Chemically modified adeno-associated virus

Abstract

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

Description

[Technical field]

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

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

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

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

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

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

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

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

[0009] 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 allow it to avoid 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] Chemical modification of viral capsids has also been suggested to introduce ligands into the capsid or to mask certain exposed amino acids to modify the antigenicity, tropism, or transduction efficiency of AAV. As a first strategy, it was proposed to genetically incorporate unnatural amino acids with modified side chains (e.g., as in WO 2015 / 062516). Unnatural amino acids, such as azide-containing amino acids, are inserted into the capsid by genetic modification before the coupling step with a ligand by click reaction to change its tropism towards target cells. Another strategy consists in direct chemical modification of viral capsids without any prior site-directed mutagenesis of capsid proteins.

[0011] In that regard, WO 2017 / 212019 proposes a method for chemically modifying the AAV capsid by covalently coupling a ligand bearing an isothiocyanate group, which reacts with amino groups present in amino acid residues such as lysine or arginine.

[0012] International Patent Application Publication No. 2021 / 005210 [0] described a method for chemically modifying tyrosine residues present in capsids by reaction with ligands bearing aryldiazonium or PTAD moieties. [Prior art documents] [Patent documents]

[0013] [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 [Patent Document 6] PCT / EP2015 / 060805 [Patent Document 7] PCT / EP2015 / 063750 [Non-Patent Document]

[0014] [Non-Patent Document 1] Zhong et al., PNAS, 2008, 105, pp. 7827 - 7832 [Non-Patent Document 2] Markusic et al., Molecular Therapy, 2010, 18, pp. 2048 - 2056 [Non-Patent Document 3] Pulichera et al., PloSone, 2012, 7(2): e32163 [Non-Patent Document 4] Naso et al., Biodrugs, 2017, 31: pp. 317 - 334 [Non-Patent Document 5] Koerber et al., Molecular Therapy (2008), 16(10), pp. 1703 - 1709 [Non-Patent Document 6] Deverman et al., Nat Biotechnol (2016), 34(2), pp. 204 - 209 [Non-Patent Document 7] Buening et al., Curr Opin Pharmacol (2015), 24, pp. 94 - 104 [Non-Patent Document 8] Koniev, O., Wagner, A, Chem. Soc. Rev., 44, p. 5495 (2015) [Non-Patent Document 9] Kolb et al., Angew. Chem. Int. Ed. 2001, 40, pp. 2004 - 2021 [Non-Patent Document 10] Rudolf et al., Current opinion in Chemical Biology, 2013, 17: pp. 110 - 117 [Non-Patent Document 11] Sletten and Bertozzi, Angew. Chem. Int. Ed. Engl. 2009, 48(38):6974-6998 [Non-Patent Document 12] Chemical Science, 2020, 11, pp. 1122-1131 [Non-Patent Document 13] S. D'Costa et al., Molecular therapy. Methods & clinical development, 2016 [Non-Patent Document 14] Blanchard et al., Journal of Lipid Research, 2020 Summary of the Invention [Problem to be solved by the invention]

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

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

[0017] [ka]

[0018] (In the formula, - X is

[0019] [ka]

[0020] is selected from the group consisting of - 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; - k is 0 or 1, R is hydrogen, halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, which groups may be optionally substituted; - Y is a spacer, - n is 0 or 1, and - M is a functional moiety It relates to adeno-associated viruses (AAV).

[0021] In some embodiments, X is of formula (b) or formula (c).

[0022] In a preferred embodiment, the AAV comprises a chemically modified cysteine ​​residue having formula (Ic):

[0023] [ka]

[0024] (wherein Y, n, M, Z, k, R2, and R3 are as defined herein). It satisfies the following.

[0025] In some embodiments, the chemically modified cysteine ​​residue has formula (Ic), R2 is a hydrogen atom, a C1-C6 alkyl group, an aryl group containing from 6 to 14 ring atoms, or a heteroaryl group containing from 5 to 14 ring atoms, and / or R3 is selected from the group consisting of an aryl group containing from 6 to 14 ring atoms and a heteroaryl group containing from 5 to 14 ring atoms, said aryl or heteroaryl group being optionally substituted by 1 to 3 substituents, preferably selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl and C1-C3 haloalkyl; and / or - k is 0) It is.

[0026] In another embodiment, the chemically modified cysteine ​​residue has formula (Ic), R2 is a hydrogen atom or a C1-C6 alkyl group, preferably H or a C1-C3 alkyl group. - R3 is unsubstituted phenyl or phenyl substituted by 1 to 3 substituents selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl; and - k is 0) It is.

[0027] In some embodiments, Y in formula (I) is formula (II):

[0028] [ka]

[0029] (In the formula, m, p, and q are each independently 0 or 1; Y1 is selected from the group consisting of alkylene, arylene and heteroarylene groups, said groups being optionally substituted, and is preferably a phenylene group; - Y2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -NH(C=S)-, or -(C=S)-NH-, preferably -(C=O)-NH-, - Y3 is selected from the group consisting of polymers, including homopolymers, copolymers and block polymers, peptides, oligosaccharides, saturated or unsaturated, branched or linear hydrocarbon chains, optionally interrupted by one or several heteroatoms and / or by groups selected from -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)- and -(C=S)-NH- and / or by one or more C3-C6 hydrocarbon rings or C2-C6 heterocycles. It is a spacer.

[0030] In some embodiments, 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.

[0031] In some embodiments, the chemically modified cysteine ​​residue of formula (I) is: X is of formula (c), - n is 1, - M is a functional moiety, and - Y is a group represented by the formula (II) (wherein m is 0, p is 0, q is 1, and Y3 is an optionally substituted saturated or unsaturated linear or branched C2-C 40 a spacer selected from the group consisting of a hydrocarbon chain, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof. Meet the following.

[0032] In some embodiments, the chemically modified cysteine ​​residue of formula (I) is: - Y is a group represented by the formula (II) (wherein q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined in claim 2, and Y3 is an optionally substituted saturated or unsaturated linear or branched C2-C 40 and / or a spacer selected from the group consisting of a hydrocarbon chain, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof; - M comprises or consists of a targeting agent such as a click chemistry group, an oligonucleotide, 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, ScFv, spiegelmer and peptide aptamers, vitamins and a drug such as a CB1 and / or CB2 ligand. Meet the following.

[0033] In some embodiments, the chemically modified cysteine ​​residue of formula (I) is: - Y is a group represented by the formula (II) (wherein q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined in claim 2, and Y3 is a linear or branched C2-C 20 and / or a spacer selected from the group consisting of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, said polymers having from 2 to 20 monomers; and / or - "M" comprises or consists of a targeting agent, preferably 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 and Neu5Acα2-8Neu5Ac), a muscle targeting agent (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. Meet the following.

[0034] In some embodiments, the chemically modified cysteine ​​residue of formula (I) is: - M is a cell type specific ligand for specifically targeting hepatocytes and has the formula (III):

[0035] [ka]

[0036] and / or - Y is a polyethylene glycol chain containing from 2 to 10 monomers Meet the following.

[0037] In some embodiments, the at least one chemically modified cysteine ​​in the capsid has formula (Ic-1):

[0038] [ka]

[0039] It is.

[0040] In some embodiments, the AAV of the invention further comprises at least one additional chemically modified amino acid residue in the capsid that is different from a cysteine ​​residue, said amino acid residue preferably being one of the following: - Formula (V):

[0041] [ka]

[0042] (In the formula, - N* is the nitrogen of the amino group of an amino acid residue, for example a lysine or arginine residue, - Y', n', and M' each have the same definition as Y, n, and M in formula (I) as defined herein. or a modified amino group of - Formula (VI):

[0043] [ka]

[0044] (In the formula, - X" is -N=N- or

[0045] [ka]

[0046] and - Y", n", and M" each have the same definition as Y, n, and M in formula (I), as defined herein. Modified tyrosine residues in has.

[0047] In particular, the AAV may be a recombinant AAV, preferably selected from an AAV having a wild-type capsid, a naturally occurring serotype AAV, a variant AAV, a pseudotype AAV, an AAV having a hybrid, and a self-complementary AAV.

[0048] Another object of the present invention is a method for chemically modifying the capsid of AAV, more precisely for chemically modifying at least one cysteine ​​residue in the capsid of AAV, comprising a step of incubating said AAV with a chemical reagent bearing a reactive group selected from maleimides, vinylsulfonamides and 3-(carboxy derivatives)acrylamides, under conditions conducive to reaction of said reactive group with a cysteine ​​residue present in the capsid of AAV to form a covalent bond.

[0049] In some embodiments, the method comprises synthesizing the AAV according to formula (VIIc):

[0050] [ka]

[0051] to form a compound of formula (Ic):

[0052] [ka]

[0053] (In the formula, - Y is a spacer, n is 0 or 1 and M is a functional moiety; - Z is -O-, -S-, or -N(R4)-; - k is 0 or 1, and - R 2、 R3 and R4 are each independently selected from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, which may be optionally substituted. The method includes obtaining at least one chemically modified cysteine ​​residue in the capsid of the antibody.

[0054] In some embodiments, the incubation step is carried out at a pH of from 5.0 to 11, preferably from 6.0 to 10.0, such as from 7.0 to 8.0 or from 8.0 to 10.0.

[0055] In some embodiments, Y in the method is represented by formula (II):

[0056] [ka]

[0057] (In the formula, m is 0, p is 0, q is 1, and Y3 is an optionally substituted saturated or unsaturated linear or branched C2-C 40 and M is a click chemistry group, a steric shielding agent, a labeling agent, a targeting agent such as a cell type specific ligand, or a drug moiety.

[0058] The present invention also relates to an AAV obtainable by a method as defined herein.

[0059] The present invention further relates to a genetic vector, which is an AAV as defined herein and which comprises a transgene sequence in its viral genome. Preferably, such a genetic vector is used in gene therapy to deliver a transgene sequence encoding a therapeutic protein in a cell, in vivo or ex vivo.

[0060] Another object of the present invention is a pharmaceutical composition comprising an AAV as defined herein or obtainable by a process as defined herein, and at least one pharma- ceutically acceptable excipient.

[0061] The present invention also relates to an AAV as defined herein, an AAV obtainable by a method as defined herein, or a pharmaceutical composition as defined herein, for use as a diagnostic agent or as a drug, preferably in gene therapy. In some embodiments, said AAV or said pharmaceutical composition is used as a diagnostic agent in vivo or as a drug, preferably in gene therapy.

[0062] In some other embodiments, the AAV or a pharmaceutical composition containing it is used as a diagnostic agent in vitro, or as a gene vector ex vivo or in vitro. [Brief description of the drawings]

[0063] [Figure 1A] FIG. 1 shows a general strategy for coupling according to the present invention. GalNAc-benzoylacrylamide (L) corresponds to a ligand of the present invention, while GalNAc-control (C) corresponds to a control compound (lacking any reactive group specific for the thiol function of cysteine). [Figure 1B] FIG. 1B shows the relative positions of conserved cysteine ​​residues on the VP subunits of various naturally occurring AAV serotypes. Numbering is based on residues in the AAV2 VP1 subunit (from Pulichera et al., PloSone, 2012, 7(2):e32163). The peptide regions shown in FIG. 1B correspond to SEQ ID NOs: 12-29 in the Sequence Listing. [Figure 2A] FIG. 1 shows dot blot analysis of AAV2 and AAV2 after incubation with a ligand of the invention (L) or a control compound (C) using immunostaining with the A20 antibody, which recognizes assembled AAV2 capsid. [Figure 2B]Figure 1 shows a Western blot analysis using immunostaining with a polyclonal antibody directed against the capsid protein (VP) of AAV2 and AAV2 after incubation with a ligand of the invention (L) or a control compound (C). The capsid protein molecular weight is shown to the right of the image according to a protein ladder. [Figure 3A] (L) Some results of LC-MS / MS for enzymatically digested AAV2 and AAV9 capsids that were not chemically modified, showing that peptide fragments present in the VP subunits of AAV2 and AAV9 and lacking cysteines did not undergo chemical modification, as no shift in the LC peak and no change in mass was observed. [Figure 3B] Figure 1 shows several results of LC-MS / MS on enzymatically digested AAV2 capsids chemically modified with (L). It shows that the cysteine ​​at position 289 in the AAV2 VP was successfully chemically modified as evidenced by the shift in the LC peak and MS; the same result was observed for the cysteine ​​at position 394 (data not shown). The LC-MS / MS analysis clearly demonstrates the efficient and highly specific coupling of the ligands of the invention to cysteine ​​residues. [Figure 3C] Figure 1 shows several results of LC-MS / MS on enzymatically digested AAV9 capsids chemically modified with (L). It shows that the cysteine ​​at position 289 in the AAV9 VP was successfully chemically modified as evidenced by the shift in the LC peak and MS; the same result was observed for the cysteine ​​at position 394 (data not shown). The LC-MS / MS analysis clearly demonstrates the efficient and highly specific coupling of the ligands of the invention to cysteine ​​residues. [Figure 4A] FIG. 2 shows examples of "M" moieties according to the present invention. [Figure 4B] FIG. 2 shows examples of "M" moieties according to the present invention. [Figure 5A]FIG. 1 shows dot blot analysis using immunostaining with A20 antibody and staining with soybean lectin for (i) AAV2 particles, (ii) AAV2 particles that have undergone a coupling procedure with ligand L, and (iii) AAV2 that has undergone a coupling procedure with ligand L2 at pH 7.3. [Figure 5B] FIG. 1 shows dot blot analysis using immunostaining with A20 antibody and staining with soybean lectin for (i) AAV2 particles, (ii) AAV2 particles that have undergone a coupling procedure with ligand L, and (iii) AAV2 that has undergone a coupling procedure with ligand L2 at pH 9.3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Methods described in the prior art for chemically modifying the AAV capsid target amino acid residues bearing amino groups, such as arginine and lysine, or tyrosine.

[0065] The present inventors have identified cysteine ​​amino acids as potential residues of interest that can be chemically modified in the AAV capsid.

[0066] Cysteines are sparse residues in VP1, VP2, and VP3 of the AAV capsid. As an example, there are only five conserved cysteine ​​residues per VP protein of AAV2, and therefore at most 300 cysteine ​​residues for the complete AAV2 capsid. These five conserved residues are located at positions 230, 289, 361, 394, and 482 in VP1 of AAV2. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located at the opposite ends of each subunit and juxtaposed at the pentamer interface. Cys482 residue is located at the base of a surface loop within the trimer region.

[0067] As shown in the partial alignment of the VP subunits of naturally occurring AAV serotypes with the AAV2 VP1 subunit (see FIG. 1B), the cysteines at positions 230 and 394 are completely conserved, while the C289S, C361S, and C482S / C482M changes are found in AAV4, AAV5, and AAV9. As an example, the positions of the cysteine ​​residues in the AAV9 V1 subunit are Cys230, Cys291, Cys363, and Cys396, with the numbering referring to the amino acid numbering in the AAV2 VP1 subunit. Thus, for a complete AAV9 capsid, there are at most 240 cysteine ​​residues.

[0068] Little is known about the biological functions of cysteines. Pulichera et al. (PloSone, 2012, 7(2):e32163) show that none of the five cysteine ​​residues appear to be involved in disulfide bond formation, and that Cys230 and Cys394 may play roles in transduction and proteasomal degradation.

[0069] Thus, chemical modification of cysteines, particularly Cys230 and Cys394, may make it possible to modulate the transduction efficiency and proteasomal degradation of AAV2. There is no specific description of the effect of cysteine ​​residues for other AAV serotypes, such as AAV9.

[0070] Moreover, due to the low number of cysteine ​​residues available for chemical modification, the inventors are of the opinion that chemical coupling of cysteine ​​(compared to other more abundant residues such as tyrosine and arginine) allows for better control over the number of ligands coupled to AAV and allows for the introduction of larger ligands without compromising AAV functionality.

[0071] To the best of the inventors' knowledge, chemical coupling of ligands on naturally occurring cysteines in the AAV capsid has never been described or suggested in the prior art.

[0072] In that regard, the inventors have conceived a method for chemically modifying cysteine ​​residues present on the surface of the AAV capsid, which relies on the specific reaction of benzoyl acrylamide derivatives with thiol functional groups present in the side chains of cysteine ​​residues.

[0073] The present inventors prepared benzoylacrylamide ligands bearing sugar moieties for chemically modifying AAV vector capsids at naturally occurring cysteine ​​residues.

[0074] Thus, as a proof of concept, we demonstrated that N-acetylgalactosamine moieties can be covalently immobilized on the surface of AAV capsids by incubating AAV particles with N-acetylgalactosamine-bearing benzoyl acrylamide (compound L) in aqueous buffer at neutral pH and room temperature. Notably, the coupling did not compromise the overall integrity of the capsid, as evidenced by dot blot with A20 antibody immunostaining, nor did it compromise the integrity of the individual capsid proteins, i.e., VP1, VP2, and VP3, as evidenced by Western blot with anti-VP polyclonal antibody. Such results suggest that the infectivity of AAV is maintained after coupling.

[0075] Furthermore, LC-MS / MS peptide analysis after enzymatic digestion of AAV vectors showed that the chemical coupling of the present invention is highly effective, since the digested peptide candidates with cysteines showed coupling (only one mass peak was detected): cysteines at positions corresponding to cysteines 289 and 394 in AAV2 VP1 were completely chemically coupled in VP1, VP2, and VP3, as evidenced by the LC peak shift with respect to retention time and detected mass before and after chemical coupling. Notably, no peak shift was observed for peptide candidates without any cysteines, indicating that the method is highly selective, since no cross-reaction with residues other than cysteine ​​was observed (see Figures 3A-C).

