WO2024165721A1

WO2024165721A1 - Squaramide-modified adeno-associated virus vectors

Info

Publication number: WO2024165721A1
Application number: PCT/EP2024/053305
Authority: WO
Inventors: Delphine Compere; Gaëlle LEFEVRE; Lavaniya KUNALINGAM
Original assignee: Coave Therapeutics SA
Current assignee: Coave Therapeutics SA
Priority date: 2023-02-10
Filing date: 2024-02-09
Publication date: 2024-08-15
Anticipated expiration: 2025-08-10
Also published as: EP4662322A1; IL322465A; AU2024217462A1; KR20250143841A; CN120936721A

Abstract

The present invention relates to adeno-associated virus (AAV) vectors modified by the covalent coupling of at least one compound comprising a squaramide moiety to at least one amino group of an amino acid residue of the capsid of the AAV vectors. The AAV vectors are useful in transducing a cell, especially for gene therapy.

Description

SQUARAMIDE-MODIFIED ADENO-ASSOCIATED VIRUS VECTORS FIELD OF INVENTION [0001] The present invention relates to adeno-associated virus (AAV) vectors modified by the covalent coupling of a squarate ester with a ligand or functional moiety and with a primary amino group of an amino acid residue of the capsid of the AAV vectors. In some embodiments, provided AAV vectors are useful in transducing a cell, especially for gene therapy. BACKGROUND OF INVENTION [0002] Gene therapy is based on the genetic modification of cells to produce a therapeutic effect by the delivery of nucleic acid into patient's cells. Indeed, sometimes the whole or part of a gene is defective or missing from birth, or a gene can change or mutate during life. Any of these variations can disrupt how proteins are synthetized, which can contribute to health problems or diseases. By gene therapy, a defective gene or genetic sequence that causes a medical problem can be replaced with a healthy version that doesn't; genes (or sequences) can also be added to help the body fight or treat disease; or genes (or sequences) that are causing problems can be knocked down or knocked out. Thereby, gene therapy can be used to treat inherited or acquired diseases. [0003] The delivery of new genes (or sequences) into cells can be carried-out by various methods, such as using a vector which is genetically engineered to deliver the sequence (e.g., gene) of interest. Viral vectors can be used for that purpose, especially adeno- associated virus (AAV) vectors. AAV vectors have proven to be reliable, efficient, versatile, and safe tools to deliver a transgene of interest to a variety of tissues. AAV vectors present the advantage of having a relatively broad tropism, a high transduction efficacy, a persistent episomal expression, and a high safety profile, in particular because wild-type AAV is not associated with any human diseases. [0004] Clinical trials of gene therapy using AAV vectors were conducted or are ongoing for several types of diseases. Nevertheless, certain trials have shown some limitations of these AAV vectors including immunogenicity, unselective distribution, and reduced therapeutic index. [0005] In particular, it was recently evidenced that humoral immunity can preexist to certain AAV serotypes, especially AAV of serotype 2. Therefore, preexisting anti-AAV neutralizing antibodies can preclude transduction in targeted tissues, resulting in a lack of efficacy, especially upon systemic administration. Moreover, when it is necessary to re- administer the AAV vector to complete the treatment, it can be precluded by the appearance of neutralizing antibodies following the first administration. Consequently, AAV vectors capable of avoiding immune detection would be highly desirable. [0006] Another limitation of AAV vectors is linked to their broad tropism. Indeed, the broad distribution of AAV vectors leads to transgene expression in tissues other than targeted ones, thus lacking specificity. This can lead to reduced therapeutic index. Indeed, high doses of vectors may be required to achieve therapeutic efficacy in a given tissue. Such high doses pose a challenge not only for vector production but also by increasing the risk of immune response. Consequently, AAV vectors capable of ensuring cell- specific transduction would be highly desirable. [0007] Various strategies have been explored in order to improve AAV vectors in order to evade the immune system and enhance cell transduction and cell specificity, especially by modifying the capsid proteins of the vectors. Such modifications of capsid proteins can be achieved by introducing mutations in surface-exposed amino acid residues of capsids of AAV vectors. Alternatively or additionally, chemical modification of viral capsids has been proposed in order to add a specific ligand on the capsid or to mask certain exposed amino acids. Such chemical modifications can be obtained, for example, by introducing a non-natural amino acid residue comprising a reactive functional group into the capsid proteins and then selectively coupling a ligand by orthogonal reaction with said reactive functional group. Another strategy is to perform a direct chemical modification on the viral capsid, without any preliminary mutation of the capsid proteins. For example, WO2017/212019 provides surface modified AAV vectors obtained by covalently coupling a ligand bearing a certain isothiocyanate group to an amino group present in an amino acid residue of the capsid proteins of the AAV, leading to improved gene transfer into specific cells. Additionally, the modification of tyrosine residues of the AAV capsid is reported in WO2021/005210, which provides a method of chemically modifying tyrosine residues of the AAV capsid in order to modify immunogenicity of AAV vectors. On the other hand, WO2022/096681 provides surface modified vectors obtained by reaction of a compound comprising a lactam (e.g., β-lactam) with an amino group present in an amino acid residue of the capsid proteins of the AAV. [0008] However, the current strategies to modify AAV vectors still suffer from several drawbacks. For example, in certain contexts, it has been observed that coupling chemistries comprising certain isothiocyanate groups are often too reactive and thus susceptible to significant self-coupling reactions. On the other hand, even if the use of β- lactams allows solving issues related to the mentioned self-coupling reactions, its use may be linked to certain regulatory constraints that may affect the design and total cost of the industrial scale-up of these products: FDA regulations require β-lactams to be processed with complete and comprehensive separation from non- β -lactam products (see April 2013 FDA regulations, Docket Number: FDA-2011-D-0104 ). [0009] Accordingly, there is still a need for new methods for modifying the properties of AAV vectors in order to achieve efficient gene transduction, especially for gene therapy. SUMMARY [0010] The present invention relates to an adeno-associated virus (AAV) vector particle comprising a squaramide linker moiety resulting from the reaction of a squarate ester with a primary amino group present within the capsid of the AAV vector (e.g., amino group of a lysine side chain). [0011] In some aspects, the present invention refers to an adeno-associated virus (AAV) vector particle comprising a moiety of formula (II):

