WO2024149844A1 - Chemically-modified adeno-associated virus - Google Patents

Chemically-modified adeno-associated virus Download PDF

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WO2024149844A1
WO2024149844A1 PCT/EP2024/050598 EP2024050598W WO2024149844A1 WO 2024149844 A1 WO2024149844 A1 WO 2024149844A1 EP 2024050598 W EP2024050598 W EP 2024050598W WO 2024149844 A1 WO2024149844 A1 WO 2024149844A1
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aav
group
alkyl
capsid
chemically
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PCT/EP2024/050598
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French (fr)
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Sébastien GOUIN
David DENIAUD
Mathieu Mevel
Sébastien DEPIENNE
Dimitri ALVAREZ-DORTA
Mohammed BOUZELHA
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Nantes Université
INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique
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Publication of WO2024149844A1 publication Critical patent/WO2024149844A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors

Definitions

  • the invention relates to a chemically modified adeno-associated (AAV) virus and its use in gene therapy.
  • AAV adeno-associated virus
  • Background of the invention Gene therapy was originally developed to correct defective genes that underlie genetic diseases.
  • gene therapy is more and more used in the treatment of a broad range of acquired diseases such as cancers.
  • Gene therapy is based on the therapeutic delivery of nucleic acid into a patient’s cell nucleus.
  • the nucleic acids may then be inserted into the genome of the targeted cell or may remain episomal. Delivery of a therapeutic nucleic acid to a subject's target cells can be carried-out by various methods, including the use of synthetic and viral vectors.
  • recombinant adeno-associated virus is gaining popularity as a versatile vector for gene therapy, particularly for in vivo applications.
  • the main advantages of recombinant AAV (rAAV) reside in their broad tropism, their high transduction efficacy, their persistent episomal expression and their high safety profile, in particular because wild-type AAV is not associated with any human diseases.
  • Human clinical trials with rAAV have demonstrated durable expression at therapeutic levels when targeting tissues such as retina, liver or motor neurons.
  • rAAV as gene vector are ongoing for a wide type of disorders.
  • Voretigene neparvovec which is an adeno-associated viral vector serotype 2 (AAV2) capsid comprising a cDNA encoding for the human retinal pigment epithelium 65kDa protein (hRPE65), for the treatment of vision loss due to inherited retinal dystrophy caused by confirmed biallelic RPE65 mutations.
  • AAV2 adeno-associated viral vector serotype 2
  • Zolgensma® ona shogene abeparvovec-xioi
  • SMA spinal muscular atrophy
  • Zolgensma® is a AAV9 vector able to deliver a functional, non-mutated copy of the defective gene in SMA, namely the SMN1 gene, in motoneurons.
  • SMA defective gene
  • SMN1 SMN1 gene
  • motoneurons a functional, non-mutated copy of the defective gene in SMA
  • Their first limitation lies on their immunogenicity. Because of their non-integrative nature, systemic gene therapy with AAV vectors, especially in paediatric patients, might be limited by tissue proliferation inducing a dilution of the vector over time. However, the re-administration of the vectors might be precluded by persistent neutralizing anti-AAV antibodies (NAbs) triggered following the first administration of the viral vector.
  • NAbs neutralizing anti-AAV antibodies
  • AAV serotypes especially AAV of serotype 2
  • AAV anti-AAV neutralizing antibodies
  • NAbs can completely prevent transduction in a target tissue, resulting in lack of efficacy, particularly when the vector is administered directly into the bloodstream.
  • subjects seropositive to AAV-Nabs are generally excluded from gene therapy trials.
  • a further limitation of AAV lies on their broad tropism, which may result in transgene expression in other tissues other than those where transgene expression is desired.
  • AAV as gene vector may also suffer from a reduced therapeutic index. Sometimes, the administration of high dose of AAV is needed to achieve effective transduction.
  • AAV2 vectors can efficiently target the liver, the transgene expression can be restricted to a very small of the transfected hepatocytes due to intracellular proteasome-mediated degradation of the vectors, whereby high dose or AAV-2 may be required to achieve the sought therapeutic effect.
  • high doses pose a challenge not only for vector production but also increases the risk of immune response, among which the induction of NAbs.
  • Several strategies have been proposed to overcome the drawbacks of AAV, especially those of the AAV of serotype 2 (AAV2) in gene therapy. Certain of these strategies are based on AAV surface remodeling, namely modification of the AAV capsid proteins. The first option is to genetically modify the viral capsid.
  • a non-natural amino acid comprising an azido group is inserted into the capsid by genetic modification prior to a coupling step with a ligand by click reaction so as to change its tropism for the target cell.
  • Another strategy resides in the direct chemical modification of the viral capsid without any preliminary site-directed mutagenesis of the capsid proteins.
  • International patent application WO2017/212019 proposes a method for chemically modifying the AAV capsid by covalently coupling a ligand bearing an isothiocyanate group which reacts with an amino group present in an amino acid residue such as lysine or arginine.
  • the Invention relates to an adeno-associated Virus (AAV) having at least one chemically-modified tyrosine residue in its capsid, wherein said chemically-modified tyrosine residue is of formula (I): wherein: - R A is -(Y) n -M, a C 1 -C 6 alkyl, a C 6 -C 14 aryl optionally substituted, or a (C 6 -C 14 aryl)-(C 1 -C 3 alkyl) optionally substituted, - each RB is independently a group of formula -(Y)n-M, a hydrogen or a substituent selected from the group consisting of a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1
  • RA is a C1-C3 alkyl, a phenyl, or a benzyl, preferably a methyl or a benzyl, more preferably a methyl and/or one or two R B is a group of formula -(Y) n -M and the other R B are hydrogens.
  • R A is a C 1 -C 3 alkyl, a phenyl, or a benzyl, preferably a methyl or a benzyl, one R B is -(Y) n -M and the remaining R B are H.
  • RA is -(Y)n-M and RB groups are H.
  • the at least one chemically-modified tyrosine residue is such that Y is a chemical chain group comprising from 2 to 500 carbon atoms and selected from the group consisting of polymers including homopolymers, copolymers and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains optionally interrupted by one or several heteroatoms and/or by one or several cyclic or heterocyclic moieties, optionally having an heteroatom, such as S, O and NH, at least one of its extremity, and optionally substituted by one or several substituents, and combinations thereof.
  • Y is a chemical chain group comprising from 2 to 500 carbon atoms and selected from the group consisting of polymers including homopolymers, copolymers and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains optionally interrupted by one or several heteroatoms and/or by one or several cyclic or heterocyclic moieties, optionally having an heteroatom,
  • Y can be a saturated or unsaturated hydrocarbon having from 2 to 100 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -N(R)- with R being H or a C1-C3 alkyl, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and -NH-CS-; and/or - C 5 -C 20 carbocyclic moieties such as cycloalkyl, cycloalkenyl, or aromatic groups; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms, - and optionally having at least one of its extremities, an heteroatomic group
  • the AAV is such that M is a functional moiety comprising a group selected from a click-chemistry group, a steric shielding agent, a labelling agent, a targeting agent such as a cell-type specific ligand, a drug moiety, an oligonucleotide and combinations thereof.
  • M can comprise, or consist of, - a click-chemistry group, a cell targeting agent, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a aptmer, a peptide aptamer, a vitamin and small chemical molecules such as drugs e.g.
  • a cell targeting agent preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof,
  • CB1 and/or CB2 ligands or - a click-chemistry group, a cell-type specific ligand derived from a protein selected from transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ⁇ FGF, a mono- or a polysaccharide comprising one or several galactose, mannose, N-acetylgalactosamine residues, bridge GalNac, or mannose-6-phosphate, sialic acid and derivatives thereof (e.g.
  • the AAV of the Invention further has at least one additional chemically modified amino acid residue in the capsid selected from cysteine, arginine or lysine.
  • the AAV of the Invention can be of any type.
  • the AAV is a recombinant AAV, preferably selected from AAV having a wildtype capsid, naturally-occurring serotype AAV, variant AAV, pseudotype AAV, AAV with hybrid or mutated capsids, and self-complementary AAV.
  • the invention relates to a method for chemically-modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, which comprises incubating said AAV with a chemical reagent bearing a N-substituted luminol moiety in conditions conducive for reacting said chemical reagent with a tyrosine residue present in the capsid of the AAV so as to form a covalent bound, said method being preferably performed by electrochemistry.
  • the method of the invention comprises incubating said AAV with a chemical reagent of formula (X): wherein: - RA is -(Y)n-M , a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, - each R B is independently selected from a group of formula -(Y) n -M, a hydrogen or a substituent chosen from a halogen, C 1 -C 6 alkyl, C 6 -C 14 aryl, C 3 -C 6 cycloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 2 -C 6 heterocycle, C 1 -C 6 alkanoyl, C 1 -C 6 carboxy esters, C 1 -C 6 acylamino, -COOH, -CONH 2 ,
  • the method of the Invention is performed in an electrochemical system with three electrodes comprising a working electrode, a counter-electrode and a reference electrode by applying a constant potential difference between the working electrode and the reference electrode, the potential difference being preferably included in a range defined as the potential oxidation of the chemical reagent ⁇ 200 mV.
  • R A is a C 1 -C 6 alkyl and
  • at least one R B is of formula -(Y) n -M and the others RB are H.
  • the method of the invention is such that M is a click-chemistry group and the method further comprises a step of click-reaction so as to covalently bond a functional moiety preferably selected from ligands and labels on AAV capsid.
  • the click reaction can be a strain promoted alkyne-azide cycloaddition (SPAAC), which means that M can be an azido group and the functional moiety bears a strained alkyne, or vice versa.
  • SPAAC strain promoted alkyne-azide cycloaddition
  • the Invention also relates to an AAV obtainable by the method of the invention.
  • the Invention also relates to a pharmaceutical composition comprising an AAV as defined above and at least one pharmaceutically acceptable excipient.
  • the Invention relates to the use of an AAV or a pharmaceutical composition of the Invention as a diagnostic agent in vivo or as a drug ex vivo or in vivo, preferably in gene therapy.
  • the Invention also relates to the use of an AAV as defined herein as a research tool in vitro, for instance as a gene transfection agent or as an imaging agent in vitro.
  • the Invention further relates to the use of an AAV as defined herein in the manufacture of a diagnostic agent or a drug, in particular for gene therapy ex vivo or in vivo.
  • the Invention also relates to the use of a compound of formula (X): defined herein as an agent for chemically modifying the capsid of an AVV by electrochemical bioconjugation.
  • Figure 1 Experimental setup and electrochemical cell assembled for AAV electro- bioconjugation. Anode, cathode and reference electrodes are clipped to the electrodes holder (A) and dipped into a low binding vial filled with AAV and luminol solution (B) inserted into the 5 mL glass vial to form (ABC). Alligator clips connect electrical connections from (A) to potentiostat. The latter is USB-controlled by a computer (software EC-Lab).
  • Figure 2A shows cyclic voltammetry of N-methyl luminol GalNAc derivative 12 (LumGalNAc) at scan rates 25, 50, 75 and 100 mV/s highlighted a coherent electrochemical process controlled by diffusion.
  • Figure 3A Top: Dot blot analysis of viral vectors (2.10 10 vg) to detect the assembled capsid using anti-capsid A20 antibody.
  • FIG. 3B show Western blot analysis of viral vectors (2.10 10 vg) after denaturation to detect (left) the three constitutive viral proteins (VP1/VP2/VP31:1:10) using a polyclonal anti-VPs antibody, or (right) surface-conjugated LumMan using a labelled Concanavalin A (Man-binding lectin).
  • Figure 5A and 5B show some examples of “M” moieties according to the invention(non- exhaustive list, for illustration only)
  • Figure 6 Coupling with N-methyl luminol azido derivatives (LumN 3 ), Dot blot analysis of viral vectors (2.10 10 vg) to detect the assembled capsid using anti-capsid A20 antibody and the surface- conjugated N3 using DBCO-fluorescein and mass spectroscopy analyses to determine the number of Azido derivatives by VP3 molecule.
  • N-methyl luminol azido derivatives LiN 3
  • Dot blot analysis of viral vectors (2.10 10 vg) to detect the assembled capsid using anti-capsid A20 antibody and the surface- conjugated N3 using DBCO-fluorescein and mass spectroscopy analyses to determine the number of Azido derivatives by VP3 molecule.
  • the electrochemical bioconjugation developed by the Applicants is based on the use of a specific reagent, namely a N-substituted luminol derivative, preferably N-methyl-luminol (NMeLum) derivative, which is electrochemically activated in situ (namely electro-oxidized) and then specifically reacts with the aromatic ring present in the side chain of tyrosine residues in the AAV capsids.
  • a specific reagent namely a N-substituted luminol derivative, preferably N-methyl-luminol (NMeLum) derivative
  • electrochemically activated in situ namely electro-oxidized
  • electrochemical bioconjugation has been mostly used to chemically modify isolated proteins (see for instance Depienne et al., Chem. Sci., 2021, 12, 15374–15381) but not proteins present in complex system comprising genetic element such as viruses.
  • the Applicants identified NMeLum as being a coupling moiety of interest in the context of AAV surface remodeling.
  • NMeLum-containing ligands can be electro-activated at low potential regardless the substituent present on NMeLum aromatic ring.
  • the potential used to oxidize NMeLum is low enough to avoid side reactions with most chemical groups other than phenol residue, which means that a large variety of ligands can be coupled through NMeLum moiety on AAV capsid.
  • NMeLum moiety at low potential enables the formation of a stable nitrogen-centered radical which can specifically react through radical coupling with tyrosine residues in the AAV capsids while avoiding the formation of highly reactive side-products and side-reactions with other amino acid residues (e.g. lysine, arginine) present in the capsid of the AAV.
  • N-substituted luminol derivatives bearing a sugar moiety (namely N-acetylgalactosamine (GalNAc), or a mannose (Man) sugar) for chemically modifying the capsids of AAV vectors on naturally-occurring tyrosine residues.
  • a sugar moiety namely N-acetylgalactosamine (GalNAc), or a mannose (Man) sugar
  • AAV2 capsid incubated with a triethylene glycol derivatized GalNAc could not be detected by SBA, precluding non-covalent ligand adsorption of the NMeLum derivatives at the capsid surface during the electrochemical bioconjugation.
  • the Applicants further showed that the electrochemical coupling with NMeLum derivatives did not alter capsid protein subunits of AAV2 as evidenced by dot blot analysis with anti-capsid A20 antibody staining (Figure 3A).
  • the Applicants also decorated the surface of AAV2 with various entities (nanobodies - also called VHH, fluorescein, biotin) in a two-step method comprising the electrochemical bioconjugation of AAV2 with an azido derivative of N-methyl luminol followed by SPAAC (Strain-Promoted Alkyne-Azide Cycloaddition) reaction with DBCO-ligands.
  • SPAAC Stra-Promoted Alkyne-Azide Cycloaddition
  • N-substituted luminol derivatives can be used to modify the surface-exposed tyrosine residues in AAV capsids with specificity and efficacy and without impairing the structural integrity and the transduction efficacity of the viruses.
  • This method enables to decorate the AAV capsids with a wide variety of ligands such as sugar moieties, biotin, fluorescent labels and proteins (e.g. nanobodies).
  • the electrochemical bioconjugation of the invention avoids using multiple chemical entities (such as oxidants, catalysts and/or scavengers), and at the end of the reaction, unreacted ligands can be easily removed by standard methods (such as dialysis).
  • electrodes used in the electrochemical bioconjugation do not produce waste, can be reused several times, and allows to easily implement the process from a laboratory to industrial process scale.
  • the Invention relates to an Adeno-Associated Virus (AAV) having at least one chemically-modified tyrosine residue, preferably at least one electrochemically-modified tyrosine residue in its capsid.
  • AAV Adeno-Associated Virus
  • Said chemically-modified tyrosine residue results from the reaction of a tyrosine in the capsid with a functional moiety bearing N-substituted luminol group.
  • the tyrosine residue to be chemically modified is a naturally-occurring residue in the capsid, i.e. the tyrosine has not been introduced by mutagenesis.
  • the chemically modified adeno-Associated Virus comprises a functional moiety, for instance a ligand, covalently linked to the aromatic ring present in the side chain of a tyrosine residue in its capsid via the following moiety: wherein RA is -(Y)n-M (as defined further below) or a hydrocarbon substituent, typically a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, preferably a methyl or a benzyl, more preferably a methyl.
  • RA is -(Y)n-M (as defined further below) or a hydrocarbon substituent, typically a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, preferably
  • the Invention also relates to a method for preparing a chemically-modified AAV comprising the step of contacting the AAV with a functional moiety bearing a N-substituted luminol group in condition enabling the reaction of said a functional moiety bearing a N-substituted luminol group with the aromatic ring of a tyrosine residue present in the AAV so as to covalently link said functional moiety to the AAV.
  • the N-substituted luminol is preferably a N-(C1-C6 alkyl) luminol, a N-(C 6 -C 14 aryl optionally substituted) luminol or a N-[(C 6 -C 14 aryl)-(C 1 -C 3 alkyl) optionally substituted] luminol, most preferably a N-methyl luminol or a N-benzyl luminol, more preferably a N-methyl luminol.
  • such a method is performed by electrochemistry, namely by subjecting the reaction medium to a potential difference sufficient to electro-activate (i.e.
  • the invention also relates to the use of the resulting chemically-modified AAV, preferably the electrochemically-modified AAV, in particular in gene therapy.
  • the Invention is described in more details hereunder:
  • the expression “about X” with X being a physical value (such as a voltage) corresponds to X ⁇ 5%.
  • the term C1-C6 it means that the corresponding hydrocarbon chain may comprise from 1 to 6 carbon atoms, especially 1, 2, 3, 4, 5 or 6 carbon atoms. If, for example, the term C 2 -C 5 is used, it means that the corresponding hydrocarbon chain may comprise from 2 to 5 carbon atoms, especially 2, 3, 4, or 5 carbon atoms.
  • 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.
  • alkyne or “alkynyl” refers to an unsaturated, linear or branched aliphatic group, having at least one carbon-carbon triple bond.
  • a preferred alkyne is “C2-C6 alkyne”, which refers to an alkyne having 2 to 6 carbon atoms.
  • alkyne examples include for instance ethynyl, propynyl, butynyl, pentynyl, or hexynyl, preferably ethynyl (-C ⁇ CH).
  • alkoxy refers to an alkyl as defined herein, attached to the remainder of the molecule via an ether bond (-O-). In other words, an alkoxy can be written “-O-alkyl”.
  • a preferred alkoxy is a C 1 -C 6 alkoxy, which has 1 to 6 carbon atoms.
  • alkoxy examples include for instance, methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy, hexyloxy.
  • alkylthio refers to an alkyl as defined herein, attached to the remainder of the molecule via a thioether bond (-S-). In other words, an alkylthio can be written “-S-alkyl”.
  • a preferred alkylthio is a C1-C6 alkylthio, which has 1 to 6 carbon atoms.
  • alkylthio examples include for instance, methylthio, ethylthio, propylthio, isopropylthio, butylthio, pentylthio, hexylthio.
  • alkylamino refers to an alkyl as defined herein, attached to the remainder of the molecule via an amino bond (-NH-). In other words, an alkylamino can be written “-NH-alkyl”.
  • a preferred alkylamino is a C1-C6 alkylamino, which has 1 to 6 carbon atoms.
  • cycloalkyl refers to a saturated mono-, bi- or tri-cyclic aliphatic group. It also includes fused, bridged, or spiro-connected cycloalkyl groups.
  • C 3 -C 6 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, cyclohexyl.
  • cycloalkyl may also refer to a bridged carbocyclyl such as bicyclo[2,2,1]heptanyl, bicyclo[2,2,2]octanyl, or adamantyl.
  • cycloalkenyl refers to an unsaturated mono-, bi- or tri-cyclic aliphatic group, comprising at least one carbon-carbon double bond. It also includes fused, bridged, or spiro- connected cycloalkenyl groups.
  • C 3 -C 6 cycloalkenyl refers to a cycloalkenyl having 3 to 6 carbon atoms.
  • the heterocycle comprises between 3 and 20 ring atoms, for instance between 3 and 6 ring atoms, wherein at least one of the ring atoms is a heteroatom such as nitrogen, oxygen or sulphur atom.
  • the “heterocycle” is a heterocycloalkyl, a heterocycloalkenyl, or a heteroaryl.
  • the heterocycle is a heterocycloalkyl, a heterocycloalkenyl, or a heteroaryl, fused with one or more carbocyclic or heterocyclic moieties (for instance, a heteroaryl fused with a cycloalkyl).
  • heterocycloalkyl corresponds to a cycloalkyl group as above defined in which at least one carbon atom has been replaced with a heteroatom such as nitrogen, oxygen, or sulphur atom.
  • heterocycloalkenyl corresponds to a cycloalkenyl group as above defined in which at least one carbon atom has been replaced with a heteroatom such as nitrogen, oxygen, or sulphur atom.
  • heterocycles which are heterocycloalkyl or heterocycloalkenyl, include, but are not limited to, aziridinyl, azepanyl, diazepanyl, dioxolanyl, benzo [1,3] dioxolyl, azetidinyl, oxetanyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4-dioxanyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, pyrimidinyl, oxozolinyl, oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiooxetanyl, thio
  • aryl refers to an aromatic ring system, which preferably has 6-14 atoms, having at least one ring having a conjugated pi electron system and which optionally may be substituted.
  • An “aryl” may contain more than one aromatic ring such as fused ring systems or an aryl group substituted with another aryl group.
  • Aryl encompass, without being limited to, phenyl, anthracenyl, naphthyl, indenyl, divalent biphenyl.
  • Heteroaryl refers to a heteroaryl group.
  • Heteroaryl refers to a chemical group, preferably having 5-14 ring atoms, wherein 1 to 4 heteroatoms are ring atoms in the aromatic ring and the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and selenium.
  • heterocycles which are heteroaryl groups, include triazolyl, furanyl, thienyl, pyridyl, pyrrolyl, N-alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinazolinyl, and quinolinyl.
  • a preferred (C 6 -C 14 aryl)-(C 1 - C 3 alkyl) is phenylmethyl (namely benzyl).
  • alkanoyl refers to an alkyl as defined herein, attached to the remainder of the molecule via an oxo group (-C(O)-). In other words, an alkanoyl can be written “-C(O)- alkyl”.
  • a preferred alkanoyl is a C1-C6 alkanoyl, which has an alkyl chain of 1 to 6 carbon atoms.
  • alkanoyl examples include for instance, methanoyl, ethanoyl, propanoyl, isopropanoyl, butanoyl, pentanoyl, hexanoyl.
  • acylamino refers to a group of formula R-C(O)-NH- wherein R is a hydrocarbon group such as C 1 -C 6 alkyl, a C 3 -C 12 cycloalkyl or an aryl.
  • a preferred acylamino is a C 1 -C 6 acyl amino, which has a hydrocarbon chain of 1 to 6 carbon atoms.
  • ester refers to a -C(O)OR’ or R’C(O)O- group, wherein R’ is any hydrocarbon group, such as a C1-C6 alkyl, a C3-C12 cycloalkyl or an aryl.
  • R is any hydrocarbon group, such as a C1-C6 alkyl, a C3-C12 cycloalkyl or an aryl.
  • a preferred ester is a C1-C6 ester, which has a hydrocarbon chain of 1 to 6 carbon atoms.
  • an “alkoxycarbonyloxy” refers to a R”-C(O)-O- group where R” is an alkoxy.
  • halogen includes chlorine, fluorine, iodine, bromine, preferably chlorine or fluorine.
  • aminoalkyl refers to an alkyl as defined above, substituted by one or more (preferably one) amino (-NH 2 ) group.
  • alkylaminoalkyl refers to an alkyl as defined above, substituted by one or more (preferably one) alkylamino group as defined above.
  • hydroxyalkyl refers to an alkyl as defined above, substituted by one or more (preferably one) hydroxy (-OH) group.
  • alkoxyalkyl refers to an alkyl as defined above, substituted by one or more alkoxy as defined above.
  • thioalkyl refers to an alkyl as defined above, substituted by one or more (preferably one) -SH group.
  • haloalkyl refers to an alkyl as defined above, substituted by one or more halogen atoms. "Substituted” or “optionally substituted” includes groups substituted by one or several substituents, typically 1, 2, 3, 4, 5 or 6 substituents.
  • the substituents may be independently selected from C 1 -C 6 alkyl, aryl, C 3 -C 6 cycloalkyl, C 3 -C 6 cycloalkenyl, C 2 -C 6 heterocycle, C 1 -C 6 alkoxy, C 1 - C 6 alkylamino, C 1 -C 6 aminoalkyl-, C 1 -C 6 alkylaminoalkyl-, -N 3 , -NH 2 , –F, -I, -Br, -Cl, -CN, C 1 - C 6 alkanoyl, C 1 -C 6 carboxy esters, C 1 -C 6 acylamino, -COOH, -CONH 2 , OH, -NO 2 , -SO 3 H, C 1 -C 6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl,
  • a N-methyl luminol refers to a compound of formula (B) wherein R A is a methyl.
  • a N-(C6-C14 aryl optionally substituted) luminol refers to a compound of formula (B) wherein RA is a C6-C14 aryl optionally substituted.
  • a N-[(C6-C14 aryl)-(C1-C3 alkyl) optionally substituted] luminol refers to a compound of formula (B) wherein R A is (C 6 -C 14 aryl)-(C 1 -C 3 alkyl) optionally substituted.
  • N-benzyl luminol refers to a compound of formula (B) wherein R A is a benzyl.
  • N-methyl luminol refers to the compound wherein R A is a methyl.
  • N-methyl luminol can also be called “2,3-Dihydro-2-methyl-1,4-phthalazinedione” and has the following CAS number: 18393- 54-9.
  • a N-substituted luminol derivative refers to N-substituted luminol as defined above, having one or several substituents on the benzene ring of the phthalazinedione group.
  • the Invention relates to an Adeno-Associated Virus (AAV) having at least one chemically-modified tyrosine residue in its capsid. More precisely, the chemically modified AAV of the Invention comprises at least one chemical moiety of formula (A): wherein RA is -(Y)n-M, a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1- C3 alkyl) optionally substituted.
  • AAV Adeno-Associated Virus
  • the one or more substituents present on C 6 -C 14 aryl or (C 6 -C 14 aryl)-(C 1 -C 3 alkyl) may be of any type.
  • said substituents are selected from the group consisting of halogens, - NO 2 , C 1 - C 3 alkyl, C 1 -C 3 alkoxy, C 1 -C 3 hydroxyalkyl, and C 1 -C 3 haloalkyl.
  • R A is a C 1 -C 3 alkyl, a phenyl optionally substituted, or a benzyl optionally substituted. More preferably, R A is a methyl, phenyl or a benzyl.
  • R A is a methyl.
  • R A is a methyl.
  • R A is linked to the aromatic ring of a tyrosine residue in the AAV capsid.
  • AAV Adeno-Associated Virus
  • Wild- type AAV has two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs).
  • ORFs open reading frames
  • ITRs inverted terminal repeats
  • the 5’ and 3’ ORFs encode replication and capsid proteins, respectively.
  • the ITR contains 145 nucleotides and serves as the AAV genome replication origin and packaging signal.
  • a recombinant AAV refers to an AAV wherein an exogenous nucleic acid sequence e.g. a transgene sequence has been introduced in the viral genome.
  • Said exogenous nucleic acid sequence may be of any type and is selected in view of the intended use of the AAV.
  • said nucleic acid may comprises any RNA or DNA sequence.
  • the AAV of the invention is a recombinant AAV.
  • said recombinant AAV is to be used as a gene vector for in vivo or in vitro applications that means that the AAV of the invention is a recombinant AAV vector.
  • AAV as vector in gene therapy
  • a recombinant AAV for use as vector in gene therapy may comprise an exogenous gene expression cassette replacing the viral ORFs and placed between the two ITRs.
  • Said cassette may comprise a promoter, the gene of interest and a terminator. The promoter and the gene of interest are selected depending on the targeted tissue/organ and the condition to treat.
  • the recombinant AAV for use in gene therapy may comprise a DNA template for homologous recombination in cells.
  • a recombinant AAV can be used in combination with gene editing tools, for promoting homologous recombination in targeted cells, in vivo, in vitro or ex vivo.
  • the gene editing tools can be of any type, and encompass, without being limited to, CRISPR/Cas9, Zinc Finger Nuclease, meganuclease as well as RNA and DNA encoding said proteins.
  • the term “AAV” include all types of AAV, including wild-type AAV and recombinant or variant AAV.
  • AAV variants encompass, without being limited to, AAV having a mutated or a synthetic capsid such as AAV with hybrid capsid, pseudotype AAV as well as self-complementary AAV (scAAV).
  • the capsid of a wildtype AAV is composed of three overlapping capsid proteins called viral protein 1 (VP1), VP2, and VP3. More precisely, the capsid of a wildtype AAV is composed of total 60 copies of the viral protein subunits VP1, VP2 and VP3 in the ratio 1 ⁇ 1 ⁇ 10.
  • Genetic engineering of the capsid refers to amino acid modifications of said capsid protein(s), e.g. in their hypervariable loops.
  • an AAV having a genetically engineered capsid or “An AAV having a mutated capsid” refers to an AAV wherein one or several amino acid modifications has(ve) been introduced in at least one capsid protein (namely VP1, and/or VP2 and/or VP3) as compared to the wild-type version of said capsid protein.
  • an amino acid modification encompass the insertion, deletion or substitution of one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 20, 30, 40, 50, or 100) amino acids.
  • the AAV has a genetically mutated capsid, wherein the mutation (s) has(ve) not been performed on naturally occurring tyrosine residues or have not resulted in the insertion of tyrosine residues.
  • the AAV of the invention may have a wildtype capsid or may have a mutated capsid wherein the naturally occurring tyrosine residues are conserved.
  • the AAV of the invention is selected from wild-type AAV and recombinant or variant AAV with a wildtype AAV capsid.
  • the AAV of the invention is a recombinant AAV with a wildtype capsid.
  • the AAV is a recombinant AAV having a mutated capsid, namely one or more amino acid modifications in at least one capsid protein as compared to the corresponding parent capsid protein.
  • the AAV is a recombinant AAV having a mutated capsid, wherein the amino acid modification(s) is/are not concerned with any tyrosine residues naturally present in wild type capsid proteins.
  • a chemically-modified tyrosine residue means that at least one tyrosine present in the capsid of the virus has been chemically modified by covalent coupling with a chemical entity, typically by the covalent coupling of a said chemical entity on the phenyl ring of the tyrosine.
  • Said tyrosine is typically a surface exposed residue present in VP1, VP2 or VP3. For instance, there are around 360 tyrosine residues which are considered as exposed in AAV2 capsid.
  • a surface exposed tyrosine means that the tyrosine is reachable for covalent coupling.
  • tyrosine residues can be identified by molecular modelling of the capsid proteins or that of the whole capsid itself.
  • “at least one chemically-modified tyrosine residue” encompasses at least 1, 2, 3, 4, 5, 6, 7, 8, 910, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more chemically-modified tyrosine residue(s).
  • the chemically-modified AAV of the invention comprises several chemically modified tyrosine residues in its capsid. Said chemically-modified tyrosine may be present on VP1, and/or VP2 and/or VP3.