[0076] Moreover, the inventors demonstrated that AAV2 chemically modified with benzoylacrylamide ligands efficiently transduced HeLa cells and thus remained infectious at a similar order of magnitude as non-chemically modified AAV2.

[0077] Covalent coupling of AAV2 with ligand L was effective at both acidic and basic pH (Example 5). However, we showed that the percentage of chemically modified cysteine ​​residues could be tuned by adjusting the pH of the coupling step: the higher the pH, the higher the coupling rate, as evidenced by staining in dot blot analysis (Figures 5A and 5B).

[0078] In other words, with respect to the benzoylacrylamide ligand, it is possible to control the proportion of chemically modified cysteines in the capsid through the pH of coupling, a property that could be of great interest depending on the functional moieties that can be covalently coupled to the surface of the AAV.

[0079] Without being bound by any theory, the inventors are of the opinion that the results obtained with benzoyl acrylamide ligands can be applied to vinylsulfonamide groups. To some extent, maleimide-bearing ligands can also be designed to chemically modify the cysteine ​​residues present in AAV capsids. However, the maleimide ligand L2 does not seem to be very effective and produces less stable coupling functional groups than those obtained with acrylamide ligands.

[0080] Thus, the present invention relates to an adeno-associated virus (AAV) having at least one chemically modified cysteine ​​residue in the capsid.

[0081] In a particular embodiment, the present invention relates to an adeno-associated virus (AAV) whose capsid comprises a functional moiety as described herein, said functional moiety being covalently attached to a cysteine ​​residue of the capsid, in other words, said cysteine ​​residue is chemically modified.

[0082] Chemically modified cysteine ​​residues present in the AAV capsid typically result from the reaction of a cysteine ​​in the capsid with a functional moiety that has a thiol-specific reactive group, including a benzoylacrylamide group, a maleimide group, or a vinylsulfonamide group.

[0083] Preferably, the cysteine ​​residue that may be chemically modified is a naturally occurring residue in the capsid, ie, the cysteine ​​has not been introduced by mutagenesis.

[0084] Thus, in certain embodiments, the chemically modified adeno-associated virus (AAV) comprises the following moiety:

[0085] [ka]

[0086] wherein "S" represents the first thiol atom in the cysteine ​​residue.

[0087] The present invention also relates to a method for preparing a chemically modified AAV, comprising the step of contacting the AAV with a functional moiety having a thiol-specific reactive group, such as a benzoylacrylamide group, a maleimide group, or a vinylsulfonamide group, under conditions that allow reaction of a thiol functional group of a cysteine ​​residue present in the AAV capsid with the reactive group to covalently link the chemical moiety to the AAV.

[0088] The invention also relates to uses of the resulting AAV, particularly in gene therapy.

[0089] The invention is described in more detail below: - General definitions As used in this disclosure, the term “C x ~C y " (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 may 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 may contain from 2 to 5 carbon atoms, particularly 2, 3, 4, or 5 carbon atoms.

[0090] 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.

[0091] 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, preferably ethenyl (-CH=CH2).

[0092] 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, preferably ethynyl (-C≡CH).

[0093] As used herein, the term "alkoxy" refers to an alkyl, as defined herein, attached to the remainder of the molecule through an ether bond (-O-). In other words, alkoxy can be written as "-O-alkyl". Preferred alkoxy is a C1-C6 alkoxy having 1-6 carbon atoms. Examples of alkoxy (or C1-C6 alkoxy) include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy, hexyloxy.

[0094] As used herein, the term "alkylthio" refers to an alkyl, as defined herein, attached 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 1-6 carbon atoms. Examples of alkylthio (or C1-C6 alkylthio) include, for example, methylthio, ethylthio, propylthio, isopropylthio, butylthio, pentylthio, and hexylthio.

[0095] As used herein, the term "alkylamino" refers to an alkyl, as defined herein, attached to the remainder of the molecule via an amino bond (-NH-). In other words, alkylamino can be written as "-NH-alkyl". Preferred alkylamino is C1-C6 alkylamino having 1-6 carbon atoms. Examples of alkylamino (or C1-C6 alkylamino) include, for example, methylamino, ethylamino, propylamino, isopropylamino, butylamino, pentylamino, and hexylamino.

[0096] As used herein, the term "hydrocarbon ring" refers to a saturated or unsaturated, aliphatic or aromatic, monocyclic, bicyclic, or tricyclic group. The hydrocarbon ring may be, inter alia, cycloalkyl, cycloalkenyl, or aryl.

[0097] 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.

[0098] 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.

[0099] As used herein, the term "heterocycle" corresponds to a saturated or unsaturated, aliphatic or aromatic, monocyclic, bicyclic, or tricyclic group containing at least one heteroatom, such as a nitrogen, oxygen, or sulfur atom. In the case of a bicyclic or tricyclic ring, the rings may be fused, bridged, or have a spiro configuration. Advantageously, the heterocycle contains between 3 and 6 ring atoms, at least one of the ring atoms being a heteroatom, such as a nitrogen, oxygen, or sulfur atom. In some embodiments, the "heterocycle" is a heterocycloalkyl, heterocycloalkenyl, or heteroaryl.

[0100] As used herein, the term "heterocycloalkyl" corresponds to a cycloalkyl group, as defined above, in which at least one carbon atom is replaced with a heteroatom, such as a nitrogen, oxygen, or sulfur atom.

[0101] As used herein, the term "heterocycloalkenyl" corresponds to a cycloalkenyl group, as defined above, in which at least one carbon atom has been replaced with a heteroatom, such as a nitrogen, oxygen, or sulfur atom.

[0102] 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, thioxetanyl, thiopyranyl, thiomorpholinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, dihydrofuranyl, dihydrothiopyranyl, dihydrothiophenyl, dihydropiperidinyl, tetrahydropiperidinyl, tetrahydrothiopyranyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothiophenyl.

[0103] As used herein, the term "aryl" refers to an aromatic ring system, preferably having 6-14 atoms, with at least one ring having a conjugated pi-electron system, which may be optionally substituted. "Aryl" may contain more than one aromatic ring, such as a fused ring system, or an aryl group substituted with another aryl group. Aryl includes, but is not limited to, phenyl, anthracenyl, naphthyl, indenyl, and divalent biphenyl.

[0104] "Heteroaryl" refers to a heteroaryl group. "Heteroaryl" refers to a chemical group, preferably having 5 to 14 ring atoms, in which 1 to 4 heteroatoms are ring atoms in an aromatic ring, and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and selenium. Examples of heterocycles that are heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinazolinyl, and quinolinyl.

[0105] Examples of bicyclic heteroaryl groups include, but are not limited to, 1H-indazolyl, benzo[l,2,3]thiadiazolyl, benzo[l,2,5]thiadiazolyl, benzothiophenyl, imidazo[l,2-a]pyridyl, quinolinyl, indolyl, and isoquinolinyl groups.

[0106] 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 1-6 carbon atoms. Examples of alkanoyl (or C1-C6 alkanoyl) include, for example, methanoyl, ethanoyl, propanoyl, isopropanoyl, butanoyl, pentanoyl, and hexanoyl.

[0107] 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 acylamino group is a hydrocarbon group such as cycloalkyl, aryl, etc. Preferred acylamino groups are C1-C6 acylamino groups having a hydrocarbon chain of 1-6 carbon atoms.

[0108] 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.

[0109] As used herein, "alkoxycarbonyloxy" refers to the group R"-C(O)-O-, where R" is alkoxy.

[0110] As used herein, the term "alkylene" refers to a divalent alkyl group, where "alkyl" is as defined herein. Preferred alkylenes are "(C1-C6) alkylenes" having 1 to 6 carbon atoms. "(C1-C6) alkylenes" particularly include those of the formula -(CH2) q where q is an integer from 1 to 6. "(C1-C6)alkylene" includes, by way of example, methylene, ethylene, propylene, butylene, isobutylene, pentylene, isopentylene, or hexylene.

[0111] As used herein, the term "arylene" refers to a divalent aryl group, with "aryl" as defined herein. Preferred arylenes are arylenes having 6 to 14 ring atoms. Arylenes include, by way of example, phenylene, anthracenylene, naphthylene, indenylene, and divalent biphenylene, preferably phenylene. Preferred phenylenes are para-phenylenes, i.e., phenylenes attached at two positions, para to the rest of the molecule.

[0112] As used herein, the term "heteroarylene" refers to a divalent heteroaryl group, with "heteroaryl" as defined herein. Preferred heteroarylenes are heteroarylenes having 5 to 14 ring atoms. Heteroarylenes include, by way of example, furanylene, thienylene, pyridylene, pyrrolylene, N-alkylpyrrolylene, pyridylene-N-oxide, pyrimidylene, pyrazinylene, imidazolylene, benzimidazolylene, benzofuranylene, benzothiophenylene, quinazolinylene, and quinolinylene.

[0113] As used herein, the term "halogen" includes chlorine, fluorine, iodine and bromine, preferably chlorine or fluorine.

[0114] As used herein, the term "aminoalkyl" refers to an alkyl, as defined above, substituted with one or more (preferably one) amino (-NH2) groups.

[0115] 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.

[0116] As used herein, the term "hydroxyalkyl" refers to an alkyl, as defined above, substituted with one or more (preferably one) hydroxy (-OH) groups.

[0117] As used herein, the term "alkoxyalkyl" refers to an alkyl, as defined above, substituted by one or more alkoxy, as defined above.

[0118] As used herein, the term "haloalkyl" refers to an alkyl, as defined above, substituted by one or more halogen atoms.

[0119] "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, -NO2, -SO3H, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C2 ... -N3, -NH2, -F, -I, -Br, -Cl, -CN, C1-C6 alkanoyl, C1-C6 carboxy ester, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C2-C6 alkoxy, C1-C6 alkylamino, -N3, -NH2, -F, -I, -Br, -Cl, -CN, C1-C6 10They may be independently selected from alkoxyalkyl, C2-C6 alkoxycarbonyloxy, -CN, -CF3, and C2-C6 alkoxyalkyl.

[0120] Preferred substituents are halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0121] The phrase "optionally substituted" may be interchanged with the phrase "substituted or unsubstituted" throughout this application.

[0122] Chemically modified AAV of the present invention Thus, the present invention relates to an adeno-associated virus (AAV) having at least one chemically modified cysteine ​​residue in the capsid.

[0123] As used herein, adeno-associated virus (AAV) refers to a small, non-enveloped virus of the dependoparvovirus family, with a single-stranded linear DNA genome of approximately 5 kb in length. Wild-type AAV has two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The 5' and 3' ORFs code for replication and capsid proteins, respectively. The ITRs contain 145 nucleotides and serve as the AAV genome replication origin and packaging signal. In recombinant AAV, the viral ORFs are replaced by exogenous gene expression cassettes, while replication and capsid proteins are provided in trans.

[0124] Thus, 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 may be of any type and is selected taking into account the intended use of the AAV. By way of example, the nucleic acid may comprise any RNA or DNA sequence.

[0125] In a preferred embodiment, the AAV of the present invention is a recombinant AAV.Typically, the recombinant AAV will be used as a gene vector for in vivo or in vitro applications, which means that the AAV of the present invention is a recombinant AAV vector.For a review on AAV as a vector in gene therapy, see Naso et al., Biodrugs, 2017, 31:317-334, the contents of which are incorporated herein by reference.

[0126] By way of example only, a recombinant AAV for use as a vector in gene therapy may contain an exogenous gene expression cassette that replaces the viral ORF and is placed between two ITRs. The cassette may contain a promoter, a gene of interest, and a terminator. The promoter and the gene of interest are selected depending on the tissue / organ to be targeted and the condition to be treated. As another example, a recombinant AAV for use in gene therapy may contain a DNA template for homologous recombination in cells. Such a recombinant AAV may be used in combination with a gene editing tool to promote homologous recombination in targeted cells in vivo, in vitro, or ex vivo. The gene editing tool may be of any type, including but not limited to CRISPR / Cas9, zinc finger nucleases, meganucleases, as well as RNA and DNA encoding the proteins.

[0127] In the context of the present invention, the term "AAV" includes all types of AAV, including wild-type AAV and recombinant or variant AAV, including, but not limited to, AAV with mutated or synthetic capsids, such as AAV with hybrid capsids, pseudotyped AAV, as well as self-complementary AAV (scAAV).

[0128] The wild-type AAV capsid is composed of three overlapping capsid proteins called viral protein 1 (VP1), VP2, and VP3. Capsid engineering refers to amino acid modifications of the capsid proteins, for example in their hypervariable loops.

[0129] As used herein, an "AAV having a genetically engineered capsid" or an "AAV having a mutant capsid" refers to an AAV in which one or several amino acid modifications have been introduced into at least one capsid protein (i.e., VP1 and / or VP2 and / or VP3) compared to the wild-type version of said capsid protein.

[0130] As used herein, an "amino acid modification" encompasses one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 20, 30, 40, 50, or 100) amino acid insertions, deletions, or substitutions.

[0131] In some embodiments, the capsid of the AAV lacks any tetracysteine ​​moiety. In particular, the capsid of the AAV has not been mutated or engineered to introduce a tetracysteine ​​moiety. In particular, the AAV of the present invention is not AAV8 in which a tetracysteine ​​moiety is introduced at a position selected from the group consisting of VP1 position 34, VP1 or VP2 position 138, or VP1, VP2, or VP3 position 583 or 589, and combinations thereof (amino acid positions refer to the amino acid numbering in AAV8 VP1).

[0132] In another embodiment, the AAV has a genetically mutated capsid in which no mutation is made at the cysteine ​​residue or does not result in the insertion of a tetracysteine ​​moiety. In other words, the AAV of the present invention may have a wild-type capsid or a mutated capsid in which the naturally occurring cysteine ​​residues are conserved. In a specific embodiment, the AAV of the present invention is selected from a wild-type AAV and a recombinant or variant AAV having a wild-type AAV capsid.

[0133] As used herein, "chemically modified cysteine ​​residue" means that at least one cysteine ​​present in the capsid of the virus is chemically modified by covalent coupling of a chemical entity, typically by covalent coupling of said chemical entity on the phenyl ring of the cysteine. The cysteine ​​is typically a surface-exposed residue present in VP1, VP2, or VP3. A surface-exposed cysteine ​​means that the cysteine ​​is accessible for covalent coupling. Such cysteine ​​residues can be identified by molecular modeling of the capsid protein or the entire capsid itself. There are five conserved cysteine ​​residues in VP1, VP2, and VP3. The conserved cysteine ​​residues are at positions 230, 289, 361, 394, and 482 in the AAV2 VP1 subunit. These residues are also conserved in the AAV2 VP2 and VP3 subunits. The amino acid positions of the cysteine ​​residues are 93, 152, 224, 257, and 345 in the AAV2 VP2 subunit, and 28, 87, 159, 192, and 280 in the AAV2 VP3 subunit.

[0134] As mentioned above, cysteines in VP are well conserved among AAV serotypes. For example, for AAV9, cysteine ​​residues are located at positions 230, 291, 363, and 396 in the VP1 subunit, 93, 154, 226, and 259 in the VP2 subunit, and 28, 89, 161, and 194 in the VP3 subunit. These cysteine ​​residues in AAV9 VP are located at positions corresponding to positions 230, 289, 361, and 394 in the AAV2 VP1 subunit.

[0135] Thus, for clarity, unless otherwise indicated, the cysteine ​​positions indicated for a given VP protein are provided with reference to the amino acid position numbering in AAV2 VP1, which correspondence can be made by performing a partial sequence alignment between the VP protein of interest and AAV2 VP1, for example, as shown in Figure 1B.

[0136] The capsid is composed of a total of 60 copies of viral protein subunits VP1, VP2, and VP3 in a ratio of 1:1:10. Therefore, the capsid contains at most 300 and 240 cysteine ​​residues for AAV2 and AAV9, respectively. At least the cysteine ​​residues at positions 289 and 394 for AAV2 and 291 and 396 for AAV9 have been shown to be chemically modified.

[0137] As used herein, "at least one chemically modified cysteine ​​residue" encompasses at least 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or more chemically modified cysteine ​​residues.

[0138] In some embodiments, the chemically modified AAV of the present invention comprises several chemically modified cysteine ​​residues in its capsid. The chemically modified cysteines may be present in VP1 and / or VP2 and / or VP3. The chemically modified cysteines may be present at positions 230, 289, 361, 394, and / or 482 in the VP subunits (the amino acid positions refer to the amino acid numbering in AAV2 VP1).

[0139] In some embodiments, the chemically modified AAV of the present invention comprises a chemically modified cysteine ​​at position 394 in VP1 and / or VP2 and / or VP3 (said amino acid positions refer to the amino acid numbering in AAV2 VP1).

[0140] In some additional embodiments, the chemically modified AAV of the present invention comprises a chemically modified cysteine ​​at position 289 in VP1 and / or VP2 and / or VP3 (said amino acid positions refer to the amino acid numbering in AAV2 VP1).

[0141] In some further embodiments, the chemically modified AAV of the present invention is characterized in that the cysteines at positions 289 and 394 in the VP subunit are chemically modified with a ligand of the present invention (the amino acid positions refer to the amino acid numbering in AAV2 VP1).

[0142] The AAV can be of any serotype. In some specific embodiments, the chemically modified AAV of the present invention is an AAV2 serotype and comprises at least one chemically modified cysteine ​​residue at positions 289 and 394 of VP1 and / or VP2 and / or VP3 (said amino acid positions refer to the amino acid numbering in AAV2 VP1).

[0143] In some other embodiments, the chemically modified AAV of the present invention is an AAV9 serotype and comprises at least one chemically modified cysteine ​​residue at positions 291 and 396 of VP1 and / or VP2 and / or VP3 (the amino acid positions refer to the amino acid numbering in VP1 of AAV9).