wherein N* is a nitrogen atom of a primary amino group from a surface-exposed amino acid residue of a capsid polypeptide from the AAV vector; ---- represents the point of attachment to the AAV vector's capsid; and R^L-NH- is a functional moiety, as defined and described in classes and subclasses in the present invention, comprising a nitrogen containing group -NH-. [0012] The AAV vector particles defined according to the present invention comprise a squaramide linker of formula (I):

wherein the functional moiety R^L-NH- and an amino acid residue of a capsid from the AAV vector, of the AAV vector particles disclosed in the present invention, are covalently linked to form the squaramide linker of formula (I); and wherein N is a nitrogen atom belonging to the functional moiety, which is thus indicated as R^L-NH- as defined in the present invention, and wherein N* is a nitrogen atom of a primary amino group from a surface-exposed amino acid residue of a capsid polypeptide from the AAV vector. [0013] In some aspects, the present invention relates to technologies for and/or methods of manufacturing a provided AAV vector particle. [0014] In some aspects, the surface-exposed amino acid residue comprising at least one primary amino group is lysine. [0015] In some aspects, N* is a nitrogen atom of an amino group of a lysine residue of the AAV vector's capsid. [0016] The functional moiety R^L-NH- includes a group -NH- which forms part of the squaramide linker of formula (I) as defined in the present invention, and a functional group R^L comprising a steric shielding agent, a labelling agent, a cell-type specific ligand, a drug moiety and combinations thereof. Accordingly, in some aspects, R^L-NH- is a functional moiety comprising or consisting of a group selected from a steric shielding agent, a labelling agent, a cell-type specific ligand, a drug moiety and combinations thereof. [0017] In some aspects, R^L-NH- comprises a labeling agent. In some aspects the labeling agent comprises or is a fluorescent dye such as fluoroalanine, fluorescein, rhodamine, boron-dipyrromethene (Bodipy^®) dyes, and Alexa fluor^®, or a radionuclide. [0018] In some aspects, R^L-NH- comprises a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, glycoproteins, proteins or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties. [0019] In some aspects, R^L-NH- comprises a steric shielding agent selected from the group consisting of polyethylene glycol, pHPMA, and polysaccharides. [0020] In some aspects, the functional moiety R^L-NH- comprises a group Z and one or more spacers L, and the adeno-associated virus (AAV) vector particle comprises a moiety represented by formula (IIa):

