to 3, wherein Y is a chemical chain group comprising 2 to 500 carbon atoms and selected from the group consisting of polymers, including homopolymers, copolymers, and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains, which may optionally be interrupted by one or several heteroatoms and / or by one or several cyclic or heterocyclic moieties, which may optionally have a heteroatom such as S, O, or NH at at least one of the termini, and which may optionally be substituted by one or several substituents, and combinations thereof.

5. Y is, - -O-, -S-, -N(R)- (wherein R is H or C 1 ~C 3 alkyl), one or more heteroatoms selected from —C(O)—, —NHC(O)—, —OC(O)—, —C(O)—OC(O)—, —NH—CO—NH—, —O—CO—NH—, NH—(CS)—NH—, and —NH—CS—; and / or - C such as cycloalkyl, cycloalkenyl, or aromatic groups 5 ~C 20 a carbocyclic moiety; and / or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyl or heteroaryl having 5 to 20 ring atoms; may be optionally interrupted by - and -O-, -S-, -N(R)- (wherein R is H or C) at least one of its termini 1 ~C 3 alkyl), -ON(R)- (R is H or C 1 ~C 3 alkyl), -N(C 1 ~C 3 optionally having a heteroatom selected from the group consisting of (alkoxy)-, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH- and NH-CS-; 4. The AAV of any one of claims 1 to 3, which is a saturated or unsaturated hydrocarbon having from 2 to 100 carbon atoms.

6. 6. The AAV of any one of claims 1 to 5, wherein M is a functional moiety comprising a group selected from a click chemistry group, a steric shielding agent, a labeling agent, a targeting agent such as a cell type-specific ligand, a drug moiety, an oligonucleotide, and combinations thereof.

7. 6. The AAV of any one of claims 1 to 5, wherein M comprises or consists of a moiety selected from a click chemistry group, preferably 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, e.g. a small chemical molecule such as a CB1 and / or CB2 ligand.

8. 6. The AAV of claim 1, wherein M comprises or consists of a moiety selected from a click chemistry group, in particular an azide group or a strained alkyl group; 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, sialic acid and its derivatives (e.g., Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5Ac); an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7; a VHH; a fluorescent label; and a cell-type specific ligand derived from a vitamin such as folic acid.

9. The AAV of any one of claims 1 to 8, further comprising at least one additional chemically modified amino acid residue in the capsid selected from a chemically modified cysteine, arginine, or lysine.

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

11. A method for chemically modifying an AAV capsid, more precisely for chemically modifying at least one tyrosine residue in an AAV capsid, comprising incubating the AAV with a chemical reagent having an N-substituted luminol moiety under conditions conducive to reacting the chemical reagent with a tyrosine residue present in the AAV capsid to form a covalent bond.

12. The method of claim 11 , which is carried out by an electrochemical method.

13. The AAV has formula (X): 【Chemistry 2】 (In the formula, -R A is -(Y) n -M, C 1 ~C 6 Alkyl, optionally substituted C 6 ~C 14 aryl, or optionally substituted (C 6 ~C 14 Aryl)-(C 1 ~C 3 alkyl), - Each R B is the formula -(Y) n -M group, hydrogen, or halogen, C 1 ~C 6 Alkyl, C 6 ~C 14 Aryl, C 3 ~C 6 Cycloalkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Alkylamino, C 2 ~C 6 Heterocycle, C 1 ~C 6 Alkanoyl, C 1 ~C 6 Carboxy ester, C 1 ~C 6 Acylamino, -COOH, -CONH 2 , -NO 2 , -SO 3 H, -CN, -CF 3 , C 1 ~C 6 Hydroxyalkyl, C 1 ~C 6 Haloalkyl, C 1 ~C 6 Alkylthio, C 1 ~C 6 Thioalkyl, C 2 ~C 10 Alkoxyalkyl, and C 2 ~C 6 independently selected from the group consisting of substituents selected from alkoxycarbonyloxy; However, R A and R B At least one of the groups is -(Y) n -M, - n is 0 or 1, Y is a spacer, and - M is a functional moiety in the presence of a potential difference that allows electroactivation of said chemical reagent of formula (X) to an oxidized form capable of reacting with tyrosine residues, resulting in a compound of formula (I): 【Transformation 3】 (In the formula, -R A and R B is as defined in formula (X), - k is 1 or 2 The method of claim 11 or 12, comprising the step of obtaining at least one chemically modified tyrosine residue in the AAV capsid.

14. 14. The method of claim 13, wherein the method is carried out in an electrochemical system having three electrodes, including a working electrode, a counter electrode, and a reference electrode, by applying a constant potential difference between the working electrode and the reference electrode, wherein the constant potential difference is preferably within a range defined by the oxidation potential of the chemical reagent of formula (X) ±200 mV.

15. R A C 1 ~C 6 alkyl and at least one R B is the formula -(Y) n -M and other R B 15. The method of claim 13 or 14, wherein is H.

16. 16. The method of any one of claims 13 to 15, wherein M is a click chemistry group and the method further comprises the step of click reaction, thereby covalently attaching a functional moiety, preferably selected from a ligand and a label, to the AAV capsid.

17. A pharmaceutical composition comprising an AAV according to any one of claims 1 to 9 and at least one pharmaceutically acceptable excipient.

18. An AAV according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 17 for use as a diagnostic agent in vivo or as a drug in vivo or ex vivo, preferably in gene therapy.

19. 10. Use of an AAV as defined in any one of claims 1 to 9 as an in vitro research tool, for example as an in vitro gene transfection agent or as an imaging agent.

20. 10. Use of an AAV as defined in any one of claims 1 to 9 in the manufacture of a diagnostic agent or a drug, in particular for ex vivo or in vivo gene therapy.

21. Formula (X) as defined in claim 13 or 15 as a substance for chemically modifying the capsid of AAV by electrochemical bioconjugation: 【Chemistry 4】 Use of compounds.

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