[0144] In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and even 100% of the surface-exposed cysteine ​​residues of the capsid are chemically modified.

[0145] In some embodiments, at least 30%, preferably at least 50%, of the cysteine ​​residues present in the capsid, i.e., in the VP1, VP2, and VP3 proteins, are chemically modified.

[0146] In certain embodiments, at least 50%, e.g., at least 60%, 70%, 80%, or 90% of the cysteines at positions 289 and 394 in the VP subunit are chemically modified (the amino acid positions refer to the amino acid numbering in VP1 of AAV9).

[0147] In the context of the present invention, AAV can be either an AAV with a wild-type capsid or an AAV with a mutated and / or synthetic capsid.

[0148] 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.

[0149] In a particular embodiment, the AAV is a recombinant AAV having a mutant capsid, wherein the amino acid modifications do not involve any cysteine ​​residues present in the capsid protein.

[0150] In another embodiment, the cysteine ​​residue in the AAV capsid is a wild-type, i.e., a naturally occurring cysteine ​​residue.

[0151] In another embodiment, the AAV capsid does not contain any mutations that introduce cysteine ​​moieties, particularly tetracysteine ​​moieties.

[0152] 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.

[0153] A "serotype" is traditionally defined based on the absence of cross-reactivity between antibodies to one virus compared to another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). AAV includes a variety of natural and synthetic (e.g., hybrid, chimeric, or shuffled serotypes) serotypes.

[0154] Such non-limiting serotypes include AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10 (such as -cy10 or -rh10), -11, -rh74, or engineered AAV capsid variants, such as AAV-2i8, AAV2G9, -LK3, -DJ, and -Anc80. In the context of the present invention, synthetic serotypes also include pseudotyped AAV, i.e., AAV resulting from a mixture of capsids and genomes from different viral serotypes, such as AAV2 / 5, AAV2 / 7, and AAV2 / 8, as well as AAV with hybrid capsids from multiple different serotypes, such as AAV-DJ, which contains hybrid capsids from eight serotypes.

[0155] Synthetic serotypes also encompass certain variants in which new glycan binding sites have been introduced into the AAV capsid, as described in particular in International Patent Application Publication No. WO2014144229 (which in particular discloses the AAV2G9 serotype). Other AAV serotypes include those disclosed in EP2292779 and EP1310571. In addition, other AAV serotypes include those obtained by shuffling as described in Koerber et al. (Molecular Therapy (2008), 16(10), pp. 1703-1709), by peptide insertion (e.g., Deverman et al., Nat Biotechnol (2016), 34(2), pp. 204-209), or by rational capsid design (reviewed in Buening et al., Curr Opin Pharmacol (2015), 24, pp. 94-104).

[0156] 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, more preferably AAV-2. By way of example, the AAV of the present invention can be of the AAV-2 or AAV-9 serotype.

[0157] AAV can target a wide variety of cells, tissues, and organs. Examples of cells targeted by AAV include, but are not limited to, liver cells; cells of the retina, i.e., photoreceptors, retinal pigment epithelium (RPE); muscle cells, i.e., myoblasts, satellite cells; cells of the central nervous system (CNS), i.e., neurons, glia; cells of the heart; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells, lymphocytes, blood cells such as hematopoietic cells, including hematopoietic stem cells, induced pluripotent stem cells (iPS), and the like. Examples of tissues and organs that can be targeted by AAV include liver, muscle, cardiac muscle, smooth muscle, brain, bone, connective tissue, heart, kidney, lung, lymph node, mammary gland, myelin, prostate, testis, thymus, thyroid, trachea, and the like. Preferred cell types are liver cells, retinal cells, muscle cells, cells of the CNS, cells of the PNS, and hematopoietic cells. Preferred tissues and organs are liver, muscle, heart, eye, and brain.

[0158] The tropism of AAVs can vary depending on their serotypes. For example, AAV-2 may be used to transduce the central nervous system (CNS), kidney, and photoreceptor cells, while AAV-8 is effective in transducing the CNS, heart, liver, photoreceptor cells, retinal pigment epithelium (RPE), and skeletal muscle.

[0159] AAV can be produced by any method known in the art, for example, by transient transfection in a cell line of interest, for example, HEK293 cells, as described in the Examples section. In this regard, reference can be made to Naso et al., Biodrugs, 2017, 31:317-334, which provides an overview on AAV as a vector in gene therapy and describes traditional methods for producing AAV on an industrial scale.

[0160] The AAV of the present invention may have other amino acids of the capsid that have been chemically modified. By way of example, the AAV may comprise one or several amino groups of the capsid that have been modified by the method disclosed in International Patent Application Publication No. 2017 / 212019, i.e., by reacting said amino groups in the capsid with a ligand that has an isothiocyanate reactive group. Alternatively or additionally, the AAV of the present invention may have one or several arginine residues of the capsid that have been modified by glycosylation, e.g., by reaction with methylglyoxal as described in Horowitz (supra). Alternatively or additionally, the AAV of the present invention may comprise one or more tyrosine residues in the capsid that have been modified by the method disclosed in International Patent Application Publication No. 2021 / 005210[0], i.e., by reacting the tyrosine residues in the capsid with a ligand having an aryldiazonium or 4-phenyl-1,2,4-triazole-3,5-dione (PTAD) reactive group.

[0161] Typically, at least one chemically modified cysteine ​​residue in the capsid has the following formula (I):

[0162] [ka]

[0163] (In the formula, - X is:

[0164] [ka]

[0165] (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 may be optionally substituted; - k is 0 or 1, - R is hydrogen, halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, which may be optionally substituted. is selected from the group consisting of - Y is a spacer, - n is 0 or 1, and - M is a functional moiety It is.

[0166] As mentioned in the definition above, in some embodiments: - the alkyl group is preferably a C1 to C6 alkyl group, the aryl group is preferably an aryl group having from 6 to 14 ring atoms, the heteroaryl group is preferably an aryl group having from 5 to 14 ring atoms, and The alkoxy group is preferably a C1 to C6 alkoxy group.

[0167] In the formulas described in this application (e.g., formula (I)), the following moieties:

[0168] [ka]

[0169] (In the formula,

[0170] [ka]

[0171] represents the bond by which that cysteine ​​is attached to the rest of the protein) represents a cysteine ​​within a capsid protein (i.e., VP1, VP2, or VP3).

[0172] "n is 1" means that the spacer Y is present. "n is 0" means that the spacer Y is absent.

[0173] -X X is of formula (a), (b) or (c) as above. Preferably, X is of formula (b) or (c). More preferably, X is of formula (c).

[0174] In formula (a), R is preferably a hydrogen atom, halogen, or C1 to C6 alkoxy, more preferably a hydrogen atom.

[0175] In formula (b), R1 is preferably C1-C6 alkyl, aryl, or heteroaryl. By way of example, R1 may be selected from C1-C6 alkyl, aryl groups containing 6 to 14 ring atoms, such as phenyl groups, and heteroaryl groups containing 5 to 14 ring atoms. In a preferred embodiment, R1 is C1-C6 alkyl, and even more preferably C1-C3 alkyl, such as methyl.

[0176] In formula (c): R2 is a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl, preferably a C1-C3 alkyl group or hydrogen, more preferably hydrogen. - R3 is preferably an aryl or heteroaryl group (e.g., phenyl), which may be optionally substituted. By way of example, the aryl or heteroaryl group may be substituted with 1 to 3 substituents, preferably selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl. In a particular embodiment, R3 is selected from an aryl group containing 6 to 14 ring atoms or a heteroaryl group containing 5 to 14 ring atoms, which may be optionally substituted. By way of example, the aryl or heteroaryl group may be substituted with 1 to 3 substituents, preferably selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl. In some preferred embodiments, R3 is a phenyl group, optionally substituted with one or three substituents selected from -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl. The substituents may be in the ortho, meta, or para positions, preferably in the ortho or para positions. Preferably, R4 is hydrogen or C1-C3 alkyl, more preferably H or CH3.

[0177] In certain embodiments, k is 1. In preferred embodiments, k is 0, which means that Z is absent.

[0178] In certain embodiments, R2 is a C1-C3 alkyl or hydrogen; R3 is a phenyl group optionally substituted with one or three substituents selected from -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl; k is 0 or 1 (preferably, k is 0); and Z, if present, is -O-, -S-, -N(CH3)-, or -NH-.

[0179] In a more particular embodiment, R2 is H and R3 is unsubstituted or substituted phenyl, preferably unsubstituted phenyl.

[0180] In a preferred embodiment, R2 is H, R3 is phenyl (unsubstituted or substituted, preferably unsubstituted phenyl), and k is 0.

[0181] - Spacer Y Y is a spacer that links X and the functional moiety M. Y can be present (when n is 1) or absent (when n is 0). When Y is absent, X and M are directly linked to each other.

[0182] Y can be any chemical chain that may contain heteroatoms as well as cyclic moieties such as aromatic groups including cycloalkyl, cycloalkenyl, heterocycloalkyl, or heteroaryl. Y can contain up to 1000 and even more carbon atoms. The length and chemical nature of the spacer can be optimized depending on the functional moiety "M" intended to be coupled to the cysteine ​​residue and the biological effect sought. Indeed, in addition to its linking function, Y can be used to refine the properties of the functional moiety "M". As an 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.

[0183] In some embodiments, Y is a chemical chain group comprising 2 to 1000 carbon atoms, preferably 2 to 500 carbon atoms, 2 to 300 carbon atoms, e.g., 2 to 100 carbon atoms, 2 to 40 carbon atoms, 4 to 30 carbon atoms, or 4 to 20 carbon atoms.

[0184] In certain embodiments, Y is of formula (II):

[0185] [ka]

[0186] (In the formula, m, p, and q are each independently 0 or 1; Y1 is selected from the group consisting of alkylene, arylene and heteroarylene groups, said groups being optionally substituted, preferably a phenylene group; - Y2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -NH(C=S)- or -(C=S)-NH-, preferably -(C=O)-NH-, and - Y3 is selected from the group consisting of polymers, including homopolymers, copolymers and block polymers, peptides, oligosaccharides, saturated or unsaturated, branched or linear hydrocarbon chains, optionally interrupted by one or several heteroatoms and / or by groups selected from -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 / or by one or more C3-C6 hydrocarbon rings or C2-C6 heterocycles, optionally bearing a heteroatom at at least one of its termini, and optionally substituted by one or several substituents, and combinations thereof. It is a spacer.

[0187] In such embodiments, the at least one chemically modified cysteine ​​residue in the capsid typically has the following formula (I-II):

[0188] [ka]

[0189] (wherein X, Y1, Y2, Y3, M, m, p, and q are as defined herein). It can be expressed as:

[0190] In certain embodiments, m is 0, p is 0 and q is 1. In such embodiments, the spacer Y is Y3.

[0191] In another specific embodiment, m is 1, p is 1 and q is 1.

[0192] In some embodiments, Y1 is an unsubstituted or substituted C1-C6 alkylene. For example, the C1-C6 alkylene may be substituted with 1 to 3 substituents, which may be independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0193] In some embodiments, Y1 is selected from arylene and heteroarylene containing from 5 to 14 ring atoms, e.g., from 6 to 10 ring atoms, where the arylene or heteroarylene may be optionally substituted. By way of example, the arylene or heteroarylene group may include 1, 2, or 3 substituents independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0194] In some particular embodiments, Y1 is selected from the group consisting of substituted or unsubstituted phenylene, pyridylene, naphthylene, and anthracenylene. The group may contain from 1 to 3 substituents, preferably independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl. Preferably, Y1 is phenylene, optionally substituted with 1 to 3 substituents, which may be independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0195] In embodiments where Y1 is phenylene (optionally substituted with 1 to 3 substituents which may be independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl), X and -(Y2) p -(Y3) q -M is preferably attached at the para position on the phenylene.

[0196] In certain embodiments, Y2 is -(C=O)-NH-.

[0197] As mentioned above, Y3 is selected from polymers, including homopolymers, copolymers, and block polymers, peptides, oligosaccharides, optionally containing one or several heteroatoms (e.g., S, O, Se, P, or NH) and / or -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 / or one or more C3 to C6 hydrocarbon rings or C2 to C6 heterocycles, optionally having heteroatoms such as S, O, and NH at at least one of its termini, and optionally substituted with one or several substituents such as hydroxyl, halogen, C1 to C3 alkoxy, -CN, -CF3, or C1 to C3 alkyl, and combinations thereof.

[0198] By way of example, Y3 may be selected from the group consisting of polymers, including homopolymers, copolymers, and block polymers, peptides, oligosaccharides, saturated or unsaturated, branched or linear hydrocarbon chains.

[0199] As used herein, "combination" means that Y3 can include several (e.g., 2, 3, 4, 5, or 6) hydrocarbon, oligomeric, or polymeric chains linked by any suitable group, such as, for example, -O-, -S-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, NH-CS-, phosphodiester, or phosphorothioate groups.

[0200] In some embodiments, Y3 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), D,L-lactic acid-co-glycolic acid copolymers (PLGA), polymers of alkyldiamines, unsaturated or saturated branched or unbranched hydrocarbon chains, optionally having heteroatoms such as O, NH, and S at at least one terminus, and combinations thereof.

[0201] As used herein, alkyldiamine refers to NH2-(CH2) r -NH2, where r is an integer from 2 to 20, for example, an integer from 2 to 10, such as 2, 3, 4, and 5. Polymers of alkyl diamines (also known as polyamines) are represented by the formula NH2-[(CH2) r -NH] t -H, where r is as defined above and t is an integer of at least 2, e.g., at least 3, 4, 5, 10 or more. Polymers of alkyldiamines of interest are, by way of example, spermidine and spermine. By way of example only, Y3 may comprise at least one polyethylene glycol moiety comprising from 2 to 40 monomers, e.g., from 2 to 10 or from 2 to 6 monomers. By way of example only, Y3 may comprise from 2 to 10 triethylene glycol blocks linked together by a linker. As another example, Y3 may comprise C 12 Alternatively, Y3 may be a hydrophilic triethylene glycol ethylamine derivative. Alternatively, Y3 may be a saturated or unsaturated C2-C 40 Hydrocarbon chains, especially C 10 ~C 20 C2-C alkyl chain or C6 alkyl chain 10 It may be an alkyl chain, which may have a group such as NH, S, or O at at least one end.

[0202] As a further example, Y3 may be selected from spermidine, putrescine, spermine, and combinations thereof.

[0203] In certain embodiments, Y3 is an optionally substituted, saturated or unsaturated, linear or branched C2-C 40 In certain embodiments, Y3 is selected from the group consisting of a hydrocarbon chain, a polyethylene glycol, a polypropylene glycol, pHPMA, PLGA, a polymer of an alkyl diamine, and combinations thereof. 20 It is selected from the group consisting of alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diamino alkyl polymers, and combinations thereof. Preferably, the polyethylene glycol, polypropylene glycol, PLGA, pHPMA, and alkyl diamine polymers contain 2 to 40 monomers, preferably 2 to 10 or 10 to 20 monomers. By way of example, Y3 may contain one or several (e.g., 2, 3, 4, or 5) triethylene glycol blocks.

[0204] - X+Y part In certain embodiments, X is of formula (a), n=1, and Y is of formula (II), - m=p=q=1 - Y1 is as defined herein, preferably a C1-C6 alkylene group or a phenylene group, more preferably a phenylene group, said groups being optionally substituted by 1 to 3 substituents which may be independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl; - Y2 is as defined herein, preferably Y2 is -(C=O)-NH-, and - Y3 is as defined herein, preferably a linear or branched C2-C 20alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diaminoalkyl polymers, and combinations thereof) It is.

[0205] In another particular embodiment, X is of formula (b), n=1, and Y is of formula (II), - m=p=q=1 - Y1 is as defined herein, preferably a phenylene group, said group being optionally substituted by 1 to 3 substituents which may be independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl; - Y2 is as defined herein, preferably Y2 is -(C=O)-NH-, and - Y3 is as defined herein, preferably a linear or branched C2-C 20 alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diaminoalkyl polymers, and combinations thereof) It is.

[0206] In a preferred embodiment, X is of formula (c), n=1, and Y is of formula (II), m=0, p=0 or 1 (preferably 0), q=1, and - Y2 is as defined herein; - Y3 is as defined herein, preferably a linear or branched C2-C 20 alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diaminoalkyl polymers, and combinations thereof) It is.

[0207] Functional part M The functional moiety "M" can be of any type. "M" is typically selected depending on the biological effect that is desired when the AAV capsid is chemically modified.

[0208] By way of example, "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.

[0209] 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.

[0210] In other embodiments, "M" comprises or consists of a steric shielding agent, e.g., an agent capable of masking certain epitopes of the capsid, thereby avoiding binding of neutralizing antibodies. By way of example, "M" can be polyethylene glycol (PEG), pHPMA, or a polysaccharide.

[0211] In certain embodiments, "M" comprises or consists of a steric shielding agent capable of masking cysteine ​​residues, thereby avoiding proteasomal degradation of AAV in cellulo.

[0212] 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 a partial meroduplex (mdRNA).

[0213] 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, e.g. a cell surface protein, receptor, or oligosaccharide, e.g. a cell surface protein present on the surface of a particular cell line or tumor cell.

[0214] By way of example, a targeting agent may be a cell type specific ligand, ie a ligand that allows for targeting of a particular type of cell.

[0215] Such ligands may make it possible to modify the tropism of AAV, i.e., its ability to selectively infect and / or transduce a given cell line, tissue, or organ.