Wherein N*, ------, Z and L, are as defined and described in classes and subclasses in the present invention. [0021] In some aspects, the functional moiety R^L-NH- comprises a group Z and one or more spacers L, wherein Z is H or comprises a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins, or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties. [0022] In some aspects, the functional moiety R^L-NH- does not comprise one or more spacers L and the functional moiety R^L-NH- consists of a group Z-NH-. [0023] In some aspects, the functional moiety R^L-NH- comprises one or more groups Z and one or more spacers L. In some aspects, the functional moiety R^L-NH- comprises 1 to 3 groups Z, each of said groups Z linked to one or more spacers L. [0024] In some aspects, Z is a saccharide. In other aspects Z is a peptide. [0025] In some aspects, Z is or comprises a saccharide selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and derivatives thereof. [0026] In some aspects, the saccharide is selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, desosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose and P1-fructose. In some preferred aspects, the saccharide is selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid and P1-fructose, more preferably mannose. [0027] In some aspects Z is or comprises a linear or a cyclic peptide, wherein said peptide may be peptide featuring biological activity, in particular a blood brain barrier penetrating peptide (BBPs). In particular, the peptide is a blood brain barrier (BBB) shuttle peptide (also referred as a BBB-penetrating peptide) with an enhanced transduction activity across the blood brain barrier. The BBB shuttle peptides have the ability to cross the BBB and are thus molecules capable of transporting a variety of cargoes into the brain parenchyma without disrupting the BBB integrity. Therefore, the BBB shuttle peptide have the potential to increase the ability of AAV to cross the BBB and enhance brain transduction, especially in neuronal cells. In some preferred aspects, the BBB shuttle peptide is selected from the group consisting of a peptide THR or a peptide with a RGD motif, including a cyclic RGD peptide. Typically, the peptide THR is capable of binding to and internalizing with the human transferrin receptor (hTfR), as described in Lee et al., 2004 (Eur J Biochem. 2001 Apr;268(7):2004-12. doi: 10.1046/j.1432-1327.2001.02073.x. PMID: 11277922) and the international patent application published under No. WO02/44329 (A2). BBB shuttle peptides have also been described in Sánchez-Navarro et al., 2022 (Pharmaceutics.2022 Sep 5;14(9):1874. Doi: 10.3390/pharmaceutics14091874. PMID: 36145622; PMCID: PMC9505527) and for instance in the international patent applications published under No. WO2008/025867 (A1), WO2012/007625 (A1), WO2013/127829 (A1) and WO 2015/001015 (A1). [0028] In some aspects, the spacer group L comprises one or more groups selected from the group consisting of an arylene or a heteroarylene group Ar; an optionally substituted group comprising saturated or unsaturated, linear or branched C₁-C₄₀ hydrocarbon chains; an alkylene amine containing group; an acyl containing group; an amino acid; an alkyl ether group such as an ethylene glycol or a propylene glycol group; a polyether such as a polyethylene glycol (PEG) or a polypropylene glycol (PPG), or a polyether of a branched polyol; a polyamide such as a β-alanine polymer; a vinylic polymer such as pHPMA; a polyester, such as PLGA; polymers of alkylene diamines; and combinations thereof. In particular, the spacer group L comprises one or more groups selected from the group consisting of an arylene or a heteroarylene group, an optionally substituted group comprising saturated or unsaturated, linear or branched, C₁-C₄₀ hydrocarbon chains, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; an acyl group, an amino acid moiety, a polyether of a branched polyol, a β-alanine polymer, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof. [0029] In some aspects, when Z is a peptide, Z is covalently linked to one C_1-6 alkylene group, or to a β-alanine moiety, or to a group Ar as defined herein, or to an acyl group, or to the squaramide moiety of formula (I), by an amide moiety or a bioisostere moiety thereof, wherein either the acyl group or the nitrogen atom of said amide moiety corresponds to, respectively, the acyl group of the C-terminal group of the peptide and to the nitrogen atom of the N-terminal group of the peptide. [0030] In some aspects, when Z is a saccharide, Z is covalently linked to a PEG or PPG group, or to a C_1-6 alkylene group, by an ether bond, wherein the oxygen atom of said ether bond corresponds to an oxygen atom belonging