  • At least 0.1%, for instance at least 0.5, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40% or more of the surface exposed tyrosine residues of the capsid are chemically modified.
  • They are various serotypes of AAV which can be either wildtype or synthetic. All serotypes are contemplated in the framework of the invention.
  • a "serotype" is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes).
  • AAV includes various naturally and synthetic (e.g.
  • 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.
  • synthetic serotypes also include pseudotyped AAV, namely AAV resulting from the mixing of a capsid and genome from different viral serotypes, such as AAV2/5, AAV2/7, and AAV2/8 as well as AAV with hybrid capsids derived from multiple different serotypes such as AAV-DJ, which contains a hybrid capsid derived from eight serotypes.
  • Synthetic serotypes also encompass specific variants wherein a new glycan binding site is introduced into the AAV capsid are in particular described in WO2014144229 (disclosing in particular the AAV2G9 serotype).
  • Other AAV serotypes include those disclosed in EP2292779, and EP1310571.
  • AAV serotypes include those obtained by shuffling, as described in Koerber et al. (Molecular Therapy (2008), 16(10), 1703–1709), peptide insertion (e.g. Deverman et al., Nat Biotechnol (2016), 34(2), 204-209), or rational capsid design (reviewed in Büning et al., Curr Opin Pharmacol (2015), 24, 94-104).
  • the AAV is selected from naturally-occurring serotypes, preferably from the group consisting of AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9 and AAVrh10, more preferably AAV-2.
  • the AAV of the invention may be of AAV-2 or AAV-9 serotype.
  • the AAV can target a large variety of cells, tissues and organs. Examples of cells targeted by AAV encompasses, but are not limited to, hepatocytes; cells of the retina; i.e. photoreceptors, retinal pigmented epithelium (RPE),; muscle cells, i.e. myoblasts, satellite cells; cells of the central nervous system (CNS), i.e.
  • tissue and organs which 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, testes, thymus, thyroid, trachea, and the like.
  • Preferred cell types are hepatocytes, retinal cells, muscle cells, cells of the CNS, cells of the PNS and hematopoietic cells.
  • AAV liver, muscle, heart, eye, and brain.
  • the tropism of AAV can vary depending on their serotype.
  • AAV-2 can be used to transduce the central nervous system (CNS), kidney, and photoreceptor cells while AAV-8 is effective for transducing the CNS, heart, liver, photoreceptor cells, retinal pigment epithelium (RPE) and skeletal muscle.
  • the AAV can be produced by any methods known in the art, such as transient transfection in cell lines of interest e.g. in HEK293 cells as described in the Example section.
  • the AAV of the invention may have other amino acid(s) of the capsid which has been chemically modified.
  • the AAV may comprise one or several amino groups of the capsid which have been modified by the method disclosed in WO2017/212019, namely by reacting said amino group(s) in the capsid with a ligand bearing an isothiocyanate reactive groups.
  • the AAV of the invention may have one or several arginine residues of the capsid modified by glycation, e.g.
  • the AAV of the invention may comprise one or several cysteine residues of the capsid which have been modified by reacting said cysteine residue with a chemical reagent bearing a reactive group selected from a maleimide, a vinyl sulfonamide and a 3-(carboxy derivative)acrylamide.
  • the Invention relates to an adeno-associated Virus (AAV) having at least one chemically-modified tyrosine residue in its capsid, wherein said chemically-modified tyrosine residue is of formula (I): wherein: - R A is-(Y) n -M, a C 1 -C 6 alkyl, a C 6 -C 14 aryl optionally substituted, or a (C 6 -C 14 aryl)-(C 1 -C 3 alkyl) optionally substituted, - each R B is independently selected from the group consisting of a group of formula -(Y) n -M, a hydrogen or a substituent selected from the group consisting of a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C
  • AAV
  • RA is a C1-C3 alkyl, a phenyl optionally substituted, or a benzyl optionally substituted. More preferably, RA is a methyl, phenyl or a benzyl. Even more preferably, RA is a methyl.
  • the following moiety represents a tyrosine within a protein of the capsid (i.e. VP1, VP2, or VP3): , wherein represents a bond by which the tyrosine is attached to the rest of the protein.
  • the N-substituted luminol moiety(ies) can be added at position ortho of the phenol group in the tyrosine residue.
  • “k is 1” means that one moiety of formula (II) is attached to the tyrosine residue: wherein RA and RB are as defined herein.
  • the at least one chemically-modified tyrosine residue in the capsid can be represented by the formula (Ia): wherein RA and RB are as defined herein.
  • “k is 2” means that two moieties of formula (II) as defined above is attached to the tyrosine residue.
  • the at least one chemically-modified tyrosine residue in the capsid can be represented by the formula (Ib): wherein R A and R B are as defined herein.
  • each R A and R B in a moiety of formula (II) can be identical to or different from those of the other moiety of formula (II), preferably identical.
  • the two moieties of formula (II) attached to the tyrosine residue in formula (Ib) can be different or identical, preferably identical.
  • the chemically-modified AAV of the invention may have one or several chemically modified tyrosine residues of formula (Ia) and one or several chemically modified tyrosine residues of formula (Ib).
  • the tyrosine residues of formula (Ia) represent in mean at least 50% e.g. at least 60%, 70%, 80%, 90% or 95% of the total number of chemically-modified tyrosine residues found in a population of chemically-modified AAVs of the invention.
  • RA is a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted.
  • RA is: --(Y) n -M as defined below, - a C 1 -C 6 alkyl; - a C 6 -C 14 aryl optionally substituted by one or more (preferably one) groups chosen from halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1- C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, , -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, C2-C6 alkoxycarbonyloxy and (C6-C14 aryl)- (C1-C3 alkyl); or
  • said substituents are selected from the group consisting of halogens, C1-C3 alkyl, C1- C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.
  • RA is a C1-C3 alkyl (such as methyl), a phenyl, or a benzyl. More preferably, RA is a methyl or a benzyl. Even more preferably, R A is a methyl.
  • each R B is independently a group of formula -(Y) n -M (where Y, n and M are as defined above), a hydrogen or a substituent chosen from a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, with proviso that at least
  • At least one RB is –(Y)n-M.
  • RA is –(Y)n-M.
  • RB are not –(Y)n-M.
  • RA is – (Y)n-M and all RB are H.
  • the chemically-modified tyrosine residue of formula (I) is such that one or two (preferably one) R B is a group of formula -(Y) n -M. More preferably, the chemically-modified tyrosine residue of formula (I) is such that one or two (preferably one) R B is a group of formula -(Y) n -M, and the other R B are hydrogens.
  • each RB is independently a hydrogen or a substituent chosen from a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C 1 -C 6 carboxy esters, C 1 -C 6 acylamino, -COOH, -CONH 2 , -NO 2 , -SO 3 H, -CN, -CF 3 , C 1 -C 6 hydroxyalkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkylthio, C 1 -C 6 thioalkyl, C 2 -C 10 alkoxyalkyl, and C 2 -C 6 alkoxy
  • RA is as defined above, preferably not –(Y)n-M. “n is 1” means that the spacer Y is present. “n is 0” means that the spacer Y is absent.
  • the chemically-modified tyrosine residue of formula (I) is such that: - R A is a methyl or a benzyl; and - one or two (preferably, one) R B is a group of formula -(Y) n -M, and the other R B are hydrogens.
  • the moiety of formula (II) as defined above is of formula (II-c) or (II- d):
  • RA is a C1-C3 alkyl, a phenyl optionally substituted, or a C1-C3 alkyl-phenyl optionnally substituted, preferably a methyl or a benzyl.
  • Spacer Y Y is a spacer that links the N-substituted luminol moiety and the functional moiety M. Y may be present (when n is 1) or absent (when n is 0). When Y is absent, the N-substituted luminol moiety and M are directly linked to each other.
  • Y may be any chemical chain (e.g. hydrocarbon chain) which can comprise heteroatoms as well as cyclic moieties such as cycloalkyl, cycloalkenyl, aromatic groups, or heterocyclic moieties such as heterocycloalkyl or heteroaryl.
  • Y may comprise up to 1000 carbon atoms and even more.
  • the length and the chemical nature of the spacer may be optimized depending on the functional moiety “M” which is intended to be coupled with the tyrosine residues and the biological effect which is sought. Indeed, further to its linking function, Y may be used to refine the properties of the functional moiety “M”.
  • Y may decrease the steric hindrance of M with respect to the capsid, improve the accessibility of M for binding with a biological entity of interest, improve the binding of M with an entity of interest and/or increase the solubility of M.
  • Y is a chemical chain group comprising from 2 to 1000 carbon atoms, preferably from 2 to 500 carbon atoms, from 2 to 300 carbon atoms, e.g. from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 4 to 30 carbon atoms or from 4 to 20 carbon atoms.
  • Y is selected from the group consisting of polymers including homopolymers, copolymers and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains optionally interrupted by one or several heteroatomic groups (e.g.
  • spacer group Y may comprise several hydrocarbon chains, oligomer chains or polymeric chains (e.g.
  • any appropriate group such as –O-, –S-, -N(R)- with R being H or C1-C3 alkyl, -C(O)-, –NHC(O)-, - OC(O)-, -C(O)-O-C(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, NH-CS-, phosphodiester or phosphorothioate groups as well as cyclic or heterocyclic groups.
  • the group(s) (also called connectors) used to link the several hydrocarbon chains, oligomer chains or polymeric chains together result from the reactions used to connect these different chains together.
  • the connector may be -NHC(O)- in case of amide coupling reaction, “N” in case of reductive amination or a triazole derivative in case of click chemistry involving the reaction of an azido with an alkyne group.
  • Y may be selected from the group consisting of polyethers such as polyethylene glycol (PEG) and polypropylene glycol, polyvinyl alcohol (PVA), polyesters such as polylactate, polyacrylate, polymethacrylate, polysilicone, polyamide such as polycaprolactone and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), poly(D,L-lactic-co-glycolic acid) (PLGA), polymers of alkyl diamines, unsaturated or saturated, branched or unbranched, hydrocarbon chains optionally having an heteroatom such as O, NH and S on at least one end, and combinations thereof.
  • polyethers such as polyethylene glycol (PEG) and polypropylene glycol
  • PVA polyvinyl alcohol
  • polyesters such as polylactate, polyacrylate, polymethacrylate, polysilicone
  • polyamide such as polycaprolactone and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA)
  • alkyl diamine refers to NH2-(CH2)r-NH2 with r is an integer from 2 to 20, for instance from 2 to 10 such as 2, 3, 4, and 5.
  • a polymer of alkyl diamines (also known as polyamines) refers to a compound of formula NH2-[(CH2)r-NH]t-H with r being as defined above and t is an integer of at least 2, for example of at least 3, 4, 5, 10 or more.
  • Polymers of alkyl diamines of interest are, for instance, spermidine, and spermine.
  • Y can comprise at least one polyethylene glycol moiety comprising from 2 to 40 monomers, e.g. from 2 to 10 or 2 to 6 monomers.
  • Y may comprise from 2 to 10 triethyleneglycol blocks linked together by linkers.
  • Y may be a C12 hydrophilic triethylene glycol ethylamine derivative.
  • Y may be a saturated or unsaturated C2-C40 hydrocarbon chain, in particular a C10-C20 alkyl chain or a C2-C10 alkyl chain such as a C6 alkyl chain.
  • the alkyl chain may have a group such as NH, S or O on at least one end.
  • Y may be putrescine.
  • Y is selected from the group consisting of saturated or unsaturated, linear or branched C 2 -C 40 hydrocarbon chains, optionally substituted, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines and combinations thereof.
  • Y is selected from the group consisting of linear or branched C2-C20 alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of diamino alkyl and combinations thereof.
  • polyethylene glycol, polypropylene glycol, PLGA, pHPMA and polymer of alkyl diamines comprise from 2 to 40 monomers, preferably from 2 to 10 or from 10 to 20 monomers.
  • Y may comprise one or several (e.g.2, 3, 4 or 5) triethylene glycol blocks.
  • Y is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O- C(O)-, -N(R)- with R being H or a C1-C3 alkyl, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and - NH-CS-; and/or - C 5 -C 20 carbocyclic moieties such as cycloalkyl, cycloalkenyl, or aromatic groups; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyl
  • Y is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -N(R)- with R being H or a C1-C3 alkyl , -C(O)-, -NHC(O)-, and -OC(O)-; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms; and optionally having an heteroatomic group chosen from -O-, -S-, -N(R)- with R being H or C1- C 3 alkyl, -O-N(R)- with R being H or C 1 -C 3 alkyl, -N(C 1 -
  • the heterocycle may be a triazolyl or a triazolyl fused with another cycle such as those selected from the following groups:
  • the symbol represents the bond by which such heterocyclic moieties are attached to the remainder part of the spacer Y.
  • Such heterocyclic moieties can be present in the spacer Y when its synthesis route comprises a step wherein an azido reacts with a strained alkyne (e.g. DBCO).
  • Y is a spacer of formula (III): Y 1 -Y 2 -Y 3 (III), wherein - each of Y1 and Y3 is independently 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 (e.g.
  • each of Y1 and Y3 is selected from the group consisting of polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of diamino alkyl, linear or branched C 2 -C 20 alkyl chains (optionally interrupted by one or several heteroatoms (e.g.
  • Y2 is a triazolyl or is chosen among the above heterocyclic moieties (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii).
  • Y has at one of its extremities (typically, the extremity linked to M) a heteroatomic group chosen from -O-N(R)- with R being H or C 1 -C 3 alkyl, and -N(C 1 -C 3 alkoxy)- , preferably chosen from -O-N(Me)-, -O-NH- -N(OMe)-.
  • Y may be one of the following formulae: wherein q is an integer from 2 to 10, and R is H or methyl.
  • ⁇ Functional moiety M The functional moiety “M” may be of any type. “M” is typically selected depending on the biological effect which is sought when chemically modifying the capsid of the AAV. Alternatively, M may be a reactive group selected so as to enable a subsequent step of coupling, such as a click- chemistry reactive group. M may be also a ligand enabling the specific non-covalent coupling of another entity at the surface of the AAV such as biotin/strept(avidin), cation binding groups (e.g.
  • M may be also a labelling moiety.
  • M may comprise a moiety selected from a chemical reactive group such as a click- chemistry reactive group, a targeting agent, a steric shielding agent, a labelling agent, an oligonucleotide, or a drug.
  • M may be also a (nano)-particle, including a magnetic (nano-) particle and a quantum dot.
  • M may be an iron, stain, silicium, gold or carbon (nano)-particle.
  • M comprises, or consists of, a labeling agent, e.g. a fluorescent dye such as fluorescein, rhodamine, boron-dipyrromethene (Bodipy) dyes, and alexa fluor, or a radionuclide.
  • a labeling agent e.g. a fluorescent dye such as fluorescein, rhodamine, boron-dipyrromethene (Bodipy) dyes, and alexa fluor, or a radionuclide.
  • a steric shielding agent e.g. an agent able to mask certain epitopes of the capsid, whereby avoiding the binding of neutralizing antibodies.
  • M may be a polyethylene glycol (PEG), pHPMA or a polysaccharide.
  • M comprises, or consists of, a steric shielding agent able to mask tyrosine residues, whereby proteasome-degradation of the AAV in cellulo is avoided.
  • M may be an oligonucleotide such as messenger RNA (mRNa) or antisense oligonucleotides such as small interferent RNA (siRNA), shRNA, snoRNA and meroduplex (mdRNA).
  • M comprises, or consists of, a targeting agent, namely a ligand enabling to target a specific organ, tissue, cell, or a protein of interest, such as a cell surface protein, receptor or oligosaccharides, e.g. a cell surface protein that is present at the surface of a particular cell line or a tumoral cell.
  • the targeting agent can be a cell-type specific ligand, namely a ligand enabling to target a specific type of cell.
  • a ligand may enable to modify the tropism of the AAV, namely its capacity to selectively infect and/or transduce a given cell line, tissue or organ.
  • M may be a ligand which specifically binds to a membrane biological entity (e.g. a membrane receptor) of the targeted cell.
  • Said ligand may be of any type e.g. a peptide a protein, an oligosaccharide or a small chemical entity.
  • M may be a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, Angiopep-2, muscle targeting peptides a membrane receptor or a fragment thereof, CB1 and CB2 ligands, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH (also called nanobody), a ScFv, a aptmer, a peptide aptamer, a small chemical molecule known to bind to the targeted biological entity and the like.
  • M is an antibody including a full length antibody or an antigen-binding domain derived from an antibody.
  • the term “antibody” refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding domain, regardless whether it is 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, mutated, and grafted antibodies.
  • the term “antibody” also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments (e.g.
  • VHH from single-chain antibody
  • antigen-binding domain or “antigen-binding fragment” refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. Where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as "epitope" or "antigenic determinant.”
  • An antigen-binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).
  • an antigen-binding fragment or domain contains at least a portion of the variable regions (heavy and light) of the antibody sufficient to form an antigen binding site (e.g., one or more CDRs, and generally all CDRs) and thus retains the binding specificity and/or activity of the antibody.
  • an antigen binding site e.g., one or more CDRs, and generally all CDRs
  • a “full-length antibody” also called herein immunoglobulin of Ig refers to a protein having the structure that constitutes the natural biological form of an antibody, including variable and constant regions.
  • Full length antibody covers both monoclonal and polyclonal full- length antibodies and also encompasses wild-type full-length antibodies, chimeric full-length antibodies, humanized full-length antibodies, the list not being limitative.
  • the structure of full-length antibodies is generally a tetramer. Said tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (typically having a molecular weight of about 25 kDa) and one "heavy” chain (typically having a molecular weight of about 50-70 kDa).
  • light chains are classified as kappa 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.
  • isotype as used herein is meant any of the classes of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • the known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.
  • “M” comprises, or consists of, a cell-type specific ligand derived from proteins such as transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ⁇ FGF.
  • “M” comprises, or consists of, a cell-type specific ligand derived from mono- or polysaccharides, e.g. comprising one or several galactose, mannose, mannose-6- phosphate, N-acetylgalactosamine (GalNac) and bridged GalNac and sialic acid and derivatives thereof (such as Neu5Ac, Neu5Ac ⁇ 2-6Gal, Neu5Ac ⁇ 2-8Neu5Ac).
  • the mono- or polysaccharides can be natural or synthetic.
  • “M” comprises, or consists of, a cell-type specific ligand derived from vitamins such as folic acid.
  • the cell-type specific ligand included in “M” may be derived from, or may consist in, a muscle targeting peptide (MTP).
  • Said ligand may comprise an amino acid sequence selected from the group consisting of: ASSLNIA (SEQ ID NO: 1); WDANGKT (SEQ ID NO: 2); GETRAPL (SEQ ID NO: 3); CGHHPVYAC (SEQ ID NO: 4); HAIYPRH (SEQ ID NO: 5), cyclic CQLFPLFRC (SEQ ID NO: 6) or the sequence of SEQ ID NO:7 as shown below: RXRRXRRXRFQILYRXRXRXRX (SEQ ID NO:7) wherein X is either an amino hexanoic acid residue or a beta-alanine residue as shown in the sequence listing.
  • cyclic CQLFPLFRC of SEQ ID NO:6 refers to:
  • M is a cancer cell targeting peptide and comprises a peptide such as RGD, including cyclic RGD.
  • M is a cell type targeting ligand selected from antibodies and fragments thereof.
  • M comprises a peptide moiety, such as a muscle targeting peptide (MTP)
  • said peptide moiety may comprise a chemical modification at its N-terminus or C-terminus.
  • “M” comprises, or consists of, a cell-type specific ligand derived from small molecules or hormones such as naproxen, ibuprofen, cholesterol, progesterone or estradiol.
  • “M” comprises, or consists of, a CB1 and/or a CB2 ligand, for instance: Galactose- derived ligands, which are recognized by asialoglycoprotein receptor (ASPGPr), can be used to specifically target hepatocytes.
  • ASPGPr asialoglycoprotein receptor
  • M is a ligand for specifically targeting hepatocytes and comprises at least one moiety of formula (Va), (Vb) or (Vc):
  • M is a ligand for targeting muscle cells, in particular skeletal muscle cells and comprises at least one mannose-6-phosphate moiety:
  • M is a ligand for photoreceptors or neuronal cells and comprises at least one mannose moiety of formula (Vf):
  • M is a ligand for Siglecs proteins (sialic-acid-bending immunoglobulin-like lectins).
  • M is sialic acid moiety or a derivative thereof.
  • sialic acid moiety and derivatives thereof include a moiety comprising one or more N-acylated neuraminic acid units and optionally one or more other saccharide units such as a galactose moiety. More specifically, M may be a sialic acid moiety or a derivative thereof, said moiety comprising or consisting of at least one moiety of formula (Vg): wherein R 5 is an alkyl, an aryl, a heteroaryl, haloalkyl (such as -CH 2 -Hal, where Hal is a halogen), -OR6, -NR7R8, -SR9, -CH2OR10, -CH2NR11R12, or -CH2SR13 where R6, R7, R8, R9, R10, R11, R12, and R13 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group.
  • Vg formula
  • R5 is an alkyl, -OR6, -CH2OR10 , -CH2-Hal, where Hal, R6, R10 are as defined herein.
  • R 5 is methyl, -CH 2 OH, or -CH 2 -F.
  • M is chosen from a Neu5Ac, Neu5Ac ⁇ ⁇ ⁇ ⁇ Gal and Neu5Ac ⁇ 2- 8Neu5Ac moiety.
  • Neu5Ac moiety refers to N-acetylneuraminic acid moiety.
  • Neu5Ac can be represented by the following formula (Vh): As used herein, Neu5Ac ⁇ ⁇ ⁇ ⁇ Gal moiety refers to a moiety consisting of a N-acetylneuraminic acid unit and a galactose unit bonded by a ⁇ ⁇ ⁇ ⁇ ⁇ bond.
  • Neu5Ac ⁇ ⁇ ⁇ ⁇ Gal moiety can be represented by the following formula (Vi): As used herein, Neu5Ac ⁇ 2-8Neu5Ac moiety refers to a moiety consisting of two N- acetylneuraminic acid units bonded by a ⁇ ⁇ ⁇ ⁇ bond.
  • Neu5Ac ⁇ 2-8Neu5Ac can be represented by the following formula (Vj):
  • M is multivalent, which means 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.
  • M may comprise a polyfunctional linker bearing several (e.g. at least 2, 3, 4, 5, or 6) cell-type ligands.
  • the cell-type ligands can be the same or different.
  • “M” may comprise a moiety of formula (VI): with n is a enter from 1 to 100, preferably from 1 to 20.
  • M is an oligosaccharide chosen from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, ,galactan and mannan.
  • M may comprise both a labelling moiety such as a fluorescent label or a radionuclide and a cell-type specific ligand.
  • M may be: namely a muscle targeting peptide of SEQ ID NO:1 linked to K-FITC.
  • M comprises, or consists of a cell targeting agent, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a aptmer, a peptide aptamer, a vitamin and small chemical molecules such as drugs e.g.
  • a cell targeting agent preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy
  • 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 ⁇ FGF, a mono- or a polysaccharide comprising one or several galactose, mannose, N- acetylgalactosamine residues, bridge GalNac, or mannose-6-phosphate, sialic acid and derivatives thereof (e.g.
  • EGF Epidermal Growth Factor
  • ⁇ FGF basic Fibroblast Growth Factor
  • M is a chemical reactive group, more preferably a “biocompatible chemical reactive group”.
  • M can enable to create a covalent interaction between the AAV and an entity of interest, without significantly altering the functionality of the AAV (and thus in a biocompatible way).
  • the functional moiety may comprise a chemical reactive group which can promote the formation of a covalent bond with the entity of interest.
  • the functional moiety may comprise a chemical reactive group suitable to create a covalent bond by click-chemistry or by bioconjugation reaction.
  • Bioconjugation reactions encompass reactions between amino acids such as lysine, cysteine or tyrosine with reactive groups as detailed in Koniev, O., Wagner, A, Chem. Soc. Rev., 44, 5495 (2015).
  • M is a click-chemistry reactive group, also called hereunder a “click-chemistry group”.
  • a “click-chemistry group” refers to any reactive chemical group that can be involved in a click chemistry reaction.
  • M is not a thiol (-SH).
  • “Click-reaction” or “Click-chemistry” is a concept introduced by Sharpless in 2001.
  • “Click chemistry” generally refers to chemical reactions characterized by high yields, high chemoselectivity, which are simple to conduct and which generate inoffensive by-products.
  • “Click reactions” can be typically conducted in complex media with high efficiency. Click reactions are typically used to create covalent heteroatom links (C-X-C) between two entities of interest.
  • Click chemistry one can refer to Kolb et al., Angew. Chem. Int. Ed.2001, 40, 2004- 2021) and to Rudolf et al., Current opinion in Chemical Biology, 2013, 17:110-117.
  • click chemistry reactions include, but are not limited to, Staudinger Ligation, azido- ene or azido-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.
  • the click chemistry reaction is not thiol-ene or thiol-maleimide reaction.
  • M may comprise, or consist of, an azido (-N 3 ), phosphine such as a triarylphosphine, aldehyde, ketone, hydrazide, oxyamine, nitrile oxide, oxime, hydroxymoyl chloride, chlororoxime, nitrile imine, hydrazone, hydrazonoyl chloride, chlorohydrazone, tetrazine, tetrazole isonitrile, aryl halide, aryl boronate, oligo-histidine, nickel- complex or nickel ligand.
  • M is N3.
  • the spacer Y when present, is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -NH-, -C(O)-, -NHC(O)-, and - OC(O)-; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms, preferably a triazolyl optionally fused with a dibenzoazepinyl; and optionally having at least one of its extremities, an heteroatomic group chosen from - O-, -S-, -NH-, -C(O)-, -NHC(O)-, and -OC(
  • Y is of formula (III) as described above.
  • the spacer Y when present, is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -NH-, -C(O)-, -NHC(O)-, and - OC(O)-; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms, preferably a triazolyl optionally fused with a dibenzoazepinyl; and optionally having at least one of its extremities, an heteroatomic group chosen from - O-, -S-,
  • Neu5Ac Neu5Ac ⁇ 2-6Gal, Neu5Ac ⁇ 2-8Neu5Ac), a MTP selected from SEQ ID NO:1 to SEQ ID NO:7, and vitamins such as folic acid.
  • Y is of formula (III) as described above.
  • ⁇ Y has at one of its extremities (typically, the extremity linked to M) a heteroatomic group chosen from -O-N(R)- with R being H or C1-C3 alkyl, and -N(C1-C3 alkoxy)-, preferably chosen from -O-N(Me)-, -O-NH- -N(OMe)-; and ⁇ M is a saccharide, a disaccharide or an oligosaccharide preferably chosen from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, galactan and mannan.
  • Y may be a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms: ⁇ having at one of its extremities (typically, the extremity linked to M) a heteroatomic group chosen from -O-N(R)- with R being H or C 1 -C 3 alkyl, and -N(C 1 -C 3 alkoxy)-, preferably chosen from -O-N(Me)-, -O-NH- -N(OMe)-; ⁇ optionally having at the other one of its extremities, an heteroatomic group chosen from -O-, - S-, -NH-, -C(O)-, -NHC(O)-, and -OC(O)-, preferably -O-; ⁇ optionally interrupted by one or more heteroatomic groups chosen from -O-, -S-, -NH-,
  • the -(Y) n -M may be any one of the following formulae: with p is an integer from 2 to 10, R is H or methyl, Ms is a monosaccharide (e.g. Mannose of GalNAc), a disaccharide moiety, or an oligosaccharide preferably chosen from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, galactan and mannan.
  • p is an integer from 2 to 10
  • R is H or methyl
  • Ms is a monosaccharide (e.g. Mannose of GalNAc), a disaccharide moiety, or an oligosaccharide preferably chosen from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligo
  • the AAV is preferably a recombinant AVV, more preferably a recombinant AAV vector.
  • the AAV may have a “naturally-occurring” capsid or a genetically modified capsid, namely comprising one or several mutations in at least one capsid protein, namely VP1, VP2 and/or VP3.
  • the AAV may be of a serotype selected from AAV1, AAV4, AAV6, AAV7, AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9 and AAVrh10, preferably AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9 and AAVrh10 and more preferably AAV-2 or AAV- 9.
  • the AAV is of a synthetic serotype.
  • the AAV of the invention may have at least one additional chemically modified amino acid residue in the capsid, which is different from a tyrosine residue, e.g. an arginine or a lysine residues.
  • said amino acid residue bears a modified amino group of formula (VIII) in its side chain: wherein: - N* being the nitrogen of the amino group of the side chain of an amino acid residue, e.g of a lysine residue or arginine residue, and - Y’ has the same definition as Y, n’ is 0 or 1, and M’ has the same definition as M. It is understood that Y’, n’, and M’ can be the same or different as those present in the at least one chemically-modified tyrosine as described above. Said modification on the amino group can be introduced as described in WO2017212019, the content of which being incorporated herein by reference.
  • said amino acid residue is a modified cysteine residue of formula (IX): wherein: - X” is selected from the group consisting of: wherein - Z is -O-, -S-, or -N(R4)-, - R 1 , R 2 , R 3 , and R 4 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, said group being optionally substituted, - w is 0 or 1, - R0 is a hydrogen, a halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, said group being optionally substituted, - Y” has the same definition as Y, n” is 0 or 1, and M” has the same definition as M.
  • - X is selected from the group consisting of: wherein - Z is -O-, -S-, or -N(R4)-, - R 1 , R 2
  • Y”, n”, and M can be the same or different as those present in the at least one chemically-modified tyrosine as described above.
  • the chemical modification(s) of the capsid of the AAV may modify one or several biological functionalities and/or properties.
  • said chemically-modified AAV may have one or several modified biological properties as compared to the same but non-chemically modified AAV, such as: - A modified tropism e.g.
  • an increased selectivity of the AAV towards a specific organ, tissue or cell (either administered in vivo or transducing tissues or cells in culture) or a shifted selectivity of the AAV from one tissue/organ/cell to another, and/or -
  • An altered immunoreactivity of the AAV e.g. a decreased immunogenicity of the AAV and/or a decreased affinity for neutralizing antibodies, and/or said AAV triggers an altered humoral response when administered in vivo, e.g. do not generate AAV-directed neutralizing antibodies - An increased infectivity efficiency of the AAV particles, and/or - An increased transduction efficacy of the AAV towards a specific cell, tissue or organ.
  • the chemically-modified AAV of the invention may have a higher transduction efficiency, which may result from increased intracellular trafficking to the nuclei, a decrease in proteasome-degradation, more efficient intranuclear decapsidation, more rapid vector genome stabilization and/or from a decrease in interaction with neutralizing antibodies and/or from a reduction of antibody-mediated clearance of AAV in vivo, as compared to the non-chemically modified AAV.
  • the AAV may have a higher infectivity efficiency and/or an increase in selectivity for a given cell, tissue or organ as compared to the non-chemically modified AAV either in vivo or in vitro.
  • the AAV when used as a drug, e.g. as a gene vector, such modified properties may result in an improvement in the therapeutic index of the AAV, which may result from decrease in the dose to administer to the patient to achieve the sought therapeutic effect and/or a decrease in the toxicity of the AAV.