[0216] By way of example, "M" may be a ligand that specifically binds to a biological entity (e.g., a membrane receptor) of the membrane of the targeted cell. The ligand may be, by way of example, a mono- or polysaccharide, a hormone, e.g., a steroid hormone, a peptide such as RGD peptide, angiopep-2, a muscle targeting peptide, a protein or a fragment thereof, a membrane receptor or a fragment thereof, CB1 and CB2 ligands, an aptamer, an antibody including heavy chain antibodies and fragments thereof such as Fab, Fab', and VHH, an ScFv, a spiegelmer, a peptide aptamer, a small chemical molecule known to bind to the targeted biological entity, and the like.

[0217] In certain embodiments, M is a full length antibody or an antibody that comprises an antigen-binding domain derived from an antibody.

[0218] As used herein, the term "antibody" refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising 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')2, Fv, scFv, Fd, dAb, and other antibody fragments (e.g., VHH from single-chain antibodies). Typically, such fragments comprise an antigen-binding domain. The term "antigen-binding domain" or "antigen-binding fragment" refers to a portion of an antibody that comprises the amino acids responsible for the specific binding between the antibody and the antigen. If the antigen is large, the antigen-binding domain may only bind a portion of the antigen. The part of the antigen molecule that is responsible for the specific interaction with the antigen-binding domain is called the "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, for example, 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, typically all CDRs) and thus retains the binding specificity and / or activity of the antibody.

[0219] As used herein, a "full-length antibody" (also referred to herein as immunoglobulin or Ig) refers to a protein having a structure that constitutes the natural biological form of an antibody, including variable and constant regions. "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, the list of which is not limiting. In most mammals, including humans and mice, the structure of a full-length antibody is generally a tetramer. 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 the case of human immunoglobulins, the 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 classes of immunoglobulins 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.

[0220] 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.

[0221] 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 several 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 may be natural or synthetic.

[0222] In another embodiment, "M" comprises or consists of a cell type specific ligand derived from a vitamin such as folic acid.

[0223] According to one embodiment, the cell type specific ligand included in "M" may be derived from or may be in muscle targeting peptide (MTP). The ligand may be ASSLNIA (SEQ ID NO:1); WDANGKT (SEQ ID NO:2); GETRAPL (SEQ ID NO:3); CGHHPVYAC (SEQ ID NO:4); HAIYPRH (SEQ ID NO:5); CYCLICCQLFPLFRC (SEQ ID NO:6), or the sequence of SEQ ID NO:7 as shown below: RXRRBRRXRFQILYRXRBRXR-B (SEQ ID NO: 7)

[0224] where X is an aminohexanoic acid residue and B is a beta-alanine residue. The amino acid sequence may be selected from the group consisting of:

[0225] As used herein, the cyclic CQLFPLFRC of SEQ ID NO:6 is

[0226] [ka]

[0227] Refers to...

[0228] In certain embodiments, "M" is a cancer cell targeting peptide, including peptides such as RGD, including cyclic RGD.

[0229] In some other embodiments, M is a cell-type targeting ligand selected from antibodies and fragments thereof.

[0230] When "M" comprises a peptide moiety, such as a muscle targeting peptide (MTP), the peptide moiety may comprise a chemical modification at its M-terminus or C-terminus. By way of example, the N-terminus of the peptide moiety may be acylated or coupled to a moiety such as -C(=O)-(PEG moiety)-NH2.

[0231] 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.

[0232] In further embodiments, "M" is a CB1 and / or CB2 ligand, such as:

[0233] [ka]

[0234] It comprises or consists of:

[0235] Galactose-derived ligands recognized by the asialoglycoprotein receptor (ASPGPr) can be used to specifically target hepatocytes. Thus, in some embodiments, "M" is a ligand for specifically targeting hepatocytes and has the following formula (IIIa), (IIIb), or (IIIc):

[0236] [ka]

[0237] Contains at least one portion of.

[0238] In some other embodiments, "M" is a ligand for targeting muscle cells, particularly skeletal muscle cells, and contains at least one of the following mannose-6-phosphate moieties:

[0239] [ka]

[0240] Includes.

[0241] In some other embodiments, "M" is a ligand for a photoreceptor or neuronal cell and comprises at least one mannose moiety of the following formula (IIIf):

[0242] [ka]

[0243] Includes.

[0244] In some other embodiments, "M" is a ligand of 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 a galactose moiety.

[0245] More specifically, M can be a sialic acid moiety or a derivative thereof, said moiety having the formula (IIIg):

[0246] [ka]

[0247] (wherein R5 is alkyl, aryl, heteroaryl, haloalkyl (e.g., -CH2-Hal (wherein Hal is halogen), -OR6, -NR7R8, -SR9, -CH2OR10 , -CH2NR 11 R 12 , or -CHSR 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.

[0248] In certain embodiments, R5 is alkyl, -OR6, -CH2OR 10、 -CH2-Hal, wherein Hal, R6, R 10 is as defined herein.

[0249] In more specific embodiments, R5 is methyl, -CH2OH, or -CH2-F.

[0250] In certain embodiments, M is selected from Neu5Ac, Neu5Acα2-6Gal, and Neu5Acα2-8Neu5Ac moieties.

[0251] As used herein, a Neu5Ac moiety refers to an N-acetylneuraminic acid moiety. Neu5Ac has the following formula (IIIh):

[0252] [ka]

[0253] It can be expressed as follows:

[0254] 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 (IIIi):

[0255] [ka]

[0256] It can be expressed as follows:

[0257] 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 (IIIj):

[0258] [ka]

[0259] It can be expressed as follows:

[0260] In some embodiments, "M" is multivalent, meaning that it comprises at least two (e.g., 2, 3, 4, 5, or 6) ligand moieties of interest, such as cell type specific ligands as described above. By way of example, M may comprise a multifunctional linker having several (e.g., at least 2, 3, 4, 5, or 6) cell type specific ligands. The cell type specific ligands may be the same or different. By way of example, "M" may be represented by the following formula (IV):

[0261] [ka]

[0262] (wherein n is an integer from 1 to 100, preferably from 1 to 20). may include a portion of

[0263] As another example of a multivalent ligand, "M" may comprise a moiety of formula (IV) 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, for example, Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5AcNeu5Ac), CB1 and / or CB2 ligands or peptides.

[0264] In certain embodiments, "M" may include both a labeling moiety, such as a fluorescent label or a radionuclide, and a cell type specific ligand. By way of example only, M may be:

[0265] [ka]

[0266] That is, it can be the muscle targeting peptide of SEQ ID NO:1 linked to K-FITC.

[0267] Other examples of chemical moieties that can be used as the "M" moiety are provided in Figures 4A and 4B.

[0268] In some embodiments, M is a chemically reactive group, more preferably a biocompatible chemically reactive group. As used herein, M can allow the covalent interaction of AAV with the entity of interest to occur without significantly changing the functionality of 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. By way of example, the functional moiety can include a chemically reactive group suitable for forming a covalent bond by click chemistry or by a bioconjugation reaction. Bioconjugation reactions include reactions between amino acids such as lysine, cysteine, or tyrosine and reactive groups, as detailed in Koniev, O., Wagner, A, Chem. Soc. Rev., 44, 5495 (2015).

[0269] Preferably, M is a click chemistry reactive group, hereinafter also referred to as a "click chemistry group".

[0270] 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).

[0271] "Click reaction" or "click chemistry" is a concept introduced by Sharpless in 2001. "Click chemistry" generally refers to a chemical reaction characterized by high yield, high functional group selectivity, easy to carry out, and generates harmless by-products. "Click reactions" can typically be carried out with high efficiency in complex media. Click reactions are typically used to generate covalent heteroatom linkages (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.

[0272] 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.

[0273] Examples of M include azides (-N3), alkenes, alkynes (particularly cyclooctynes ​​(OCT), aryl-less cyclooctynes ​​(ALO), monofluorocyclooctynes ​​(MOFO), difluorocyclooctynes ​​(DIFO), dibenzocyclooctynes ​​(DIBO), dimethoxyazacyclooctynes ​​(DIMAC), biarylazacyclooctynes ​​(BARAC), bicyclononynes (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctynes ​​(DIFBO), oxa-diphenylphosphine (PDP), cyclooctynes ​​(CD ... The aryl group may comprise or consist of a strained alkyne such as benzocyclooctyne (ODIBO), carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne), 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 isonitrile, an aryl halide, an arylboronic acid, an oligohistidine, a nickel complex, or a nickel ligand.

[0274] In certain embodiments, M is a click chemistry group that includes an azide group (-N3) or an alkyne group (e.g., a -C≡CH group or a strained alkyne such as cyclooctyne (OCT), arylless cyclooctyne (ALO), monofluorocyclooctyne (MOFO), difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctyne (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa-dibenzocyclooctyne (ODIBO), carboxymethyl monobenzocyclooctyne (COMBO), or benzocyclononyne).

[0275] As mentioned above, preferred chemically modified AAVs are those that contain at least one chemically modified cysteine ​​of formula (I) or (I-II), where X is formula (c). Indeed, as illustrated in the Examples section, acylamide ligands allow for significantly higher coupling rates than maleimide ligands and are therefore more effective. Moreover, the proportion of chemically modified cysteine ​​residues in the AAV capsid can be adjusted by adjusting the pH of coupling with respect to acrylamide ligands. Finally, cysteine ​​residues modified with acrylamide ligands are expected to be more stable than those modified with maleimide ligands.

[0276] Thus, in a preferred embodiment, the present invention provides a compound of formula (Ic):

[0277] [ka]

[0278] (wherein Y, n, M, Z, k, R2, and R3 are as defined herein) and at least one chemically modified cysteine ​​present in the capsid.

[0279] It is understood that when Z is as defined herein, and when Z is NR4, R4 is as defined herein. More particularly, the present invention provides compounds of formula (I-IIc):

[0280] [ka]

[0281] (wherein Y1, Y2, Y3, m, p, q, M, Z, k, R2, and R3 are as defined herein).

[0282] In some embodiments of the invention, the chemically modified cysteine ​​present in the capsid has formula (Ic) or (I-IIc) and has the following characteristics: R2 is hydrogen; R3 is an aryl or heteroaryl group, preferably phenyl, said group being optionally substituted by 1 to 3 substituents, preferably selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl and C1-C3 haloalkyl; - k is 0 or 1 (preferably, k is 0), - Z, if present, is -O-, -S-, -N(CH3)-, or -NH-; - M is as defined above. M may be 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; The compound may be further characterized by one or more of the following:

[0283] In some other embodiments of the invention, the chemically modified cysteine ​​present in the capsid has formula (Ic) or (I-IIc) and has the following characteristics: - R2 is a hydrogen atom, a C1-C6 alkyl group, an aryl group containing from 6 to 14 ring atoms, or a heteroaryl group containing from 5 to 14 ring atoms, and R3 is selected from an aryl group containing 6 to 14 ring atoms or a heteroaryl group containing 5 to 14 ring atoms, said aryl or heteroaryl group being optionally substituted. By way of example, said aryl or heteroaryl group may be substituted by 1 to 3 substituents, preferably selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl. It is further characterized by: Preferably, k is 0.

[0284] In some still further embodiments of the present invention, the chemically modified cysteine ​​present in the capsid is of formula (Ic) or (I-IIc) and has the following characteristics: R2 is C1-C3 alkyl or hydrogen, preferably hydrogen, and R3 is phenyl or a 6-membered heteroaryl, preferably phenyl unsubstituted or substituted by 1 to 3 substituents selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl; It is further characterized by: Preferably, k is 0.

[0285] In another embodiment of the invention, the chemically modified cysteine ​​present in the capsid is of formula (Ic) or (I-IIc) and has the following characteristics: - R2 is C1-C3 alkyl or hydrogen; - R3 is phenyl, unsubstituted or substituted with a group selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl; and - k is 0 It is further characterized by:

[0286] In all embodiments described herein, M may comprise or consist of a cell targeting agent, preferably selected from mono- or polysaccharides, hormones, such as steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTP) or angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, ScFv, spiegelmers, peptide aptamers, vitamins, and drugs, such as CB1 and / or CB2 ligands. In another embodiment, M comprises or consists of a click chemistry reactive group, for example comprising an azide or an alkyne, or an oligonucleotide, for example as defined above.

[0287] In some embodiments of the present invention, the chemically modified cysteine ​​present in the capsid has formula (I-IIc) and the following characteristics: - m=0, - p=0, - q=1, - Y3 is as defined herein, preferably a linear or branched C2-C 20 selected from the group consisting of alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, diaminoalkyl polymers, and combinations thereof; R2 is hydrogen; R3 is an aryl or heteroaryl group, preferably phenyl, which is optionally substituted by 1 to 3 substituents, preferably selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl; - k is 0 or 1 (preferably, k is 0), - Z, if present, is -O-, -S-, -N(CH3)-, or -NH-; - M is as defined above. In particular, M comprises or consists of a click chemistry reactive group (e.g. comprising an azide or an alkyne), an oligonucleotide, 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 a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, a 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. The compound may be further characterized by one or more of the following:

[0288] In some embodiments, Y, when present, is represented by formula (II), where q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined above in formula (II), and Y3 is an optionally substituted, saturated or unsaturated, linear or branched C2-C 40 and / or M comprises or consists of a click chemistry group, an oligonucleotide, preferably a mono- or polysaccharide, a peptide such as a hormone, e.g. a steroid hormone, an RGD peptide, a muscle targeting peptide (MTP) or angiopep-2, a protein or fragment thereof, a membrane receptor or fragment thereof, an aptamer, an antibody including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, an ScFv, a spiegelmer, a peptide aptamer, a vitamin, and a drug such as a CB1 and / or CB2 ligand.

[0289] In some other embodiments, Y, when present, is represented by formula (II), where q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined above in formula (II), and Y3 is an optionally substituted, saturated or unsaturated, linear or branched C2-C 40 and / or M comprises or consists of a cell type specific ligand derived from a protein selected from transferrin, epidermal growth factor (EGF) and basic fibroblast growth factor βFGF, a mono- or polysaccharide containing one or several galactose, mannose, N-acetylgalactosamine residues, cross-linked GalNAc or mannose-6-phosphate, sialic acid and its derivatives (e.g. Neu5Ac, Neu5Acα2-6Gal, Neu5Acα2-8Neu5Ac, etc.), an MTP selected from SEQ ID NO: 1 to SEQ ID NO: 7, and a vitamin such as folic acid.

[0290] In some embodiments, Y, when present, is a group represented by formula (II) where q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined above in formula (II), and Y3 is a linear or branched C2-C 40 and / or M comprises or consists of a click chemistry group, a cell type specific targeting ligand, preferably selected from mono- or polysaccharides, hormones, e.g. steroid hormones, peptides such as RGD peptides, muscle targeting peptides (MTP) or angiopep-2, proteins or fragments thereof, membrane receptors or fragments thereof, aptamers, antibodies including heavy chain antibodies and fragments thereof such as Fab, Fab' and VHH, ScFv, spiegelmers, peptide aptamers, vitamins and drugs such as CB1 and / or CB2 ligands.

[0291] In some other embodiments, Y, when present, is a group represented by formula (II) where q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined above in formula (II), and Y3 is a linear or branched C2-C 20 and 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.

[0292] In some other embodiments, Y, when present, is represented by formula (II), where q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined above in formula (II), and Y3 is an optionally substituted, saturated or unsaturated, linear or branched C2-C 40 and M is a spacer selected from the group consisting of a hydrocarbon chain, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof, and M comprises or consists of a click chemistry group, including, for example, an azide or an alkyne.

[0293] In some other embodiments, Y, when present, is a group represented by formula (II) where q is 1, m is 0 or 1, p is 0 or 1, Y1 and Y2 are as defined above in formula (II), and Y3 is a linear or branched C2-C 20and M is a spacer selected from the group consisting of an alkyl chain, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, a polymer of an alkyl diamine, and combinations thereof, said polymer comprising 2-40 monomers), and M comprises or consists of a click chemistry group, e.g., including an azide or an alkyne.

[0294] By way of example only, the AAV of the invention may have formula (Ia-1), (Ib-1), or (Ic-1):

[0295] [ka]

[0296] The capsid may comprise at least one chemically modified cysteine.

[0297] Preferably, the AAV of the present invention has the formula (Ic-1):

[0298] [ka]

[0299] at least one chemically modified cysteine ​​in the capsid.

[0300] In all of the above embodiments, particularly those in which at least one chemically modified cysteine ​​is of formula (I), (I-II), (Ia), (Ib), (Ic), (I-IIc), (Ia-1), (Ib-1), or (Ic-1), the AAV is preferably a recombinant AAV, more preferably a recombinant AAV vector.

[0301] As mentioned above, the AAV may have a "naturally occurring" capsid, or a genetically modified capsid, i.e. a capsid comprising one or several mutations in at least one capsid protein, namely VP1, VP2, and / or VP3. Preferably, said mutations do not introduce any additional cysteine ​​residues, in particular any tetracysteine ​​moieties, in VP1, VP2, and / or VP3.

[0302] 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.

[0303] Alternatively, the AAV is a synthetic serotype.

[0304] In some further embodiments, the AAV of the invention may have at least one additional chemically modified amino acid residue in the capsid that is different from a cysteine ​​residue, e.g., a tyrosine, arginine, or lysine residue. In some embodiments, the amino acid residue has a structure of formula (V) in its side chain:

[0305] [ka]

[0306] (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). In particular, Y′ has a modified amino group of the following formula: -(Y 1' ) m' -(Y 2' ) p' -(Y 3' )q' -(In the formula, Y 1' , Y 2' , Y 3'、 m', p', and q' are Y1, Y2, Y 3、 m, p, and q, each of which has the same definition as Y', n', Y 1'、 Y 2'、 Y 3'、 It is understood that m', p', and q' and M' can be the same or different from those present in at least one chemically modified cysteine ​​as described above. The modifications on the amino group can be introduced as described in International Patent Application Publication No. 2017212019, the contents of which are incorporated herein by reference.