to the saccharide. [0031] In some aspects, L comprises a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers. In some aspects, L comprises a polyethylene glycol (PEG), comprising 1 to 10 ethylene glycol monomers. In some aspects, the polyethylene glycol (PEG) is PEG1, PEG2, PEG3, PEG4, or PEG5. [0032] In some aspects, L comprises a β-alanine polymer, or a β-alanine moiety, comprising 1 to 40 β-alanine monomers or β-alanine units. In some aspects, the β-alanine polymer comprises 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 8 or 10 β-alanine monomers. [0033] In some aspects, L comprises a polyethylene glycol (PEG) and a β-alanine polymer. [0034] In some aspects, L comprises a polyether of a branched polyol, such as a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, more preferably a branched C₄ polyol. [0035] In some aspects, L comprises an amino acid moiety. For the purposes of the present disclosure the term amino acid refers to a molecule including both an amine group and a carboxylic acid group, including accordingly also, but not only, proteogenic and non-proteogenic amino acids. In some aspects L comprises an arginine moiety, a β- alanine moiety. In particular, said amino acid moieties are linked to another part of the spacer L, or to the squaramide linker of formula (I) or to a functional moiety by the carboxylate group or/and by the amine group thereof. [0036] In some aspects, L comprises one or more arylene or a heteroarylene groups Ar. [0037] In some aspects, L comprises a C_1-6 alkylene group, which may be a linear C_1-6 alkylene group or a branched C_3-6 alkylene group, more preferably L comprises a -CH₂- group or a branched C₄ alkylene group. In some aspects, L comprises a C_1-6 alkylenamine group, preferably -CH₂-CH₂-NH- group. In some aspects, L comprises a C_1-6 acyl group, preferably a -CH₂-CH₂-C(O)- group. In some aspects between a PEG group and a β- alanine polymer an acyl group –(CH₂)n-C(O), where n is 1 to 6, preferably a group -CH₂- CH₂-C(O)-, is present so that the PEG and the β-alanine polymer are linked by an amide moiety. [0038] In some aspects, the arylene or a heteroarylene group Ar is a bivalent aromatic radical (or bivalent aromatic moiety), i.e. which is linked to two different groups of the moiety of formula (II) of the AAV vector particles of the present invention (i.e. forming a bridge between two parts of the moiety of formula (II)), and which may additionally comprise one or more optional substitutions. Preferably, said arylene or a heteroarylene group Ar is a 6- to 10-membered arylene group or a 5- or 12-membered heteroarylene group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se. In some particular aspects, the arylene or the heteroarylene group Ar is a phenylene or pyridylene group, optionally comprising one or more substitutions. In some aspects, said arylene or heteroarylene group Ar comprises one or more substitutions selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1- ₆ alkoxy. [0039] In some aspects, Z is a saccharide or a peptide and L comprises a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers. [0040] In other aspects, Z is a saccharide or a peptide and L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and an arylene or a heteroarylene group Ar, preferably wherein said PEG and Ar groups are covalently linked by an amide moiety or a bioisostere moiety thereof. [0041] In other aspects, Z is a saccharide or a peptide and L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, one or more C_1-6 alkylene groups and an arylene or a heteroarylene group Ar, preferably wherein said PEG and Ar groups are covalently linked by an amide moiety or a bioisostere moiety thereof, or wherein said PEG and C_1-6 alkylene group are covalently linked by an amide moiety or a bioisostere moiety thereof. [0042] In other aspects, the functional moiety R^L-NH- comprises 1 to 3 groups, Z; wherein Z is a saccharide or a peptide, and L comprises one or more polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, one or more C_1-6 alkylene group, a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and an arylene or a heteroarylene group Ar; preferably wherein each Z is linked to a first C_1-6 alkylene group or to a PEG, being said first C_1-6 alkylene group linked to the group Z by an amide moiety, and being said PEG covalently linked to a second C_1-6 alkylene group by an amide moiety or a bioisostere moiety thereof; wherein said second C_1-6 alkylene group is linked to the branched C_3-12 polyol by an ether bond, and wherein the branched C_3-12 polyol is linked to an arylene or a heteroarylene group Ar by an amide moiety or a bioisostere moiety thereof; or wherein the arylene or a heteroarylene group