  • the chemically-modified AAV of the invention shows a preferential tropism for an organ or cell selected from liver, heart, brain, joints, retina and skeletal muscle.
  • the chemically-modified AAV of the invention shows a preferential tropism for cultured cells selected from, but not limited to, hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS).
  • HSC hematopoietic stem cells
  • iPS induced pluripotent stem cells
  • an object of the present invention is a method for chemically-modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, which comprises incubating said AAV with a chemical reagent bearing a N- substituted luminol moiety under conditions conducive for reacting said chemical reagent with a tyrosine residue present in the capsid of the AAV so as to form a covalent bound, wherein the N- substituted luminol moiety is a moiety of N-(C1-C6 alkyl) luminol, N-(C6-C14 aryl optionally substituted) luminol or N-[(C6-C14 aryl)-(C1-C3 alkyl) optionally
  • the coupling conditions typically enable the in-situ activation of N-substituted luminol moiety into an oxidized entity able to react with phenyl group in tyrosine residues.
  • the coupling conditions enable the formation of nitrogen-centered radical in the N-substituted luminol moiety.
  • Such an oxidation can be performed by any methods known by the skilled artisan to activate N- substituted luminol preferably into single radical, for instance by an enzymatic system such as horse radish peroxidase/H2O2 or laccase/O2 or by electrochemistry.
  • the Applicants showed that chemical reagents bearing N-substituted luminol have an oxidation potential of about 0.6 V versus Saturated Calomel Electrode (SCE) regardless the substituent present on the nitrogen atom or on the aromatic ring of the N-substituted luminol and that the resulting oxidized entity formed at said potential is reactive towards the phenyl group of tyrosine.
  • SCE Saturated Calomel Electrode
  • the Applicants showed that subjecting AAV to such a low voltage potential does not impair its structural and functional integrity.
  • the Applicants managed to specifically and efficiently modify tyrosine residues in the AAV capsid by incubating the AAV particles with a chemical reagent bearing a N-substituted luminol moiety during a short time under a constant low potential difference.
  • the chemically-modified AAV of the invention is preferably prepared by electrochemistry.
  • electrochemistry refers to branch of chemistry wherein the reaction between entities of interest is triggered by subjected said entities to an electrical potential difference.
  • the method of the Invention refers to a method for chemically- modifying the capsid of an AAV with a chemical reagent bearing a N-substituted luminol moiety by electrochemistry, namely by contacting the AAV and the chemical reagent under a potential difference enabling the electro-activation of the chemical reagent.
  • the potential difference is such that it does not significantly impair the integrity of the AAV particles.
  • the “electro-activation of the chemical reagent of the invention” refers to the oxidation of chemical reagent of the invention into an oxidized form able to react with the phenyl group present in tyrosine residues by means of a potential difference.
  • the Invention refers to a method for electro-chemically modifying an adeno-associated Virus (AAV), by contacting the AAV with a chemical reagent of formula (X) wherein: R A and R B are as described above, under electrochemical conditions conducive for the reaction of the chemical reagent of formula (X) with a tyrosine residue present in the capsid of the AAV so as to form a covalent bound.
  • AAV adeno-associated Virus
  • Preferred chemical reagents of formula (X) are: wherein R A is a C 1 -C 3 alkyl, a phenyl optionally substituted, or a phenyl-(C 1 -C 3 alkyl) optionnally substituted, preferably a methyl or a benzyl, and Y, n and M being as defined above. More particularly, such chemical reagent of formula (X) is incubated with the AAV so as to obtain at least one chemically-modified tyrosine residue in the capsid of formula (I) as defined herein.
  • the electrochemical conditions are selected so as to enable the oxidation of the chemical reagent of formula (X) without impairing the integrity of the AAV.
  • the “potential conditions” refer to a potential difference with respect to a reference electrode which enables to oxidize the chemical reagent preferably into a radical. This potential difference is close to the oxidation potential of the chemical reagent (determined with respect to said reference electrode).
  • the potential difference to apply is generally included in a range from the oxidation potential of the chemical reagent (OP) minus 200 mV to OP plus 500 mV, preferably from OP minus 150 mV to OP plus 400 mV.
  • the potential difference to apply is equal or substantially close to the oxidation potential (OP) of the chemical reagent, e.g. equal to the oxidation potential of the chemical reagent ⁇ 200mV or ⁇ 150 mV, more preferably ⁇ 100 mV such as ⁇ 50 mV or ⁇ 25 mV.
  • the potential difference to apply to oxidize the chemical reagent in the method of the invention varies, among others, depending on the reference potential of the reference electrode used to implement the method.
  • the oxidation potential can be identified by standard proceeding well known by the skilled artisan, such as cyclic voltammetry . One can refer to the method described in the Example section (see Example 2).
  • the AAV and the chemical reagent are subjected to a potential difference (or equivalently a “voltage”).
  • Any suitable electrochemical device may be used to apply the voltage.
  • the voltage is applied by means of an electrochemical device comprising a three-electrode system, namely a working electrode, an auxiliary electrode and a reference electrode.
  • the working electrode refers to the electrode on which the reaction of interest occurs. Depending on whether the reaction on the electrode is a reduction or an oxidation, the working electrode is called cathodic or anodic, respectively.
  • the working electrode is the anode.
  • the auxiliary electrode also called counter electrode
  • the electrochemical device can further comprise: a vial, electrical connector means and a mean to control the potential difference between the reference electrode and the working electrode, typically a potentiostat.
  • a vial e.g. a plastic or glass vial
  • the AAV and the chemical reagent are mixed in an appropriate buffer and the resulting mixture is placed in an appropriate vial (e.g. a plastic or glass vial) in which three electrodes are plunged.
  • the three electrodes are connected with appropriate electrical connector means to a potentiostat.
  • the auxiliary electrode may be isolated e.g. by using a glass frit in order to avoid the formation of byproducts.
  • the potentiostat is used to maintain the potential difference between the reference electrode and the working electrode at a constant value enabling the oxidation of the chemical reagent selectively.
  • anodes, cathodes and reference electrodes that can be used in electrochemical processes are well-known to the skilled artisan.
  • material from which anodes can be made include, but are not limited to, carbon (e.g. glassy carbon or graphite), lead bronze, tungsten, niobium, copper, magnesium, titanium, zinc, stainless steel, platinum, gold, silver, aluminum, boron doped diamond, tin, nickel, cobalt, preferably carbon anode (e.g. glassy carbon or graphite).
  • Examples of material from which cathodes can be made include, but are not limited to, nickel, platinum, silver, lead bronze, tungsten, niobium, copper, magnesium, titanium, zinc, stainless steel, gold, aluminum, boron doped diamond, tin, nickel, cobalt, preferably platinum cathode.
  • Examples of reference electrodes include, but are not limited to, Standard hydrogen electrode (SHE), Normal hydrogen electrode (NHE), Reversible hydrogen electrode (RHE), Saturated calomel electrode (SCE), Copper-copper(II) sulfate electrode (CSE), Silver chloride electrode, Palladium-hydrogen electrode, dynamic hydrogen electrode (DHE), and Mercury- mercurous sulfate electrode (MSE), preferably silver chloride electrode.
  • the electrochemical device used to apply the voltage comprises: - a silver chloride electrode as a reference electrode, - a platinum electrode as a catho
  • the shape and the morphology of the electrodes is not particularly limited.
  • a carbon electrode can be reticulated, laminar or crucible.
  • Examples of electrode shapes include, but are not limited to, a plate, a wire, or a flat rod.
  • the size of the electrodes can be adjusted by the skilled artisan, depending on the scale of the process, in particular the volume of the incubation medium.
  • the surface of the electrodes in contact with the incubation medium can also be adjusted by the skilled artisan. Preferably, at least 20% 30%, 40%, 50%, 60% , 70%, 80%, 90% of the total surface of each electrode is in contact with the incubation medium.
  • the voltage may be set and controlled by any suitable device, typically a potentiostat connected to the electrodes (i.e. cathode, anode, reference electrode) of the electrochemical device.
  • the voltage is set at a value that allows the activation (e.g. the oxidation) of the N-substituted luminol moiety of the chemical reagent.
  • the voltage to be applied can be easily determined by the skilled artisan, in particular through the determination of the oxidation potential of the chemical reagent versus a reference electrode, in the incubation medium of interest (i.e herein the buffer of interest) by cyclovoltammetry.
  • the potential difference to apply during the incubation is generally included in a range from the oxidation potential of the chemical reagent (OP) minus 200 mV to OP plus 500 mV, preferably from OP minus 150 mV to OP plus 400 mV.
  • the potential difference to apply is equal or substantially close to the oxidation potential (OP) of the chemical reagent, e.g. equal to the oxidation potential of the chemical reagent ⁇ 150 mV, more preferably ⁇ 100 mV such as ⁇ 50 mV or ⁇ 25 mV.
  • the incubation is carried out under a voltage equal to such oxidation potential of the chemical reagent ⁇ 150 mV, preferably ⁇ 100 mV or ⁇ 50 mV, more preferably ⁇ 40 mV or ⁇ 30 mV, such as ⁇ 20 mV or ⁇ 10 mV.
  • the oxidation potential of the chemical reagent used in the Invention is about 0.75 V vs. Ag/AgCl in saturated KCl.
  • the incubation may be carried out under a voltage selected from values in the range from + 0.55 V to 1.25 V vs. Ag/AgCl in saturated KCl, preferably between+ 0.60 V to 1.0 V vs.
  • the incubation may be carried out under a voltage between +0.55 and +0.65 V vs. SCE, preferably between +0.57 and +0.63 V vs. SCE, more preferably about + 0.6 V vs. SCE, wherein the reference potential of SCE is +0.241 V.
  • the incubation may be performed in an aqueous buffer having a pH from 5 to 11, preferably from 7 to 10, e.g. from 7.0 to 8.0, e.g. about 7.4.
  • the concentration in buffer agent is at least 30 mM, preferably at least 50 mM and up to 1 M.
  • the buffer may be selected from appropriate biocompatible buffers, e.g TRIS buffer, sodium carbonate - sodium bicarbonate buffer, phosphate buffer e.g. PBS or Dulbecco's phosphate- buffered saline (dPBS), or Good’s buffer.
  • the incubation time during which the potential difference is applied may vary depending on several parameters such as (i) the volume of the buffer and the dimension of the vial, (ii) the surface of the electrodes being in contact with the incubation medium (iii) the amounts of chemical reagent and the amount of AAV to chemically modify, (iv) the chemical reagent, (v) the solubility of the chemical reagent, (vi) the voltage and (vii) the stirring rate.
  • the incubation may last from few seconds to several hours, for instance from 5 seconds to 120 min, for instance from 10 seconds to 60 min, e.g. from 15 seconds to 30 minutes, e.g. from 30 seconds to 10 minutes, e.g.
  • an effective coupling is obtained within 5 min, e.g. within 1 min of incubation under potential difference.
  • the temperature of incubation is typically from 10°C to 40°C.
  • the incubation is performed at room temperature, i.e. at a temperature from 18°C to 30°C, e.g. at around 20°C.
  • the reaction is performed under stirring, preferably under orbital stirring. In some other embodiments, the reaction is performed without stirring.
  • the AAV titer is typically from 1E11 to 1E14 vg/mL, for instance 1E12 vg/ml
  • concentration of the chemical reagent may be from 0.01 mM to 50 mM, for instance from 0.1 mM and 20 mM, such as from 0.1 to 10.0 mM or such as 0.5 to 5.0 mM.
  • the ratio of the chemical reagent concentration to the AAV particles may be from 3E2 to 3E7.
  • the method of the Invention refers to a method for chemically- modifying the capsid of an AAV with a chemical reagent bearing a N-substituted luminol moiety, using an enzymatic system, namely by incubating the AAV and the chemical reagent in the presence of an enzymatic system enabling the activation of the chemical reagent.
  • the chemical reagent is a compound of formula (X) as described above.
  • the enzymatic system may in particular consist of a combination of an enzyme and oxidant, such as a peroxidase (e.g. horseradish peroxidase) with H 2 O 2 , or a laccase with O 2 .
  • the pH, buffer, temperature and duration of incubation, and the concentration of the chemical reagent and of the AAV may be similar to those described above for the electrochemical conditions.
  • the method of the invention may comprise one or several additional steps prior to, or after the step of incubation (e.g. electrochemical or enzymatic incubation) as described above.
  • the method of the invention may comprise a step of providing or producing the AAV particles to be chemically modified.
  • the method of the invention may also comprise a step of providing or preparing the chemical reagent.
  • the chemical reagent can be produced by synthetic routes.
  • a chemical reagent of formula (X) may be prepared from methyl-hydrazine and a phtalic anhydride or phtalate, which can be substituted by a functionalizable group.
  • a functionalizable group As illustration only, one can refer to the synthesis of compounds 7, or 12 described in the Example section.
  • a chemical reagent of formula (X) wherein Y has, at its the extremity linked to M, a heteroatomic group chosen from -O-N(R)- with R being H or C 1 -C 3 alkyl, and -N(C 1 -C 3 alkoxy)-, and M is an oligosaccharide, a saccharide or a disaccharide, the corresponding precursor having an end function being -O-NH(R) and -NH(C1-C3 alkoxy) respectively, can be contacted with said oligosaccharide.
  • the method of the Invention can also comprise a step of determining the oxidation potential of the chemical reagent versus a reference electrode such as Ag/AgCl in saturated KCl or saturated calomel electrode (SCE).
  • the method of the invention may also comprise one or several additional steps following the step of incubation, such as: - a step of removing the unreacted reagent, e.g.
  • the method of the invention may further comprise a step of chemically modifying an amino acid residue other than tyrosine residue of the capsid of the AAV.
  • said additional chemically-modified amino acid residue may bear an amino group (e.g. lysine, arginine) or may be a cysteine.
  • the method of the invention may comprise a step of incubating the AAV with a chemical reagent of formula (XI): under conditions conducive to promote for reacting said chemical reagent with the amino group of an amino acid residue, e.g. a lysine residue or an arginine residue, present in the capsid of the AAV so as to form a covalent bound.
  • a step enables to chemically modify an amino group present in an amino acid residue of the capsid according to the following formula: wherein: - N* being the nitrogen of the amino group of an amino acid residue, e.g.
  • a step may be performed in an aqueous buffer, such as TRIS buffer, at a pH from 8 to 10, e.g at a pH of about 9.3 and a temperature from 10°C to 50°C, e.g. at room temperature. More details concerning the implementation of such a step can be found in WO2017/212019, the content of which being incorporated herein by reference.
  • the method of the invention may comprise a step of incubating the AAV with a chemical reagent of formula (XIIa), (XIIb) or (XIIc): wherein - Z is -O-, -S-, or -N(R 4 )-, - R 1 , R 2 , R 3 , and R 4 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, said group being optionally substituted, - w is 0 or 1, - R0 is a hydrogen, a halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, said group being optionally substituted, - Y” has the same definition as Y, n” is 0 or 1, and M
  • Y”, n”, and M can be the same or different as those present in the at least one chemically-modified tyrosine as described above.
  • a step enables to chemically modify a cysteine residue of the capsid according to the following formula: wherein: - X” is selected from the group consisting of: wherein - Z is -O-, -S-, or -N(R 4 )-, - R1, R2, R3, and R4 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, said group being optionally substituted, - w is 0 or 1, - R0 is a hydrogen, a halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, said group being optionally substituted, - Y” has the same definition as Y, n” is 0 or 1, and M” has the same definition as M.
  • This step can be performed prior or after: - the step of chemically-modifying at least one tyrosine residue in the capsid of the AAV, as described herein, and/or - the step of chemically-modifying at least one amino group (e.g. from arginine or lysine) in the capsid of the AAV, as described above.
  • the functional group M of the chemical reagent e.g. the chemical reagent of formula (X) (Xa), (Xb) or (Xc)
  • the incubation step e.g. electrochemical or enzymatic incubation
  • the functional group M in the chemical reagent should not be oxidized under the oxidation conditions of the incubation step or should not react with the activated N-substituted luminol.
  • the functional group M of the chemical reagent does not comprise any phenol moiety.
  • such a functional group is not the MTP of SEQ ID NO:1 linked to K-FITC as defined above, the MTP of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7 as defined above, or the ligand of formula (IVb) as defined above.
  • the group M present in the chemical reagent of formula (X), (Xa), (Xb) or (Xc) may not contain any chemical group having an oxidation potential equal or lower that of the N- substituted luminol moiety.
  • the introduction of a functional group on the surface of the AAV having a chemical group which is not compatible with electrochemical activation as described above can be performed in two steps namely: the introduction of a chemical moiety on the surface of the AAV able to undergo a click chemistry reaction, and then the introduction of the functional moiety of interest having such an incompatible chemical moiety through click chemistry.
  • the chemically-modified AAV can undergo a supplementary step aiming at coupling a functional group Z’ by reaction with M group.
  • the implementation of this supplementary step is particularly suitable when Z’ is a functional group incompatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above.
  • the incompatibility of the functional group may for instance be due to the ability of such functional group to be oxidized under the oxidation conditions (i.e.
  • Z’ may be for instance a functional group comprising a phenol moiety or other moieties that can be found in certain fluorophores incompatible with electrochemistry. It is the case, for instance, of xanthene derivatives such as fluorescein and rhodamine, Cyanine 3/5/7 (Cy3, Cy5, Cy7). Other examples are and peptides/polypeptides comprising tyrosine residues e.g. therapeutic antibodies and therapeutic proteins.
  • Z be a functional group comprising a phenol moiety, such as the MTP of SEQ ID NO:1 linked to K-FITC as defined above, the MTP of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7 as defined above, or the ligand of formula (IVb) as defined above.
  • the invention also relates to a method for grafting a functional moiety Z’ on a tyrosine residue in AAV capsid, said method comprising a step of: ⁇ preparing a chemically-modified AAV having at least one chemically modified tyrosine with a functional M group, said functional M group being a click-chemistry group, preferably by the method as described above, and ⁇ coupling a functional group Z’ by click reaction with M group.
  • M is a click-chemistry group compatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above, namely a click chemistry group that cannot be oxidized under the oxidation conditions (i.e.
  • M is an azido, phosphine, aldehyde, ketone, hydrazide, oxyamine, nitrile oxide, oxime, hydroxymoyl chloride, chlororoxime, nitrile imine, hydrazone, hydrazonoyl chloride, chlorohydrazone, tetrazine, isonitrile, aryl halide, aryl boronate, oligo-histidine, nickel-complex or nickel ligand.
  • M is an azido.
  • the chemically-modified AAV of the invention can be reacted with a compound of the following formula (XIII): Q-(W) r -Z’ (XIII), wherein: ⁇ Q is a click-chemistry group that is able to react with M through a click chemistry reaction, ⁇ r is 0 or 1, ⁇ W is a spacer, and ⁇ Z’ is a functional group different from a click-chemistry group.
  • Q may comprise, or consist of, an azido (-N3), an alkene, an alkyne (in particular a strained alkyne, such as cyclooctyne (OCT), aryl-less cyclooctyne (ALO), monofluorocyclooctyne (MOFO),difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa- dibenzocyclooctyne (ODIBO), carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne), pho
  • M and Q can be permuted, with the proviso that M is a click-chemistry group compatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above. All the above-mentioned chemical reactions result in a covalent link.
  • M is an azido (-N3) and Q is an alkyne (such as a -C ⁇ CH group or a strained alkyne such as those mentioned above).
  • M and Q are not thiol group (-SH), and the click chemistry reaction is not thiol-ene or thiol-maleimide reaction.
  • Preferred click-reactions are free-metal reactions, i.e. click-reactions which do not require the presence of a metal catalyzer such as copper salt.
  • the click reaction of interest is a strain promoted alkyne-azide cycloaddition (SPAAC), which means that M can be an azido group and Q can be a strained alkyne as described above.
  • W is spacer that has typically the same definition as Y in formula (I).
  • Z’ is a functional group that has typically the same definition as M in formula (I) as defined herein, except that it is not a click-chemistry group.
  • Z’ can be a targeting agent such as a cell-type targeting ligand, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a aptmer, a peptide aptamer, an oligonucleotide, a vitamin and small chemical molecules such as drugs e.g.
  • a targeting agent such as a cell-type targeting ligand, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2,
  • Z’ can be a cell-type specific ligand derived from proteins such as transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ⁇ FGF, muscle targeting peptides as described above and from mono- or polysaccharides, e.g. comprising one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine, or bridge GalNac, sialic acid and derivatives thereof (e.g. Neu5Ac, Neu5Ac ⁇ 2-6Gal, Neu5Ac ⁇ 2-8Neu5Ac), CB1 and/or CB2 ligands and vitamins such as folic acid.
  • proteins such as transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ⁇ FGF
  • muscle targeting peptides as described above and from mono- or polysaccharides, e.g. comprising one or several galactose, mannose, mannose-6-phosphate,
  • Z' may be preferably a functional group incompatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above, in particular a functional group comprising a phenol moiety and/or having an oxidation potential lower than or equal to the N-substituted luminol, such as the MTP of SEQ ID NO:1 linked to K-FITC as defined above, the MTP of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7 as defined above, or the ligand of formula (IVb) as defined above.
  • the Invention also relates to the chemically-modified AAV obtainable, or obtained, by the method of the invention as described above.
  • the invention also relates to a method for modifying one or several biological properties of the AAV, more precisely that of a recombinant AAV intended to be used as gene vector in gene therapy.
  • a method for chemically- modifying the capsid of an AAV more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, as described above may enable to: - Modify the tropism e.g.
  • AAV AAV- Alter immunoreactivity of the AAV, e.g. a decrease immunogenicity of the AAV and/or decrease affinity for neutralizing antibodies, and/or said AAV triggers an altered humoral response when administered in vivo, e.g. do not generate AAV-directed neutralizing antibodies, and/or - Increase infectivity efficiency of the AAV particles, and/or - Reduce off-target effects i.e. transduce cells that are not necessary for providing a benefit of the drug and could be even detrimental.
  • Alter immunoreactivity of the AAV e.g. a decrease immunogenicity of the AAV and/or decrease affinity for neutralizing antibodies
  • said AAV triggers an altered humoral response when administered in vivo, e.g. do not generate AAV-directed neutralizing antibodies, and/or - Increase infectivity efficiency of the AAV particles, and/or - Reduce off-target effects i.e. transduce cells that are not necessary for providing a benefit of the drug and could be even detrimental.
  • AAV of the invention can be used as a research tool or as a medicament, for instance as vectors for the delivery of therapeutic nucleic acids such as DNA or RNA and or as a diagnostic mean e.g. as an imaging agent or combination of both, including theragnostic use.
  • the chemically-modified AAV of the invention is used for delivering a nucleic acid into a cell, in particular an exogenous nucleic acid such as a transgene, and is thus a recombinant AAV.
  • the recombinant AAV can be administered to the cell in vivo, ex vivo or in vitro.
  • the cell may be derived from any mammal including humans, primates, cows, mice, sheeps, goats, pigs, rats, and the like.
  • the cell may be of any type, including hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS).
  • the recombinant AAV of the invention may be used to deliver a therapeutic nucleic acid of interest in a subject.
  • the invention thus relates to a method for delivering a therapeutic nucleic acid of interest in a subject in need thereof comprising administering the chemically-modified AAV of the invention to a subject in need thereof.
  • the recombinant AAV of the invention can be delivered by any appropriate route to the subject.
  • Appropriate administration routes encompass, without being limited to, inhalational, topical, intra-tissue (e.g. intramuscular, intracardiac, intrahepatic, intrarenal), conjunctical (e.g. intraretinal, subretinal), mucosal (e.g. buccal, nasal), intra-articular, intravitreal, intracranial, intravascular (e.g. intravenous), intraventricular, intracisternal, intraperitoneal and intralymphatic routes.
  • intra-tissue e.g. intramuscular, intracardiac, intrahepatic, intrarenal
  • conjunctical e.g. intraretinal, subretinal
  • mucosal e.g. buccal, nasal
  • intra-articular e.g. intravitreal, intracranial, intravascular (e.g. intravenous), intraventricular, intracisternal, intraperitoneal and intralymphatic routes.
  • the route of administration is selected depending on the targeted tissue/
  • the dose of AAV to administer to the subject is typically determined by the skilled artisan in view of the specific features of the subject, the therapeutic effect sought and the targeted tissue/organ.
  • One single administration or several administrations of the AAV may be requested to achieve the sought therapeutic effect.
  • the AAV of the invention is typically administered in the form of a pharmaceutical composition, namely as a mixture with one or several pharmaceutical excipients.
  • the conditions to be treated by the administration of the AAV may be of any type, and includes genetic disorders as well as acquired disorders. Genetic disorders of interest encompass genetic muscle disorders such as Duchenne Muscular Dystrophy, leukodystrophy, spinal muscular atrophy (SMA), hemophilia, sickle disease, and inherited retinal dystrophy.
  • the chemically-modified AAV may also be used for treating disorders such as cancers, arthritis, arthrosis, congenital and acquired cardiac diseases, Parkinson disease, Alzheimer’s disease as well as infectious diseases such as hepatitis C.
  • Another object of the invention is a pharmaceutical composition comprising a chemically-AAV of the invention and at least one pharmaceutically acceptable excipient.
  • the pharmaceutical excipients may be selected from well-known excipients such as carriers, preservatives, antioxidants, surfactants, buffer, stabilizer agents, and the like.
  • the invention further relates to an in vivo or ex vivo method for delivering a nucleic acid of interest in a cell comprising contacting the chemically-modified AAV of the invention with the cell.
  • the cell may be from the patient.
  • the cell may be transplanted to the patient in need thereof.
  • the cell may be, for instance, hematopoietic stem cells.
  • the nucleic acid of interest may be of any type and is selected depending on the sought effect.
  • the AAV may comprise an exogenous gene expression cassette. Said cassette may comprise a promoter, the gene of interest and a terminator.
  • the AAV of the invention may comprise a DNA template for homologous recombination in cells. Such a recombinant AAV can be used in combination with gene editing tools, for promoting homologous recombination in targeted cells.
  • the gene editing tools can be of any type, and encompass, without being limited to, CRISPR/Cas9, Zinc Finger Nuclease, meganuclease as well as RNA and DNA encoding said proteins.
  • the Invention also relates to a host cell transfected with a chemically modified AAV of the invention, said host cell can be of any type.
  • said host cell may be hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS).
  • Electro-bioconjugation Aqueous buffers were obtained from Sigma Aldrich® or Thermofisher®. Fluorescent cyclooctynes were purchased from Jena Biosciences®. Nanobody cyclooctynes and anti-nanobody antibody were purchased from NanoTag®. SP-50 potentiostat was purchased from BioLogic®. Electrosynthesis equipments including ElectraSyn 2.0, electrodes and vials were purchased from IKA®. Chronocoulometric experiments were performed with a three-electrode system connected to SP-50 potentiostat for voltage control. All data were recorded using EC-Lab software.
  • Electrode system was typically graphite plate as anode, platinum plate as cathode, and the reference was Ag/AgCl (a thin silver rod submerged with saturated aqueous KCl solution and protected from electrolysis mixture by a porous frit glass).
  • electrodes used were thoroughly washed with EtOH, SDS and distillated water, and working electrode was re-polished on high grit sand paper ( ⁇ 1200 grit) to prevent potential passivation.
  • EXAMPLE 1 Synthesis of N-Methyl luminol derivatives
  • Compound 1 azido derivative of N-Methyl luminol Azido derivative was prepared in 5 steps from dimethyl 4-hydroxyphthalte as previously reported (S.
  • EXAMPLE 3 Production and purification of AAV2 and electrochemical bioconjugation AAV2 vectors were produced from two plasmids: (i) pHelper, PDP2-KANA encoding AAV Rep2- Cap2 and adenovirus helper genes (E2A, VA RNA, and E4); and (ii) the pVector ss-CAG-eGFP containing the ITRs. All vectors were produced by transient transfection of HEK293 cells using the calcium phosphate-HeBS method. AAV2 transfected cells were harvested 48 h after transfection and treated with Triton-1% and benzonase (25 U/mL) for 1 h at 37 °C.
  • the resulting bulk was subjected to freeze-thaw cycles to release vector particles.
  • the cellular debris were removed by centrifugation at 2500 rpm for 15 min.
  • Cell lysates were precipitated with PEG overnight and clarified by centrifugation at 4000 rpm for 1 h.
  • the precipitates were then incubated with benzonase for 30 min at 37 °C and collected after centrifugation at 10,000 g for 10 min at 4 °C.
  • Vectors were purified by double cesium chloride (CsCl) gradient ultracentrifugation.
  • the viral suspension was then subjected to four successive rounds of dialysis with mild stirring in a 10 kDa MWCO Slide-a-Lyzer cassette (Pierce) against dPBS (containing Ca2+ and Mg2+).
  • Electro- In a low-binding vial 100 ⁇ L of AAV2-GFP (1E12 vg in dPBS pH 7.4) were added to 900 ⁇ L dPBS pH 7.4 containing the appropriate quantity of N-methyl luminol derivative (LumGalNAc).
  • the electrochemical setup was assembled ( Figure 1) and 750 mV vs Ag/AgCl were applied during the studied time at room temperature under gentle orbital shaking.
  • EXAMPLE 4 Characterization of the chemically modified AAV with GalNAc derivative of N-methyl luminol - Material and methods * Titration of viral genomes (vg) : To determine the titer (vg/mL) of all AAV samples, 3 ⁇ L were treated with 20 units of DNase I (Roche #04716728001) at 37 °C for 45 min to remove residual DNA in vector samples.
  • qPCR Quantitative real time PCR
  • qPCR was carried out with an initial denaturation step at 95 °C for 20 seconds, followed by 45 cycles of denaturation at 95 °C for 1 second and annealing/extension at 56 °C for 20 seconds. Plasmid standards were generated with seven serial dilutions (containing 108 to 102 plasmid copies).
  • Dot Blot, Western Blot and Silver Staining For dot blot analysis, nitrocellulose membrane was soaked briefly in PBS prior to assembling the dot blot manifold (BioRad), then AAV vectors (2.10 10 vg) were loaded. The obtained nitrocellulose membrane was then treated for the appropriate characterization (see below: capsid integrity or carbohydrate detection).
  • membrane was washed thrice 15 min at RT with PBS-0.1%Tween, and probed with secondary antibody anti-mouse-HRP (from Dako®, diluted in milk solution 1:2000) during 1h30 at RT.
  • Membrane was finally washed thrice 15 min at RT with PBS-0.1%Tween and detection of bands was performed by local treatment with H2O2/luminol during 1 min followed by chemiluminescence visualization on X-ray films.
  • Viral capsid proteins detection Membrane was saturated for 2 h at RT with PBS containing 5% semi-skimmed milk and 0.1% tween.
  • membrane was probed with primary antibody to rabbit polyclonal anti-AAV capsid proteins (from PROGEN Biotechnik®, diluted in milk solution 1:2000) overnight at 4 °C. Then, membrane was washed thrice 15 min at RT with PBS-0.1%Tween, and probed with secondary antibody anti-rabbit-HRP (from Jackson®, diluted in milk solution 1:20000) during 1h30 at RT. Membrane was finally washed thrice 15 min at RT with PBS-0.1%Tween and detection of bands was performed by local treatment with H2O2/luminol during 1 min followed by chemiluminescence visualization on X-ray films.