[0307] In some embodiments, the amino acid residue has at its side chain the formula (VI):

[0308] [ka]

[0309] (In the formula, - X" is -N=N- or

[0310] [ka]

[0311] and - Y" has the same definition as Y, n" is 0 or 1, and M" has the same definition as M). In particular, Y" has the formula: -(Y 1" ) m" -(Y 2" ) p" -(Y 3" ) q" -(In the formula, Y 1" , Y 2" , Y 3"、 m", p", and q" are Y1, Y2, Y 3、 m, p, and q, each with the same definition as Y", n", Y1" , Y 2" , Y 3"、 It is understood that m", p", and q" and M" can be the same or different from those present in at least one chemically modified cysteine ​​as described above. In a preferred embodiment, Y" is a group of the formula -(Y 1" ) m" -(Y 2" ) p" -(Y 3" ) q" - (wherein m=1, and Y 1" is selected from arylene and heteroarylene containing from 5 to 14 ring atoms, e.g., from 6 to 10 ring atoms, where the arylene or heteroarylene may be optionally substituted. By way of example, the arylene or heteroarylene group may contain one, two, or three substituents independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl. Modifications on the tyrosine group may be introduced as described in International Patent Application Publication No. 2021005210, the contents of which are incorporated herein by reference.

[0312] In a specific embodiment, the AAV of the present invention has at least one additional chemically modified arginine or lysine residue in the capsid having a modified amino group of formula (V) as defined above, and / or at least one additional chemically modified tyrosine residue in the capsid having a modified tyrosine group of formula (VI) as defined above.

[0313] Chemical modification of the capsid of an AAV may alter one or several biological functionalities and / or properties. Depending on the nature of "M" covalently attached to the surface of the chemically modified AAV, said chemically modified AAV may have one or several altered biological properties, such as the following biological properties, compared to the same but non-chemically modified AAV: - altered tropism, e.g., increased selectivity of the AAV for a particular organ, tissue, or cell (either administered in vivo or by transducing tissues or cells in culture), or shifted selectivity of the AAV from one tissue / organ / cell to another; and / or - altered immune reactivity of the AAV, e.g., decreased immunogenicity and / or decreased affinity for neutralizing antibodies of the AAV, and / or the AAV elicits an altered humoral response when administered in vivo, e.g., failure to generate neutralizing antibodies directed against the AAV - increased infection efficiency of AAV particles, and / or - Increased transduction efficiency of AAV into specific cells, tissues, or organs - Reduced cytotoxicity when transducing cells in culture - Inducing targeted killing of cancer cells - Visualization / monitoring of AAV particles during in vivo administration or in vitro modification of cells with these AAV particles. - Theragnostic applications; e.g. combining therapeutic and diagnostic agents may have:

[0314] In some embodiments, the chemically modified AAV of the present invention may have a higher transduction efficiency, which may be due to increased intracellular trafficking to the nucleus, decreased proteasomal degradation, more efficient intranuclear decapsidation, more rapid vector genome stabilization, and / or decreased interaction with neutralizing antibodies, and / or decreased antibody-mediated clearance of AAV in vivo, compared to non-chemically modified AAV. In some other embodiments, the AAV may have a higher infection efficiency and / or increased selectivity for a given cell, tissue, or organ, either in vivo or in vitro, compared to non-chemically modified AAV.

[0315] In some other embodiments, when AAV is used as a drug, e.g., as a gene vector, such altered properties may result in an improved therapeutic index of the AAV, which may result in a reduction in the dose that needs to be administered to a patient to achieve the desired therapeutic effect and / or reduced toxicity of the AAV.

[0316] In certain embodiments, the chemically modified AAV of the present invention exhibits preferential tropism for an organ or cell selected from the liver, heart, brain, joints, retina, and skeletal muscle. In alternative or additional embodiments, the chemically modified AAV of the present invention exhibits preferential tropism for a cultured cell selected from, but not limited to, hepatocytes, cardiomyocytes, muscle cells, neurons, motor neurons, retinal pigment cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC), or induced pluripotent stem cells (iPS).

[0317] Methods for preparing chemically modified AAV of the present invention The present invention also relates to a method for chemically modifying the capsid of AAV, more precisely for chemically modifying at least one cysteine ​​residue in the capsid of AAV, comprising a step of incubating said AAV with a chemical reagent having a reactive group selected from maleimides, vinylsulfonamides and 3-(carboxy derivatives)acrylamides under conditions conducive to reacting said reactive group with a cysteine ​​residue present in the capsid of AAV to form a covalent bond.

[0318] As used herein, "3-(carboxy derivative)acrylamide" refers to an acrylamide substituted at the 3-position with a carboxy derivative. The carboxy derivative is typically a group of the formula: -C(O)-R3, where R3 is a hydrogen atom, an alkyl group, an aryl group, or a heteroaryl group, which may be optionally substituted.

[0319] In a specific embodiment, the method of the invention is for obtaining a chemically modified AAV comprising at least one chemically modified cysteine ​​residue in the capsid, wherein the chemically modified cysteine ​​residue has formula (I):

[0320] [ka]

[0321] and wherein the AAV is:

[0322] [ka]

[0323] R, R1, R2, R 3、 It is understood that Z and k are as defined herein for formulas (a), (b), (c), and X, Y, n, and M are as defined herein for formula (I), and that when X is formula (a), the chemical reagent is formula (VIIa), when X is formula (b), the chemical reagent is formula (VIIb), and when X is formula (c), the chemical reagent is formula (VIIc). In certain embodiments, Y in formula (I) and formula (VIIa), (VIIb), or (VIIc) is formula (II) as defined herein.

[0324] Typically, AAV particles are incubated with the chemical reagent under conditions suitable to promote the formation of a covalent bond between the thiol of a cysteine ​​residue and the chemical reagent without compromising the structural integrity of the AAV.

[0325] The incubation may be carried out in an aqueous buffer having a pH of from 4 to 12, preferably from 5 to 11, for example from 6 to 10 or from 6 to 9. The pH of the aqueous buffer may be from 5 to 6, from 6 to 8, or from 8 to 10.

[0326] The inventors have shown that a basic pH increases the coupling rate, especially when an acrylamide reagent (i.e., a reagent of formula (VIIc)) is used. If a high rate of chemical coupling of cysteine ​​residues is desired, it may be advantageous to carry out the coupling at a pH of 8.0 to 11, for example 8.5 to 10.5. If a low rate of chemical coupling is desired, the coupling may be carried out at a neutral pH, typically 6.5 to 8.5 or 7 to 8.

[0327] For the maleimide and sulfonamide reagents of formulae (VIIa) and (VIIb), respectively, the incubation is preferably carried out at a basic pH, typically at a pH of from 8.0 to 10.0, such as around pH 9.5.

[0328] The buffer may be selected from any suitable buffer for biological applications, such as TRIS buffer, Tris-buffered saline (TBS), sodium carbonate-sodium bicarbonate buffer, Good's buffer (e.g., AMPSO, HEPES), borate buffer, phosphate buffer, such as PBS or Dulbecco's phosphate-buffered saline (dPBS), and may preferably be dPBS or TBS.

[0329] The incubation may last from a few minutes to a few hours, for example from 1 minute to 6 hours, for example from 3 to 5 hours. Typically, the incubation is terminated when a sufficient yield of coupling is achieved.

[0330] The incubation temperature is typically from 10° C. to 50° C. Preferably, the incubation is carried out at room temperature, ie from 18° C. to 30° C., for example at about 20° C.

[0331] The incubation may be carried out with stirring.

[0332] The molar ratio of chemical reagent to AAV particles ranged from 1:10 to 1:10 8 For example, 1:105 From 1:10 7 It could be up to.

[0333] In some embodiments, the methods of the invention do not contain any prior step in which AAV is incubated with a reducing agent, such as dithiothreitol (DTT), to reduce potential cysteine ​​disulfides present in AAV.

[0334] In some embodiments, the methods of the invention involve treating an AAV with formula (VIIa):

[0335] [ka]

[0336] to form a compound of formula (Ia):

[0337] [ka]

[0338] wherein Y is a spacer, n is 0 or 1, M is a functional moiety, and R is as defined herein for formula (a), providing at least one chemically modified cysteine ​​residue in the capsid of formula (a):

[0339] In some embodiments, the methods of the invention involve treating an AAV with formula (VIIb):

[0340] [ka]

[0341] to form a compound of formula (Ib):

[0342] [ka]

[0343] (In the formula, - Y is a spacer, n is 0 or 1, M is a functional moiety, and - R1 is as defined herein for formula (b). obtaining at least one chemically modified cysteine ​​residue in the capsid.

[0344] In some embodiments, the methods of the invention involve treating an AAV with formula (VIIc):

[0345] [ka]

[0346] to form a compound of formula (Ic):

[0347] [ka]

[0348] (In the formula, - Y is a spacer, n is 0 or 1, M is a functional moiety, and - R2, R3, Z and k are as defined herein for formula (c). obtaining at least one chemically modified cysteine ​​residue in the capsid.

[0349] In certain embodiments, Y in formula (I) and formula (VIIa), (VIIb), or (VIIc) is formula (II) as defined herein.

[0350] In certain embodiments, the AAV prepared by the methods of the invention comprises at least one chemically modified cysteine ​​of formula (I-IIc) as defined herein. In such embodiments, the chemical reagent has the formula (VII-IIc):

[0351] [ka]

[0352] (wherein R2, R3, Z, k, Y1, Y2, Y3, m, p, q, and M are as defined herein).

[0353] The method of the invention may comprise one or several additional steps before or after the incubation step as described above.

[0354] By way of example, a method of the invention may include a step of providing or producing AAV particles, which may be chemically modified.

[0355] The present invention may also include the step of providing or preparing a chemical reagent.

[0356] The chemical reagent can be produced by a synthetic route. For example, the chemical reagent of formula (VIIc) can be prepared from an azide derivative, which is reduced to NH2, for example by hydrogenation with Pd / C as a catalyst, and then, for example, the NH2 group is coupled with a 3-(carboxy derivative) acrylate ester to form a 3-(carboxy derivative) acrylamide. By way of example only, reference can be made to the synthesis of compound 7 described in the Examples section.

[0357] The method of the present invention may also include, after the incubation step, - destroying any unreacted chemical reagents at the end of the incubation step; and / or - removing unreacted reagents, for example by dialysis or tangential flow filtration, and / or - harvesting the chemically modified AAV particles; and / or - purifying the chemically modified AAV particles; and / or - recovering the chemically modified AAV particles; and / or - formulating and / or packaging the chemically modified AAV The method may include one or several additional steps.

[0358] The methods of the present invention may further comprise a step of chemically modifying amino acid residues other than cysteine ​​residues of the AAV capsid.

[0359] By way of example, the additional chemically modified amino acid residue may have an amino group (eg, lysine, arginine) or a tyrosine group (eg, tyrosine) in its side chain.

[0360] In certain embodiments, the methods of the invention involve administering an AAV to a mammalian subject of formula (VIII):

[0361] [ka]

[0362] Such a step may include incubating a compound of formula (I) with a chemical reagent of formula (I) under conditions conducive to promoting reaction of said chemical reagent with an amino group of an amino acid residue, such as a lysine residue or an arginine residue, present in the capsid of AAV to form a covalent bond. Such a step may include incubating a compound of formula (I) with a chemical reagent of formula (I)

[0363] [ka]

[0364] (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. In particular, Y′ is a compound represented by the formula: -(Y 1' ) m' -(Y 2' ) p' -(Y 3' ) q' -(In the formula, Y 1' , Y 2' , Y 3'、m', p', and q' can be the same as Y1, Y2, Y3, m, p, and q, respectively. Y', n', Y 1' , Y 2' , Y 3'、 It is understood that m', p', and q', as well as M', can be the same or different from those present in at least one chemically modified cysteine ​​as defined above.

[0365] Typically, such steps may be carried out in an aqueous buffer such as TRIS buffer, at a pH of 8 to 10, e.g., a pH of about 9.3, and at a temperature of 10° C. to 50° C., e.g., at room temperature. Details regarding the performance of such steps can be found in International Patent Application Publication No. WO 2017 / 212019, the contents of which are incorporated herein by reference.

[0366] This step can be carried out before, simultaneously with, or after the step of chemically modifying at least one cysteine ​​residue in the AAV capsid as described above.

[0367] In another specific embodiment, the method of the invention comprises administering to the patient an AAV comprising the gene of formula (IX) or (X):

[0368] [ka]

[0369] Such a step may include incubating a compound of formula (I) with a chemical reagent of formula (I) under conditions conducive to promoting reaction of said chemical reagent with an amino group of an amino acid residue, such as a lysine residue or an arginine residue, present in the capsid of AAV to form a covalent bond.

[0370] [ka]

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

[0372] [ka]

[0373] and - Y" has the same definition as Y, n" is 0 or 1, and M" has the same definition as M). In particular, Y" can be chemically modified according to the following formula: -(Y 1" ) m" -(Y 2" ) p" -(Y 3" ) q" -(In the formula, Y 1" , Y 2" , Y 3" , m", p", and q" can be Y1, Y2, Y3, m, p, and q, respectively, with the same definitions. Y", n", Y 1" , Y 2" , Y 3" It is understood that m", p", and q" and M" may be the same or different from those present in at least one chemically modified cysteine ​​as described above. A chemical reagent of formula (IX) allows for obtaining a modified residue of formula (VI) where X" is -N=N, while a chemical reagent of formula (X) allows for obtaining a modified residue of formula (VI) where X" is -N=N.

[0374] [ka]

[0375] It is also understood that it is possible to obtain modified residues of the formula:

[0376] In a preferred embodiment, Y" is a group represented by the formula: -(Y 1" ) m" -(Y 2" ) p" -(Y 3" ) q" - (wherein m=1, and Y 1"is selected from arylene and heteroarylene containing from 5 to 14 ring atoms, e.g., from 6 to 10 ring atoms, where the arylene or heteroarylene may be optionally substituted. By way of example, the arylene or heteroarylene group may contain 1, 2, or 3 substituents independently selected from halogen, -OH, NH2, NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.

[0377] The counter anion present in the aryl diazonium salt reagent of formula (IX) can be of any type, preferably TsO - , BF4 - , Cl - , AcO - , PF6 - , TfO - , or CF3CO2 - It could be.

[0378] Typically, such steps may be carried out in an aqueous buffer, such as TRIS buffer, at a pH of 7 to 10, and at a temperature of 10° C. to 50° C., e.g., room temperature. Further details regarding the performance of such steps can be found in International Patent Application Publication No. WO2021005210, the contents of which are incorporated herein by reference.

[0379] This process includes the following steps: - chemically modifying at least one cysteine ​​residue in the capsid of the AAV as described above, and / or - chemically modifying at least one amino group (e.g., from arginine or lysine) in the capsid of the AAV as described above; It can be performed before, simultaneously with, or after.

[0380] In the case where M is a click chemistry group, the chemically modified AAV may undergo an additional step aimed at coupling a functional group Z' by reaction with the M group. Thus, the present invention also relates to a method for grafting a functional moiety Z' onto a cysteine ​​residue in an AAV capsid, said method comprising the steps of: - preparing a chemically modified AAV having at least one chemically modified cysteine ​​comprising a functional M group, preferably by a method as described above, wherein said functional M group is a click chemistry group; and - coupling the functional group Z' with the M group by a click reaction; The present invention relates to a method comprising the steps of:

[0381] Typically, the chemically modified AAV of the present invention has the following formula (XI): Q-(W) r -Z' (XI) (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. It can react with compounds of the formula:

[0382] Q is typically selected from among the same click chemistry groups as M defined above, i.e. Q is selected from among azide (-N3), alkenes, alkynes (in particular cyclooctynes ​​(OCT), aryllesscyclooctynes ​​(ALO), monofluorocyclooctynes ​​(MOFO), difluorocyclooctynes ​​(DIFO), dibenzocyclooctynes ​​(DIBO), dimethoxyazacyclooctynes ​​(DIMAC), biarylazacyclooctynes ​​(BARAC), bicyclononynes (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctynes ​​(BDOC), diphenyl ether (DMTH ... The compound may comprise or consist of a strained alkyne such as dibenzocyclooctyne (DIFBO), oxa-dibenzocyclooctyne (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.

[0383] 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 hydroximoyl chloride or chlorooxime, and Click chemistry groups include, but are not limited to, M=alkene or alkyne), 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=aryl boronic acid), or His tag (M=oligohistidine and Q=nickel complex or nickel ligand). In the above mentioned list of click chemistry groups involved in click chemistry reactions, it is understood that M and Q can be interchanged. All the above mentioned chemical reactions result in covalent linkages.

[0384] In certain embodiments, M is an azide (-N3) and Q is an alkyne (e.g., a -C≡CH group, or a strained alkyne such as those mentioned above), or Q is an azide (-N3) and M is an alkyne (e.g., a -C≡CH group, or a strained alkyne such as those mentioned above). 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.

[0385] W is typically a spacer having the same definition as Y in formula (I).

[0386] 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' may be a targeting agent such as a cell type specific targeting ligand, 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, ScFvs, spiegelmers, peptide aptamers, oligonucleotides, vitamins, and small chemical molecules such as drugs, e.g., CB1 and / or CB2 ligands.

[0387] 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 galactose, 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.