Ar is linked to one or more C_1-6 alkylene groups different from the first and second C_1-6 alkylene groups, and wherein the branched C_3-12 polyol is linked to one of the one or more C_1-6 alkylene groups by an amide moiety or a bioisostere moiety thereof. In one aspect the functional moiety R^L-NH- comprises 1 to 3 groups, Z; wherein Z is a saccharide. In another aspect the functional moiety R^L-NH- comprises 1 to 3 groups, Z; wherein each Z is independently a saccharide or a peptide. [0043] In other aspects Z is a peptide and L comprises a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers and one or more C_1-6 alkylene groups; or one or more C_1-6 alkylenamine groups, or one or more C_1-6 acyl groups. [0044] In other aspects, Z is a peptide and L comprises a β-alanine polymer comprising 1 to 40 β-alanine monomers. [0045] In other aspects, Z is a peptide and L comprises a β-alanine polymer comprising 1 to 40, preferably 1 to 10, β-alanine monomers and one or more C_1-6 alkylene groups, preferably a C_1-2 alkylene group, or one or more C_1-6 alkylenamine groups, preferably a - CH₂-CH₂-NH- group, or one or more C_1-6 acyl groups, preferably a -CH₂-CH₂-C(O)- group. [0046] In other aspects, Z is a peptide and L comprises a β-alanine polymer comprising 1 to 40, preferably 1 to 10, β-alanine monomers, one or more C_1-6 alkylene groups, preferably a C_1-2 alkylene group, or one or more C_1-6 alkylenamine groups, preferably a - CH₂-CH₂-NH- group, or one or more C_1-6 acyl groups, preferably a -CH₂-CH₂-C(O)- group, and an arylene or a heteroarylene group Ar; wherein said PEG and Ar group are covalently linked by an amide moiety or a bioisostere moiety thereof, or wherein said PEG and one C_1-6 alkylene group are covalently linked by an amide moiety or a bioisostere moiety thereof.. [0047] In other aspects, Z is a peptide and L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, one or more C_1-6 alkylene groups, preferably a C_1-2 alkylene group, or one or more C_1-6 alkylenamine groups, preferably a - CH₂-CH₂-NH- group, or one or more C_1-6 acyl groups, preferably a -CH₂-CH₂-C(O)- group; and an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, more preferably an arginine moiety. [0048] In other aspects, Z is a peptide and L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, a β-alanine polymer comprising 1 to 40 β- alanine monomers, one or more C_1-6 alkylene groups, preferably a C_1-2 alkylene group, or one or more C_1-6 alkylenamine groups, preferably a -CH₂-CH₂-NH- group, or one or more C_1-6 acyl groups, preferably a -CH₂-CH₂-C(O)- group, and an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, more preferably an arginine moiety. [0049] In some aspects, the peptide Z and the spacer L, in particular a spacer group C1- 6 alkylene, are covalently linked by an amide moiety, for example and amide -N(R₁)C(O)- , or a bioisostere moiety thereof; wherein R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z-(OCH₂–CH₂)n–, Z-C(O)NH-(CH₂)q–(OCH₂-CH₂)n–, and Z- NHC(O)–(CH₂)q–(OCH₂-CH₂)n–, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂–CH₂)n–,wherein n is selected from 1 to 40 and more preferably R₁ is H or Z-(OCH₂–CH₂)n–,wherein n is selected from 1 to 40, even more preferably R₁ is H. [0050] In some aspects, L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and an arylene or a heteroarylene group Ar, as defined in the present invention, wherein said PEG and Ar groups are covalently linked by an amide moiety, for example an amide -N(R₁)C(O)-, or a bioisostere moiety thereof; or L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, one or more C_1-6 alkylene groups and an arylene or a heteroarylene group Ar, as defined in the present invention, wherein said PEG and Ar groups are covalently linked by an amide moiety, for example an amide -N(R₁)C(O)-, or a bioisostere moiety thereof or wherein said PEG and C_1-6 alkylene group are covalently linked by an amide moiety, , for example an amide -N(R₁)C(O)-, or a bioisostere moiety thereof; wherein R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z-(OCH₂–CH₂)n–, Z-C(O)NH- (CH₂)q–(OCH₂-CH₂)n–, and Z-NHC(O)–(CH₂)q–(OCH₂-CH₂)n–, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂–CH₂)n–,wherein n is selected from 1 to 40 and more preferably R₁ is H or Z-(OCH₂–CH₂)n–,wherein n is selected from 1 to 40, even more preferably R₁ is H. [0051] In some aspects, the one or more spacer L is selected from the group consisting of L₁, L₂ and L₃ and said AAV vector particle comprises a moiety selected from the group consisting of formula (IIa₁), (IIa₂), (IIa₃), (IIa₄), (IIa₅), (IIa₆), (IIa₇), (IIa₈), (IIa₉), (IIa₁₀), (IIa₁₁), (IIa₁₂), (IIa₁₃), (IIa₁₄), (IIa₁₅), (IIa₁₆), (IIa₁₇), (IIa₁₈), (IIa₁₉) and (IIa₂₀):