  • rabbit polyclonal anti-AAV capsid proteins from PROGEN Biotechnik®, diluted in milk solution 1:2000
  • Membrane was saturated for 2 h at RT with PBS containing 1% gelatin, 0.1% igepal and 0.1% tween. After saturation, the membrane was probed with Soybean Agglutinin-Fluorescein lectin (from Vector Laboratories®, diluted in PBS-0.1%Tween solution 1:200) for GalNAc detection or Concanavalin A-Fluorescein lectin (from Vector Laboratories®, diluted in PBS-0.1%Tween solution 1:200) for mannose detection, overnight at 4 °C.
  • Soybean Agglutinin-Fluorescein lectin from Vector Laboratories®, diluted in PBS-0.1%Tween solution 1:200
  • GalNAc detection from Vector Laboratories®, diluted in PBS-0.1%Tween solution 1:200
  • Concanavalin A-Fluorescein lectin from Vector Laboratories®, diluted in PBS-0.1%Tween solution 1
  • membrane was washed thrice 15 min at RT with PBS-0.1%Tween, and probed with secondary antibody anti-Fluorescein-HRP (from abcam®, diluted in PBS-0.1%Tween solution 1:5000) during 1h30 at RT.
  • Membrane was finally washed thrice 15 min at RT with PBS-0.1%Tween and detection of bands was performed by local treatment with H 2 O 2 /luminol during 1 min followed by chemiluminescence visualization on X-ray films.
  • AAV2-LumGalNAc (1 min) showed improved transduction capacity, as compared to AAV2, on HuH-7 cells expressing GalNAc receptors (example in Figure 4, right). This supports the relevance of viral vectors functionalization to promote specific cell lines transduction potentially mediated by ligand-receptor interactions.
  • EXAMPLE 6 Production and purification of AAV2, electrochemical bioconjugation with LumBiotin and LumN 3 , and SPAAC reaction The same protocol as that described in Example 3 was used for AVV2 production and purification and electrochemical bioconjugation except that LumBiotin and LumN 3 were used.
  • the resulting AAV2-LumN 3 were subjected to SPAAC reaction so as to decorate the AAV2 with nanobody or fluorescein moieties.
  • DBCO-fluorescein, DBCO-biotin, DBCO-CD62L nanobody or DBCO-CD45 nanobody were added to the AAV2-LumN3 at a concentration of 10, 15 or 50 ⁇ M and the reaction was done during one or four hours at RT or 37°C under gentle shaking.

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Abstract

The invention relates to a chemically modified adeno-associated (AAV) virus, a method for preparing it and its use in gene therapy. The chemically modified AAV is preferably prepared by incubating the AAV with a chemical reagent bearing a N-substituted luminol moiety in conditions conducive for reacting said chemical reagent with the surface exposed tyrosine residues of AAV capsid, preferably by electrochemistry.

Description

  Chemically-modified adeno-associated virus Field of the invention The invention relates to a chemically modified adeno-associated (AAV) virus and its use in gene therapy. Background of the invention Gene therapy was originally developed to correct defective genes that underlie genetic diseases. Nowadays, gene therapy is more and more used in the treatment of a broad range of acquired diseases such as cancers. Gene therapy is based on the therapeutic delivery of nucleic acid into a patient’s cell nucleus. The nucleic acids may then be inserted into the genome of the targeted cell or may remain episomal. Delivery of a therapeutic nucleic acid to a subject's target cells can be carried-out by various methods, including the use of synthetic and viral vectors. Among the many viral vectors available (e.g, retrovirus, lentivirus, adenovirus, and the like), recombinant adeno-associated virus (AAV) is gaining popularity as a versatile vector for gene therapy, particularly for in vivo applications. The main advantages of recombinant AAV (rAAV) reside in their broad tropism, their high transduction efficacy, their persistent episomal expression and their high safety profile, in particular because wild-type AAV is not associated with any human diseases. Human clinical trials with rAAV have demonstrated durable expression at therapeutic levels when targeting tissues such as retina, liver or motor neurons. Several clinical trials using rAAV as gene vector are ongoing for a wide type of disorders. The FDA and the EMA authorized Voretigene neparvovec, which is an adeno-associated viral vector serotype 2 (AAV2) capsid comprising a cDNA encoding for the human retinal pigment epithelium 65kDa protein (hRPE65), for the treatment of vision loss due to inherited retinal dystrophy caused by confirmed biallelic RPE65 mutations. As a further example, Zolgensma® (onasemnogene abeparvovec-xioi) was approved by the FDA for the treatment of paediatric patients less than 2 years of age with spinal muscular atrophy (SMA). Zolgensma® is a AAV9 vector able to deliver a functional, non-mutated copy of the defective gene in SMA, namely the SMN1 gene, in motoneurons.       In spite of these success, certain clinical trials have shown some limitations of these vectors, in the treatment of certain diseases. Their first limitation lies on their immunogenicity. Because of their non-integrative nature, systemic gene therapy with AAV vectors, especially in paediatric patients, might be limited by tissue proliferation inducing a dilution of the vector over time. However, the re-administration of the vectors might be precluded by persistent neutralizing anti-AAV antibodies (NAbs) triggered following the first administration of the viral vector. Moreover, it was further shown that preexisting humoral immunity to certain AAV serotypes, especially AAV of serotype 2, are highly prevalent in humans. Anti-AAV neutralizing antibodies (NAbs) can completely prevent transduction in a target tissue, resulting in lack of efficacy, particularly when the vector is administered directly into the bloodstream. As a result, subjects seropositive to AAV-Nabs are generally excluded from gene therapy trials. A further limitation of AAV lies on their broad tropism, which may result in transgene expression in other tissues other than those where transgene expression is desired. AAV as gene vector may also suffer from a reduced therapeutic index. Sometimes, the administration of high dose of AAV is needed to achieve effective transduction. For instance, although AAV2 vectors can efficiently target the liver, the transgene expression can be restricted to a very small of the transfected hepatocytes due to intracellular proteasome-mediated degradation of the vectors, whereby high dose or AAV-2 may be required to achieve the sought therapeutic effect. Such high doses pose a challenge not only for vector production but also increases the risk of immune response, among which the induction of NAbs. Several strategies have been proposed to overcome the drawbacks of AAV, especially those of the AAV of serotype 2 (AAV2) in gene therapy. Certain of these strategies are based on AAV surface remodeling, namely modification of the AAV capsid proteins. The first option is to genetically modify the viral capsid. For instance, it was shown that mutations in surface-exposed tyrosine residues on AAV2 enable to circumvent 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). Another option is to introduce chemical modifications in the viral capsids in order to introduce a ligand on the capsid or in order to mask certain exposed amino acids so as to modify the antigenicity, the tropism or the transduction efficacity of AAV. For this purpose, it was proposed to genetically incorporate unnatural amino acids with modified side chains (e.g. as in       WO2015/062516). For instance, a non-natural amino acid comprising an azido group is inserted into the capsid by genetic modification prior to a coupling step with a ligand by click reaction so as to change its tropism for the target cell. Another strategy resides in the direct chemical modification of the viral capsid without any preliminary site-directed mutagenesis of the capsid proteins. In that matter, International patent application WO2017/212019 proposes a method for chemically modifying the AAV capsid by covalently coupling a ligand bearing an isothiocyanate group which reacts with an amino group present in an amino acid residue such as lysine or arginine. International patent application WO2021/005210 describes a method for chemically modified tyrosine residues present in the capsids by reaction with a ligand bearing an aryl diazonium. However, there is still a need for new methods enabling to modulate the properties of AAV gene delivery vectors in gene therapy. Summary of the invention The Invention relates to an adeno-associated Virus (AAV) having at least one chemically-modified tyrosine residue in its capsid, wherein said chemically-modified tyrosine residue is of formula (I):
Figure imgf000004_0001
wherein:       - RA is -(Y)n-M, a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, - each RB is independently a group of formula -(Y)n-M, a hydrogen or a substituent selected from the group consisting of a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, or C2-C6 alkoxycarbonyloxy, with proviso that at least one group among RB groups and RA is a group of formula -(Y)n-M, - k is 1 or 2, - n is 0 or 1, - Y is a spacer, and - M is a functional moiety. In some embodiments, RA is a C1-C3 alkyl, a phenyl, or a benzyl, preferably a methyl or a benzyl, more preferably a methyl and/or one or two RB is a group of formula -(Y)n-M and the other RB are hydrogens. In an additional embodiment, RA is a C1-C3 alkyl, a phenyl, or a benzyl, preferably a methyl or a benzyl, one RB is -(Y)n-M and the remaining RB are H. In some other embodiments, RA is -(Y)n-M and RB groups are H. In certain embodiments, the at least one chemically-modified tyrosine residue is such that Y is a chemical chain group comprising from 2 to 500 carbon atoms and selected from the group consisting of polymers including homopolymers, copolymers and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains optionally interrupted by one or several heteroatoms and/or by one or several cyclic or heterocyclic moieties, optionally having an heteroatom, such as S, O and NH, at least one of its extremity, and optionally substituted by one or several substituents, and combinations thereof. In particular, Y can be a saturated or unsaturated hydrocarbon having from 2 to 100 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -N(R)- with R being H or a C1-C3 alkyl, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and -NH-CS-; and/or - C5-C20 carbocyclic moieties such as cycloalkyl, cycloalkenyl, or aromatic groups; and/or       - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms, - and optionally having at least one of its extremities, an heteroatomic group chosen from -O-, - S-, -N(R)- with R being H or C1-C3 alkyl, -O-N(R)- with R being H or C1-C3 alkyl, -N(C1-C3 alkoxy)-, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)- NH-, NH-CS-. In certain embodiments, the AAV is such that M is a functional moiety comprising a group selected from a click-chemistry group, a steric shielding agent, a labelling agent, a targeting agent such as a cell-type specific ligand, a drug moiety, an oligonucleotide and combinations thereof. For instance, M can comprise, or consist of, - a click-chemistry group, a cell targeting agent, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a spiegelmer, a peptide aptamer, a vitamin and small chemical molecules such as drugs e.g. CB1 and/or CB2 ligands; or - a click-chemistry group, a cell-type specific ligand derived from a protein selected from transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ^FGF, a mono- or a polysaccharide comprising one or several galactose, mannose, N-acetylgalactosamine residues, bridge GalNac, or mannose-6-phosphate, sialic acid and derivatives thereof ( e.g. Neu5Ac, Neu5Ac ^2-6Gal, Neu5Ac ^2-8Neu5Ac), a MTP selected from SEQ ID NO:1 to SEQ ID NO:7, and vitamins such as folic acid. In some additional embodiments, the AAV of the Invention further has at least one additional chemically modified amino acid residue in the capsid selected from cysteine, arginine or lysine. The AAV of the Invention can be of any type. For instance, the AAV is a recombinant AAV, preferably selected from AAV having a wildtype capsid, naturally-occurring serotype AAV, variant AAV, pseudotype AAV, AAV with hybrid or mutated capsids, and self-complementary AAV.       In other aspect, the invention relates to a method for chemically-modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, which comprises incubating said AAV with a chemical reagent bearing a N-substituted luminol moiety in conditions conducive for reacting said chemical reagent with a tyrosine residue present in the capsid of the AAV so as to form a covalent bound, said method being preferably performed by electrochemistry. In certain embodiment, the method of the invention comprises incubating said AAV with a chemical reagent of formula (X):
Figure imgf000007_0001
wherein: - RA is -(Y)n-M , a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, - each RB is independently selected from a group of formula -(Y)n-M, a hydrogen or a substituent chosen from a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, with proviso that at least one group among RB groups and RA is -(Y)n-M, - n is 0 or 1, - Y is a spacer, and - M is a functional moiety; in the presence of a potential difference enabling the electro-activation of said chemical reagent of formula (X) into an oxidized form able to react with the tyrosine residues so as to obtain at least one chemically-modified tyrosine residue in the AAV capsid of formula (I):      
Figure imgf000008_0001
wherein: - RA and RB are as defined in formula (X), - k is 1 or 2. In a particular embodiment, the method of the Invention is performed in an electrochemical system with three electrodes comprising a working electrode, a counter-electrode and a reference electrode by applying a constant potential difference between the working electrode and the reference electrode, the potential difference being preferably included in a range defined as the potential oxidation of the chemical reagent ± 200 mV. In some embodiments, (i) RA is a C1-C6 alkyl and (ii) at least one RB is of formula -(Y)n-M and the others RB are H. In other embodiments, the method of the invention is such that M is a click-chemistry group and the method further comprises a step of click-reaction so as to covalently bond a functional moiety preferably selected from ligands and labels on AAV capsid. For instance, the click reaction can be a strain promoted alkyne-azide cycloaddition (SPAAC), which means that M can be an azido group and the functional moiety bears a strained alkyne, or vice versa.       The Invention also relates to an AAV obtainable by the method of the invention. In an additional aspect, the Invention also relates to a pharmaceutical composition comprising an AAV as defined above and at least one pharmaceutically acceptable excipient. In a further aspect, the Invention relates to the use of an AAV or a pharmaceutical composition of the Invention as a diagnostic agent in vivo or as a drug ex vivo or in vivo, preferably in gene therapy. The Invention also relates to the use of an AAV as defined herein as a research tool in vitro, for instance as a gene transfection agent or as an imaging agent in vitro. The Invention further relates to the use of an AAV as defined herein in the manufacture of a diagnostic agent or a drug, in particular for gene therapy ex vivo or in vivo. The Invention also relates to the use of a compound of formula (X):
Figure imgf000009_0001
defined herein as an agent for chemically modifying the capsid of an AVV by electrochemical bioconjugation. Figure 1: Experimental setup and electrochemical cell assembled for AAV electro- bioconjugation. Anode, cathode and reference electrodes are clipped to the electrodes holder (A) and dipped into a low binding vial filled with AAV and luminol solution (B) inserted into the 5 mL glass vial to form (ABC). Alligator clips connect electrical connections from (A) to potentiostat. The latter is USB-controlled by a computer (software EC-Lab). Figure 2A shows cyclic voltammetry of N-methyl luminol GalNAc derivative 12 (LumGalNAc) at scan rates 25, 50, 75 and 100 mV/s highlighted a coherent electrochemical process controlled by diffusion. Figure 2B shows multicyclic voltammetry (n= 6) of LumGalNAc at 100 mV/s outlining a clean reversible oxidation process.       Figure 3A Top: Dot blot analysis of viral vectors (2.1010 vg) to detect the assembled capsid using anti-capsid A20 antibody. Bottom: Dot blot analysis of viral vectors (2.1010 vg) to detect surface- conjugated LumGalNAc using a labelled soybean agglutinin (GalNAc-binding lectin). Figure 3B show Western blot analysis of viral vectors (2.1010 vg) after denaturation to detect (left) the three constitutive viral proteins (VP1/VP2/VP31:1:10) using a polyclonal anti-VPs antibody, or (right) surface-conjugated LumMan using a labelled Concanavalin A (Man-binding lectin). Figure 4: (left) % of GFP + cells quantified by flow cytometry after 48 h incubation of electro- conjugated carbohydrates-coated viral vectors AAV2-LumMan and AAV2-LumGalNAc (carrying a GFP reporter gene) on HEK293 cells at a MOI of 5.103. Results from 3 independent electro-conjugation experiments and transduction assays. (right) Comparison of transduction efficiencies of AAV2 and AAV2-LumGalNAc (1min) on HEK293 and HuH-7 (expressing GalNAc receptors) cell lines, at a MOI of 1.103. % of GFP + cells quantified by flow cytometry after 48 h of incubation (n = 3). Figure 5A and 5B: show some examples of “M” moieties according to the invention(non- exhaustive list, for illustration only) Figure 6: Coupling with N-methyl luminol azido derivatives (LumN3), Dot blot analysis of viral vectors (2.1010 vg) to detect the assembled capsid using anti-capsid A20 antibody and the surface- conjugated N3 using DBCO-fluorescein and mass spectroscopy analyses to determine the number of Azido derivatives by VP3 molecule. Figure 7: Western blot and silver staining analysis of viral vectors (2.1010 vg) after denaturation to validate the SPAAC reaction using labelled anti-fluorescein antibody, streptavidin, anti-CD62L and anti-CD45 nanobody antibodies, and to detect the constitutive viral proteins. Detailed description for the Invention Surface exposed tyrosine residues have been proposed to play a pivotal role in the immunogenicity as well as in the proteasome degradation of AAV in cell. Accordingly, surface exposed tyrosine residues were identified as residues to target as to refine AAV properties. In International patent application WO2021/005210, the Applicants describe the chemical modification of surface exposed tyrosine residues by chemical coupling using a ligand bearing an aryl diazonium function which specifically reacts with aromatic ring of tyrosine side chain.       The Applicants have now developed a new strategy to chemically modify the surface exposed tyrosine residues in the AAV. This strategy is different from that disclosed in previous application WO2021/005210 as it relies on the use of a distinct reactive entity and can be implemented by electrochemical bioconjugation at low potential. The electrochemical bioconjugation developed by the Applicants is based on the use of a specific reagent, namely a N-substituted luminol derivative, preferably N-methyl-luminol (NMeLum) derivative, which is electrochemically activated in situ (namely electro-oxidized) and then specifically reacts with the aromatic ring present in the side chain of tyrosine residues in the AAV capsids. To the knowledge of the Applicants, electrochemical bioconjugation has never been described or suggested in the prior art for externally modify the surface of viruses, in particular for chemically modifying tyrosine residues in AAV capsid proteins. Indeed, to the knowledge of the Inventors, electrochemical bioconjugation has been mostly used to chemically modify isolated proteins (see for instance Depienne et al., Chem. Sci., 2021, 12, 15374–15381) but not proteins present in complex system comprising genetic element such as viruses. The Applicants surprisingly showed that subjecting AAV2 particles to an electrochemical potential during one hour did not alter the viral capsid integrity as well as the infectious and transduction capacity of the viruses. In other words, both the viral proteins and genome were not altered by exposure to electrochemical potential. Furthermore, the Applicants identified NMeLum as being a coupling moiety of interest in the context of AAV surface remodeling. NMeLum-containing ligands can be electro-activated at low potential regardless the substituent present on NMeLum aromatic ring. Of note, the potential used to oxidize NMeLum is low enough to avoid side reactions with most chemical groups other than phenol residue, which means that a large variety of ligands can be coupled through NMeLum moiety on AAV capsid. Besides, without to be bound by any theory, the Applicants are of the opinion that electro-activation of NMeLum moiety at low potential enables the formation of a stable nitrogen-centered radical which can specifically react through radical coupling with tyrosine residues in the AAV capsids while avoiding the formation of highly reactive side-products and side-reactions with other amino acid residues (e.g. lysine, arginine) present in the capsid of the AAV.       As a proof of concept, the Applicants prepared N-substituted luminol derivatives bearing a sugar moiety (namely N-acetylgalactosamine (GalNAc), or a mannose (Man) sugar) for chemically modifying the capsids of AAV vectors on naturally-occurring tyrosine residues. The Applicants showed that such sugar moieties can be efficiently and covalently coupled on the surface of the AAV particles of serotype 2 by incubating the viral particles with the N-substituted luminol derivative in a biocompatible buffer (e.g. dPBS, pH 7.4) and under a low potential sufficient to oxidize the N-substituted luminol derivative into a possible radical (e.g. 750 mV vs Ag/AgCl as reference electrode). An effective coupling was obtained for both derivatives after a short time (typically after few minutes and even within 1 minute) as evidenced by dot blot analysis with soybean agglutinin (SBA lectin– a GalNAc binding protein) and concanavalin A lectin (a Mannose binding protein) detection (Figure 3A). Of note, AAV2 capsid incubated with a triethylene glycol derivatized GalNAc (without N-substituted luminol moiety) could not be detected by SBA, precluding non-covalent ligand adsorption of the NMeLum derivatives at the capsid surface during the electrochemical bioconjugation. The Applicants further showed that the electrochemical coupling with NMeLum derivatives did not alter capsid protein subunits of AAV2 as evidenced by dot blot analysis with anti-capsid A20 antibody staining (Figure 3A). On the other hand, denaturation of the AAV2 capsid followed by SDS-PAGE separation of VP1, VP2 and VP3 evidenced a mass shift for each VP protein, which confirmed that tyrosine residues in the VP proteins have been coupled with the NMeLum derivatives (Example 3B). The Applicants further showed that the infection and transduction efficacy of the AAV2 is not impaired by electrochemical coupling: The electrochemically modified AAV2 vectors carrying GFP reporter gene displayed a transduction efficacy close to that of non-chemically modified vectors in hepatic cell line HuH7 and in HEK cell as evidenced by a similar level of fluorescence using flow cytometry (Example 4 and Figure 4). The Applicants also decorated the surface of AAV2 with various entities (nanobodies - also called VHH, fluorescein, biotin) in a two-step method comprising the electrochemical bioconjugation of AAV2 with an azido derivative of N-methyl luminol followed by SPAAC (Strain-Promoted Alkyne-Azide Cycloaddition) reaction with DBCO-ligands. As shown in Example 6 and illustrated in Figures 6 and 7, each ligand was efficiently coupled on viral capsid proteins without denaturation of the capsid.       To sum-up, the Applicants demonstrated that N-substituted luminol derivatives can be used to modify the surface-exposed tyrosine residues in AAV capsids with specificity and efficacy and without impairing the structural integrity and the transduction efficacity of the viruses. This method enables to decorate the AAV capsids with a wide variety of ligands such as sugar moieties, biotin, fluorescent labels and proteins (e.g. nanobodies). The Applicants developed an electrochemical bioconjugation process displaying many advantages such as a high reaction kinetics and conversion yield, a high chemo-selectivity towards tyrosine residues, few generations of by-products and implementation in biocompatible conditions. Besides, the electrochemical bioconjugation of the invention avoids using multiple chemical entities (such as oxidants, catalysts and/or scavengers), and at the end of the reaction, unreacted ligands can be easily removed by standard methods (such as dialysis). In addition, electrodes used in the electrochemical bioconjugation do not produce waste, can be reused several times, and allows to easily implement the process from a laboratory to industrial process scale. Accordingly, the Invention relates to an Adeno-Associated Virus (AAV) having at least one chemically-modified tyrosine residue, preferably at least one electrochemically-modified tyrosine residue in its capsid. Said chemically-modified tyrosine residue results from the reaction of a tyrosine in the capsid with a functional moiety bearing N-substituted luminol group. Preferably, the tyrosine residue to be chemically modified is a naturally-occurring residue in the capsid, i.e. the tyrosine has not been introduced by mutagenesis. Accordingly, the chemically modified adeno-Associated Virus (AAV) comprises a functional moiety, for instance a ligand, covalently linked to the aromatic ring present in the side chain of a tyrosine residue in its capsid via the following moiety:
Figure imgf000013_0001
wherein RA is -(Y)n-M (as defined further below) or a hydrocarbon substituent, typically a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, preferably a methyl or a benzyl, more preferably a methyl.       Wherein the “-N” is linked to the aromatic ring of said tyrosine residue in the AAV capsid. The Invention also relates to a method for preparing a chemically-modified AAV comprising the step of contacting the AAV with a functional moiety bearing a N-substituted luminol group in condition enabling the reaction of said a functional moiety bearing a N-substituted luminol group with the aromatic ring of a tyrosine residue present in the AAV so as to covalently link said functional moiety to the AAV. The N-substituted luminol is preferably a N-(C1-C6 alkyl) luminol, a N-(C6-C14 aryl optionally substituted) luminol or a N-[(C6-C14 aryl)-(C1-C3 alkyl) optionally substituted] luminol, most preferably a N-methyl luminol or a N-benzyl luminol, more preferably a N-methyl luminol. In preferred embodiments, such a method is performed by electrochemistry, namely by subjecting the reaction medium to a potential difference sufficient to electro-activate (i.e. oxidize) the N- substituted luminol group to promote its reaction with the tyrosine residue. The invention also relates to the use of the resulting chemically-modified AAV, preferably the electrochemically-modified AAV, in particular in gene therapy. The Invention is described in more details hereunder:
Figure imgf000014_0001
As used herein, the expression “about X” with X being a physical value (such as a voltage) corresponds to X ± 5%. The term “Cx-Cy” in which x and y are integers, as used in the present disclosure, means that the corresponding hydrocarbon chain comprises from x to y carbon atoms. If, for example, the term C1-C6 is used, it means that the corresponding hydrocarbon chain may comprise from 1 to 6 carbon atoms, especially 1, 2, 3, 4, 5 or 6 carbon atoms. If, for example, the term C2-C5 is used, it means that the corresponding hydrocarbon chain may comprise from 2 to 5 carbon atoms, especially 2, 3, 4, or 5 carbon atoms. 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, hexyl. 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 alkene (or C2-C6 alkene) include for instance ethenyl, propenyl, butenyl, pentenyl, or hexenyl, preferably ethenyl (-CH=CH2). 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 “C2-C6 alkyne”, which refers to an alkyne having 2 to 6 carbon atoms. Examples of alkyne (or C2-C6 alkyne) include for instance ethynyl, propynyl, butynyl, pentynyl, or hexynyl, preferably ethynyl (-C≡CH). As used herein, the term “alkoxy” refers to an alkyl as defined herein, attached to the remainder of the molecule via an ether bond (-O-). In other words, an alkoxy can be written “-O-alkyl”. A preferred alkoxy is a C1-C6 alkoxy, which has 1 to 6 carbon atoms. Examples of alkoxy (or C1-C6 alkoxy) include for instance, methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy, hexyloxy. As used herein, the term “alkylthio” refers to an alkyl as defined herein, attached to the remainder of the molecule via a thioether bond (-S-). In other words, an alkylthio can be written “-S-alkyl”. A preferred alkylthio is a C1-C6 alkylthio, which has 1 to 6 carbon atoms. Examples of alkylthio (or C1-C6 alkylthio) include for instance, methylthio, ethylthio, propylthio, isopropylthio, butylthio, pentylthio, hexylthio. 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, an alkylamino can be written “-NH-alkyl”. A preferred alkylamino is a C1-C6 alkylamino, which has 1 to 6 carbon atoms. Examples of alkylamino (or C1-C6 alkylamino) include for instance, methylamino, ethylamino, propylamino, isopropylamino, butylamino, pentylamino, hexylamino. As used herein, the term “carbocycle” (or “carbocyclic group”) refers to a saturated or unsaturated, aliphatic or aromatic, mono-, bi- or tri-cyclic hydrocarbon group. The carbocyclic group may be in particular a cycloalkyl, a cycloalkenyl, or an aryl. As used herein, the term “cycloalkyl” refers to a saturated mono-, bi- or tri-cyclic aliphatic group. It also includes fused, bridged, or spiro-connected 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, cyclohexyl. The term “cycloalkyl” may also refer to a bridged carbocyclyl such as bicyclo[2,2,1]heptanyl, bicyclo[2,2,2]octanyl, or adamantyl. As used herein, the term “cycloalkenyl” refers to an unsaturated mono-, bi- or tri-cyclic aliphatic group, comprising at least one carbon-carbon double bond. It also includes fused, bridged, or spiro-       connected 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. As used herein, the term “heterocycle” corresponds to a saturated or unsaturated, aliphatic or aromatic, mono-, or polycyclic (e.g. bi-, tri-, or tetra-cyclic) group, comprising at least one heteroatom such as nitrogen, oxygen, or sulphur atom. In the case of a bi- or tricycle, wherein the cycles can be fused, bridged or have a spiro configuration. Advantageously, the heterocycle comprises between 3 and 20 ring atoms, for instance between 3 and 6 ring atoms, wherein at least one of the ring atoms is a heteroatom such as nitrogen, oxygen or sulphur atom. In some embodiments, the “heterocycle” is a heterocycloalkyl, a heterocycloalkenyl, or a heteroaryl. In some embodiments, the heterocycle is a heterocycloalkyl, a heterocycloalkenyl, or a heteroaryl, fused with one or more carbocyclic or heterocyclic moieties (for instance, a heteroaryl fused with a cycloalkyl). As used herein, the term “heterocycloalkyl” corresponds to a cycloalkyl group as above defined in which at least one carbon atom has been replaced with a heteroatom such as nitrogen, oxygen, or sulphur atom. As used herein, the term “heterocycloalkenyl” corresponds to a cycloalkenyl group as above defined in which at least one carbon atom has been replaced with a heteroatom such as nitrogen, oxygen, or sulphur atom. Examples of heterocycles, which are heterocycloalkyl or heterocycloalkenyl, include, but are not limited to, aziridinyl, azepanyl, diazepanyl, dioxolanyl, benzo [1,3] dioxolyl, azetidinyl, oxetanyl, pyrazolinyl, pyranyl, thiomorpholinyl, pyrazolidinyl, piperidyl, piperazinyl, 1,4-dioxanyl, imidazolinyl, pyrrolinyl, pyrrolidinyl, piperidinyl, imidazolidinyl, morpholinyl, 1,4-dithianyl, pyrrolidinyl, pyrimidinyl, oxozolinyl, oxazolidinyl, isoxazolinyl, isoxazolidinyl, thiooxetanyl, thiopyranyl, thiomorpholinyl, thiazolinyl, thiazolidinyl, isothiazolinyl, isothiazolidinyl, dihydropyranyl, dihydrofuranyl, dihydrothiopyranyl, dihydrothiophenyl, dihydropiperidinyl, tetrahydropiperidinyl, tetrahydrothiopyranyl, tetrahydropyranyl, tetrahydrofuranyl, and tetrahydrothiophenyl. As used herein, the term "aryl" refers to an aromatic ring system, which preferably has 6-14 atoms, having at least one ring having a conjugated pi electron system and which optionally may be substituted. An “aryl” may contain more than one aromatic ring such as fused ring systems or an       aryl group substituted with another aryl group. Aryl encompass, without being limited to, phenyl, anthracenyl, naphthyl, indenyl, divalent biphenyl. "Heteroaryl" refers to a heteroaryl group. “Heteroaryl” refers to a chemical group, preferably having 5-14 ring atoms, wherein 1 to 4 heteroatoms are ring atoms in the aromatic ring and the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus, and selenium. Examples of heterocycles, which are heteroaryl groups, include triazolyl, furanyl, thienyl, pyridyl, pyrrolyl, N-alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinazolinyl, and quinolinyl. Examples of bicyclic heteroaryl groups encompass, without being limited to bicyclic heteroaryl groups that may be mentioned include 1H-indazolyl, benzo[l ,2,3]thiadiazolyl, benzo[l,2,5]thiadiazolyl, benzothiophenyl, imidazo[l,2-a]pyridyl, quinolinyl, indolyl and isoquinolinyl groups. As used herein, a (C6-C14 aryl)-(C1-C3 alkyl) refers to a C1-C3 alkyl as defined herein, substituted by at least one (preferably, one only) C6-C14 aryl as defined herein. A preferred (C6-C14 aryl)-(C1- C3 alkyl) is phenylmethyl (namely benzyl). As used herein, the term “alkanoyl” refers to an alkyl as defined herein, attached to the remainder of the molecule via an oxo group (-C(O)-). In other words, an alkanoyl can be written “-C(O)- alkyl”. A preferred alkanoyl is a C1-C6 alkanoyl, which has an alkyl chain of 1 to 6 carbon atoms. Examples of alkanoyl (or C1-C6 alkanoyl) include for instance, methanoyl, ethanoyl, propanoyl, isopropanoyl, butanoyl, pentanoyl, hexanoyl. As used herein, the term “acylamino” refers to a group of formula R-C(O)-NH- wherein R is a hydrocarbon group such as C1-C6 alkyl, a C3-C12 cycloalkyl or an aryl. A preferred acylamino is a C1-C6 acyl amino, which has a hydrocarbon chain of 1 to 6 carbon atoms. As used herein, the term “ester” or “carboxy ester” refers to a -C(O)OR’ or R’C(O)O- group, wherein R’ is any hydrocarbon group, such as a C1-C6 alkyl, a C3-C12 cycloalkyl or an aryl. A preferred ester is a C1-C6 ester, which has a hydrocarbon chain of 1 to 6 carbon atoms. As used herein, an “alkoxycarbonyloxy” refers to a R”-C(O)-O- group where R” is an alkoxy. As used herein, the term “halogen” includes chlorine, fluorine, iodine, bromine, preferably chlorine or fluorine.       As used herein, the term “aminoalkyl” refers to an alkyl as defined above, substituted by one or more (preferably one) amino (-NH2) group. As used herein, the term “alkylaminoalkyl” refers to an alkyl as defined above, substituted by one or more (preferably one) alkylamino group as defined above. As used herein, the term “hydroxyalkyl” refers to an alkyl as defined above, substituted by one or more (preferably one) hydroxy (-OH) group. As used herein, the term “alkoxyalkyl” refers to an alkyl as defined above, substituted by one or more alkoxy as defined above. As used herein, the term “thioalkyl” refers to an alkyl as defined above, substituted by one or more (preferably one) -SH group. As used herein, the term “haloalkyl” refers to an alkyl as defined above, substituted by one or more halogen atoms. "Substituted" or "optionally substituted" includes groups substituted by one or several substituents, typically 1, 2, 3, 4, 5 or 6 substituents. For instance, the substituents may be independently selected from 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 esters, C1-C6 acylamino, -COOH, -CONH2, OH, -NO2, -SO3H, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, C2-C6 alkoxycarbonyloxy, -CN, -CF3 and C2-C6 alkoxyalkyl. Preferred substituents are halogens, -NO2, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1- C3 haloalkyl. As used herein, N-substituted luminol refers to the compound of the following formula:
Figure imgf000018_0001
wherein RA is a hydrocarbon substituent or -(Y)n-M as defined further below.       As used herein, a N-(C1-C6 alkyl) luminol refers to a compound of formula (B) wherein RA is C1- C6 alkyl. As used herein, a N-methyl luminol refers to a compound of formula (B) wherein RA is a methyl. As used herein, a N-(C6-C14 aryl optionally substituted) luminol refers to a compound of formula (B) wherein RA is a C6-C14 aryl optionally substituted. As used herein, a N-[(C6-C14 aryl)-(C1-C3 alkyl) optionally substituted] luminol refers to a compound of formula (B) wherein RA is (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted. As used herein, a N-benzyl luminol refers to a compound of formula (B) wherein RA is a benzyl. N-methyl luminol refers to the compound wherein RA is a methyl. N-methyl luminol can also be called “2,3-Dihydro-2-methyl-1,4-phthalazinedione” and has the following CAS number: 18393- 54-9. As used herein, “a N-substituted luminol derivative” refers to N-substituted luminol as defined above, having one or several substituents on the benzene ring of the phthalazinedione group. The phrase "optionally substituted" can be replaced with the phrase "substituted or unsubstituted" throughout this application. - Chemically-modified AAV of the invention According to a first aspect, the Invention relates to an Adeno-Associated Virus (AAV) having at least one chemically-modified tyrosine residue in its capsid. More precisely, the chemically modified AAV of the Invention comprises at least one chemical moiety of formula (A):
Figure imgf000019_0001
wherein RA is -(Y)n-M, a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1- C3 alkyl) optionally substituted. The one or more substituents present on C6-C14 aryl or (C6-C14 aryl)-(C1-C3 alkyl) may be of any type. Preferably, said substituents are selected from the group consisting of halogens, - NO2, C1- C3 alkyl, C1-C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl.       Preferably, RA is a C1-C3 alkyl, a phenyl optionally substituted, or a benzyl optionally substituted. More preferably, RA is a methyl, phenyl or a benzyl. Even more preferably, RA is a methyl. Wherein the “-N” is linked to the aromatic ring of a tyrosine residue in the AAV capsid. As used herein, an Adeno-Associated Virus (AAV) refers to a small, nonenveloped virus of the dependoparvovirus family having a single-stranded linear DNA genome of about 5kb long. Wild- type AAV has two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The 5’ and 3’ ORFs encode replication and capsid proteins, respectively. The ITR contains 145 nucleotides and serves as the AAV genome replication origin and packaging signal. In recombinant AAV, viral ORFs are replaced by the exogenous gene expression cassette, while the replication and capsid proteins are provided in trans. Accordingly, in the context of the Invention, a recombinant AAV refers to an AAV wherein an exogenous nucleic acid sequence e.g. a transgene sequence has been introduced in the viral genome. Said exogenous nucleic acid sequence may be of any type and is selected in view of the intended use of the AAV. For instance, said nucleic acid may comprises any RNA or DNA sequence. In preferred embodiments, the AAV of the invention is a recombinant AAV. Typically, said recombinant AAV is to be used as a gene vector for in vivo or in vitro applications that means that the AAV of the invention is a recombinant AAV vector. For review concerning AAV as vector in gene therapy, one can refer to Naso et al., Biodrugs, 2017, 31:317-334, the content of which being incorporated herein by reference. For illustration only, a recombinant AAV for use as vector in gene therapy may comprise an exogenous gene expression cassette replacing the viral ORFs and placed between the two ITRs. Said cassette may comprise a promoter, the gene of interest and a terminator. The promoter and the gene of interest are selected depending on the targeted tissue/organ and the condition to treat. As another example, the recombinant AAV for use in gene therapy may comprise a DNA template for homologous recombination in cells. Such a recombinant AAV can be used in combination with gene editing tools, for promoting homologous recombination in targeted cells, in vivo, in vitro or ex vivo. The gene editing tools can be of any type, and encompass, without being limited to, CRISPR/Cas9, Zinc Finger Nuclease, meganuclease as well as RNA and DNA encoding said proteins.       In the context of the invention, the term “AAV” include all types of AAV, including wild-type AAV and recombinant or variant AAV. AAV variants encompass, without being limited to, AAV having a mutated or a synthetic capsid such as AAV with hybrid capsid, pseudotype AAV as well as self-complementary AAV (scAAV). The capsid of a wildtype AAV is composed of three overlapping capsid proteins called viral protein 1 (VP1), VP2, and VP3. More precisely, the capsid of a wildtype AAV is composed of total 60 copies of the viral protein subunits VP1, VP2 and VP3 in the ratio 1∶1∶10. Genetic engineering of the capsid refers to amino acid modifications of said capsid protein(s), e.g. in their hypervariable loops. As used herein, “an AAV having a genetically engineered capsid” or “An AAV having a mutated capsid” refers to an AAV wherein one or several amino acid modifications has(ve) been introduced in at least one capsid protein (namely VP1, and/or VP2 and/or VP3) as compared to the wild-type version of said capsid protein. As used herein, “an amino acid modification” encompass the insertion, deletion or substitution of one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 9, 10, 15, 20, 30, 40, 50, or 100) amino acids. In some embodiments, the AAV has a genetically mutated capsid, wherein the mutation (s) has(ve) not been performed on naturally occurring tyrosine residues or have not resulted in the insertion of tyrosine residues. In other words, the AAV of the invention may have a wildtype capsid or may have a mutated capsid wherein the naturally occurring tyrosine residues are conserved. In a particular embodiment, the AAV of the invention is selected from wild-type AAV and recombinant or variant AAV with a wildtype AAV capsid. In some embodiments, the AAV of the invention is a recombinant AAV with a wildtype capsid. In other embodiment, the AAV is a recombinant AAV having a mutated capsid, namely one or more amino acid modifications in at least one capsid protein as compared to the corresponding parent capsid protein. In a particular embodiment, the AAV is a recombinant AAV having a mutated capsid, wherein the amino acid modification(s) is/are not concerned with any tyrosine residues naturally present in wild type capsid proteins. As used herein, “a chemically-modified tyrosine residue” means that at least one tyrosine present in the capsid of the virus has been chemically modified by covalent coupling with a chemical entity, typically by the covalent coupling of a said chemical entity on the phenyl ring of the tyrosine. Said tyrosine is typically a surface exposed residue present in VP1, VP2 or VP3. For       instance, there are around 360 tyrosine residues which are considered as exposed in AAV2 capsid. A surface exposed tyrosine means that the tyrosine is reachable for covalent coupling. Such tyrosine residues can be identified by molecular modelling of the capsid proteins or that of the whole capsid itself. As used herein, “at least one chemically-modified tyrosine residue” encompasses at least 1, 2, 3, 4, 5, 6, 7, 8, 910, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more chemically-modified tyrosine residue(s). In some embodiments, the chemically-modified AAV of the invention comprises several chemically modified tyrosine residues in its capsid. Said chemically-modified tyrosine may be present on VP1, and/or VP2 and/or VP3. In some embodiments at least 0.1%, for instance at least 0.5, 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40% or more of the surface exposed tyrosine residues of the capsid are chemically modified. They are various serotypes of AAV which can be either wildtype or synthetic. All serotypes are contemplated in the framework of the invention. A "serotype" is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes). AAV includes various naturally and synthetic (e.g. hybrid, chimera or shuffled serotypes) serotypes. 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 invention, synthetic serotypes also include pseudotyped AAV, namely AAV resulting from the mixing of a capsid and genome from different viral serotypes, such as AAV2/5, AAV2/7, and AAV2/8 as well as AAV with hybrid capsids derived from multiple different serotypes such as AAV-DJ, which contains a hybrid capsid derived from eight serotypes. Synthetic serotypes also encompass specific variants wherein a new glycan binding site is introduced into the AAV capsid are in particular described in WO2014144229 (disclosing in particular 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), 1703–1709), peptide insertion (e.g. Deverman et al., Nat Biotechnol (2016), 34(2), 204-209), or rational capsid design (reviewed in Büning et al., Curr Opin Pharmacol (2015), 24, 94-104).       In some embodiments, the AAV is selected from naturally-occurring serotypes, preferably from the group consisting of AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9 and AAVrh10, more preferably AAV-2. For instance, the AAV of the invention may be of AAV-2 or AAV-9 serotype. The AAV can target a large variety of cells, tissues and organs. Examples of cells targeted by AAV encompasses, but are not limited to, hepatocytes; cells of the retina; i.e. photoreceptors, retinal pigmented epithelium (RPE),; muscle cells, i.e. myoblasts, satellite cells; cells of the central nervous system (CNS), i.e. neurons, glial; cells of the heart; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells, blood cells such as lymphocytes, hematopoietic cells including hematopoietic stem cells, induced pluripotent stem cells (iPS) and the like. Examples of tissues and organs which 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, testes, thymus, thyroid, trachea, and the like. Preferred cell types are hepatocytes, retinal cells, muscle cells, cells of the CNS, cells of the PNS and hematopoietic cells. Preferred tissue and organs are liver, muscle, heart, eye, and brain. The tropism of AAV can vary depending on their serotype. For instance, AAV-2 can be used to transduce the central nervous system (CNS), kidney, and photoreceptor cells while AAV-8 is effective for transducing the CNS, heart, liver, photoreceptor cells, retinal pigment epithelium (RPE) and skeletal muscle. The AAV can be produced by any methods known in the art, such as transient transfection in cell lines of interest e.g. in HEK293 cells as described in the Example section. To that matter one can refer to Naso et al., Biodrugs, 2017, 31:317-334 which provide a review on AAV as vectors in gene therapy, and describe the traditional methods for producing AAV at the industrial scale. The AAV of the invention may have other amino acid(s) of the capsid which has been chemically modified. For instance, the AAV may comprise one or several amino groups of the capsid which have been modified by the method disclosed in WO2017/212019, namely by reacting said amino group(s) in the capsid with a ligand bearing an isothiocyanate reactive groups. Alternatively or additionally, the AAV of the invention may have one or several arginine residues of the capsid modified by glycation, e.g. by reaction with methylglyoxal as described in Horowitz et al. (Bioconj Chem, 2011, 22(4):529-532). Alternatively, or additionally, the AAV of the invention may comprise one or several cysteine residues of the capsid which have been modified by reacting said       cysteine residue with a chemical reagent bearing a reactive group selected from a maleimide, a vinyl sulfonamide and a 3-(carboxy derivative)acrylamide. In some embodiments, the Invention relates to an adeno-associated Virus (AAV) having at least one chemically-modified tyrosine residue in its capsid, wherein said chemically-modified tyrosine residue is of formula (I):
Figure imgf000024_0001
wherein: - RA is-(Y)n-M, a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, - each RB is independently selected from the group consisting of a group of formula -(Y)n-M, a hydrogen or a substituent selected from the group consisting of a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 thioalkyl, C1-C6 alkylthio, C2-C10 alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, with proviso that at least one group among RA and RB groups is -(Y)n-M, - k is 1 or 2, - n is 0 or 1,       - Y is a spacer, and - M is a functional moiety. Preferably, RA is a C1-C3 alkyl, a phenyl optionally substituted, or a benzyl optionally substituted. More preferably, RA is a methyl, phenyl or a benzyl. Even more preferably, RA is a methyl. In the formulae described in the present application (such as in formula (I)), the following moiety represents a tyrosine within a protein of the capsid (i.e. VP1, VP2, or VP3):
Figure imgf000025_0001
, wherein
Figure imgf000025_0002
represents a bond by which the tyrosine is attached to the rest of the protein. The N-substituted luminol moiety(ies) can be added at position ortho of the phenol group in the tyrosine residue. In formula (I) above: “k is 1” means that one moiety of formula (II) is attached to the tyrosine residue:
Figure imgf000025_0003
wherein RA and RB are as defined herein. Typically, when k is 1, the at least one chemically-modified tyrosine residue in the capsid can be represented by the formula (Ia):      
Figure imgf000026_0001
wherein RA and RB are as defined herein. “k is 2” means that two moieties of formula (II) as defined above is attached to the tyrosine residue. Typically, when k is 2, the at least one chemically-modified tyrosine residue in the capsid can be represented by the formula (Ib):
Figure imgf000026_0002
      wherein RA and RB are as defined herein. In such formula (Ib), each RA and RB in a moiety of formula (II) can be identical to or different from those of the other moiety of formula (II), preferably identical. In other words, the two moieties of formula (II) attached to the tyrosine residue in formula (Ib) can be different or identical, preferably identical. It goes without saying that the chemically-modified AAV of the invention may have one or several chemically modified tyrosine residues of formula (Ia) and one or several chemically modified tyrosine residues of formula (Ib). In some embodiments, the tyrosine residues of formula (Ia) represent in mean at least 50% e.g. at least 60%, 70%, 80%, 90% or 95% of the total number of chemically-modified tyrosine residues found in a population of chemically-modified AAVs of the invention. - RA In the above formula (I), RA is a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted. In some embodiments, RA is: --(Y)n-M as defined below, - a C1-C6 alkyl; - a C6-C14 aryl optionally substituted by one or more (preferably one) groups chosen from halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1- C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, , -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, C2-C6 alkoxycarbonyloxy and (C6-C14 aryl)- (C1-C3 alkyl); or - a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted by one or more (preferably one) groups chosen from halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino,-CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, C2-C6 alkoxycarbonyloxy and (C6-C14 aryl)-(C1-C3 alkyl). Preferably, said substituents are selected from the group consisting of halogens, C1-C3 alkyl, C1- C3 alkoxy, C1-C3 hydroxyalkyl, and C1-C3 haloalkyl. Preferably, RA is a C1-C3 alkyl (such as methyl), a phenyl, or a benzyl. More preferably, RA is a methyl or a benzyl. Even more preferably, RA is a methyl.       - RB In the above formula (I), each RB is independently a group of formula -(Y)n-M (where Y, n and M are as defined above), a hydrogen or a substituent chosen from a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, with proviso that at least one group among RA and RB groups is -(Y)n-M, In some embodiments RA is selected from methyl, phenyl or benzyl, preferably methyl. In such embodiments, at least one RB is –(Y)n-M. In some other embodiments, RA is –(Y)n-M. Preferably RB are not –(Y)n-M. For instance, RA is – (Y)n-M and all RB are H. In preferred embodiments, the chemically-modified tyrosine residue of formula (I) is such that one or two (preferably one) RB is a group of formula -(Y)n-M. More preferably, the chemically-modified tyrosine residue of formula (I) is such that one or two (preferably one) RB is a group of formula -(Y)n-M, and the other RB are hydrogens. -(Y)n-M can be at any position of the aromatic ring (namely, any RB). Preferably, the moiety of formula
Figure imgf000028_0001
wherein each RB is independently a hydrogen or a substituent chosen from a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6       hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, preferably each RB is a hydrogen. RA is as defined above, preferably not –(Y)n-M. “n is 1” means that the spacer Y is present. “n is 0” means that the spacer Y is absent. In preferred embodiment, the chemically-modified tyrosine residue of formula (I) is such that: - RA is a methyl or a benzyl; and - one or two (preferably, one) RB is a group of formula -(Y)n-M, and the other RB are hydrogens. In a preferred embodiment, the moiety of formula (II) as defined above is of formula (II-c) or (II- d):
Figure imgf000029_0001
Wherein RA is a C1-C3 alkyl, a phenyl optionally substituted, or a C1-C3 alkyl-phenyl optionnally substituted, preferably a methyl or a benzyl. ^ Spacer Y Y is a spacer that links the N-substituted luminol moiety and the functional moiety M. Y may be present (when n is 1) or absent (when n is 0). When Y is absent, the N-substituted luminol moiety and M are directly linked to each other. Y may be any chemical chain (e.g. hydrocarbon chain) which can comprise heteroatoms as well as cyclic moieties such as cycloalkyl, cycloalkenyl, aromatic groups, or heterocyclic moieties such as heterocycloalkyl or heteroaryl. Y may comprise up to 1000 carbon atoms and even more. The length and the chemical nature of the spacer may be optimized depending on the functional moiety “M” which is intended to be coupled with the tyrosine residues and the biological effect which is       sought. Indeed, further to its linking function, Y may be used to refine the properties of the functional moiety “M”. For instance, Y may decrease the steric hindrance of M with respect to the capsid, improve the accessibility of M for binding with a biological entity of interest, improve the binding of M with an entity of interest and/or increase the solubility of M. In some embodiments, Y is a chemical chain group comprising from 2 to 1000 carbon atoms, preferably from 2 to 500 carbon atoms, from 2 to 300 carbon atoms, e.g. from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 4 to 30 carbon atoms or from 4 to 20 carbon atoms. Typically, Y is selected from the group consisting of polymers including homopolymers, copolymers and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains optionally interrupted by one or several heteroatomic groups (e.g. S, O, Se, P, -C(O)-, -NHC(O)-, -OC(O)-, -N(R)- with R being H or C1-C3 alkyl), and/or by one or several cyclic or heterocyclic moieties, and/or optionally having an heteroatomic group (such as S, O, - C(O)-, -NHC(O)-, -OC(O)-, -N(R)- with R being H or C1-C3 alkyl, -O-N(R)- with R being H or C1-C3 alkyl, or -N(C1-C3 alkoxy)-) at least one of its extremity, and/or optionally being substituted by one or several substituents (e.g. hydroxyl, halogens, C1-C3 alkoxy, -CN, -CF3, or C1-C3 alkyl), and combinations thereof. As used herein, “combinations” means that the spacer group Y may comprise several hydrocarbon chains, oligomer chains or polymeric chains (e.g. 2, 3, 4, 5 or 6) linked (or connected) by any appropriate group, such as –O-, –S-, -N(R)- with R being H or C1-C3 alkyl, -C(O)-, –NHC(O)-, - OC(O)-, -C(O)-O-C(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, NH-CS-, phosphodiester or phosphorothioate groups as well as cyclic or heterocyclic groups. Typically, the group(s) (also called connectors) used to link the several hydrocarbon chains, oligomer chains or polymeric chains together result from the reactions used to connect these different chains together. For instance, the connector may be -NHC(O)- in case of amide coupling reaction, “N” in case of reductive amination or a triazole derivative in case of click chemistry involving the reaction of an azido with an alkyne group. In some embodiments, Y may be selected from the group consisting of polyethers such as polyethylene glycol (PEG) and polypropylene glycol, polyvinyl alcohol (PVA), polyesters such as polylactate, polyacrylate, polymethacrylate, polysilicone, polyamide such as polycaprolactone and poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), poly(D,L-lactic-co-glycolic acid) (PLGA), polymers of alkyl diamines, unsaturated or saturated, branched or unbranched,       hydrocarbon chains optionally having an heteroatom such as O, NH and S on at least one end, and combinations thereof. As used herein, alkyl diamine refers to NH2-(CH2)r-NH2 with r is an integer from 2 to 20, for instance from 2 to 10 such as 2, 3, 4, and 5. A polymer of alkyl diamines (also known as polyamines) refers to a compound of formula NH2-[(CH2)r-NH]t-H with r being as defined above and t is an integer of at least 2, for example of at least 3, 4, 5, 10 or more. Polymers of alkyl diamines of interest are, for instance, spermidine, and spermine. For instance, Y can comprise at least one polyethylene glycol moiety comprising from 2 to 40 monomers, e.g. from 2 to 10 or 2 to 6 monomers. For illustration only, Y may comprise from 2 to 10 triethyleneglycol blocks linked together by linkers. As another example, Y may be a C12 hydrophilic triethylene glycol ethylamine derivative. Alternatively, Y may be a saturated or unsaturated C2-C40 hydrocarbon chain, in particular a C10-C20 alkyl chain or a C2-C10 alkyl chain such as a C6 alkyl chain. The alkyl chain may have a group such as NH, S or O on at least one end. For instance, Y may be putrescine. In a particular embodiment, Y is selected from the group consisting of saturated or unsaturated, linear or branched C2-C40 hydrocarbon chains, optionally substituted, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines and combinations thereof. In a particular embodiment, Y is selected from the group consisting of linear or branched C2-C20 alkylene chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of diamino alkyl and combinations thereof. Preferably said polyethylene glycol, polypropylene glycol, PLGA, pHPMA and polymer of alkyl diamines comprise from 2 to 40 monomers, preferably from 2 to 10 or from 10 to 20 monomers. For instance, Y may comprise one or several (e.g.2, 3, 4 or 5) triethylene glycol blocks. In some embodiments, Y is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O- C(O)-, -N(R)- with R being H or a C1-C3 alkyl, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and - NH-CS-; and/or - C5-C20 carbocyclic moieties such as cycloalkyl, cycloalkenyl, or aromatic groups; and/or       - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms; and optionally having at least one of its extremities, an heteroatomic group chosen from -O-, -S-, -N(R)- with R being H or C1-C3 alkyl, -O-N(R)- with R being H or C1-C3 alkyl, -N(C1-C3 alkoxy), -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, NH-CS-. In some embodiments, Y is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -N(R)- with R being H or a C1-C3 alkyl , -C(O)-, -NHC(O)-, and -OC(O)-; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms; and optionally having an heteroatomic group chosen from -O-, -S-, -N(R)- with R being H or C1- C3 alkyl, -O-N(R)- with R being H or C1-C3 alkyl, -N(C1-C3 alkoxy)-, -C(O)-, -NHC(O)-, and - OC(O)- at least one of its extremities. The heterocycle (or “heterocyclic moiety”) may be a triazolyl or a triazolyl fused with another cycle such as those selected from the following groups:      
Figure imgf000033_0001
When the above heterocyclic moieties (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii) are part of the spacer Y, the symbol
Figure imgf000033_0002
represents the bond by which such heterocyclic moieties are attached to the remainder part of the spacer Y. Such heterocyclic moieties can be present in the spacer Y when its synthesis route comprises a step wherein an azido reacts with a strained alkyne (e.g. DBCO). In some embodiments, Y is a spacer of formula (III): Y1-Y2-Y3 (III), wherein - each of Y1 and Y3 is independently 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 (e.g. O, N, S) and/or by a group chosen from -C(O)-, -C(=O)-NH, -C(=O)-O, -C(=O)- O-C(=O)-, O-(C=O)-, NH-C(=O)-, NH-C(=O)-NH, -O-C(=O)-O-, -NH(C=S)-, or -(C=S)-NH-, optionally having at least one of its extremities an heteroatomic group (such as -O-, -S-, -N(R)-       with R being H or C1-C3 alkyl, -O-N(R)- with R being H or C1-C3 alkyl, -N(C1-C3 alkoxy)-, -C(O)- , -NHC(O)-, and -OC(O)-), and optionally substituted by one or several substituents, and combinations thereof; and - Y2 is a cyclic or a heterocyclic moiety having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms. In a particular embodiment, each of Y1 and Y3 is selected from the group consisting of polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of diamino alkyl, linear or branched C2-C20 alkyl chains (optionally interrupted by one or several heteroatoms (e.g. O, N, S) and/or by a group chosen from -C(O)-, -C(=O)-NH, -C(=O)-O, -C(=O)-O-C(=O)-, O-(C=O)-, NH- C(=O)-, NH-C(=O)-NH, -O-C(=O)-O-, -NH(C=S)-, or -(C=S)-NH-, optionally having an heteroatomic group at least one of its extremities (such as -O-, -S-, -N(R)- with R being H or C1- C3 alkyl, -O-N(R)- with R being H or C1-C3 alkyl, -N(C1-C3 alkoxy)-, -C(O)-, -NHC(O)-, and - OC(O)-), and combinations thereof. In a particular embodiment, Y2 is a triazolyl or is chosen among the above heterocyclic moieties (i), (ii), (iii), (iv), (v), (vi), (vii) and (viii). In some embodiments, Y has at one of its extremities (typically, the extremity linked to M) a heteroatomic group chosen from -O-N(R)- with R being H or C1-C3 alkyl, and -N(C1-C3 alkoxy)- , preferably chosen from -O-N(Me)-, -O-NH- -N(OMe)-. For instance, Y may be one of the following formulae:
Figure imgf000034_0001
     
Figure imgf000035_0001