[0388] The conditions performed for click chemistry reactions are well known to those of skill in the art. Click reactions can be "bioorthogonal" or "biocompatible", meaning that the reagents involved in the click reaction can react selectively and rapidly with each other in the presence of multiple biological entities. In some embodiments, the click reaction can be performed in a medium containing living cells without interfering with cellular processes. By way of example, biocompatible or bioorthogonal click reactions include metal-free click reactions (i.e., do not require a metal catalyst). An example of a metal-free click reaction is depicted below:

[0389] [ka]

[0390] Other metal-free click reactions of interest are, for example, iminosydnone or sydnone derivative-strained alkyne cycloadditions, as described in PCT / EP2015 / 060805 and PCT / EP2015 / 063750, the disclosures of which are incorporated herein by reference. For a review of bioorthogonal chemistry, including click chemistry, see Sletten and Bertozzi, Angew. Chem. Int. Ed. Engl. 2009, 48(38):6974-6998, the disclosures of which are incorporated herein by reference.

[0391] 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.

[0392] 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, or vice versa, as described above.

[0393] The present invention also relates to a chemically modified AAV obtainable or obtainable by the method of the invention as described above.

[0394] In a further aspect, the present invention also relates to a method for modifying one or several biological properties of AAV, more precisely of a recombinant AAV intended to be used as a gene vector in gene therapy. Indeed, depending on the nature of the "M" part, a method for chemically modifying the capsid of AAV, more precisely for chemically modifying at least one cysteine ​​residue in the capsid of AAV as described above, consists in: - modifying the tropism, e.g., increasing the selectivity of the AAV for a particular organ, tissue, or cell (either administered in vivo or transducing a tissue or cell in culture), or shifting the selectivity of the AAV from one tissue / organ / cell to another; and / or - altering the immune reactivity of the AAV, e.g. reducing the immunogenicity of the AAV and / or reducing the affinity for neutralizing antibodies, and / or eliciting an altered humoral response when administered in vivo, e.g. not generating neutralizing antibodies directed against the AAV; and / or - increasing the infection efficiency of AAV particles; and / or - Reduce off-target effects, i.e. transducing cells that are not necessary to provide the benefit of the drug and may even be harmful - Increase the transduction efficiency of AAV into specific cells, tissues, or organs - Reduces cytotoxicity when transducing cells in culture - Induce targeted killing of cancer cells - Allows visualization / monitoring of AAV particles during in vivo administration or in vitro modification of cells with these AAV particles - Combining therapeutic and diagnostic agents in AAV This may make it possible.

[0395] Uses of the AAVs of the Invention The chemically modified AAV of the present invention may be used as a research tool or as a pharmaceutical agent, e.g., as a vector for delivery of therapeutic nucleic acids such as DNA or RNA, and / or as a diagnostic tool, e.g., as an imaging agent, or a combination of both, including theragnostic uses.

[0396] 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 thus a recombinant AAV.

[0397] The recombinant AAV can be administered to cells in vivo, ex vivo, or in vitro. The cells can be from any mammal, including humans, primates, cows, mice, sheep, goats, pigs, rats, and the like. The cells can be of any type, including hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigment cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPS).

[0398] The recombinant AAV of the present invention can be used to deliver a therapeutic nucleic acid of interest in a subject. Thus, the present invention relates to a method for delivering a therapeutic nucleic acid of interest in a subject in need, comprising administering a chemically modified AAV of the present invention to a subject in need. The recombinant AAV of the present invention can be delivered to a subject by any suitable route. Suitable administration routes include, but are not limited to, inhalation, topical, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal), conjunctival (e.g., intraretinal, subretinal), mucosal (e.g., buccal, nasal), intra-articular, intravitreal, intracranial, intravascular (e.g., intravenous), intraventricular, intracisternal, intraperitoneal, and intralymphatic routes. Typically, the administration route is selected depending on the tissue / organ to be targeted, i.e., depending on the tissue / organ for which transduction is sought.

[0399] The dose of AAV administered to a subject is typically determined by a skilled artisan, taking into account the specific characteristics of the subject, the therapeutic effect desired, and the tissue / organ to be targeted. A single dose or several doses of AAV may be required to achieve the desired therapeutic effect. The AAV of the present invention is typically administered in the form of a pharmaceutical composition, i.e., in a mixture with one or several pharmaceutical excipients.

[0400] The condition that can be treated by administering AAV can be of any type, including genetic disorders as well as acquired disorders.Genetic disorders of interest include inherited muscle disorders such as Duchenne muscular dystrophy, leukodystrophy, spinal muscular atrophy (SMA), hemophilia, sickle cell disease, and inherited retinal dystrophy.Chemically modified AAV can also be used to treat disorders such as cancer, arthritis, osteoarthritis, congenital and acquired heart disease, Parkinson's disease, Alzheimer's disease, and infectious diseases such as Hepatitis C.

[0401] Another object of the present invention is a pharmaceutical composition comprising the chemically modified AAV of the present invention and at least one pharma- ceutically acceptable excipient, which may be selected from well-known excipients such as carriers, preservatives, antioxidants, surfactants, buffers, stabilizers, and the like.

[0402] The present invention further relates to an in vivo or ex vivo method for delivering a nucleic acid of interest in a cell, comprising contacting the cell with the chemically modified AAV of the present invention. The cell may be derived from a patient. After transduction, the cell may be transplanted into a patient in need thereof. The cell may be, for example, a hematopoietic stem cell. The nucleic acid of interest may be of any type and is selected depending on the effect desired.

[0403] As an example, the AAV may contain an exogenous gene expression cassette. The cassette may include a promoter, a gene of interest, and a terminator. As another example, the AAV of the present invention may contain a DNA template for homologous recombination in a cell. Such recombinant AAV may be used in combination with a gene editing tool to promote homologous recombination in a targeted cell. The gene editing tool may be of any type, including but not limited to CRISPR / Cas9, zinc finger nucleases, meganucleases, as well as RNA and DNA encoding the protein.

[0404] The present invention also relates to a host cell transfected with the chemically modified AAV of the present invention, said host cell may be of any type.

[0405] 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).

[0406] Further aspects and advantages of the present invention are disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application. EXAMPLES

[0407] Example 1 Synthesis of the Ligands According to the Invention 1. Synthesis of the ligand according to the invention: The following formula:

[0408] [ka]

[0409] The GalNAc-acrylamide ligand (L) was prepared from 3-benzoylacrylic acid according to the following reaction steps:

[0410] [ka]

[0411] 3-Benzoylacrylic acid (500 mg, 2.8 mmol) was dissolved in anhydrous DCM, and a mixture of pentafluorophenyl trifluoroacetate (530 μL, 3.1 mmol) and DIPEA (990 μL, 5.7 mmol) was added with stirring. The reaction mixture was stirred at room temperature for 1 h 30 min. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (50% DCM / petroleum ether) to give compound A (793 mg, 2.31 mmol) as a yellow solid. Yield=66.4%. 1 H NMR (300 MHz, CDCl3, 298.15 K): δ H 8.17 (d, 1H, 3 J = 15.5Hz, H 3 ), 8.04 (m, 2H, H arom ), 7.67 (m, 1H, H arom ), 7.56 (m, 2H, H arom ), 7.12 (d, 1H, 3 J = 15.5 Hz, H 2 ) 13 C NMR (100.62 MHz, CDCl3, 298.15 K): δ C 188.54 (1C), 161.58 (1C), 140.34 (1C), 136.24 (1C), 134.53 (1C), 129.24 (2C), 129.12 (2C), 128.71 (1C) HRMS (ESI + ): m / z C 16 H9F5O3[M+H] + Calculated value: 343.0389 Measured value: 343.0394

[0412] Preparation of Compound 1

[0413] [ka]

[0414] N-Acetylgalactosamine (2 g, 9.04 mmol) was dissolved in acetic anhydride and pyridine (1 / 1, 2.5 mL / mmol) along with 4-dimethylaminopyridine (110 mg, 0.904 mmol). The mixture was stirred at room temperature, concentrated in vacuo, dissolved in dichloromethane (100 mL), washed with an aqueous solution of HCl (1 M), a saturated solution of aqueous NaHCO3, water, and brine. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo to give the product as a colorless amorphous solid (3.51 g, quantitative yield). 1H NMR (400 MHz, MeOD, 298.15 K): δ H 6.17 (d, 1H, 3 J = 3.6 Hz, H 1 ), 5.48 (dd, 1H, 3 J = 3.1Hz and 1.3Hz, H 4 ), 5.21 (dd, 1H, 3 J = 11.7Hz, and 3.2Hz, H 3 ), 4.57 (ddd, 1H, 3 J = 11.7Hz, 9.1Hz and 3.6Hz, H 2 ), 4.38 (dd, 1H, 3 J = 1.1Hz and 6.7Hz, H 5 ), 4.13 (dd, 1H, 3 J = 11.2 Hz and 6.7Hz, H 6a ), 4.05 (dd, 1H 3 J = 11.2Hz and 6.6Hz, H 6b ), 2.16 (s, 3H, CH3), 2.15 (s, 3H, CH3), 2.01 (s, 3H, CH3), 1.97 (s, 3H, CH3), 1.92 (s, 3H, CH3) HRMS (ESI + ): m / z C 16 H 23 NO 10 Na [M+Na] + Calculated value: 412.1216 Measured value: 412.1220

[0415] Preparation of compound 2

[0416] [ka]

[0417] TMSOTf (0.47 mL, 2.62 mmol) was added to a stirred solution of compound 1 (300 mg, 0.77 mmol) in anhydrous DCM (7.7 mL) at room temperature under N2. The reaction mixture was stirred at 45 °C overnight and then quenched by the addition of NEt3 (0.22 mL, 1.54 mmol) at 0 °C. The mixture was diluted with CH2Cl2 (100 mL), washed with a saturated solution of aqueous NaHCO3, water, and brine, dried over MgSO4, filtered, and concentrated in vacuo. The resulting crude oxazoline (brown oil) (231 mg, 91% yield) was used without further purification. 1 H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 6.00 (d, 1H, 3 J = 6.7 Hz, H 1 ), 5.46 (t, 1H, 3 J= 3Hz, H 4 ), 4.92 (dd, 1H, 3 J = 7.4 Hz and 3.3 Hz, H 3 ), 4.24 (dd, 1H, 3 J = 6.8 Hz and 2.9 Hz, H 5 ), 4.20 (dd, 1H, 3 J = 11.1 Hz and 5.8 Hz, H 6a ), 4.11 (dd, 1H, 3 J = 11.1 Hz and 6.9 Hz, H 6b ), 4.00 (td, 1H, 3 J = 7.3 Hz and 1.3 Hz, H 2 ), 2.12 (s, 3H, CH3), 2.06 (s, 9H, 3x CH3) HRMS (ESI + ): m / z C 14 H 20 NO8[M+H] + Calculated value: 330.1188 Measured value: 330.1189

[0418] Preparation of compound 3

[0419] [ka]

[0420] 2-(2-(2-chloroethoxy)ethoxy)ethanol (1.36 mL, 9.38 mmol) was added to a stirred solution of compound 2 (2.81 g, 8.53 mmol) in anhydrous DCM (85 mL) under N2 at room temperature, followed by TMSOTf (0.77 mL, 4.27 mmol) at 0 °C. The reaction mixture was stirred at room temperature overnight and then quenched by the addition of TEA (0.75 mL, 5.55 mmol) at 0 °C. The mixture was diluted in DCM (100 mL), washed with water, brine, dried over MgSO4, filtered and concentrated under vacuum to give the β-anomer (4.23 g, quantitative yield) as a brown liquid oil. 1 H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 6.27 (d, 1H, 3 J= 9.5 Hz, NH), 5.29 (dd, 1H, 3 J = 3.4 Hz and 0,6 Hz H 4 ), 4.98 (dd, 1H, J= 11,2 Hz and 3,4 Hz, H 3 ), 4.78 (d, 1H, J= 8,6 Hz, H 1 ), 4.25 (m, 1H, H 2 ), 4,13 (t , 2H, J= 6,5 Hz, 2x H 6 ), 3.92-3.58 (m, 13H, 5xCH2, H 6 and H 5 ), 2.15 (s, 3H, CH3), 2.03 (s, 3H, CH3), 1.97-1.98 (s, 6H, 2×CH3) 13 C NMR (100.62 MHz, CDCl3, 298.15 K): δ C 170.77 (1C), 170.70 (1C), 170.64 (1C), 170.55 (1C), 102.53 ( 1C, C 1), 71.99 (1C), 71.42 (1C, C 3 ), 71.20 (1C, C 5 ), 70.76 (1C), 70.68 (1C), 70.40 (1C), 68.64 (1C), 66.79 (1C, C 4 ), 61.72 (1C, C 6 ), 50.62 (1C, C 2 ), 43.06 (1C), 23.43 (1C), 20.90 (1C), 20.85 (1C), 20.80 (1C)

[0421] Preparation of compound 4

[0422] [ka]

[0423] Compound 3 (2.083 g, 4.18 mmol) was dissolved in dry DMF (41.8 mL) and sodium azide (1.36 g, 20.9 mmol) was added along with sodium iodide (63 mg, 0.42 mmol) at room temperature under N2 atmosphere. The mixture was stirred at 70° C. for 16 h, then concentrated in vacuo, dissolved in dichloromethane (150 mL), washed with a saturated solution of aqueous NaHCO3, water, and brine. The organic layer was dried over MgSO4, filtered, concentrated in vacuo, and purified by column chromatography to give the corresponding azide (1.27 mg, 60% yield) as a brown liquid oil. 1 H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 6.06 (d, 1H, 3 J= 6.14 Hz, NH), 5.31 (dd, 1H, 3 J = 3.3 Hz and 0.8 Hz, H 4 ), 5.07 (dd, 1H, 3 J = 11.2 Hz and 3.4 Hz, H 3 ), 4.77 (d, 1H, 3 J = 8.6 Hz, H 1), 4.18-4.03 (m, 1H, H 2 ), 4.08 (m, 2H, H 6 ), 3.93-3.78 (m, 3H, CH2,H 5 ), 3.76-3.59 (m, 8H, 4 x CH2, ), 3.45 (t, 2H, 3 J = 4.9 Hz, H 13 ), 2.08 (s, 3H, CH3), 1.98 (s, 3H, CH3), 1.93-1.91 (s ,6H, 2 x CH3). 13 C NMR (100.62 MHz, CDCl3, 298.15 K): δ C 170.72 (1C), 170.57 (1C), 170.55 (1C), 170.48 (1C), 102.33 (1C, C 1 ), 71.72 (1C), 71.01 (1C, C 3 ), 70.85 (1C, C 5 ), 70.79 (1C), 70.54 (1C), 69.93 (1C), 68.71 (1C), 66.88 (1C, C 4 ), 61.71 (1C, C 6 ), ,50.95 (1C, C 2 ), 50.55 (1C, C 13 ), 23.33 (1C), 20.86 (1C), 20.80 (2C) HRMS (ESI + ): m / z C 20 H 32 N4O 11 Na [M+Na] + Calculated value 527.1965 Measured value 527.1984

[0424] Modulation of Compound 5

[0425]

change

[0426] To a solution of compound 4 (980 mg, 1.94 mmol) in methanol was added sodium methoxide (0.1 equiv.) in methanol, stirred at room temperature for 6 h, neutralized with Dowex ion exchange (H+) resin, filtered, and concentrated in vacuo to give the corresponding azide 5. 1 H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 4.44 (d, 1H, 3 J = 8.4 Hz, H 1 ), 4.00-3.86 (m, 3H, CH2 and H 2 ), 3.83 (dd, 1H, J= 3.2 Hz and 0.4 , H 4 ), 3.80-3.62 (m, 10H, 4xCH2 and H 6 ), 3.58 ( dd, 1H, 3 J = 10.7 Hz and 3.3 Hz, H 3 ), 3.47 ( td, 1H, 3 J = 5.3 Hz and 0.8 Hz, H 5 ), 3.39 (t, 2H, 3 J = 5.0 Hz, H 13 ), 1.99 (s, 3H, CH3) 13 C NMR (100.62 MHz, CDCl3, 298.15 K): δ C 174.17 (1C), 103.09 (1C, C 1 ), 76.75 (1C, C 5 ), 73.58 (1C, C 3 ), 71.66 (1C), 71.59 (1C), 71.55 (1C), 71.07 (1C), 69.78 (1C), 69.71 (1C, C 4 ), 62.52 (1C, C 6 ), 54.27 (1C, C 2 ), 51.77 (1C, C 13 ), 19.01 (1C) HRMS (ESI + ): m / z C 14 H26 N4O8Na [M+Na] + Calculated value: 401.1648 Measured value: 401.1659.