wherein N*, ----, Z are as defined and described in classes and subclasses disclosed in the present invention and wherein L₁ is one or more groups selected from the group consisting of a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and a β-alanine polymer comprising 1 to 40 β-alanine monomers, or a mixture thereof; L₂ comprises one or more arylene or a heteroarylene groups Ar; and L₃ is one or more groups selected from the group consisting of an amino acid moiety, C_1-6 alkylene amine group, a C_1-6 alkylene acyl group and a group C_1-6 alkylene group, being said C_1-6 alkylene a linear C_1-6 alkylene group or a branched C_3-6 alkylene group; and wherein preferably Z is a peptide or a saccharide. [0052] In some aspects, L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and wherein, L₁ and L₂, or L₁ and L₃ are covalently linked by an amide moiety or a bioisostere moiety thereof; or wherein L₁ and L₃ covalently linked by an ether bond. [0053] In some aspects, the polyethylene glycol (PEG) is PEG1, PEG2, PEG3, PEG4, or PEG5. In some aspects, the β-alanine polymer comprises 1 to 10 β-alanine monomers, In some aspects, L₂ comprises a phenylene group or a pyridylene group. In some aspects, L₃ is a C_1-3 alkylene, a -CH₂-CH₂-NH- group, a - CH₂-CH₂-C(O)- group or an aminoacid moiety, preferably an arginine moiety or a β-alanine moiety. [0054] In some aspects, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers or a β-alanine polymer comprising 1 to 40 β-alanine monomers, or a mixture thereof, L₂ is an arylene or a heteroarylene group Ar; L₁ and L₂ are covalently linked by an amide moiety or a bioisostere moiety thereof. [0055] In some aspects, L₁ and L₃ are covalently linked by an amide moiety or a bioisostere moiety thereof. [0056] In some aspects, L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group. [0057] In some aspects, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers or a β-alanine polymer comprising 1 to 40 β-alanine monomers, or a mixture thereof, L₂ is an arylene or a heteroarylene group Ar, and L₃ one or more groups selected from the group consisting of an amino acid moiety, C_1-6 alkylene amine group, a C_1-6 alkylene acyl group and a group C_1-6 alkylene group; wherein L₁ and L₂ or L₁ and L₃ are covalently linked by an amide moiety, or a bioisostere moiety thereof. [0058] In some aspects, L₁ is a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and L₃ is a group C_1-6 alkylene; being L₁ and L₃ covalently linked by an ether bond. [0059] In some aspects, L₁ is a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and L₃ is a group C_1-6 alkylene; being L₁ and L₃ covalently linked by an amide moiety or a bioisostere moiety thereof. [0060] In some aspects, L₁ is a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and L₂ comprises an arylene or a heteroarylene group Ar; being L₁ and L₂ covalently linked by an amide moiety or a bioisostere moiety thereof. [0061] In some aspects, when L₂ comprises an arylene or a heteroarylene group Ar, L₁ and the squaramide linker of formula (I) are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para, or when L₂ comprises an arylene or a heteroarylene group Ar and L₃ is present, L₁ and L₃ are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para. [0062] In some aspects, when L₂ comprises an arylene or a heteroarylene group Ar and L₃ is a group C_1-6 alkylene, L₃ and the squaramide linker of formula (I) are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para. [0063] In some aspects, when L₂ comprises an arylene or a heteroarylene group Ar and L₃ is a group C_1-6 alkylene, one or more groups L₃ are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para. [0064] In some aspects, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers; L₂ comprises one or more arylene or a heteroarylene groups; L₃ is a C_1-6 alkylene group, L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and L₁ and L₂ are covalently linked by an amide moiety or by a bioisostere moiety thereof. [0065] In some aspects, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers; L₂ comprises one or more arylene or a heteroarylene groups; L₃ is a C_1-6 alkylene group, L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and L₁ and L₃ are covalently linked by an amide moiety or by a bioisostere moiety thereof. [0066] In some aspects, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers; L₂ comprises one or more arylene or a heteroarylene groups; L₃ is a C_1-6 alkylene group, L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and L₁ and L₂ are covalently linked by an amide moiety or by a bioisostere moiety thereof or L₁ and L₃ are covalently linked by an amide moiety or by a bioisostere moiety thereof. [0067] In some aspects, Z is a saccharide and L₁ is or comprises a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, being Z and L₁ covalently linked by an ether bond. [0068] In some aspects, Z is a peptide and L₃ is a C_1-6 alkylene group, being Z and the C_1-6 alkylene group covalently linked by an amide moiety or a bioisostere moiety thereof, for example an amide moiety N(R₁)C(O)-, or a bioisostere moiety thereof, as defined in the claims and embodiments of the present specification. [0069] In some aspects, Z is a peptide, L₁ is or comprises a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers and L₃ is an arginine derivative, being L₁ and L₃ covalently linked by an amide moiety or a bioisostere moiety thereof. [0070] In some aspects, the AAV vector particle comprising a moiety of formula (II) is an AAV vector particle comprising a moiety selected from the group consisting of formula (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (IIm), (IIn), (IIp), (IIq), (IIr), (IIs), (IIt), (IIv), (IIw) and (IIx):