wherein q is an integer from 2 to 10, and R is H or methyl. ^ Functional moiety M The functional moiety “M” may be of any type. “M” is typically selected depending on the biological effect which is sought when chemically modifying the capsid of the AAV. Alternatively, M may be a reactive group selected so as to enable a subsequent step of coupling, such as a click- chemistry reactive group. M may be also a ligand enabling the specific non-covalent coupling of another entity at the surface of the AAV such as biotin/strept(avidin), cation binding groups (e.g. nitrilotriacetate (NTA), for binding to His tags), protein binding tags or ligand/anti-ligand couples (e.g. antibody/antigen such as biotin/anti-biotin antibody and digoxigenin/anti-digoxigenin antibody, or ligand/receptor). M may be also a labelling moiety. Accordingly, “M” may comprise a moiety selected from a chemical reactive group such as a click- chemistry reactive group, a targeting agent, a steric shielding agent, a labelling agent, an oligonucleotide, or a drug. “M” may be also a (nano)-particle, including a magnetic (nano-) particle and a quantum dot. For instance, M may be an iron, stain, silicium, gold or carbon (nano)-particle. In some embodiments, “M” comprises, or consists of, a labeling agent, e.g. a fluorescent dye such as fluorescein, rhodamine, boron-dipyrromethene (Bodipy) dyes, and alexa fluor, or a radionuclide. In other embodiments, “M” comprises, or consists of, a steric shielding agent, e.g. an agent able to mask certain epitopes of the capsid, whereby avoiding the binding of neutralizing antibodies. For instance, “M” may be a polyethylene glycol (PEG), pHPMA or a polysaccharide. In a specific embodiment, “M” comprises, or consists of, a steric shielding agent able to mask tyrosine residues, whereby proteasome-degradation of the AAV in cellulo is avoided.       In another embodiment, “M” may be an oligonucleotide such as messenger RNA (mRNa) or antisense oligonucleotides such as small interferent RNA (siRNA), shRNA, snoRNA and meroduplex (mdRNA). In some embodiments, M comprises, or consists of, a targeting agent, namely a ligand enabling to target a specific organ, tissue, cell, or a protein of interest, such as a cell surface protein, receptor or oligosaccharides, e.g. a cell surface protein that is present at the surface of a particular cell line or a tumoral cell. For instance, the targeting agent can be a cell-type specific ligand, namely a ligand enabling to target a specific type of cell. Such a ligand may enable to modify the tropism of the AAV, namely its capacity to selectively infect and/or transduce a given cell line, tissue or organ. For instance, “M” may be a ligand which specifically binds to a membrane biological entity (e.g. a membrane receptor) of the targeted cell. Said ligand may be of any type e.g. a peptide a protein, an oligosaccharide or a small chemical entity. For instance, M may be a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, Angiopep-2, muscle targeting peptides a membrane receptor or a fragment thereof, CB1 and CB2 ligands, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH (also called nanobody), a ScFv, a spiegelmer, a peptide aptamer, a small chemical molecule known to bind to the targeted biological entity and the like. In a particular embodiment, M is an antibody including a full length antibody or an antigen-binding domain derived from an antibody. As used herein, the term "antibody" refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen-binding domain, regardless whether it is 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, mutated, and grafted antibodies. The term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments (e.g. VHH from single-chain antibody) that retain antigen-binding function, i.e., the ability to bind their target specifically. Typically, such fragments would comprise an antigen-binding domain. The terms "antigen-binding domain," or "antigen-binding fragment," refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody       and the antigen. Where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as "epitope" or "antigenic determinant." An antigen-binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). However, it does not necessarily comprise both (see e.g. the antigen-binding domain 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) of the antibody sufficient to form an antigen binding site (e.g., one or more CDRs, and generally all CDRs) and thus retains the binding specificity and/or activity of the antibody. As used herein, a “full-length antibody” (also called herein immunoglobulin of Ig) refers to a protein having the structure that constitutes the natural biological form of an antibody, including variable and constant regions. "Full length antibody" covers both monoclonal and polyclonal full- length antibodies and also encompasses wild-type full-length antibodies, chimeric full-length antibodies, humanized full-length antibodies, the list not being limitative. In most mammals, including humans and mice, the structure of full-length antibodies is generally a tetramer. Said tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (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, light chains are classified as kappa 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 is meant any of the classes of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. In some embodiments, “M” comprises, or consists of, a cell-type specific ligand derived from proteins such as transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ^FGF. In some other embodiments, “M” comprises, or consists of, a cell-type specific ligand derived from mono- or polysaccharides, e.g. comprising one or several galactose, mannose, mannose-6- phosphate, N-acetylgalactosamine (GalNac) and bridged GalNac and sialic acid and derivatives       thereof (such as Neu5Ac, Neu5Ac ^2-6Gal, Neu5Ac ^2-8Neu5Ac). The mono- or polysaccharides can be natural or synthetic. In another embodiment, “M” comprises, or consists of, a cell-type specific ligand derived from vitamins such as folic acid. According to one embodiment, the cell-type specific ligand included in “M” may be derived from, or may consist in, a muscle targeting peptide (MTP). Said ligand may comprise an amino acid sequence selected from the group consisting of: ASSLNIA (SEQ ID NO: 1); WDANGKT (SEQ ID NO: 2); GETRAPL (SEQ ID NO: 3); CGHHPVYAC (SEQ ID NO: 4); HAIYPRH (SEQ ID NO: 5), cyclic CQLFPLFRC (SEQ ID NO: 6) or the sequence of SEQ ID NO:7 as shown below: RXRRXRRXRFQILYRXRXRXRX (SEQ ID NO:7) wherein X is either an amino hexanoic acid residue or a beta-alanine residue as shown in the sequence listing. As used herein, cyclic CQLFPLFRC of SEQ ID NO:6 refers to:
Figure imgf000038_0001
In certain embodiments, “M” is a cancer cell targeting peptide and comprises a peptide such as RGD, including cyclic RGD. In some other embodiments, M is a cell type targeting ligand selected from antibodies and fragments thereof. When “M” comprises a peptide moiety, such as a muscle targeting peptide (MTP), said peptide moiety may comprise a chemical modification at its N-terminus or C-terminus. For instance, the N-terminus of the peptide moiety can be acylated or coupled to a moiety such as -C(=O)-(PEG moiety)-NH2.       In another embodiments, “M” comprises, or consists of, a cell-type specific ligand derived from small molecules or hormones such as naproxen, ibuprofen, cholesterol, progesterone or estradiol. In an additional embodiment, “M” comprises, or consists of, a CB1 and/or a CB2 ligand, for instance:
Figure imgf000039_0001
Galactose- derived ligands, which are recognized by asialoglycoprotein receptor (ASPGPr), can be used to specifically target hepatocytes. Accordingly, in some embodiments “M” is a ligand for specifically targeting hepatocytes and comprises at least one moiety of formula (Va), (Vb) or (Vc):
Figure imgf000039_0003
In some other embodiments, “M” is a ligand for targeting muscle cells, in particular skeletal muscle cells and comprises at least one mannose-6-phosphate moiety:
Figure imgf000039_0002
  In some other embodiments, “M” is a ligand for photoreceptors or neuronal cells and comprises at least one mannose moiety of formula (Vf):      
Figure imgf000040_0001
    In some other embodiments, “M” is a ligand for Siglecs proteins (sialic-acid-bending immunoglobulin-like lectins). In some embodiments, M is sialic acid moiety or a derivative thereof. As used herein, “sialic acid moiety and derivatives thereof” include a moiety comprising one or more N-acylated neuraminic acid units and optionally one or more other saccharide units such as a galactose moiety. More specifically, M may be a sialic acid moiety or a derivative thereof, said moiety comprising or consisting of at least one moiety of formula (Vg):
Figure imgf000040_0002
wherein R5 is an alkyl, an aryl, a heteroaryl, haloalkyl (such as -CH2-Hal, where Hal is a halogen), -OR6, -NR7R8, -SR9, -CH2OR10, -CH2NR11R12, or -CH2SR13 where R6, R7, R8, R9, R10, R11, R12, and R13 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group. In a particular embodiment, R5 is an alkyl, -OR6, -CH2OR10 , -CH2-Hal, where Hal, R6, R10 are as defined herein. In a more particular embodiment, R5 is methyl, -CH2OH, or -CH2-F. In a particular embodiment, M is chosen from a Neu5Ac, Neu5Ac ^ ^ ^ ^Gal and Neu5Ac ^2- 8Neu5Ac moiety.  As used herein, Neu5Ac moiety refers to N-acetylneuraminic acid moiety. Neu5Ac can be represented by the following formula (Vh):
Figure imgf000040_0003
      As used herein, Neu5Ac ^ ^ ^ ^Gal moiety refers to a moiety consisting of a N-acetylneuraminic acid unit and a galactose unit bonded by a ^ ^ ^ ^ ^bond. Neu5Ac ^ ^ ^ ^Gal moiety can be represented by the following formula (Vi):
Figure imgf000041_0001
As used herein, Neu5Ac ^2-8Neu5Ac moiety refers to a moiety consisting of two N- acetylneuraminic acid units bonded by a ^ ^ ^ ^ ^bond. Neu5Ac ^2-8Neu5Ac can be represented by the following formula (Vj):
Figure imgf000041_0002
In some embodiments, “M” is multivalent, which means 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. For instance, M may comprise a polyfunctional linker bearing several (e.g. at least 2, 3, 4, 5, or 6) cell-type ligands. The cell-type ligands can be the same or different. For instance, “M” may comprise a moiety of formula (VI):
Figure imgf000041_0003
with n is a enter from 1 to 100, preferably from 1 to 20.       As another example of multivalent ligands, “M” may comprise a moiety of formula (VI) wherein the GalNac groups are replaced by mannose, phosphate-6-mannose, bridged GalNac, sialic acid or derivatives thereof (e.g. Neu5Ac, Neu5Ac ^ ^ ^ ^Gal, Neu5Ac ^2-8Neu5Ac for instance as shown above), CB1 and/or CB2 ligands or peptides. In some embodiments, M is an oligosaccharide chosen from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, ,galactan and mannan. In some particular embodiment, “M” may comprise both a labelling moiety such as a fluorescent label or a radionuclide and a cell-type specific ligand. For illustration only, M may be:
Figure imgf000042_0001
namely a muscle targeting peptide of SEQ ID NO:1 linked to K-FITC. Other examples of chemical moieties which can be used as “M” moieties are provided in Figure 5A and in Figure 5B. In a particular embodiment, M comprises, or consists of a cell targeting agent, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a spiegelmer, a peptide aptamer, a vitamin and small chemical molecules such as drugs e.g. CB1 and/or CB2 ligands. In another embodiment, M comprises, or consists of, a cell-type specific ligand derived from a protein selected from transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ^FGF, a mono- or a polysaccharide comprising one or several galactose, mannose, N-       acetylgalactosamine residues, bridge GalNac, or mannose-6-phosphate, sialic acid and derivatives thereof ( e.g. Neu5Ac, Neu5Ac ^2-6Gal, Neu5Ac ^2-8Neu5Ac), a MTP selected from SEQ ID NO:1 to SEQ ID NO:7, and vitamins such as folic acid. In a particular embodiment, M is a chemical reactive group, more preferably a “biocompatible chemical reactive group”. As used herein, M can enable to create a covalent interaction between the AAV and an entity of interest, without significantly altering the functionality of the AAV (and thus in a biocompatible way). In other words, the functional moiety may comprise a chemical reactive group which can promote the formation of a covalent bond with the entity of interest. For instance, the functional moiety may comprise a chemical reactive group suitable to create a covalent bond by click-chemistry or by bioconjugation reaction. Bioconjugation reactions encompass reactions between amino acids such as lysine, cysteine or tyrosine with reactive groups as detailed in Koniev, O., Wagner, A, Chem. Soc. Rev., 44, 5495 (2015). Preferably, M is a click-chemistry reactive group, also called hereunder a “click-chemistry group”. As used herein, a “click-chemistry group” refers to any reactive chemical group that can be involved in a click chemistry reaction. Preferably, M is not a thiol (-SH). “Click-reaction” or “Click-chemistry” is a concept introduced by Sharpless in 2001. “Click chemistry” generally refers to chemical reactions characterized by high yields, high chemoselectivity, which are simple to conduct and which generate inoffensive by-products. “Click reactions” can be typically conducted in complex media with high efficiency. Click reactions are typically used to create covalent heteroatom links (C-X-C) between two entities of interest. For review about click chemistry, one can refer to Kolb et al., Angew. Chem. Int. Ed.2001, 40, 2004- 2021) and to Rudolf et al., Current opinion in Chemical Biology, 2013, 17:110-117. Examples of click chemistry reactions include, but are not limited to, Staudinger Ligation, azido- ene or azido-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 thiol-ene or thiol-maleimide reaction. For instance, M may comprise, or consist of, an azido (-N3), phosphine such as a triarylphosphine, aldehyde, ketone, hydrazide, oxyamine, nitrile oxide, oxime, hydroxymoyl chloride,       chlororoxime, nitrile imine, hydrazone, hydrazonoyl chloride, chlorohydrazone, tetrazine, tetrazole isonitrile, aryl halide, aryl boronate, oligo-histidine, nickel- complex or nickel ligand. In a preferred embodiment, M is N3. In some embodiments of the invention: - the spacer Y, when present, is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -NH-, -C(O)-, -NHC(O)-, and - OC(O)-; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms, preferably a triazolyl optionally fused with a dibenzoazepinyl; and optionally having at least one of its extremities, an heteroatomic group chosen from - O-, -S-, -NH-, -C(O)-, -NHC(O)-, and -OC(O)- - M comprises, or consists of, a click-chemistry group, a cell targeting agent, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a spiegelmer, a peptide aptamer, a vitamin and small chemical molecules such as drugs e.g. CB1 and/or CB2 ligands. Preferably Y is of formula (III) as described above. In some other embodiments: - the spacer Y, when present, is a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -NH-, -C(O)-, -NHC(O)-, and - OC(O)-; and/or       - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms, preferably a triazolyl optionally fused with a dibenzoazepinyl; and optionally having at least one of its extremities, an heteroatomic group chosen from - O-, -S-, -NH-, -C(O)-, -NHC(O)-, and -OC(O)-; and - M comprises, or consists of, a click-chemistry group, a cell-type specific ligand derived from a protein selected from transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ^FGF, a mono- or a polysaccharide comprising one or several galactose, mannose, N- acetylgalactosamine residues, bridge GalNac, or mannose-6-phosphate, sialic acid and derivatives thereof ( e.g. Neu5Ac, Neu5Ac ^2-6Gal, Neu5Ac ^2-8Neu5Ac), a MTP selected from SEQ ID NO:1 to SEQ ID NO:7, and vitamins such as folic acid. Preferably Y is of formula (III) as described above. In some embodiments: ‐ Y has at one of its extremities (typically, the extremity linked to M) a heteroatomic group chosen from -O-N(R)- with R being H or C1-C3 alkyl, and -N(C1-C3 alkoxy)-, preferably chosen from -O-N(Me)-, -O-NH- -N(OMe)-; and ‐ M is a saccharide, a disaccharide or an oligosaccharide preferably chosen from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, galactan and mannan. In such embodiment, Y may be a hydrocarbon chain (for instance an alkyl chain) having from 2 to 100 carbon atoms, 2 to 40 carbon atoms, from 2 to 30 carbon atoms, or from 2 to 20 carbon atoms: ‐ having at one of its extremities (typically, the extremity linked to M) a heteroatomic group chosen from -O-N(R)- with R being H or C1-C3 alkyl, and -N(C1-C3 alkoxy)-, preferably chosen from -O-N(Me)-, -O-NH- -N(OMe)-; ‐ optionally having at the other one of its extremities, an heteroatomic group chosen from -O-, - S-, -NH-, -C(O)-, -NHC(O)-, and -OC(O)-, preferably -O-; ‐ optionally interrupted by one or more heteroatomic groups chosen from -O-, -S-, -NH-, -C(O)- , -NHC(O)-, and -OC(O)-, preferably -NHC(O)-.       For illustration only, the -(Y)n-M may be any one of the following formulae:
Figure imgf000046_0001
with p is an integer from 2 to 10, R is H or methyl, Ms is a monosaccharide (e.g. Mannose of GalNAc), a disaccharide moiety, or an oligosaccharide preferably chosen from gangliosides, globosides, sialylated oligosaccharides, fucosylated oligosaccharides, sulfated oligosaccharides, blood group antigens, Lewis antigens, alginates, galactan and mannan. In all the embodiments described above, the AAV is preferably a recombinant AVV, more preferably a recombinant AAV vector. As mentioned above, the AAV may have a “naturally-occurring” capsid or a genetically modified capsid, namely comprising one or several mutations in at least one capsid protein, namely VP1, VP2 and/or VP3. In some additional or alternate embodiments, the AAV may be of a serotype selected from AAV1, AAV4, AAV6, AAV7, AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9 and AAVrh10, preferably AAV-2, AAV-3b, AAV-5, AAV-8, AAV-9 and AAVrh10 and more preferably AAV-2 or AAV- 9. Alternatively, the AAV is of a synthetic serotype.       In some further embodiments, the AAV of the invention may have at least one additional chemically modified amino acid residue in the capsid, which is different from a tyrosine residue, e.g. an arginine or a lysine residues. In some embodiments, said amino acid residue bears a modified amino group of formula (VIII) in its side chain:
Figure imgf000047_0001
wherein: - N* being the nitrogen of the amino group of the side chain of an amino acid residue, e.g of a lysine residue or arginine residue, and - Y’ has the same definition as Y, n’ is 0 or 1, and M’ has the same definition as M. It is understood that Y’, n’, and M’ can be the same or different as those present in the at least one chemically-modified tyrosine as described above. Said modification on the amino group can be introduced as described in WO2017212019, the content of which being incorporated herein by reference. In some embodiments, said amino acid residue is a modified cysteine residue of formula (IX):
Figure imgf000047_0002
wherein: - X” is selected from the group consisting of:
Figure imgf000047_0003
wherein - Z is -O-, -S-, or -N(R4)-,       - R1, R2, R3, and R4 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, said group being optionally substituted, - w is 0 or 1, - R0 is a hydrogen, a halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, said group being optionally substituted, - Y” has the same definition as Y, n” is 0 or 1, and M” has the same definition as M. It is understood that Y”, n”, and M” can be the same or different as those present in the at least one chemically-modified tyrosine as described above. The chemical modification(s) of the capsid of the AAV may modify one or several biological functionalities and/or properties. Depending on the nature of “M” which is covalently bound on the surface of the chemically-modified AAV, said chemically-modified AAV may have one or several modified biological properties as compared to the same but non-chemically modified AAV, such as: - A modified tropism e.g. an increased selectivity of the AAV towards a specific organ, tissue or cell (either administered in vivo or transducing tissues or cells in culture) or a shifted selectivity of the AAV from one tissue/organ/cell to another, and/or - An altered immunoreactivity of the AAV, e.g. a decreased immunogenicity of the AAV and/or a decreased affinity for neutralizing antibodies, and/or said AAV triggers an altered humoral response when administered in vivo, e.g. do not generate AAV-directed neutralizing antibodies - An increased infectivity efficiency of the AAV particles, and/or - An increased transduction efficacy of the AAV towards a specific cell, tissue or organ. - A reduced cellular toxicity when transducing cells in culture - Induce cellular targeted mortality into cancer cells - Visualization/monitoring of the AAV particle upon in vivo administration or upon modification of cells in vitro with these AAV particles - Theragnostic applications; e.g. combining a therapeutic agent and a diagnostic agent In some embodiments, the chemically-modified AAV of the invention may have a higher transduction efficiency, which may result from increased intracellular trafficking to the nuclei, a decrease in proteasome-degradation, more efficient intranuclear decapsidation, more rapid vector       genome stabilization and/or from a decrease in interaction with neutralizing antibodies and/or from a reduction of antibody-mediated clearance of AAV in vivo, as compared to the non-chemically modified AAV. In some other embodiments, the AAV may have a higher infectivity efficiency and/or an increase in selectivity for a given cell, tissue or organ as compared to the non-chemically modified AAV either in vivo or in vitro. In some other embodiments, when the AAV is used as a drug, e.g. as a gene vector, such modified properties may result in an improvement in the therapeutic index of the AAV, which may result from decrease in the dose to administer to the patient to achieve the sought therapeutic effect and/or a decrease in the toxicity of the AAV. In a particular embodiment, the chemically-modified AAV of the invention shows a preferential tropism for an organ or cell selected from liver, heart, brain, joints, retina and skeletal muscle. In another or additional embodiment, the chemically-modified AAV of the invention shows a preferential tropism for cultured cells selected from, but not limited to, hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS). Methods for preparing the chemically-modified AAV of the invention The invention also relates to a method to modify the capsid of an AAV with a chemical reagent bearing a N-substituted luminol moiety. As mentioned above, such a chemical reagent can be a functional moiety “M” as described above linked directly or via a spacer Y to the N-substituted luminol moiety. More specifically, an object of the present invention is a method for chemically-modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, which comprises incubating said AAV with a chemical reagent bearing a N- substituted luminol moiety under conditions conducive for reacting said chemical reagent with a tyrosine residue present in the capsid of the AAV so as to form a covalent bound, wherein the N- substituted luminol moiety is a moiety of N-(C1-C6 alkyl) luminol, N-(C6-C14 aryl optionally substituted) luminol or N-[(C6-C14 aryl)-(C1-C3 alkyl) optionally substituted] luminol, preferably a N-methyl or N-benzyl luminol moiety, more preferably N-methyl luminol moiety. The coupling conditions typically enable the in-situ activation of N-substituted luminol moiety into an oxidized entity able to react with phenyl group in tyrosine residues. Preferably the coupling       conditions enable the formation of nitrogen-centered radical in the N-substituted luminol moiety. Such an oxidation can be performed by any methods known by the skilled artisan to activate N- substituted luminol preferably into single radical, for instance by an enzymatic system such as horse radish peroxidase/H2O2 or laccase/O2 or by electrochemistry. As explained above, the Applicants showed that chemical reagents bearing N-substituted luminol have an oxidation potential of about 0.6 V versus Saturated Calomel Electrode (SCE) regardless the substituent present on the nitrogen atom or on the aromatic ring of the N-substituted luminol and that the resulting oxidized entity formed at said potential is reactive towards the phenyl group of tyrosine. Besides, the Applicants showed that subjecting AAV to such a low voltage potential does not impair its structural and functional integrity. At last, the Applicants managed to specifically and efficiently modify tyrosine residues in the AAV capsid by incubating the AAV particles with a chemical reagent bearing a N-substituted luminol moiety during a short time under a constant low potential difference. Accordingly, the chemically-modified AAV of the invention is preferably prepared by electrochemistry. As you herein, “electrochemistry” refers to branch of chemistry wherein the reaction between entities of interest is triggered by subjected said entities to an electrical potential difference. In a particular embodiment, the method of the Invention refers to a method for chemically- modifying the capsid of an AAV with a chemical reagent bearing a N-substituted luminol moiety by electrochemistry, namely by contacting the AAV and the chemical reagent under a potential difference enabling the electro-activation of the chemical reagent. Typically, the potential difference is such that it does not significantly impair the integrity of the AAV particles. As used herein, the “electro-activation of the chemical reagent of the invention” refers to the oxidation of chemical reagent of the invention into an oxidized form able to react with the phenyl group present in tyrosine residues by means of a potential difference. In a more particular embodiment, the Invention refers to a method for electro-chemically modifying an adeno-associated Virus (AAV), by contacting the AAV with a chemical reagent of formula (X)      
Figure imgf000051_0001
wherein: RA and RB are as described above, under electrochemical conditions conducive for the reaction of the chemical reagent of formula (X) with a tyrosine residue present in the capsid of the AAV so as to form a covalent bound. Preferred chemical reagents of formula (X) are:
Figure imgf000051_0002
wherein RA is a C1-C3 alkyl, a phenyl optionally substituted, or a phenyl-(C1-C3 alkyl) optionnally substituted, preferably a methyl or a benzyl, and Y, n and M being as defined above. More particularly, such chemical reagent of formula (X) is incubated with the AAV so as to obtain at least one chemically-modified tyrosine residue in the capsid of formula (I) as defined herein.       The electrochemical conditions (also called herein the potential conditions) are selected so as to enable the oxidation of the chemical reagent of formula (X) without impairing the integrity of the AAV. As used herein, the “potential conditions” refer to a potential difference with respect to a reference electrode which enables to oxidize the chemical reagent preferably into a radical. This potential difference is close to the oxidation potential of the chemical reagent (determined with respect to said reference electrode). Typically the potential difference to apply is generally included in a range from the oxidation potential of the chemical reagent (OP) minus 200 mV to OP plus 500 mV, preferably from OP minus 150 mV to OP plus 400 mV. In a particular embodiment, the potential difference to apply is equal or substantially close to the oxidation potential (OP) of the chemical reagent, e.g. equal to the oxidation potential of the chemical reagent ± 200mV or ± 150 mV, more preferably ± 100 mV such as ± 50 mV or ± 25 mV. The potential difference to apply to oxidize the chemical reagent in the method of the invention varies, among others, depending on the reference potential of the reference electrode used to implement the method. For a given chemical reagent, the oxidation potential can be identified by standard proceeding well known by the skilled artisan, such as cyclic voltammetry . One can refer to the method described in the Example section (see Example 2). Typically, the AAV and the chemical reagent are subjected to a potential difference (or equivalently a “voltage”). Any suitable electrochemical device may be used to apply the voltage. In some embodiments, the voltage is applied by means of an electrochemical device comprising a three-electrode system, namely a working electrode, an auxiliary electrode and a reference electrode. The working electrode refers to the electrode on which the reaction of interest occurs.  Depending on whether the reaction on the electrode is a reduction or an oxidation, the working electrode is called cathodic or anodic, respectively. In the context of the invention, the working electrode is the anode. In the context of the invention, the auxiliary electrode (also called counter electrode) is the cathode. The electrochemical device can further comprise: a vial, electrical connector means and a mean to control the potential difference between the reference electrode and the working electrode, typically a potentiostat.       For instance, at the laboratory scale, the AAV and the chemical reagent are mixed in an appropriate buffer and the resulting mixture is placed in an appropriate vial (e.g. a plastic or glass vial) in which three electrodes are plunged. The three electrodes are connected with appropriate electrical connector means to a potentiostat. The auxiliary electrode may be isolated e.g. by using a glass frit in order to avoid the formation of byproducts. The potentiostat is used to maintain the potential difference between the reference electrode and the working electrode at a constant value enabling the oxidation of the chemical reagent selectively. The anodes, cathodes and reference electrodes that can be used in electrochemical processes are well-known to the skilled artisan. Examples of material from which anodes can be made include, but are not limited to, carbon (e.g. glassy carbon or graphite), lead bronze, tungsten, niobium, copper, magnesium, titanium, zinc, stainless steel, platinum, gold, silver, aluminum, boron doped diamond, tin, nickel, cobalt, preferably carbon anode (e.g. glassy carbon or graphite). Examples of material from which cathodes can be made include, but are not limited to, nickel, platinum, silver, lead bronze, tungsten, niobium, copper, magnesium, titanium, zinc, stainless steel, gold, aluminum, boron doped diamond, tin, nickel, cobalt, preferably platinum cathode. Examples of reference electrodes include, but are not limited to, Standard hydrogen electrode (SHE), Normal hydrogen electrode (NHE), Reversible hydrogen electrode (RHE), Saturated calomel electrode (SCE), Copper-copper(II) sulfate electrode (CSE), Silver chloride electrode, Palladium-hydrogen electrode, dynamic hydrogen electrode (DHE), and Mercury- mercurous sulfate electrode (MSE), preferably silver chloride electrode. The potentials of reference electrodes are easily available in reference handbooks. Examples of reference potentials, defined with respect to the SHE, are herein provided (non-exhaustive list): - Standard hydrogen electrode (SHE): E = 0.000 V - Normal hydrogen electrode (NHE): E ≈ 0.000 V, - Reversible hydrogen electrode (RHE): E = 0.000 V, - Saturated calomel electrode (SCE): E = +0.241 V saturated, - Copper-copper (II) sulfate electrode (CSE): E = +0.314 V, - Silver chloride electrode: E = +0.197 V in saturated KCl, - Silver chloride electrode: E = +0.210 V in 3.0 mol KCl/kg, - Silver chloride electrode: E = +0.22249 V in 3.0 mol KCl/L,       - Preferably, the electrochemical device used to apply the voltage comprises: - a silver chloride electrode as a reference electrode, - a platinum electrode as a cathode, and - a carbon (e.g. graphite) electrode as an anode. The shape and the morphology of the electrodes is not particularly limited. For instance, a carbon electrode can be reticulated, laminar or crucible. Examples of electrode shapes include, but are not limited to, a plate, a wire, or a flat rod. The size of the electrodes can be adjusted by the skilled artisan, depending on the scale of the process, in particular the volume of the incubation medium. The surface of the electrodes in contact with the incubation medium can also be adjusted by the skilled artisan. Preferably, at least 20% 30%, 40%, 50%, 60% , 70%, 80%, 90% of the total surface of each electrode is in contact with the incubation medium. The voltage may be set and controlled by any suitable device, typically a potentiostat connected to the electrodes (i.e. cathode, anode, reference electrode) of the electrochemical device. The voltage is set at a value that allows the activation (e.g. the oxidation) of the N-substituted luminol moiety of the chemical reagent. The voltage to be applied can be easily determined by the skilled artisan, in particular through the determination of the oxidation potential of the chemical reagent versus a reference electrode, in the incubation medium of interest (i.e herein the buffer of interest) by cyclovoltammetry. As mentioned above, the potential difference to apply during the incubation is generally included in a range from the oxidation potential of the chemical reagent (OP) minus 200 mV to OP plus 500 mV, preferably from OP minus 150 mV to OP plus 400 mV. In a particular embodiment, the potential difference to apply is equal or substantially close to the oxidation potential (OP) of the chemical reagent, e.g. equal to the oxidation potential of the chemical reagent ± 150 mV, more preferably ± 100 mV such as ± 50 mV or ± 25 mV. In some embodiments, the incubation is carried out under a voltage equal to such oxidation potential of the chemical reagent ± 150 mV, preferably ± 100 mV or ± 50 mV, more preferably ± 40 mV or ± 30 mV, such as ± 20 mV or ± 10 mV. Generally, the oxidation potential of the chemical reagent used in the Invention is about 0.75 V vs. Ag/AgCl in saturated KCl. The incubation may be carried out under a voltage selected from values in the range from + 0.55 V to 1.25 V vs. Ag/AgCl in saturated KCl, preferably       between+ 0.60 V to 1.0 V vs. Ag/AgCl in saturated KCl, more preferably about + 0.70 V to + 0.85 V such as from 0.70 V to 0.80 V e.g. about 0.75 V vs. Ag/AgCl in saturated KCl, wherein the reference potential of Ag/AgCl in saturated KCl is +0.197 V. As another example, the incubation may be carried out under a voltage between +0.55 and +0.65 V vs. SCE, preferably between +0.57 and +0.63 V vs. SCE, more preferably about + 0.6 V vs. SCE, wherein the reference potential of SCE is +0.241 V. The incubation may be performed in an aqueous buffer having a pH from 5 to 11, preferably from 7 to 10, e.g. from 7.0 to 8.0, e.g. about 7.4. The concentration in buffer agent is at least 30 mM,  preferably at least 50 mM and up to 1 M. The buffer may be selected from appropriate biocompatible buffers, e.g TRIS buffer, sodium carbonate - sodium bicarbonate buffer, phosphate buffer e.g. PBS or Dulbecco's phosphate- buffered saline (dPBS), or Good’s buffer. The incubation time during which the potential difference is applied may vary depending on several parameters such as (i) the volume of the buffer and the dimension of the vial, (ii) the surface of the electrodes being in contact with the incubation medium (iii) the amounts of chemical reagent and the amount of AAV to chemically modify, (iv) the chemical reagent, (v) the solubility of the chemical reagent, (vi) the voltage and (vii) the stirring rate. The incubation may last from few seconds to several hours, for instance from 5 seconds to 120 min, for instance from 10 seconds to 60 min, e.g. from 15 seconds to 30 minutes, e.g. from 30 seconds to 10 minutes, e.g. 5 s to 20 s, from 20 s to 40 s, from 40 s to 60 s, from 1 min to 2 min, from 2 min to 3 min, from 3 min to 5 min, from 5 min to 7 min or from 7 min to 10 min. In some embodiments, an effective coupling is obtained within 5 min, e.g. within 1 min of incubation under potential difference. The temperature of incubation is typically from 10°C to 40°C. Preferably the incubation is performed at room temperature, i.e. at a temperature from 18°C to 30°C, e.g. at around 20°C. In some embodiments, the reaction is performed under stirring, preferably under orbital stirring. In some other embodiments, the reaction is performed without stirring. The AAV titer is typically from 1E11 to 1E14 vg/mL, for instance 1E12 vg/ml The concentration of the chemical reagent may be from 0.01 mM to 50 mM, for instance from 0.1 mM and 20 mM, such as from 0.1 to 10.0 mM or such as 0.5 to 5.0 mM. The ratio of the chemical reagent concentration to the AAV particles may be from 3E2 to 3E7.       