[0427] Preparation of compound 6

[0428] [ka]

[0429] To the azide 5 (636 mg, 1.68 mmol) in methanol (17 mL) was added PTSA (289 mg, 1.68 mmol) followed by 10% Pd-C (10% w). The resulting suspension was stirred under H2 atmosphere for 18 h. Pd / C was removed by filtration through Celite® and the filtrate was evaporated under reduced pressure to give the ammonium salt as confirmed by 1H NMR. The crude product of the reaction was dissolved in a mixture 1:1 HO / MeOH (20 mL) followed by the addition of Amberlite IRN78 basic resin. After stirring at 20° C. for 1 h, the reaction mixture was filtered and evaporated under reduced pressure to give the corresponding amine 6 (550 mg, 93% yield) as a white solid which was used in the next step without further purification. 1 H NMR (300.13 MHz, MeOD, 298.15 K): δ H 4.43 (d, 1H, 3 J = 8.4 Hz, H 1 ), 3.95 (m, 2H, CH2), 3.92 (m, 1H, H 2 ), 3.82 (m, 1H, H 4 ), 3.79 - 3.45 (m, 12H, 4xCH2 and H 3 and H 5 and 2xH 6 ), 2.79 (t, 2H, 3 J = 5.5Hz, H 13 ), 1.99 (s, 3H, CH3) 13 C NMR (100.62 MHz, MeOD, 298.15 K): δC 174.13 (1C, C), 103.57 (1C, C 1 ), 76.77 (1C, C 5 ), 73.48 (1C, C 3 ), 73.45 (1C, CH2), 71.61 (1C, CH2), 71.52 (1C, CH2), 69.79 (1C, C 6 ), 69.69 (1C, C 4 ), 62.54 (1C, CH2), 56.28 (1C, CH2), 54.48 (1C, C 2 ), 42.06 (1C, C 13 ), 22.88 (1C, CH3) HRMS (ESI + ): m / z C 14 H 24 Calculated value of N2O8Na [M+Na]: 375.1743 Found value: 375.1743

[0430] Final step to obtain the ligand of the invention: Compound GalNAc-acrylamide (L)

[0431] [ka]

[0432] Compound 6 (100 mg, 0.28 mmol) was dissolved in anhydrous DMF, and a mixture of compound A (86 mg, 0.25 mmol) and DIPEA (90 μL, 0.50 mmol) in anhydrous DCM was added with stirring. The reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (DCM / MeOH 85 / 15) to give GalNAc-acrylamide (L) (47 mg, 92 μmol) as an oil. Yield=33%. 1 H NMR (300.13 MHz, MeOD, 298.15 K): δ H 8.03 ( m, 2H, H arom ), 7.88 (d, 1H, 3J = 15.4Hz, H 14 ), 7.66 ( m, 1H, H arom ), 7.66 (m, 2H, H arom ),7.06 ( d, 1H, 3 J = 15.4 Hz, H 15 ), 4.44 (d, 1H, 3 J = 8.4 Hz, H 1 ), 4.00-3.90 ( m, 3H, H 2及び CH2), 3.84 ( m, 1H, H 4 ), 3.81-3.56 (m, 11H, H 3 Andび2xH 6 and び4xCH2), 3.55-3.46 (m, 3H and びH 5 And びH 12 ), 1,98 (s, 3H, CH3) 13 C NMR (100.62 MHz, MeOD, 298.15 K): δ C 191.6 (1C, C 16 ), 174.2 (1C, C 17 ),166.8 (1C, C 13 ), 138.30 (1C, C arom ), 136.6 (1C, C 15 ), 134.9 ( 1C, C arom ), 134.1 ( 1C, C 14 ), 130.0 (1C, 2xC arom ), 129.8 (1C, 2xC arom ), 103.1 (1C, C 1 ), 76.75 (1C, C 5 ), 73.44 (1C, C 3 ), 71.6-71.4 ( 3C, 2xCH2 and びC 6 ), 70.4 (1C, CH2), 69.8 (1C, CH2), 69.71 (1C, C 4 ), 62.6 (1C, CH2), 54.3 (1C, C 2 ), 40,8 (1C, CH2), 23,1 ( 1C, CH3) HRMS (ESI + ): m / z C 24 H 34 N2O 10 Na [M+Na] + Calculated value 533,2112 Measured value 533,2111

[0433] 2. Synthesis of Compound C (Comparative)

[0434] [ka]

[0435] The synthesis of compound C was prepared as described in Chemical Science, 2020, 11, pp. 1122-1131.

[0436] Example 2 Preparation of chemically modified AAV - AAV2 and AAV9 production and purification AAV vectors were generated from two plasmids: (i) pHelper, PDP2-KANA encoding AAV Rep2-Cap2 and adenovirus helper genes (E2A, VA RNA, and E4) for AAV2 vectors, or PDP9-KANA encoding AAV Rep2-Cap9 and adenovirus helper genes (E2A, VA RNA, and E4) for AAV9 vectors, and (ii) pVector ss-CAG-eGFP containing the ITRs. All vectors were generated by transient transfection of HEK293 cells with the calcium phosphate-HeBS method. AAV2 transfected cells were harvested 48 hours after transfection and treated with Triton-1% and benzonase (25 U / mL) for 1 hour at 37°C. AAV9 transfected cells were harvested 96 hours after transfection and only the supernatant was PEG precipitated at 5+ / -3°C overnight. The precipitated supernatant is then centrifuged. The supernatant is discarded and the PEG-pellet is resuspended in TBS prior to benzonase digestion. The vector was purified by two-fold cesium chloride (CsCl) gradient ultracentrifugation. The virus suspension was then spun in dPBS (CaCl) with gentle agitation in a Slide-a-Lyzer cassette (Pierce). ++ and Mg ++ The cells were subjected to four consecutive rounds of dialysis against 100 mM NaCl (containing

[0437] - Coupling and purification The general procedure is shown in Figure 1. 12 vg, 2.49 nmol, 100 μL) was added to a solution of dPBS buffer (100 μL or 900 μL) containing GalNAc-acrylamide ligand (L) or comparison compound (C) at different molar ratios (3E5 or 3E6) and incubated at room temperature, pH 7.4 for 4 hours. The vector-containing solution was then dialyzed against dPBS + 0.001% Pluronic to remove free molecules not bound to the AAV capsid. For HPLC / MS analysis, the solution (200 μL) was directly lyophilized without dialysis.

[0438] Example 3 Characterization of chemically modified AAV (inventive and comparative) - Quantification of AAV vector genomes 3 μL of AAV was treated with 20 units of DNase I (Roche #04716728001) for 45 minutes at 37° C. to remove residual DNA in the vector sample. After DNase I treatment, 20 μL of Proteinase K 20 mg / mL (MACHEREY-NAGEL #740506) was added and incubated for 20 minutes at 70° C. DNA was then extracted from the purified AAV vector using extraction columns (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 20 μL final volume PCR containing primers and probes targeting the ITR2 sequence, 2 PCR Master Mix (TaKaRa), and 5 μL of template DNA (plasmid standard or sample DNA). qPCR was performed with an initial denaturation step at 95°C for 20 s, followed by 45 cycles of denaturation at 95°C for 1 s and annealing / extension at 56°C for 20 s. Plasmid standards were diluted in seven serial dilutions (10 8 ~10 2 10 copies of the plasmid.

[0439] - Western Blot and Silver Staining All vectors were denatured using Laemmli sample buffer at 100°C for 5 min and separated by SDS-PAGE 10% Tris-glycine polyacrylic gels (Life Technologies). Precision plus Protein All Blue Standards (BioRad) were used as molecular weight size markers. After electrophoresis, gels were either silver stained (PlusOne Silver Staining Kit, Protein, GE Healthcare) or transferred onto nitrocellulose membranes for Western blot analysis. The proteins were transferred to nitrocellulose membranes using transfer buffer (25 mM tris / 192 mM glycine / 0.1 (w / v) SDS / 20% MeOH) in a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) at 150 mA for 1 h, and then the membranes were saturated with 5% semi-skimmed milk in PBS-Tween (0.1%) or 1% gelatin, 0.1% Igepal in PBS-Tween (0.01%) for 2 h at room temperature. After saturation, the membranes were probed with the corresponding antibodies or lectins overnight at 4° C. To remove unbound reagents, three washes were performed between each step with PBS-Tween (0.1%) for 15 min at room temperature. Bands were visualized by chemiluminescence using alkaline phosphatase (AP) or horseradish peroxidase (HRP)-conjugated secondary antibodies and captured on X-ray film.

[0440] - Immuno dot blot analysis Before assembling the dot blot manifold (Bio-Rad), AAV vectors were plated onto nitrocellulose paper that had been briefly soaked in PBS for 10 min. 10 The vectors were loaded at a dose of 1000 ng / ml. The nitrocellulose membranes containing the vectors were processed as for Western blotting.

[0441] - Enzymatic digestion of AAV vectors and LC-MS / MS peptide analysis AAV sample (1 × 10 13A total of 100 μL of 100 μL of 100 mM ammonium bicarbonate, pH 8, was prepared using the ProteinWorks™ eXpress kit (Waters Corporation) as previously described [Blanchard et al., Journal of Lipid Research, 2020]. Samples (40 μL) were incubated in digestion buffer (ammonium bicarbonate 50 mM, pH 8, 100 μL) and RapidGest detergent solution (7 mg / mL, 10 μL) at 80° C. for 10 min, reduced with dithiothreitol (70 mM, 20 μL) at 60° C. for 20 min, alkylated with iodoacetamide (142 mM, 30 μL) in the dark at room temperature for 30 min, and digested with trypsin (HCl 1 mM, 7 mg / mL in 30 μL) at 37° C. overnight (approximately 16 h). Enzymatic digestion was stopped with 20% trifluoroacetic acid (TFA; 5 μL). After 15 min at 45° C., the precipitate was removed by centrifugation (15 min; 10° C.; 10,000 rpm) and the supernatant was clarified on a 30 mg Oasis HLB cartridge (Waters Corporation), which was conditioned (100% methanol; 1 mL), equilibrated (100% water; 1 mL), loaded (sample; approx. 200 μL), washed (5% methanol; 1 mL), and eluted (80% methanol; 500 μL). The eluate was dried under nitrogen (45° C.), reconstituted in 5% acetonitrile containing 0.1% formic acid (100 μL), and injected (10 μL) into the LC-HRMS system. LC-HRMS analysis was performed on a Synapt G2 HRMS Q-TOF mass spectrometer (Waters Corporation) equipped with an ESI interface and an Acquity H-Class UPLC device operated in positive mode. Samples (10 μL) were injected onto an Acquity CSH C18 peptide reversed-phase column (1.7 μm; 2.1 × 100 mm; 130 Å) held at 60° C. Digested peptides were then eluted with a linear gradient of mobile phase A (5% acetonitrile) to mobile phase B (100% acetonitrile) over 20 min, each containing 0.1% formic acid at a flow rate of 250 μL / min.Mobile phase B was held constant at 1% for 1 min, then linearly increased from 1% to 80% for 15 min, held constant for 1 min, returned to initial conditions over 1 min, and held constant for 1 min before the next injection. Full HRMS mode was applied to the peptides (scan range 100-4000 m / z) at a mass resolution of 25,000 (half-width). Ionization settings were as follows: capillary voltage, +3 kV; cone voltage, 30 V; desolvation gas (N2) flow rate, 1000 L / h; desolvation gas / source temperature, 450 / 120 °C. In the lockspray channel, leucine enkephalin solution (2 μg / mL, 50% acetonitrile) was injected at a constant flow rate of 10 μL / min to allow correction of the measured m / z throughout the batch (theoretical m / z 556.2771 in positive mode). Data acquisition and processing were achieved using MassLynx® software (version 4.1, Waters Corporation). The MS profiles of the peaks in the chromatograms allowed the peptides to be identified and their conversion to conjugated compounds to be confirmed. Tandem mass spectrometry (MS / MS) fragmentation was then performed on the major ion peaks (singly, doubly or triply charged) to identify the location of the conjugation using a collision energy gradient from 15 eV to 40 eV.

[0442] - results The results are shown in Figures 2A-3C. The integrity of the capsid after the coupling reaction with the ligand (L) of the present invention was evaluated by dot blot analysis using immunostaining with the A20 antibody. The A20 antibody recognizes the assembled AAV2 capsid. The results are shown in Figure 2A. The positive dots with the A20 antibody indicate that the AAV2 remained intact after the coupling procedure with the ligand (L).

[0443] Further evidence of capsid integrity is provided by Western blot analysis: immunostaining performed with polyclonal antibodies specific for capsid proteins shows that chemical coupling with the ligands of the invention did not compromise the integrity of the AAV2 capsid subunit (Figure 2B).

[0444] The specific coupling of the ligands of the invention with cysteine ​​residues in the capsid was demonstrated by LC-MS / MS analysis. Some results of LC-MS / MS are shown in Figure 3A, Figure 3B, Figure 3C. To confirm the specific conjugation of cysteine ​​residues in AAV2 and AAV9, a "bottom-up" strategy was used. Such a mass spectrometry-based protocol involves the analysis of a peptide mixture resulting from the enzymatic proteolysis of the protein. The peptides are then separated by liquid chromatography and detected by mass spectrometry according to their precise (±10 ppm) mass-to-charge ratio (m / z), which takes into account the expected mass shifts caused by the chemical modifications. The peptides were then fragmented using the MS / MS mode to confirm the location of the modifications. The fragmentation pattern allows to identify the location of the modifications in the peptide sequence.

[0445] Computational selection of proteotypic peptides yielded five or four candidates with cysteine ​​residues for AAV2 or AAV9, respectively. The most specific and detectable of them were selected to optimize the sensitivity and specificity of the assay. The following two peptide candidates were of particular interest because they are common to both AAV2 and AAV9 and are carried by VP1, VP2, and VP3:

[0446] [ka]

[0447] (Cysteine ​​is located at position 289 (numbering refers to amino acid position in AAV2 VP1)) and

[0448] [ka]

[0449] (The cysteine ​​is located at position 394 (numbering refers to the amino acid position in AAV2 VP1). These peptides were successfully detected by mass spectrometry at their exact m / z in the control sample (unmodified AAV) but not in the chemically modified sample. In contrast, by considering the expected mass shift (+510 Da) at these specific m / z, the two peptides were detected in the chemically modified sample but not in the control sample, suggesting a successful chemical reaction, i.e., specific chemical coupling at the cysteines at positions 289 and 394.

[0450] To ensure that the modification occurred only at the cysteine ​​residues, MS / MS fragmentation was then performed on the doubly charged precursor ions. The fragmentation patterns clearly indicated that the chemical modification occurred at the cysteine ​​residues, since a specific mass shift was observed only on the peptide fragments bearing cysteine ​​compared to the unmodified peptide.

[0451] To confirm the specificity of the chemistry, two additional peptides lacking cysteine ​​residues were also examined using the same strategy: DVYLQGPIWAK (SEQ ID NO: 10) and HPPPQILIK (SEQ ID NO: 11). The two peptides were clearly detected and sequenced without any modification in both modified and unmodified samples, providing evidence for the specificity of the chemistry to cysteine ​​residues.

[0452] Briefly, the inventors have demonstrated that the benzoyl acrylamides of the present invention allow for specific and efficient coupling at cysteine ​​residues present in the capsids of AAV2 and AAV9.

[0453] Example 3 Infectivity assay for ligand (L) - Protocol The infectivity of the AAV2-GalNAc-acrylamide (AAV2 chemically modified with ligand L) prepared in Example 2 and the infectivity of the starting AAV2 were evaluated as follows: HeLa cells were cultured at 10 x 10 in 2 mL DMEM growth medium in a 6-well culture plate. 6 The cells were seeded at a density of 10 ... The transduction unit (TU) titer was calculated using the following formula: TU / mL=(4040×NGFP×dilution×1000) / V where NGFP is the average number of GFP-positive cells per well and V is the volume (μL) of vector used to infect the cells.

[0454] - Result: The infectivity of AAV2-GalNAc-acrylamide (3E6 equivalents) was assessed by measuring the ratio of vector genome (vg) to GFP-forming units (vg / GFU) in HeLa cells. This ratio is classically used as a quality control measure to evaluate the in vitro infectivity of rAAV vectors (the higher the ratio, the less infectious the vector). Non-chemically modified AAV2 was used as a control.

[0455] [Table 1]

[0456] The Vg / GFU ratio for the AAV2-GalNAc-acrylamide vector was in the same order of magnitude as that of non-chemically modified AAV2. Thus, the results demonstrated that the AAV2-GalNAc-acrylamide vector efficiently transduced HeLa cells and that this vector remained infectious after the bioconjugation process.

[0457] Example 4 Synthesis of other ligands according to the invention - Synthesis of GalNAc-sulfonamide (L1) - Preparation of intermediate compound 9

[0458] [ka]

[0459] compound 7 To a solution of 4-(methylamino)benzoic acid (1.00 g, 6.616 mmol) in MeOH (20 mL) was added pTSA (1.51 g, 7.939 mmol). The resulting mixture was stirred under reflux for 24 h. The mixture was evaporated under reduced pressure. The residue was diluted with AcOEt and washed with a saturated solution of NaHCO3. The crude product was used in the next step without further purification.

[0460] To a solution of the crude product in dry DCM (60 mL) containing TEA (4.51 mL, 33.080 mmol) was added dropwise 2-chloroethanesulfonyl chloride (1.04 mL, 9.924 mmol) at 0° C. After stirring for 1 h, water was added to extract the compound. The crude product of the reaction was used in the next step without further purification (1.27 g, 4.962 mmol, 75% for 2 steps). 1H NMR (300.13 MHz, CDCl3): δ H 8.02 (d, 2H, J = 8.8 Hz, Ar), 7.39 (d, 2H, J = 8.8 Hz, Ar), 6.42 (dd, 1H, J = 16.5 Hz, J = 9.8 Hz, vinyl), 6.21 (d, 1H, J = 16.5 Hz, vinyl), 6.03 (d, 1H, J = 9.8 Hz, vinyl), 3.91 (s, 3H, COOMe), 3.27 (s, 3H, NHMe). 13C NMR (75.48 MHz, CDCl3): δ C 166.2 (C, COOMe), 142.2 (C, Ar), 132.0 (CH, vinyl), 130.5 (2 x CH, Ar), 129.0 (CH2, vinyl), 128.4 (C, Ar), 125.2 (2 x CH, Ar), 52.2 (CH3), 37.5 (CH3). HRMS (ESI+): m / z C 11 H 13 NO4SNa [M+Na]+ calculated value 278.0463 Found value 278.0458.

[0461] compound 9 To a solution of the crude product (500 mg, 1.958 mmol) in 1,4-dioxane / HO, 1:1 (20 mL) was added LiOH (56 mg, 2.350 mmol). The resulting mixture was stirred at room temperature for 3 h. The mixture was neutralized by adding acidic resin. After evaporation, the carboxylic acid 8 was obtained (HRMS-ESI-: 10 H 10 Calculated m / z [MH]- 240.0331 for N2O4S, measured 240.0330).

[0462] To a solution of carboxylic acid 8 in dry DMF (20 mL) containing DIPEA (1.71 mL, 9.79 mmol) was added dropwise TSTU (884 mg, 2.937 mmol) at 0 °C. After stirring at room temperature for 1 h, the mixture was washed with a saturated solution of NaHCO3. The residue was purified by flash chromatography (SiO2, DCM / AcOEt: 50 / 50 to 0 / 100) to give the vinylsulfonamide derivative 9 (325 mg, 0.959 mmol, 49%) as a white solid. 1H NMR (300.13 MHz, CDCl3): δ H 8.12 (d, 2H, J = 8.8 Hz, Ar), 7.47 (d, 2H, J = 8.8 Hz, Ar), 6.41 (dd, 1H, J = 16.5 Hz, J = 9.8 Hz, vinyl), 6.22 (d, 1H, J = 16.5 Hz, vinyl), 6.05 (d, 1H, J = 9.8 Hz, vinyl), 3.30 (s, 3H, NHMe), 2.90 (s, 4H, NHS). 13C NMR (75.48 MHz, CDCl3): δ C 169.15 (2 x C, NHS), 161.0 (C, COOH), 147.0 (C, Ar), 131.9 (CH, vinyl), 131.5 (2 x CH, Ar), 129.5 (CH2, vinyl), 124.8 (2 x CH, Ar), 122.8 (C, Ar), 37.2 (CH3), 25.6 (CH2, NHS). HRMS (ESI+): m / z C 14 H 14 Calculated value of N2O6SNa [M+Na]+ is 361.0470. Found value is 361.0460.

[0463] - Preparation of Compound GalNAc-Sulfonamide (L1) from Compounds 6 and 9

[0464] [ka]

[0465] To a solution of compound 6 (see Example 1 - 20 mg, 0.0567 mmol) containing DIPEA (30 μL, 0.1700 mmol) in DMF (1 mL) was added a solution of compound 9 (23 mg, 0.0681 mmol) in DMF (0.5 mL). The resulting mixture was stirred at room temperature for 30 min. The mixture was evaporated under reduced pressure. The residue was purified by flash chromatography (SiO2, DCM / MeOH: 90 / 10 to 80 / 20) to give GalNAc-sulfonamide (L1) (24 mg) as a white solid. A second purification by reverse phase chromatography (C18 cartridge 15 μm, gradient eluent H2O 0.1% TFA / ACN: 95 / 5→90 / 10→80 / 20) gave pure compound GalNAc-sulfonamide (L1) (24 mg, 0.0417 mmol, 74%). 1H NMR (300.13 MHz, MeOD, 298.15 K): δ H 7.85 (m, 2H, H17), 7.47 (m, 2H, H18), 6.67 (m, 1H, H21), 6.11 (m, 1H, H22), 4.41 (d, J1,2 = 8.3 Hz, 1H, H1), 3.99-3.45 (m, 18H), 3.27 (s, 3H, H20), 1.97 (s, 3H, NHAc). 13C NMR (75.48 MHz, MeOD, 298.15 K): δ C 174.2 (C, CO, NHAc), 169.4 (C, CO), 145.8 (C, Ar), 134 (C, Ar), 133.8 (CH), 129.8 (CH2), 129.2 (CH, Ar), 127.1 (CH, Ar), 103.8 (CH, C1), 76.8 (CH, C5), 73.5 (CH, C3), 71.6 (CH2) , 71.5 (CH2), 71.4 (CH2), 70.6 (CH2), 69.7 (CH, C4), 62.6 (CH2, C6), 54.3 (CH, C2), 37.7 (CH2) , 38.2 (CH3) 23.1 (CH3). HRMS (ESI+): m / z C 24 H 37 N3O 11 SNa [M+Na]+ calculated value 598.2046 Found value 598.2056.

[0466] Synthesis of compound GalNAc-maleimide (L2) from compound 6

[0467] [ka]

[0468] To a solution of compound 6 (see Example 1 - 38 mg, 0.106 mmol) containing DIPEA (55 μL, 0.318 mmol) in DMF (2 mL) was added 3-(maleimido)propionic acid N-hydroxysuccinimide ester (34 mg, 0.127 mmol). The resulting mixture was stirred at room temperature for 1 h. The mixture was evaporated under reduced pressure. The residue was purified by flash chromatography (SiO2, DCM / MeOH: 90 / 10 to 70 / 30) to give GalNAc-maleimide (L2) (43 mg) as a white solid. A second purification on reversed phase chromatography (C18 cartridge 15 μm, gradient eluent H2O 0.1% TFA / ACN: 95 / 5→90 / 10→80 / 20) gave pure compound GalNAc-maleimide (L2) (34 mg, 0.0675 mmol, 64%). 1H NMR (300.13 MHz, D2O, 298.15 K): δ H 6.84 (s, 2H, maleimide), 4.47 (d, J1,2 = 8.4 Hz, 1H, H1), 3.99 (ddd, 1H, J = 11.4 Hz, J = 5.5 Hz, J = 3.0 Hz, H5), 3.91-3.58 (m, 17H), 3.53 (t, 2H, J = 5.3 Hz), 3.29 (t, 2H, J = 5.3 Hz), 4.49 (t, 2H, J = 6.5 Hz), 2.00 (s, 3H, NHAc). 13C NMR (75.48 MHz, D2O, 298.15 K): δ C 174.6 (C, CO), 173.6 (C, CO), 172.5 (C, CO), 134.4 (CH, maleimide), 101.5 (CH, C1), 75.1 (CH, C5), 71.0 (CH), 69.7 (2 x CH2), 69.4 (CH2), 68.8 (CH2), 68.6 (CH2), 67.8 (CH), 60.9 (CH), 52.3 (CH2), 38.9 (CH2), 34.6 (CH2), 34.4 (CH2), 22.2 (CH3, NHAc). HRMS (ESI+): m / z C 21 H 33 N3O 11 Calculated value of Na [M+Na]+ 526.2013 Measured value.

[0469] Example 5 Coupling experiments with ligand L2 (maleimide ligand), comparison with ligand L (benzoylacrylamide), and effect of pH on coupling yields The same coupling protocol as shown in Example 2 was used. Two pHs were used: pH=7.3 (as used in Example 1) and pH=9.1. Briefly, AAV2-CAG-GFP (10E12vg, 2.49nmol, 100μL) was added to a solution of GalNAc-acrylamide ligand (L) (3E6 equivalents) or GalNAc-maleimide ligand (L2) (3E6 equivalents) in dPBS buffer (100μL or 900μL) or basic buffer 50 / 50 dPBS / PBS (pH=9.1) (200μL) and incubated at room temperature for 4 hours. The solution containing the vector was then dialyzed against dPBS+0.001% Pluronic to remove free molecules not bound to the AAV2 capsid. The resulting AAV2 was characterized by dot blot analysis using immunostaining with A20 antibody to assess capsid integrity and by staining with soybean lectin, which can selectively detect GalNAc, to assess coupling efficiency for each tested ligand.

[0470] result: The results of the dot blot analysis are shown in Figures 5A and 5B. The integrity of the capsid after the coupling reaction with ligand L (benzoylacrylamide ligand) or ligand L2 (maleimide ligand) was assessed by dot blot analysis using immunostaining with the A20 antibody. The A20 antibody recognizes assembled AAV2 capsids. Positive dots with the A20 antibody indicate that AAV2 remained intact after the coupling procedure with ligand L or ligand L2, regardless of the coupling pH.

[0471] Dot blot analysis with soybean lectin, which selectively binds GalNAc, showed clear positive dots for ligand L, confirming covalent coupling (as also demonstrated by HPLC / MC analysis in Example 3). Surprisingly, the intensity of the dots in soybean lectin staining was stronger for AAV2 coupled with ligand L at pH 9.1 than for AAV2 coupled with L at pH 7.4. Such results support that basic pH increases the efficiency of coupling, i.e., the number of cysteine ​​residues in the capsid chemically modified with L. In other words, by adjusting the pH of coupling, it is possible to adjust the proportion of cysteine ​​residues in the AAV capsid that are chemically modified with acrylamide ligands.

[0472] The intensity of the dots in soybean lectin staining obtained for the ligand L2 (maleimide) is lower than that observed for AAV2 chemically modified with L. At pH 7.3, the intensity of the dots for L2 is weak and even comparable to the negative control (AAV2), suggesting at best a very low proportion of chemically modified cysteine ​​residues present in the AAV2 capsid. For coupling performed with L2 at pH 9.1, the dots for L2 are more clearly visible and stronger than at pH 7.3, suggesting better chemical coupling. However, the dots for L2 at pH 9.1 are much weaker than those observed for L.

[0473] In other words, the L2 ligand (maleimide) appears to be significantly less effective than the acrylamide ligand in chemically modifying cysteine ​​residues in the AAV capsid, regardless of the pH used for coupling. The low coupling efficiency of the maleimide ligand L can be explained, at least in part, by the instability of the coupling functional group in aqueous buffers due to the retro-Michael reaction.

Claims

1. Adeno-associated virus (AAV) having at least one chemically modified cysteine ​​residue in its capsid, wherein the chemically modified cysteine ​​residue is of formula (I): 【Chemistry 1】 (In the formula, - X is, 【Chemistry 2】 Selected from the group consisting of, - Z is -O-, -S-, or -N(R 4 )- and, - R 1 , R 2 , R 3 , and R 4 Each of these is independently selected from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, and the aforementioned groups may be optionally substituted. - k is either 0 or 1, - R is hydrogen, halogen, alkyl group, aryl group, heteroaryl group, or alkoxy group, and the aforementioned groups may be optionally substituted. - Y is a spacer, - n is 0 or 1, and - M is the functional part.) This is adeno-associated virus (AAV).

2. The AAV according to claim 1, wherein X is formula (b) or formula (c).

3. Chemically modified cysteine ​​residues are given by formula (Ic): 【Transformation 3】 (In the formula, Y, n, M, Z, k, R 2 , and R 3 (This is as defined in claim 1.) The AAV according to claim 1.

4. - R 2 is a hydrogen atom, C 1 - C 6 alkyl group, aryl group containing 6 to 14 ring atoms, or heteroaryl group containing 5 to 14 ring atoms, and / or - R 3 The group is selected from the group consisting of aryl groups containing 6 to 14 ring atoms and heteroaryl groups containing 5 to 14 ring atoms, wherein the aryl or heteroaryl group is optional, preferably a halogen, -OH, or NH. 2 NO 2 , C 1 ~C 3 Alkyl, C 1 ~C 3 Alkoxy, C 1 ~C 3 Hydroxyalkyl, and C 1 ~C 3 They may be substituted with 1 to 3 substituents selected from haloalkyl groups, and / or - k is 0, The AAV according to claim 3.

5. - R 2 is a hydrogen atom or C 1 ~C 6 Alkyl alkyl groups, preferably H or C 1 ~C 3 It is an alkyl group - R 3 This refers to unsubstituted phenyl, or halogen, -OH, NH 2 NO 2 , C 1 ~C 3 Alkyl, C 1 ~C 3 Alkoxy, C 1 ~C 3 Hydroxyalkyl, and C 1 ~C 3 Phenyl substituted with 1 to 3 substituents selected from haloalkyls, and - k is 0, The AAV according to claim 3.

6. Y is given by equation (II): 【Chemistry 4】 (In the formula, - m, p, and q are each independently 0 or 1. - Y 1 The group is selected from the group consisting of alkylene groups, arylene groups, and heteroarylene groups, and the group may be optionally substituted, preferably a phenylene group. - Y 2 is -C(=O)-NH, -C(=O)-O, -C(=O)-OC(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -OC=OO-, O, NH, -NH(C=S)-, or -(C=S)-NH-, preferably -(C=O)-NH-, - Y 3 Polymers, peptides, oligosaccharides, including homopolymers, copolymers, and block polymers, optionally by one or more heteroatoms and / or by groups selected from -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)-, and -(C=S)-NH- and / or by one or more C 3 ~C 6 Hydrocarbon ring or C 2 ~C 6 (Selected from the group consisting of saturated or unsaturated branched or linear hydrocarbon chains, which may be interrupted by heterocycles.) AAV according to any one of claims 1 to 5, which is a spacer for the AAV.

7. The AAV according to any one of claims 1 to 5, wherein M is a functional moiety comprising a group selected from click chemistry groups, steric shielding agents, labeling agents, targeting agents such as cell type-specific ligands, drug moieties, oligonucleotides, and combinations thereof.

8. The aforementioned chemically modified cysteine ​​residue is given by formula (I) (wherein, - X is given by equation (c), - n is 1, - M is a functional part, and - Y is given by equation (II) (where m is 0, p is 0, q is 1, and Y 3 This is a saturated or unsaturated linear or branched C, which may be optionally substituted. 2 ~C 40 (A spacer selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, alkyldiamine polymers, and combinations thereof.) The AAV according to claim 6.

9. - Y is given by equation (II) (where q is 1, m is 0 or 1, p is 0 or 1, Y 1 and Y 2 Y is as defined in claim 6, 3 This is a saturated or unsaturated linear or branched C, which may be optionally substituted. 2 ~C 40 Spacers selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, alkyldiamine polymers, and combinations thereof, and / or - M comprises or consists of a targeting agent such as a cell type-specific ligand selected from click chemistry groups, oligonucleotides, preferably monosaccharides or polysaccharides, hormones including 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, spiegelmer, and peptide aptamers, vitamins, and drugs such as CB1 and / or CB2 ligands. The AAV according to claim 6.

10. - Y is given by equation (II) (where q is 1, m is 0 or 1, p is 0 or 1, Y 1 and Y 2 Y is as defined in claim 6, 3 C is linear or branched 2 ~C 20 A spacer selected from the group consisting of alkyl chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, alkyldiamine polymers, and combinations thereof, wherein the polymer has 2 to 20 monomers, and / or - "M" comprises or consists of a targeting agent, preferably a protein selected from transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor βFGF; a monosaccharide or polysaccharide containing one or more galactose, mannose, N-acetylgalactosamine residues, cross-linked GalNAc, or mannose-6-phosphate, sialic acid and its derivatives (e.g., Neu5Ac, Neu5Acα2-6Gal, and Neu5Acα2-8Neu5Ac); an MTP selected from SEQ ID NOs: 1 to 7; and a cell type-specific ligand derived from a vitamin such as folic acid. The AAV according to claim 6.

11. - M is a cell-type specific ligand for specifically targeting hepatocytes, and formula (III): 【Transformation 5】 Including at least one part of and / or - Y is a polyethylene glycol chain containing 2 to 10 monomers. The AAV according to any one of claims 1 to 5.

12. At least one chemically modified cysteine ​​in the capsid is given by formula (Ic-1): 【Transformation 6】 The AAV according to claim 11.

13. The capsid further has at least one additional chemically modified amino acid residue different from the cysteine ​​residue, wherein the amino acid residue is preferably as follows: - Formula (V): 【Transformation 7】 (In the formula, - N* is the nitrogen atom of the amino group of an amino acid residue, such as a lysine residue or an arginine residue. - Y', n', and M' have the same definitions as Y, n, and M in formula (I) as defined in claim 1.) Modified amino group; or - Equation (VI): 【Transformation 8】 (In the formula, - X" is -N=N- or 【Chemistry 9】 And, - Y'', n'', and M'' have the same definitions as Y, n, and M in formula (I) as defined in claim 1. Modified tyrosine residues An AAV according to any one of claims 1 to 5, having the following characteristics.

14. The AAV according to any one of claims 1 to 5, which is a recombinant AAV selected from recombinant AAV, preferably AAV having a wild-type capsid, naturally occurring serotype AAV, variant AAV, pseudotype AAV, hybrid AAV, and self-complementary AAV.

15. A method for chemically modifying the capsid of an AAV, more precisely, for chemically modifying at least one cysteine ​​residue in the capsid of an AAV, comprising the step of incubating the AAV with a chemical reagent having a reactant selected from maleimide, vinylsulfonamide, and 3-(carboxyl derivative)acrylamide, under conditions that facilitate the reaction of the reactant with a cysteine ​​residue present in the capsid of the AAV to form a covalent bond.

16. AAV is given by equation (VIIc): 【Chemistry 10】 Incubate with the chemical reagents and formula (Ic): 【Chemistry 11】 (In the formula, - Y is a spacer, n is 0 or 1, and M is a functional part. - Z is -O-, -S-, or -N(R 4 )- and, - k is 0 or 1, and - R 2、 R 3 , and R 4 Each of these groups is independently selected from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, and the groups may be optionally substituted. The method according to claim 15, comprising the step of obtaining at least one chemically modified cysteine ​​residue in the capsid.

17. - Y is given by equation (II): 【Chemistry 12】 (In the equation, m is 0, p is 0, q is 1, Y 3 This is a saturated or unsaturated linear or branched C, which may be optionally substituted. 2 ~C 40 A spacer selected from the group consisting of hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, alkyldiamine polymers, and combinations thereof, and - M is a click chemistry group, steric shielding agent, labeling agent, targeting agent such as a cell type-specific ligand, or drug moiety. The method according to claim 16.

18. The method according to any one of claims 15 to 17, wherein the incubation step is carried out at a pH of 6.0 to 10.0, preferably 7.0 to 8.0 or 8.0 to 10.

0.

19. A pharmaceutical composition comprising the AAV described in claim 1 and at least one pharmaceutically acceptable excipient.

20. A gene vector comprising an AAV as defined in any one of claims 1 to 5, wherein the viral genome contains a transgene sequence.

21. A gene vector according to claim 20 for use in gene therapy, wherein the introduced gene sequence encodes a therapeutic protein.

22. An AAV according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 19 for use as an in vivo diagnostic agent or as a drug in in vivo gene therapy.

23. Use of the AAV according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 19 as an in vitro diagnostic agent or as an ex vivo or in vitro gene vector.

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