In another particular embodiment, the method of the Invention refers to a method for chemically- modifying the capsid of an AAV with a chemical reagent bearing a N-substituted luminol moiety, using an enzymatic system, namely by incubating the AAV and the chemical reagent in the presence of an enzymatic system enabling the activation of the chemical reagent. Preferably, the chemical reagent is a compound of formula (X) as described above. The enzymatic system may in particular consist of a combination of an enzyme and oxidant, such as a peroxidase (e.g. horseradish peroxidase) with H2O2, or a laccase with O2. The pH, buffer, temperature and duration of incubation, and the concentration of the chemical reagent and of the AAV may be similar to those described above for the electrochemical conditions. The method of the invention may comprise one or several additional steps prior to, or after the step of incubation (e.g. electrochemical or enzymatic incubation) as described above. For instance, the method of the invention may comprise a step of providing or producing the AAV particles to be chemically modified. The method of the invention may also comprise a step of providing or preparing the chemical reagent. The chemical reagent can be produced by synthetic routes. For instance, a chemical reagent of formula (X) may be prepared from methyl-hydrazine and a phtalic anhydride or phtalate, which can be substituted by a functionalizable group. As illustration only, one can refer to the synthesis of compounds 7, or 12 described in the Example section. In order to prepare a chemical reagent of formula (X) wherein Y has, at its the extremity linked to M, a heteroatomic group chosen from -O-N(R)- with R being H or C1-C3 alkyl, and -N(C1-C3 alkoxy)-, and M is an oligosaccharide, a saccharide or a disaccharide, the corresponding precursor having an end function being -O-NH(R) and -NH(C1-C3 alkoxy) respectively, can be contacted with said oligosaccharide. When the electrochemical conditions described above are used, the method of the Invention can also comprise a step of determining the oxidation potential of the chemical reagent versus a reference electrode such as Ag/AgCl in saturated KCl or saturated calomel electrode (SCE).       The method of the invention may also comprise one or several additional steps following the step of incubation, such as: - a step of removing the unreacted reagent, e.g. by dialysis, tangential flow filtration, centrifugation through porous membrane, steric exclusion chromatography and/or - a step of collecting the chemically modified AAV particles and/or - a step of purifying the chemically modified AAV particles and/or - a step of recovering the chemically modified AAV particles and/or - a step of formulating and/or packaging the chemically-modified AAV. The method of the invention may further comprise a step of chemically modifying an amino acid residue other than tyrosine residue of the capsid of the AAV. For instance, said additional chemically-modified amino acid residue may bear an amino group (e.g. lysine, arginine) or may be a cysteine. In a particular embodiment, the method of the invention may comprise a step of incubating the AAV with a chemical reagent of formula (XI):
Figure imgf000057_0001
under conditions conducive to promote for reacting said chemical reagent with the amino group of an amino acid residue, e.g. a lysine residue or an arginine residue, present in the capsid of the AAV so as to form a covalent bound. Such a step enables to chemically modify an amino group present in an amino acid residue of the capsid according to the following formula:
Figure imgf000057_0002
wherein: - N* being the nitrogen of the amino group of an amino acid residue, e.g. of a lysine residue or arginine residue, - Y’ has the same definition as Y, n’ is 0 or 1, and M’ has the same definition as M; It is understood that Y’, n’, and M’ can be the same or different as those present in the at least one chemically-modified tyrosine as described above.       Typically, such a step may be performed in an aqueous buffer, such as TRIS buffer, at a pH from 8 to 10, e.g at a pH of about 9.3 and a temperature from 10°C to 50°C, e.g. at room temperature. More details concerning the implementation of such a step can be found in WO2017/212019, the content of which being incorporated herein by reference. This step can be performed prior or after the step of chemically-modifying at least one tyrosine residue in the capsid of the AAV, as described herein In a particular or alternate embodiment, the method of the invention may comprise a step of incubating the AAV with a chemical reagent of formula (XIIa), (XIIb) or (XIIc):
Figure imgf000058_0001
wherein - Z is -O-, -S-, or -N(R4)-, - R1, R2, R3, and R4 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, said group being optionally substituted, - w is 0 or 1, - R0 is a hydrogen, a halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, said group being optionally substituted, - Y” has the same definition as Y, n” is 0 or 1, and M” has the same definition as M, under conditions conducive for reacting said chemical reagent with a cysteine residue present in the capsid of the AAV so as to form a covalent bound. It is understood that Y”, n”, and M” can be the same or different as those present in the at least one chemically-modified tyrosine as described above.       Such a step enables to chemically modify a cysteine residue of the capsid according to the following formula:
Figure imgf000059_0001
wherein: - X” is selected from the group consisting of:
Figure imgf000059_0002
wherein - Z is -O-, -S-, or -N(R4)-, - R1, R2, R3, and R4 are each independently chosen from a hydrogen atom, an alkyl group, an aryl group, and a heteroaryl group, said group being optionally substituted, - w is 0 or 1, - R0 is a hydrogen, a halogen, an alkyl group, an aryl group, a heteroaryl group, an alkoxy group, said group being optionally substituted, - Y” has the same definition as Y, n” is 0 or 1, and M” has the same definition as M. This step can be performed prior or after: - the step of chemically-modifying at least one tyrosine residue in the capsid of the AAV, as described herein, and/or - the step of chemically-modifying at least one amino group (e.g. from arginine or lysine) in the capsid of the AAV, as described above. Advantageously, the functional group M of the chemical reagent (e.g. the chemical reagent of formula (X) (Xa), (Xb) or (Xc)) is compatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above. In other words, the functional group M       in the chemical reagent should not be oxidized under the oxidation conditions of the incubation step or should not react with the activated N-substituted luminol. Preferably, the functional group M of the chemical reagent does not comprise any phenol moiety. For instance, such a functional group is not the MTP of SEQ ID NO:1 linked to K-FITC as defined above, the MTP of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7 as defined above, or the ligand of formula (IVb) as defined above. More generally, the group M present in the chemical reagent of formula (X), (Xa), (Xb) or (Xc) may not contain any chemical group having an oxidation potential equal or lower that of the N- substituted luminol moiety. The introduction of a functional group on the surface of the AAV having a chemical group which is not compatible with electrochemical activation as described above can be performed in two steps namely: the introduction of a chemical moiety on the surface of the AAV able to undergo a click chemistry reaction, and then the introduction of the functional moiety of interest having such an incompatible chemical moiety through click chemistry. Indeed, when M present in the chemical reagent of formula (X), (Xa), (Xb) or (Xc) is a click- chemistry group, the chemically-modified AAV can undergo a supplementary step aiming at coupling a functional group Z’ by reaction with M group. The implementation of this supplementary step is particularly suitable when Z’ is a functional group incompatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above. The incompatibility of the functional group may for instance be due to the ability of such functional group to be oxidized under the oxidation conditions (i.e. a functional group having a oxidation potential lower than or equal to that of the N-substituted luminol) of the incubation step or its ability to react with the activated N-substituted luminol. Z’ may be for instance a functional group comprising a phenol moiety or other moieties that can be found in certain fluorophores incompatible with electrochemistry. It is the case, for instance, of xanthene derivatives such as fluorescein and rhodamine, Cyanine 3/5/7 (Cy3, Cy5, Cy7). Other examples are and peptides/polypeptides comprising tyrosine residues e.g. therapeutic antibodies and therapeutic proteins. For instance, Z be a functional group comprising a phenol moiety, such       as the MTP of SEQ ID NO:1 linked to K-FITC as defined above, the MTP of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7 as defined above, or the ligand of formula (IVb) as defined above. Accordingly, the invention also relates to a method for grafting a functional moiety Z’ on a tyrosine residue in AAV capsid, said method comprising a step of: ‐ preparing a chemically-modified AAV having at least one chemically modified tyrosine with a functional M group, said functional M group being a click-chemistry group, preferably by the method as described above, and ‐ coupling a functional group Z’ by click reaction with M group. In such embodiment, M is a click-chemistry group compatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above, namely a click chemistry group that cannot be oxidized under the oxidation conditions (i.e. having a oxidation potential higher than that of the N-substituted luminol) of the incubation step or that cannot react with the activated N-substituted luminol. Preferably, in such embodiment, M is an azido, phosphine, aldehyde, ketone, hydrazide, oxyamine, nitrile oxide, oxime, hydroxymoyl chloride, chlororoxime, nitrile imine, hydrazone, hydrazonoyl chloride, chlorohydrazone, tetrazine, isonitrile, aryl halide, aryl boronate, oligo-histidine, nickel-complex or nickel ligand. More preferably, in such embodiment, M is an azido. Typically, the chemically-modified AAV of the invention can be reacted with a compound of the following formula (XIII): Q-(W)r-Z’ (XIII), wherein: ‐ Q is a click-chemistry group that is able to react with M through a click chemistry reaction, ‐ r is 0 or 1, ‐ W is a spacer, and ‐ Z’ is a functional group different from a click-chemistry group. Q may comprise, or consist of, an azido (-N3), an alkene, an alkyne (in particular a strained alkyne, such as cyclooctyne (OCT), aryl-less cyclooctyne (ALO), monofluorocyclooctyne (MOFO),difluorocyclooctyne (DIFO), dibenzocyclooctyne (DIBO), dimethoxyazacyclooctyne (DIMAC), biarylazacyclooctynone (BARAC), bicyclononyne (BCN), tetramethylthiepinium (TMTI, TMTH), difluorobenzocyclooctyne (DIFBO), oxa- dibenzocyclooctyne (ODIBO),       carboxymethylmonobenzocyclooctyne (COMBO), or benzocyclononyne), phosphine, aldehyde, ketone, hydrazide, oxyamine, nitrile oxide, oxime, hydroxymoyl chloride, chlororoxime, nitrile imine, hydrazone, hydrazonoyl chloride, chlorohydrazone, tetrazine, isonitrile, aryl halide, aryl boronate, oligo-histidine, nickel- complex or nickel ligand. Examples of complementary click-chemistry groups and click chemistry reactions include, but are not limited to: azido-alkyne click-chemistry (M= azide and Q= alkyne (e.g. strained intracyclic 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 hydroxymoyl chloride or chlororoxime and Q= 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-Aider ligation (M= alkene and Q= tetrazine), isonitrile-tetrazine click chemistry (M= isonitrile and Q= tetrazine), Suzuki-Miyaura coupling (M= aryl halide and Q= aryl boronate). In the above-mentioned listing of click-chemistry groups involved in the click chemistry reactions, M and Q can be permuted, with the proviso that M is a click-chemistry group compatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above. All the above-mentioned chemical reactions result in a covalent link. In a particular embodiment, M is an azido (-N3) and Q is an alkyne (such as a -C≡CH group or a strained alkyne such as those mentioned above). Preferably, M and Q are not thiol group (-SH), and the click chemistry reaction is not thiol-ene or thiol-maleimide reaction. Preferred click-reactions are free-metal reactions, i.e. click-reactions which do not require the presence of a metal catalyzer such as copper salt. In a particular embodiment, the click reaction of interest is a strain promoted alkyne-azide cycloaddition (SPAAC), which means that M can be an azido group and Q can be a strained alkyne as described above. W is spacer that has typically the same definition as Y in formula (I). Z’ is a functional group that has typically the same definition as M in formula (I) as defined herein, except that it is not a click-chemistry group. For instance, Z’ can be a targeting agent such as a cell-type targeting ligand, preferably selected from a mono- or a polysaccharide, a hormone,       including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a spiegelmer, a peptide aptamer, an oligonucleotide, a vitamin and small chemical molecules such as drugs e.g. CB1 and/or CB2 ligands. For instance, Z’ can be a cell-type specific ligand derived from proteins such as transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ^FGF, muscle targeting peptides as described above and from mono- or polysaccharides, e.g. comprising one or several galactose, mannose, mannose-6-phosphate, N-acetylgalactosamine, or bridge GalNac, sialic acid and derivatives thereof (e.g. Neu5Ac, Neu5Ac ^2-6Gal, Neu5Ac ^2-8Neu5Ac), CB1 and/or CB2 ligands and vitamins such as folic acid. As mentioned above, Z' may be preferably a functional group incompatible with the conditions of the incubation step (e.g. electrochemical or enzymatic incubation) described above, in particular a functional group comprising a phenol moiety and/or having an oxidation potential lower than or equal to the N-substituted luminol, such as the MTP of SEQ ID NO:1 linked to K-FITC as defined above, the MTP of SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:7 as defined above, or the ligand of formula (IVb) as defined above. The Invention also relates to the chemically-modified AAV obtainable, or obtained, by the method of the invention as described above. In a further aspect, the invention also relates to a method for modifying one or several biological properties of the AAV, more precisely that of a recombinant AAV intended to be used as gene vector in gene therapy. Indeed, depending on the nature of “M” moiety, the method for chemically- modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, as described above may enable to: - Modify the tropism e.g. increase selectivity of the AAV towards a specific organ, tissue or cell (either administered in vivo or transducing tissues or cells in culture) or shift the selectivity of the AAV from one tissue/organ/cell to another, and/or - Alter immunoreactivity of the AAV, e.g. a decrease immunogenicity of the AAV and/or decrease affinity for neutralizing antibodies, and/or said AAV triggers an altered humoral response when administered in vivo, e.g. do not generate AAV-directed neutralizing antibodies, and/or       - Increase infectivity efficiency of the AAV particles, and/or - Reduce off-target effects i.e. transduce cells that are not necessary for providing a benefit of the drug and could be even detrimental. - Increase transduction efficacy of the AAV towards a specific cell, tissue or organ. - Reduce cellular toxicity when transducing cells in culture - Induce cellular targeted mortality into cancer cells - Enabling visualization/monitoring of the AAV particle upon in vivo administration or upon modification of cells in vitro with these AAV particles - combine a therapeutic agent and a diagnostic agent in the AAV Uses of the AAV of the invention The chemically-modified AAV of the invention can be used as a research tool or as a medicament, for instance as vectors for the delivery of therapeutic nucleic acids such as DNA or RNA and or as a diagnostic mean e.g. as an imaging agent or combination of both, including theragnostic use. In some embodiments, the chemically-modified AAV of the invention is used for delivering a nucleic acid into a cell, in particular an exogenous nucleic acid such as a transgene, and is thus a recombinant AAV. The recombinant AAV can be administered to the cell in vivo, ex vivo or in vitro. The cell may be derived from any mammal including humans, primates, cows, mice, sheeps, goats, pigs, rats, and the like. The cell may be of any type, including hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS). The recombinant AAV of the invention may be used to deliver a therapeutic nucleic acid of interest in a subject. The invention thus relates to a method for delivering a therapeutic nucleic acid of interest in a subject in need thereof comprising administering the chemically-modified AAV of the invention to a subject in need thereof. The recombinant AAV of the invention can be delivered by any appropriate route to the subject. Appropriate administration routes encompass, without being limited to, inhalational, topical, intra-tissue (e.g. intramuscular, intracardiac, intrahepatic, intrarenal), conjunctical (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 route of administration is selected       depending on the targeted tissue/organ, namely depending on the tissue/organ in which the transduction is sought. The dose of AAV to administer to the subject is typically determined by the skilled artisan in view of the specific features of the subject, the therapeutic effect sought and the targeted tissue/organ. One single administration or several administrations of the AAV may be requested to achieve the sought therapeutic effect. The AAV of the invention is typically administered in the form of a pharmaceutical composition, namely as a mixture with one or several pharmaceutical excipients. The conditions to be treated by the administration of the AAV may be of any type, and includes genetic disorders as well as acquired disorders. Genetic disorders of interest encompass genetic muscle disorders such as Duchenne Muscular Dystrophy, leukodystrophy, spinal muscular atrophy (SMA), hemophilia, sickle disease, and inherited retinal dystrophy. The chemically-modified AAV may also be used for treating disorders such as cancers, arthritis, arthrosis, congenital and acquired cardiac diseases, Parkinson disease, Alzheimer’s disease as well as infectious diseases such as hepatitis C. Another object of the invention is a pharmaceutical composition comprising a chemically-AAV of the invention and at least one pharmaceutically acceptable excipient. The pharmaceutical excipients may be selected from well-known excipients such as carriers, preservatives, antioxidants, surfactants, buffer, stabilizer agents, and the like. The invention further relates to an in vivo or ex vivo method for delivering a nucleic acid of interest in a cell comprising contacting the chemically-modified AAV of the invention with the cell. The cell may be from the patient. After the transduction, the cell may be transplanted to the patient in need thereof. The cell may be, for instance, hematopoietic stem cells. The nucleic acid of interest may be of any type and is selected depending on the sought effect. For instance, the AAV may comprise an exogenous gene expression cassette. Said cassette may comprise a promoter, the gene of interest and a terminator. As another example, the AAV of the invention may comprise a DNA template for homologous recombination in cells. Such a recombinant AAV can be used in combination with gene editing tools, for promoting homologous recombination in targeted cells. The gene editing tools can be of any type, and encompass, without being limited to, CRISPR/Cas9, Zinc Finger Nuclease, meganuclease as well as RNA and DNA encoding said proteins.       The Invention also relates to a host cell transfected with a chemically modified AAV of the invention, said host cell can be of any type. For instance, said host cell may be hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPS). 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 the present application. section 1. Generalities Organic synthesis: Most of the chemical reagents and anhydrous solvents were purchased from Sigma Aldrich®, Carbosynth®, Acros Organics®, Alfa Aesar® or TCI Chemical®. All reagents were stored according to the detailed specifications and used without further purification. Reactions requiring anhydrous conditions were performed under positive nitrogen or argon pressure. Usual reaction monitoring was carried out with thin layer chromatography (TLC) on Merck 60 F254 silica gel plates. Revelations were performed under UV light (254 nm) or by dipping in a solution of cerium molybdate, potassium permanganate, sulfuric acid or vanillin and subsequently heated. Purification by silica gel chromatography were carried on Silica 60 M 0.04 – 0.063 mm. 1H and 13C NMR were recorded on Bruker Avance 300 or Bruker Avance 400 spectrometers. NMR spectra were assigned on the basis of the following 1D and 2D experiments: 1H, 13C, DEPT-135, COSY, HSCQ, HMBC and NOESY. All chemical shifts (δ) are shown in ppm on the X-axis using the residual solvent as internal standard. Coupling constants (J) are reported in Hz and peak multiplicities are noted according to the following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m = multiplet, dd = doublet of doublet, dt = doublet of triplet, br = broad signal. Atom numbering used for NMR attribution is different from the numbers used in nomenclature of compounds. High-resolution mass spectrometry (HRMS) was recorded on a Waters Xevo GL-XS Qtof spectrometer coupled with an Acquity H- class LC apparatus. Ionization sources were performed with the available methods (ESI+, ESI-, ASAP+, ASAP-). A tolerance of 5 ppm was applied between calculated and experimental values.       Electro-bioconjugation: Aqueous buffers were obtained from Sigma Aldrich® or Thermofisher®. Fluorescent cyclooctynes were purchased from Jena Biosciences®. Nanobody cyclooctynes and anti-nanobody antibody were purchased from NanoTag®. SP-50 potentiostat was purchased from BioLogic®. Electrosynthesis equipments including ElectraSyn 2.0, electrodes and vials were purchased from IKA®. Chronocoulometric experiments were performed with a three-electrode system connected to SP-50 potentiostat for voltage control. All data were recorded using EC-Lab software. Three-electrode system was typically graphite plate as anode, platinum plate as cathode, and the reference was Ag/AgCl (a thin silver rod submerged with saturated aqueous KCl solution and protected from electrolysis mixture by a porous frit glass). Before each experiment, electrodes used were thoroughly washed with EtOH, SDS and distillated water, and working electrode was re-polished on high grit sand paper (<1200 grit) to prevent potential passivation. EXAMPLE 1: Synthesis of N-Methyl luminol derivatives Compound 1 : azido derivative of N-Methyl luminol
Figure imgf000067_0001
Figure imgf000067_0002
Azido derivative was prepared in 5 steps from dimethyl 4-hydroxyphthalte as previously reported (S. Depienne, et al., Chem. Sci., 2021, 12, 15374-15381). It is formed as a mixture of 2 regioisomers in 60/40 proportions.1H NMR (400.16 MHz, DMSO-d6, 298.15 K): ^H 8.15 (d, J=8.8 Hz, 0.4H, HAr), 7.91 (d, J=8.8 Hz, 0.6H, HAr), 7.62 (d, J=2.7 Hz, 0.6H, HAr), 7.46 (dd, J=8.8 Hz, J=2.7 Hz, 0.6H, HAr), 7.43 (dd, J=8.8 Hz, J=2.7 Hz, 0.4H, HAr), 7.33 (d, J= 2.7 Hz, 0.4H, HAr), 4.36 (doubled t, 2H, OCH2CH2N), 3.72 (doubled t, 2H, OCH2CH2N), 3.55+3.53 (doubled s, 3H, NCH3).13C NMR (100.62 MHz, DMSO-d6, 298.15 K): ^C 161.2, 160.8, 157.0, 156.9, 150.2, 149.9, 130.8, 128.7, 126.6, 122.7, 121.8, 121.0, 118.7, 108.3, 106.2, 67.4, 49.4, 37.6, 37.3. HRMS (ASAP-): m/z calculated for C11H10N5O3 [M-H]- 260.0783 found 260.0784. Synthesis of Mannose derivative and GalNAc derivative      
Figure imgf000068_0001
Compound 3 To a suspension of commercially available tBuOK (793 mg, 7.07 mmol, 1 equiv.) in anhydrous THF (25 mL), commercially available diethylene glycol 2 (1.34 mL, 14.13 mmol, 2 equiv.) was added at 0 °C and under positive nitrogen atmosphere. Mixture was stirred at room temperature during 30 min. 80% propargyl bromide solution in toluene (0.61 mL, 7.07 mmol, 1 equiv.) was then solubilized in 5 mL of anhydrous THF and added dropwise to the mixture. Reaction was stirred at room temperature during 18 h (completion monitored by TLC, Rf = 0.3 in pure AcOEt) and mixture was filtered on a celite pad washed with THF. The obtained solution was concentrated under reduced pressure and the residue purified by silica gel chromatography (2:8 CyHex/AcOEt to 100% AcOEt) to afford 3 (735 mg, 72 %) as a colourless oil. 1H NMR (300.13 MHz, CDCl3, 298.15 K): δH 4.20 (d, J=2.4 Hz, 2H, -CH2C≡CH), 3.75-3.65 (m, 6H, Hchain), 3.60 (m, 2H, Hchain), 2.44 (t, J=2.4 Hz, 1H, -CH2C≡CH) ; HRMS (ES+): m/z calculated for C7H12O3Na [M+Na]+ 167.0684 found 167.0689 Compound 5       To a solution of commercially available peracetylated mannose 4 (1.7 g, 4.39 mmol, 1 equiv.) in anhydrous MeCN (50 mL, 0.1 M), at room temperature and under positive nitrogen atmosphere, was added mono-O-propargyl chain 3 (950 mg, 6.59 mmol, 1.5 equiv.). Mixture was cooled to 0 °C and BF3.OEt2 (1.6 mL, 13.18 mmol, 3 equiv.) was added dropwise. Reaction was stirred at room temperature during 48 h (completion monitored by TLC, Rf = 0.3 in 4:6 CyHex/AcOEt) and was quenched with a saturated solution of NaHCO3. Aqueous layer was extracted three times with AcOEt and the combined organic layer was washed once with H2O and once with brine before being dried over MgSO4, filtered and concentrated under reduced pressure. The obtained crude compound was purified by silica gel chromatography (60:40 to 40:60 CyHex/AcOEt) to afford 5 (1.28 g, 63 %) as a colorless oil. 1H NMR (300.13 MHz, CDCl3, 298.15 K): δ H 5.36 (dd, J=10.0 Hz, J=3.3 Hz, 1H, H3), 5.32-5.24 (m, 2H, H4 + H2), 4.87 (d, J=1.6 Hz, 1H, H1), 4.30 (dd, J=12.8 Hz, J=5.3 Hz, 1H, H6a), 4.20 (d, J=2.4 Hz, 2H, CH2C≡CH), 4.11-4.04 (m, 2H, H5, H6b), 3.87-3.77 (m, 1H, Hchain), 3.72-3.63 (m, 7H, Hchain), 2.44 (t, J=2.4 Hz, 1H, CH2C≡CH), 2.15 (s, 3H, COCH3), 2.09 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 1.98 (s, 3H, COCH3) ; 13C NMR (75.48 MHz, CDCl3, 298.15 K): δ C 170.8, 170.2, 170.0, 169.9, 97.9, 79.8, 74.7, 70.7, 70.2, 69.8, 69.3, 69.3, 68.6, 67.5, 66.3, 62.6, 58.6, 21.0, 20.9, 20.8, 20.8 ; HRMS (ES+): m/z calculated for C21H30O12Na [M+Na]+ 497.1635 found 497.1630 Compound 6 To a solution of 5 (600 mg, 1.26 mmol, 1.1 equiv.) in dioxane (12 mL) were added prepared azide derivative 1 (300 mg, 1.15 mmol, 1 equiv.) and sodium ascorbate (273 mg, 1.38 mmol, 1.2 equiv.). An aqueous solution (3 mL) of CuSO4.H2O (123 mg, 0.69 mmol, 0.6 equiv.) was then added and reaction was heated up to 70 °C and stirred protected from light with aluminium. After 45 min, completion was monitored by TLC (Rf = 0.3 in 9:1 DCM/MeOH) and mixture was cooled to room temperature. Chelex resin® was added (4 spatula) and mixture was left stirred 10 min. Resin was filtered off and washed twice with MeOH. The obtained solution was concentrated under reduced pressure and purified by silica gel chromatography (92:8 DCM/MeOH) to afford 6 (750 mg, 89%) as a white solid. 1H NMR (400.16 MHz, MeOD-d4, 298.15 K): δH 8.17 (d, J=8.8 Hz, 0.6H, HAr), 8.12 (s, 1H, 1xC=CHtriazol), 7.94 (d, J=8.8 Hz, 0.4H, HAr), 7.67 (d, J=2.5 Hz, 0.4H, HAr), 7.42–7.35 (m, 1.6H, HAr), 5.28-5.20 (m, 3H, H2 + H3 + H4), 4.94 – 4.89 (m, 2H, ArOCH2CH2N), 4.86 (d, J=1.3 Hz, 1H, H1), 4.65 (s, 2H, Hchain) ; 4.63-4.58 (m, 2H, ArOCH2CH2N), 4.24–4.17 (m, 1H, H6a), 4.13–4.05 (m, 2H, H6b + H5), 3.83–3.77 (m, 1H, H7), 3.69–3.60 (m, 10H, H7 + NCH3 + 6xHchain) ;       13C NMR (100.62 MHz, MeOD-d4, 298.15 K): δC 172.4, 171.6, 171.5, 171.5, 163.3, 162.8, 159.9, 159.8, 152.9, 152.3, 146.3, 131.9, 129.9,127.9, 125.9, 124.1, 123.4, 122.5, 109.5, 107.6, 98.9, 71.6, 71.2, 70.8, 70.8, 70.7, 69.8, 68.4, 68.2, 67.3, 65.1, 63.6, 50.8, 38.9, 38.7, 20.7 ; HRMS (ES-): m/z calculated for C32H40N5O15 [M-H]- 734.2521 found 734.2522 Compound 7 (LumMan) To a solution of prepared 6 (100 mg, 0.14 mmol, 1 equiv.) in anhydrous 3:2 MeOH/THF (0.9 mL MeOH/0.6 mL THF, 0.1 M) a 1 M solution of MeONa in anhydrous MeOH (0.27 mL, 0.27 mmol, 2 equiv.) was added and reaction was left stirred at room temperature under nitrogen atmosphere. After 30 min, completion was monitored by TLC (Rf = 0-0.1 in 85:15 DCM/MeOH) and Dowex- 50 acidic resin® (beforehand reactivated with conc. HCl and washed with water and MeOH) was added (4 spatula). Mixture was left stirred 15 min and resin was filtered off and washed twice with MeOH. The obtained solution was concentrated under reduced pressure and lyophilized to afford pure 7 (72 mg, 94%) as a white solid that did not require further purification. 1H NMR (400.16 MHz, DMSO-d6, 298.15 K): δH 8.21+8.20 (doubled s, 1H, C=CHtriazol), 8.11 (d, J=8.7 Hz, 0.4H, HAr), 7.88 (d, J=8.7 Hz, 0.6H, HAr), 7.61 (d, J=2.5 Hz, 0.6H, HAr), 7.45-7.37 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, HAr), 7.31 (d, J=2.5 Hz, 0.4H, HAr), 4.82 (m, 2H, ArOCH2CH2N), 4.65-4.57 (m, 3H, H1 + ArOCH2CH2N), 4.53 (s, 2H, OCH2Ctriazol), 3.69-3.28 (m, 17H, 6x carb + 8Hchain + NCH3) ; 13C NMR (100.62 MHz, DMSO-d6, 298.15 K): δC 161.2, 160.7, 144.0, 130.7, 128.6, 126.6, 124.4, 122.7, 121.8, 121.1, 108.5, 99.99, 73.9, 70.9, 69.7, 69.5, 68.9, 66.9, 66.7, 63.5, 61.2, 48.8, 37.6 ; HRMS (ES-): m/z calculated for C24H32N5O11 [M-H]- 566.2098 found 566.2100 Compound 9 To a solution of commercially available peracetylated galactosamine 8 (2 g, 5.14 mmol, 1 equiv.) in anhydrous DCM (25 mL, 0.2 M) at 0 °C and under positive nitrogen atmosphere, TMSOTf (3.25 mL, 17.98 mmol, 3 equiv.) was added dropwise. The ice bath was removed and reaction was heated up to 50 °C and stirred during 5 h. Completion was monitored by TLC (Rf = 0.3 in 98:2 DCM/MeOH) and reaction was quenched with a saturated solution of NaHCO3. Aqueous layer was extracted three time with DCM and the combined organic layer was washed once with H2O and once with brine before being dried over MgSO4, filtered and concentrated under reduced pressure. The obtained crude compound 9 (1.65 g, orange oily solid) was used in next step without further purification. Compound 10       To a solution of freshly prepared crude oxazoline 9 (1.43 g, 4.34 mmol, 1.25 equiv.) in anhydrous DCM (30 mL, 0.1 M), at room temperature and under positive nitrogen atmosphere, mono-O- propargyl chain 3 (500 mg, 3.47 mmol, 1 equiv.) was added. Mixture was cooled to 0 °C and TMSOTf (0.31 mL, 1.74 mmol, 0.5 equiv.) was added. After 24 h stirring at room temperature, completion was monitored by TLC (Rf = 0.25 in 95:5 DCM/MeOH) and reaction was quenched with a saturated solution of NaHCO3. Aqueous layer was extracted once with DCM and the combined organic layer was washed once with H2O and once with brine before being dried over MgSO4, filtered and concentrated under reduced pressure. The obtained crude compound was purified by silica gel chromatography (95:5 DCM/MeOH) to afford 10 (670 mg, 42 %) as a yellow oil. 1H NMR (300.13 MHz, CDCl3, 298.15 K): δH 6.46 (d, J=9.5 Hz, 1H, NHAc), 5.29 (m, 1H, H4), 4.99 (dd, J=11.1 Hz, J=3.4 Hz, 1H, H3), 4.82 (d, J=8.7 Hz, 1H, H1), 4.34-4.21 (m, 3H, H2 + - CH2C≡CH), 4.14 (m, 2H, H6a,b), 3.92-3.56 (m, 9H, H5 + 8Hchain), 2.49 (t, J=2.4 Hz, 1H, - CH2C≡CH), 2.14 (s, 3H, COCH3), 2.03 (s, 3H, COCH3), 1.97 (s, 3H, COCH3), 1.95 (s, 3H, COCH3) ; 13C NMR (75.48 MHz, CDCl3, 298.15 K): δC 170.6, 170.5, 170.5, 170.4, 102.5, 79.3, 75.4, 72.1, 71.1, 70.6, 70.1, 69.2, 68.2, 66.7, 61.7, 58.3, 50.5, 23.17, 20.8, 20.7, 20.7 ; HRMS (ES+): m/z calculated for C21H31NO11Na [M+Na]+ 496.1495 found 496.1797 Compound 11 To a solution of prepared 10 (585 mg, 1.24 mmol, 1.1 equiv.) in dioxane (9.5 mL, final conc. 0.1 M) were added prepared azide derivative 1 (293 mg, 1.12 mmol, 1 equiv.) and sodium ascorbate (267 mg, 1.35 mmol, 1.2 equiv.). An aqueous solution (2.5 mL) of CuSO4.H2O (120 mg, 0.67 mmol, 0.6 equiv.) was then added and reaction was heated up to 70 °C and stirred protected from light with aluminium. After 45 min, completion was monitored by TLC (Rf = 0.35 in 9:1 DCM/MeOH) and mixture was cooled to room temperature. Chelex resin® was added (4 spatula) and mixture was left stirred 15 min. Resin was filtered off and washed twice with MeOH. The obtained solution was concentrated under reduced pressure and purified by silica gel chromatography (93:7 DCM/MeOH) to afford 11 (680 mg, 83%) as a white solid. 1H NMR (400.16 MHz, DMSO-d6, 298.15 K): δH 11.57 (br s, 1H, NH), 8.21+8.20 (doubled s, 1H, C=CHtriazol), 8.10 (d, J=8.7 Hz, 0.4H, HAr), 7.87 (d, J=8.7 Hz, 0.6H, HAr), 7.80 (d, J=9.2 Hz, 1H, NHAc), 7.60 (d, J=2.5 Hz, 0.6H, HAr), 7.45-7.36 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, HAr), 7.30 (d, J=2.5 Hz, 0.4H, HAr), 5.21 (d, J=3.3 Hz, 1H, H4), 4.97 (dd, J=11.2 Hz, J=3.4 Hz, 1H, H3), 4.82 (m, 2H, ArOCH2CH2N), 4.60 (m, 2H, ArOCH2CH2N), 4.57-4.50 (d + s, J=8.7 Hz, 3H, H1 +       OCH2Ctriazol), 4.02 (m, 3H, H5 + H6a,b), 3.87 (m, 1H, H2), 3.77 (m, 1H, Hchain), 3.61-3.45 (m, 10H,
Figure imgf000072_0001
2.09 (s, 3H, COCH3), 1.98 (s, 3H, COCH3), 1.89 (s, 3H, COCH3), 1.75 (s, 3H, COCH3) ; 13C NMR (100.62 MHz, DMSO-d6, 298.15 K): δC 169.9, 169.8, 169.6, 169.3, 161.3, 160.7, 144.0, 130.7, 128.6, 126.6, 124.4, 122.8, 121.8, 121.1, 108.6, 100.9, 70.5, 69.9, 69.7, 69.4, 68.9, 68.2, 66.8, 66.7, 63.4, 61.4, 49.4, 48.8, 22.7, 20.5, 20.4, 20.4 ; HRMS (ES+): m/z calculated for C32H43N6O14 [M+H]+ 735.2837 found 735.2832 Compound 12 (LumGalNAc) To a solution of prepared 11 (200 mg, 0.27 mmol, 1 equiv.) in anhydrous MeOH (3 mL, 0.1 M) was added a 1 M solution of MeONa in anhydrous MeOH (0.4 mL, 0.4 mmol, 1.5 equiv.) and reaction was left stirred at room temperature under nitrogen atmosphere. After 3 h, completion was monitored by TLC (Rf = 0-0.05 in 85:15 DCM/MeOH) and Dowex-50 acidic resin® (beforehand reactivated with conc. HCl and washed with water and MeOH) was added (4 spatula). Mixture was left stirred 15 min and resin was filtered off and washed twice with MeOH. The obtained solution was concentrated under reduced pressure and lyophilized to afford pure 12 (100 mg, 61%) as a white solid that did not require further purification.1H NMR (400.16 MHz, DMSO- d6, 298.15 K): δH 8.21+8.20 (doubled s, 1H, C=CHtriazol), 8.11 (d, J=8.7 Hz, 0.4H, HAr), 7.88 (d, J=8.7 Hz, 0.6H, HAr), 7.61 (d, J=2.5 Hz, 0.6H, HAr), 7.57 (d, J=9.0 Hz, 1H, NHAc), 7.45-7.37 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, HAr), 7.31 (d, J=2.5 Hz, 0.4H, HAr), 4.82 (m, 2H, ArOCH2CH2N), 4.61 (m, 2H, ArOCH2CH2N), 4.52 (s, 2H, OCH2Ctriazol), 4.29 (d, J=8.5 Hz, 1H, H1), 3.78 (m, 2H, 2Hchain), 3.70 (m, 1H, H2), 3.64 (m, 1H, H4), 3.58-3.48 (m, 11H, NCH3 + H6a,b + 6Hchain), 3.42 (dd, J=10.6 Hz, J=3.3 Hz, 1H, H3), 3.30 (t, J=6.1 Hz, 1H, H5), 1.77 (s, 3H, NHAc) ; 13C NMR (100.62 MHz, DMSO-d6, 298.15 K): δC 169.5, 161.1, 160.8, 144.0, 130.7, 128.7, 126.6, 124.4, 122.7, 121.8, 121.1, 108.5, 101.3, 75.3, 71.6, 69.7, 69.6, 68.9, 67.5, 66.7, 63.5, 60.5, 52.0, 48.8, 48.6, 22.9 ; HRMS (ES+): m/z calculated for C26H37N6O11 [M+H]+ 609.2520 found 609.2523 - Synthesis of LumBiotin
Figure imgf000072_0002
      Biotin-propargyl derivative was prepared as described in literature from commercially available Biotin-acid (C.-C. Lin, et al., Org. Lett., 2007, 9, 2131-2134). Then, to a solution of Biotin- propargyl (70 mg, 0.25 mmol, 1.1 equiv.) in dioxane (2 mL) were added luminol-azido derivative (59 mg, 0.22 mmol, 1 equiv.) and sodium ascorbate (54 mg, 0.27 mmol, 1.2 equiv.). An aqueous solution (0.5 mL) of CuSO4.H2O (24 mg, 0.13 mmol, 0.6 equiv.) was then added and reaction was heated up to 70 °C and stirred protected from light with aluminium. After 45 min, completion was monitored by TLC (Rf = 0.2 in 85:15 DCM/MeOH) and mixture was cooled to room temperature. Chelex resin® was added (2 spatula) and mixture was left stirred 10 min. Resin was filtered off and washed twice with MeOH. The obtained solution was concentrated under reduced pressure and purified by silica gel chromatography (90:10 to 85:15 DCM/MeOH) to afford 15 (61 mg, 50%) as a white solid.1H NMR (400.16 MHz, DMSO-d6, 298.15 K): ^H 8.25 (t, J=5.5 Hz, 1H, NHamide), 8.11 (d, J=8.7 Hz, 0.3H, HAr), 8.00+7.99 (doubled s, 1H, C=CHtriazol), 7.88 (d, J=8.7 Hz, 0.7H, HAr), 7.56 (d, J=2.5 Hz, 0.7H, HAr), 7.44-7.35 (2x dd, J=8.7 Hz, J=2.5 Hz, 1H, HAr), 7.31 (d, J=2.5 Hz, 0.4H, HAr), 6.38 (br s, 1H, NHurea), 6.33 (br s, 1H, NHurea), 4.79 (m, 2H, NCH2CH2Oluminol), 4.58 (m, 2H, NCH2CH2Oluminol), 4.28 (d, J=5.5 Hz, 2H, NCH2Ctriazol, overlapped with m, 1H, H2), 4.11 (m, 1H, H3), 3.54+3.52 (doubled s, 3H, NCH3), 3.08 (m, 1H, H4), 2.80 (dd, J=12.3 Hz, J=5.1 Hz, 1H, H1a), 2.57 (dd, J=12.3 Hz, J=2.0 Hz, 1H, H1b), 2.09 (t, J=7.5 Hz, 2H, CH2CONHamide), 1.65-1.39 (m, 4H, Hchain (including H5a + H5b)), 1.36-1.21 (m, 2H, Hchain) ; 13C NMR (100.62 MHz, DMSO-d6, 298.15 K): ^C 172.9, 163.6, 162.0, 161.6, 146.1, 131.7, 129.6, 127.5, 124.2, 123.7, 122.7, 122.0, 109.5, 67.7, 61.9, 60.1, 56.3, 49.68, 35.9, 35.0, 29.1, 28.9, 26.1 ; HRMS (ES-): m/z calculated for C24H29N8O5S [M-H]- 541.1982 found 541.1984 EXAMPLE 2: Electrochemical behavior of luminol derivatives Electrochemical behaviour of luminol derivatives was measured by cyclic voltammetry and multicyclic voltammetry at 1 mM in 1:1 MeCN/PB 100 mM pH 7.4 and recorded at a 2 mm disc glassy carbon electrode cathode, with a platinum cathode and saturated calomel electrode or silver chloride (saturated KCl) electrode as reference (example in Figure 2). For cyclic voltammetry, scan rates of 25, 50, 75, 100 and 250 mV/s were recorded between -0.1 and 1.1 V. For multicylic voltammetry, 6 cycles were recorded at 100 mV/s between -0.1 and 1.1 V. Between each experiment, GCE electrode was repolished on high grit sand paper to prevent potential passivation. Cyclic voltammetry of compounds in pure aqueous buffers using silver chloride (saturated KCl)       reference electrode were also performed to obtain their accurate oxidation potential in electro- bioconjugation conditions. EXAMPLE 3: Production and purification of AAV2 and electrochemical bioconjugation AAV2 vectors were produced from two plasmids: (i) pHelper, PDP2-KANA encoding AAV Rep2- Cap2 and adenovirus helper genes (E2A, VA RNA, and E4); and (ii) the pVector ss-CAG-eGFP containing the ITRs. All vectors were produced by transient transfection of HEK293 cells using the calcium phosphate-HeBS method. AAV2 transfected cells were harvested 48 h after transfection and treated with Triton-1% and benzonase (25 U/mL) for 1 h at 37 °C. The resulting bulk was subjected to freeze-thaw cycles to release vector particles. The cellular debris were removed by centrifugation at 2500 rpm for 15 min. Cell lysates were precipitated with PEG overnight and clarified by centrifugation at 4000 rpm for 1 h. The precipitates were then incubated with benzonase for 30 min at 37 °C and collected after centrifugation at 10,000 g for 10 min at 4 °C. Vectors were purified by double cesium chloride (CsCl) gradient ultracentrifugation. The viral suspension was then subjected to four successive rounds of dialysis with mild stirring in a 10 kDa MWCO Slide-a-Lyzer cassette (Pierce) against dPBS (containing Ca2+ and Mg2+). Electro-
Figure imgf000074_0001
In a low-binding vial, 100 ^L of AAV2-GFP (1E12 vg in dPBS pH 7.4) were added to 900 ^L dPBS pH 7.4 containing the appropriate quantity of N-methyl luminol derivative (LumGalNAc). The electrochemical setup was assembled (Figure 1) and 750 mV vs Ag/AgCl were applied during the studied time at room temperature under gentle orbital shaking. After modification, the excess of unreacted luminol anchor was removed by dialysis in four successive rounds against dPBS pH 7.4 (+0.001% Poloxamer) in a 10 kDa MWCO cassette. EXAMPLE 4: Characterization of the chemically modified AAV with GalNAc derivative of N-methyl luminol - Material and methods * Titration of viral genomes (vg) : To determine the titer (vg/mL) of all AAV samples, 3 µL were treated with 20 units of DNase I (Roche #04716728001) at 37 °C for 45 min to remove residual DNA in vector samples. After treatment with DNase I, 20 µL of proteinase K (20 mg/mL, from MACHEREY-NAGEL®) was added and the mixture was incubated at 70 °C for 20 min. An       extraction column (NucleoSpin®RNA Virus) was then used to extract DNA from purified AAV vectors. Quantitative real time PCR (qPCR) was performed with a StepOnePlus™ Real-Time PCR System Upgrade (Life Technologies). All PCRs were performed with a final volume of 20 µL, including primers and probes targeting the ITR2 sequence, PCR Master Mix (TaKaRa), and 5 µL of template DNA (plasmid standard or sample DNA). qPCR was carried out with an initial denaturation step at 95 °C for 20 seconds, followed by 45 cycles of denaturation at 95 °C for 1 second and annealing/extension at 56 °C for 20 seconds. Plasmid standards were generated with seven serial dilutions (containing 108 to 102 plasmid copies). * Dot Blot, Western Blot and Silver Staining: For dot blot analysis, nitrocellulose membrane was soaked briefly in PBS prior to assembling the dot blot manifold (BioRad), then AAV vectors (2.1010 vg) were loaded. The obtained nitrocellulose membrane was then treated for the appropriate characterization (see below: capsid integrity or carbohydrate detection). For silver nitrate or western blot, all AAV vectors (2.1010 vg) were denatured at 100 °C for 5 min using Laemmli sample buffer (5 µL) and separated by SDS-PAGE on 10% Tris-glycine polyacrylamide gels (Life Technologies). Precision Plus Protein All Blue Standards (BioRad) were used as a molecular- weight size marker. After electrophoresis at 120 V during 200 min, gels were either silver stained (PlusOne Silver Staining Kit, Protein from GE Healthcare®) or transferred onto nitrocellulose membranes for Western blot. A 25 mM Tris/192 mM glycine/0.1 (w/v) SDS/20% MeOH buffer was used to transfer proteins during 10 min at 150 mA in a Trans-Blot SD Semi-Dry Transfer Cell (from BioRad®). The obtained nitrocellulose membrane was then treated for the appropriate characterization (See below: viral capsid proteins or carbohydrate detection). * Capsid integrity: Membrane was saturated for 2 h at RT with PBS containing 5% semi-skimmed milk and 0.1% tween. After saturation, the membrane was probed with primary antibody to mouse anti-capsid A20 (from Kleinschmidt®, diluted in milk solution 1:20) overnight at 4 °C. Then, membrane was washed thrice 15 min at RT with PBS-0.1%Tween, and probed with secondary antibody anti-mouse-HRP (from Dako®, diluted in milk solution 1:2000) during 1h30 at RT. Membrane was finally washed thrice 15 min at RT with PBS-0.1%Tween and detection of bands was performed by local treatment with H2O2/luminol during 1 min followed by chemiluminescence visualization on X-ray films. * Viral capsid proteins detection: Membrane was saturated for 2 h at RT with PBS containing 5% semi-skimmed milk and 0.1% tween. After saturation, the membrane was probed with primary       antibody to rabbit polyclonal anti-AAV capsid proteins (from PROGEN Biotechnik®, diluted in milk solution 1:2000) overnight at 4 °C. Then, membrane was washed thrice 15 min at RT with PBS-0.1%Tween, and probed with secondary antibody anti-rabbit-HRP (from Jackson®, diluted in milk solution 1:20000) during 1h30 at RT. Membrane was finally washed thrice 15 min at RT with PBS-0.1%Tween and detection of bands was performed by local treatment with H2O2/luminol during 1 min followed by chemiluminescence visualization on X-ray films. * Carbohydrate detection: Membrane was saturated for 2 h at RT with PBS containing 1% gelatin, 0.1% igepal and 0.1% tween. After saturation, the membrane was probed with Soybean Agglutinin-Fluorescein lectin (from Vector Laboratories®, diluted in PBS-0.1%Tween solution 1:200) for GalNAc detection or Concanavalin A-Fluorescein lectin (from Vector Laboratories®, diluted in PBS-0.1%Tween solution 1:200) for mannose detection, overnight at 4 °C. Then, membrane was washed thrice 15 min at RT with PBS-0.1%Tween, and probed with secondary antibody anti-Fluorescein-HRP (from abcam®, diluted in PBS-0.1%Tween solution 1:5000) during 1h30 at RT. Membrane was finally washed thrice 15 min at RT with PBS-0.1%Tween and detection of bands was performed by local treatment with H2O2/luminol during 1 min followed by chemiluminescence visualization on X-ray films. - Results Carbohydrate-coating of AAV2 capsid was performed with 7 (Man) and 12 (GalNAc) derivatives of luminol in a one-step electro-conjugation procedure with varying concentration and time of voltage. The integrity of the assembled viral capsid was conserved for all coupling conditions as confirmed by dot blot staining with anti-capsid A20 antibody (example in Figure 3a, top). A similar dot blot assay using a fluorescent labelled GalNAc-binding lectin (soybean agglutinin, SBA) or Man-binding lectin (concanavalin A, Con A) evidenced the efficient electro-conjugation of 12 (example in Figure 3a, bottom) and 7 respectively, with an observable time-dependent increase of staining intensity. Of note, coupling could be detected after very short time of conjugation in both cases (20 sec). Contrariwise, when voltage was applied to a mixture of AAV2 and a GalNAc derivative with no luminol moiety (Ctrl), GalNAc could not be detected by SBA, showing the covalent anchoring of 12 to the capsid during eY-click. All electro-conjugated samples were also subjected to capsid denaturation followed by SDS-PAGE separation on gels. The three constitutive envelop proteins (VP1/VP2/VP31:1:10) could be cleanly identified by silver nitrate staining and       western blot using anti-VPs polyclonal antibody (example in Figure 3b, left). A significant mass shift of VPs bands was observed overall for >5 min eY-click conditions for both carbohydrates suggesting an efficient, progressive and tunable degree of Y-labelling. Labelled lectins SBA and ConA were also used in western blot staining to respectively detect GalNAc and Man (example in Figure 3b, right), confirming the covalent conjugation of luminol-carbohydrates on the three constitutive proteins of the capsid. EXAMPLE 5: Transduction efficacy of the chemically-modified AAV of the Invention - Material and Methods In vitro transduction: The infectivity of each sample was measured as follows. HEK293 or HuH- 7 cells were seeded in DMEM with 10% FBS serum and 1% penicillin-streptomycin in 6-well culture plates at a density of 106 cells/well. Cells were then incubated overnight at 37 °C with 5% CO2 to reach 50% confluence. Then, AAV samples were prepared by serial dilution considering the studied multiplicity of infection (MOI = virus/cells ratio, varying from 103 to 104) and 2 µL of the samples were added to separate wells in the 6-well plates. The latter were incubated at 37 °C for 48 h. AAV-GFP-infected cells were detected and quantified by fluorescence microscopy and flow cytometry. - Results Transduction efficiency of the electro-conjugated carbohydrate-coated viral particles carrying a GFP reporter gene was evaluated on the HEK293 cell line. Cells were incubated at varying virus/cell ratio during 48 h and the percentage of GFP+ cells was measured by flow cytometry. Importantly, control experiment of 1 h voltage with GalNAc derivative having no luminol moiety resulted in fully conserved infectivity of the AAV, proving that the applied potential difference does not alter transduction capacity. Both AAV2-LumGalNAc and AAV2-LumMan samples electro-conjugated in 20 sec and 1 min also showed complete conserved transduction efficacy (example in Figure 4, left). In these examples, a controlled level of carbohydrate-coating on the AAV surface was therefore reproducibly achieved within 20 sec and 1 min experiments only. Additionally, AAV2-LumGalNAc (1 min) showed improved transduction capacity, as compared to AAV2, on HuH-7 cells expressing GalNAc receptors (example in Figure 4, right). This supports       the relevance of viral vectors functionalization to promote specific cell lines transduction potentially mediated by ligand-receptor interactions. EXAMPLE 6: Production and purification of AAV2, electrochemical bioconjugation with LumBiotin and LumN3, and SPAAC reaction The same protocol as that described in Example 3 was used for AVV2 production and purification and electrochemical bioconjugation except that LumBiotin and LumN3 were used. After the electrochemical bioconjugation of AAV2 with LumN3, the resulting AAV2-LumN3 were subjected to SPAAC reaction so as to decorate the AAV2 with nanobody or fluorescein moieties. In brief, DBCO-fluorescein, DBCO-biotin, DBCO-CD62L nanobody or DBCO-CD45 nanobody were added to the AAV2-LumN3 at a concentration of 10, 15 or 50 µM and the reaction was done during one or four hours at RT or 37°C under gentle shaking. After the SPAAC reaction, the excess of DBCO compound was removed by dialysis in four successive rounds against dPBS pH 7.4 (+0.001% Poloxamer) in a 10 kDa MWCO cassette. - Results N3-coating of AAV2 capsid was performed with LumN3 in an one-step electro-conjugation procedure with varying concentration and time of voltage. The integrity of the assembled viral capsid was conserved for all coupling conditions as confirmed by dot blot staining with anti-capsid A20 antibody (Figure 6). A similar dot blot assay using DBCO-fluorescein evidenced the efficient electro-conjugation of the Azido derivatives (Figure 6). Mass spectroscopy showed that an average of four Azido derivatives per VP3 protein was coupled on tyrosine residue(Figure 6). After the SPAAC reaction with DBCO-fluorescein, DBCO-biotin, DBCO-CD62L nanobody or DBCO-CD45 nanobody in a second time, all the samples were subjected to capsid denaturation followed by SDS-PAGE separation on gels. The three constitutive envelop proteins (VP1/VP2/VP31:1:10) could be clearly identified by silver nitrate staining for AAV2 subjected to the SPAAC with DBCO-fluorescein, DBCO-CD62L nanobody and DBCO-CD45 nanobody (Figure 7). Labelled anti-fluorescein antibody, streptavidin, anti-CD62L and anti-CD45 nanobody antibodies were also used in western blot staining to respectively detect fluorescein, biotin, CD62L nanobody and CD45 nanobody (Figure 7), confirming the covalent SPAAC conjugation between the azido       moieties present on the surface of AAV2 and the DBCO derivatives. For the two nanobodies, additional bands corresponding to different molecular weight were observed on western blot and silver staining suggesting an efficient electrochemical bioconjugation labelling.    

Claims

  Claims 1. An adeno-associated Virus (AAV) having at least one chemically-modified tyrosine residue in its capsid, wherein said chemically-modified tyrosine residue is of formula (I):
Figure imgf000080_0001
wherein: - RA is -(Y)n-M, a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, - each RB is independently selected from the group consisting of a group of formula -(Y)n-M, a hydrogen or a substituent selected from the group consisting of a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1-C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, With proviso that at least one group among RA and RB groups is -(Y)n-M, - k is 1 or 2, - n is 0 or 1, - Y is a spacer, and - M is a functional moiety.       2. The AAV of claim 1, wherein RA is a C1-C3 alkyl, a phenyl, or a benzyl, preferably a methyl or a benzyl, more preferably a methyl. 3. The AAV of claim 1 or 2, wherein one or two RB is a group of formula -(Y)n-M and the other RB are hydrogens. 4.The AAV of any one of Claims 1 to 3, wherein Y is a chemical chain group comprising from 2 to 500 carbon atoms and selected from the group consisting of polymers including homopolymers, copolymers and block polymers, peptides, oligosaccharides, and saturated or unsaturated hydrocarbon chains optionally interrupted by one or several heteroatoms and/or by one or several cyclic or heterocyclic moieties, optionally having an heteroatom, such as S, O and NH, at least one of its extremity, and optionally substituted by one or several substituents, and combinations thereof. 5.The AAV of any one of Claims 1 to 3, wherein Y is a saturated or unsaturated hydrocarbon having from 2 to 100 carbon atoms, optionally interrupted by: - one or more heteroatomic groups chosen from -O-, -S-, -N(R)- with R being H or a C1-C3 alkyl, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)-NH-, and -NH-CS-; and/or - C5-C20 carbocyclic moieties such as cycloalkyl, cycloalkenyl, or aromatic groups; and/or - one or more heterocyclic moieties having 5 to 20 ring atoms, such as heterocycloalkyls or heteroaryls having 5 to 20 ring atoms, - and optionally having at least one of its extremities, an heteroatomic group chosen from -O-, - S-, -N(R)- with R being H or C1-C3 alkyl, -O-N(R)- with R being H or C1-C3 alkyl, -N(C1-C3 alkoxy)-, -C(O)-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-CO-NH-, -O-CO-NH-, NH-(CS)- NH-, NH-CS-. 6. The AAV of any one of claims 1 to 5, wherein M is a functional moiety comprising a group selected from a click-chemistry group, a steric shielding agent, a labelling agent, a targeting agent such as a cell-type specific ligand, a drug moiety, an oligonucleotide and combinations thereof.       7. The AAV of any one of claims 1 to 5, wherein M comprises, or consists of a moiety selected from a click-chemistry group, a cell targeting agent, preferably selected from a mono- or a polysaccharide, a hormone, including a steroid hormone, a peptide such as RGD peptide, muscle targeting peptides (MTP), or Angiopep-2, a protein or a fragment thereof, a membrane receptor or a fragment thereof, an aptamer, an antibody including heavy-chain antibody, and fragments thereof such as Fab, Fab’, and VHH, a ScFv, a spiegelmer, a peptide aptamer, a vitamin and small chemical molecules such as drugs e.g. CB1 and/or CB2 ligands. 8. The AAV of any one of claims 1 to 5, wherein M comprises or consists of a moiety selected from a click-chemistry group in particular an azido group or a strained alkyl group, a cell-type specific ligand derived from a protein selected from transferrin, Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor ^FGF, a mono- or a polysaccharide comprising one or several galactose, mannose, N-acetylgalactosamine residues, bridge GalNac, or mannose-6-phosphate, sialic acid and derivatives thereof ( e.g. Neu5Ac, Neu5Ac ^2-6Gal, Neu5Ac ^2-8Neu5Ac), a MTP selected from SEQ ID NO:1 to SEQ ID NO:7, a VHH, a fluorescent label and vitamins such as folic acid. 9. The AAV of any one of claims 1 to 8, which further has at least one additional chemically modified amino acid residue in the capsid selected from chemically modified cysteine, arginine or lysine. 10. The AAV according to any one of claims 1 to 9, wherein the AAV is a recombinant AAV, preferably selected from AAV having a wildtype capsid, naturally-occurring serotype AAV, variant AAV, pseudotype AAV, AAV with hybrid or mutated capsids, and self-complementary AAV. 11. A method for chemically-modifying the capsid of an AAV, more precisely for chemically modifying at least one tyrosine residue in the capsid of an AAV, which comprises incubating said AAV with a chemical reagent bearing a N-substituted luminol moiety in conditions conducive for       reacting said chemical reagent with a tyrosine residue present in the capsid of the AAV so as to form a covalent bound. 12. The method of claim 11 which is performed by electrochemistry. 13. The method according to claim 11 or 12, which comprises incubating said AAV with a chemical reagent of formula (X):
Figure imgf000083_0001
wherein: - RA is -(Y)n-M, a C1-C6 alkyl, a C6-C14 aryl optionally substituted, or a (C6-C14 aryl)-(C1-C3 alkyl) optionally substituted, - each RB is independently selected from the group consisting of a group of formula -(Y)n-M, a hydrogen or a substituent chosen from a halogen, C1-C6 alkyl, C6-C14 aryl, C3-C6 cycloalkyl, C1- C6 alkoxy, C1-C6 alkylamino, C2-C6 heterocycle, C1-C6 alkanoyl, C1-C6 carboxy esters, C1-C6 acylamino, -COOH, -CONH2, -NO2, -SO3H, -CN, -CF3, C1-C6 hydroxyalkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 thioalkyl, C2-C10 alkoxyalkyl, and C2-C6 alkoxycarbonyloxy, With proviso that at least one group among RA and RB groups is -(Y)n-M, - n is 0 or 1, - Y is a spacer, and - M is a functional moiety; in the presence of a potential difference enabling the electro-activation of said chemical reagent of formula (X) into an oxidized form able to react with the tyrosine residues so as to obtain at least one chemically-modified tyrosine residue in the AAV capsid of formula (I):      
Figure imgf000084_0001
wherein: - RA and RB are as defined in formula (X), - k is 1 or 2. 14. The method according to claim 13, wherein the method is performed in an electrochemical system with three electrodes comprising a working electrode, a counter-electrode and a reference electrode by applying a constant potential difference between the working electrode and the reference electrode, said constant potential difference being preferably included in a range defined by the oxidation potential of the chemical reagent of formula (X) ± 200 mV.. 15. The method of claim 13 or 14 wherein RA is a C1-C6 alkyl and at least one RB is of formula - (Y)n-M and the other RB are H. 16. The method of any one of claims 13 to 15 wherein M is a click-chemistry group and wherein the method further comprises a step of click-reaction so as to covalently bond a functional moiety preferably selected from ligands and labels on the AAV capsid.       17. A pharmaceutical composition comprising an AAV as defined in any one of claims 1-9 and at least one pharmaceutically acceptable excipient. 18. An AAV of claims 1-9, or a pharmaceutical composition of claim 17, for use as a diagnostic agent in vivo or as a drug in vivo or ex vivo, preferably in gene therapy. 19. Use of an AAV as defined in any one of claims 1-9 as a research tool in vitro, for instance as a gene transfection agent or as an imaging agent in vitro. 20. Use of an AAV as defined in any one of claims 1-9 in the manufacture of a diagnostic agent or a drug, in particular for gene therapy ex vivo or in vivo. 21. Use of a compound of formula (X):
Figure imgf000085_0001
defined in Claim 13 or 15 as an agent for chemically modifying the capsid of an AVV by electrochemical bioconjugation.    
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1310571A2 (en) 2001-11-13 2003-05-14 The Trustees of The University of Pennsylvania A Method of detecting and/or identifying adeno-associated virus (AVV) sequences and isolating novel sequences identified thereby
EP2292779A2 (en) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses thereof
WO2014144229A1 (en) 2013-03-15 2014-09-18 The University Of North Carolina At Chapel Hill Methods and compositions for dual glycan binding aav vectors
WO2015062516A1 (en) 2013-10-30 2015-05-07 北京大学 Adeno-associated virus with site-directed mutagenesis and site-directed modification, and preparation method and application therefor
WO2017212019A1 (en) 2016-06-09 2017-12-14 Centre National De La Recherche Scientifique (Cnrs) Raav with chemically modified capsid
WO2021005210A1 (en) 2019-07-11 2021-01-14 Centre National De La Recherche Scientifique Chemically-modified adeno-associated virus
WO2022061378A2 (en) * 2020-09-21 2022-03-24 Asklepios Biopharmaceutical, Inc. Methods for treating neurological disease

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1310571A2 (en) 2001-11-13 2003-05-14 The Trustees of The University of Pennsylvania A Method of detecting and/or identifying adeno-associated virus (AVV) sequences and isolating novel sequences identified thereby
EP2292779A2 (en) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Adeno-associated virus (AAV) clades, sequences, vectors containing same, and uses thereof
WO2014144229A1 (en) 2013-03-15 2014-09-18 The University Of North Carolina At Chapel Hill Methods and compositions for dual glycan binding aav vectors
WO2015062516A1 (en) 2013-10-30 2015-05-07 北京大学 Adeno-associated virus with site-directed mutagenesis and site-directed modification, and preparation method and application therefor
WO2017212019A1 (en) 2016-06-09 2017-12-14 Centre National De La Recherche Scientifique (Cnrs) Raav with chemically modified capsid
WO2021005210A1 (en) 2019-07-11 2021-01-14 Centre National De La Recherche Scientifique Chemically-modified adeno-associated virus
WO2022061378A2 (en) * 2020-09-21 2022-03-24 Asklepios Biopharmaceutical, Inc. Methods for treating neurological disease

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
BUNING ET AL., CURR OPIN PHARMACOL, vol. 24, 2015, pages 94 - 104
C.-C. LIN ET AL., ORG. LETT., vol. 9, 2007, pages 2131 - 2134
DEPIENNE SÉBASTIEN ET AL: "Luminol anchors improve the electrochemical-tyrosine-click labelling of proteins", CHEMICAL SCIENCE, vol. 12, no. 46, 1 January 2021 (2021-01-01), United Kingdom, pages 15374 - 15381, XP093050444, ISSN: 2041-6520, DOI: 10.1039/D1SC04809K *
DEVERMAN ET AL., NAT BIOTECHNOL, vol. 34, no. 2, 2016, pages 204 - 209
HOROWITZ ET AL., BIOCONJ CHEM, vol. 22, no. 4, 2011, pages 529 - 532
KOERBER ET AL., MOLECULAR THERAPY, vol. 16, no. 10, 2008, pages 1703 - 1709
KOLB ET AL., ANGEW. CHEM. INT. ED., vol. 40, 2001, pages 2004 - 2021
KONIEV, O.WAGNER, A, CHEM. SOC. REV., vol. 44, 2015, pages 5495
MARKUSIC ET AL., MOLECULAR THERAPY, vol. 18, 2010, pages 2048 - 2056
NASO ET AL., BIODRUGS, vol. 31, no. 18393-54-9, 2017, pages 317 - 334
RUDOLF ET AL., CURRENT OPINION IN CHEMICAL BIOLOGY, vol. 17, 2013, pages 110 - 117
S. DEPIENNE ET AL., CHEM. SCI., vol. 12, 2021, pages 15374 - 15381
SHINICHI SATO ET AL: "Tyrosine-Specific Chemical Modification with in Situ Hemin-Activated Luminol Derivatives", ACS CHEMICAL BIOLOGY, vol. 10, no. 11, 20 November 2015 (2015-11-20), pages 2633 - 2640, XP055372690, ISSN: 1554-8929, DOI: 10.1021/acschembio.5b00440 *
ZHONG ET AL., PNAS, vol. 105, 2008, pages 7827 - 7832

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