wherein R_a, R_b and R_c are each independently H or a group R’ :

being at least one of R_a, R_b and R_c a group R’, and wherein n and n’ are each independently selected from 1 to 40, mi and m2, are each independently 0, 1 or 2, m₃ and m₄, m₅ and m₆ are each independently selected from 1 to 6, preferably 1, 2 or 3, and N*, - , Z, Ar and R₁ are as defined and described in classes and subclasses disclosed in the present invention. In some particular aspects n is 3, 4 or 5; Z is or comprises a linear or a cyclic peptide, wherein the peptide may be a peptide featuring biological activity, preferably wherein the peptide is a blood brain barrier (BBB) shuttle peptide, more preferably a BBB shuttle peptide selected from the group consisting of a peptide THR or a peptide with a RGD motif, including a cyclic RGD peptide; or a saccharide, being the saccharide selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and derivatives thereof, preferably a saccharide selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, desosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6- mannose, P6-glucose, sialic acid, SI -fructose and P1 -fructose, preferably selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid and Pl- fructose, more preferably mannose; Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se, preferably a phenylene or pyridylene group, optionally comprising one or more substitutions selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy; and R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z-(OCH₂-CH₂)n-, Z-C(O)NH- (CH₂)q-(OCH₂-CH₂)n-, and Z-NHC(O)-(CH₂)q-(OCH₂-CH₂)n-, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂-CH₂)n- wherein n is selected from 1 to 40 and more preferably R₁ is H or Z-(OCH₂-CH₂)n- wherein n is selected from 1 to 40, even more preferably R₁ is H.

[0071] In some aspects, the AAV vector particle is moiety selected from those of Table 1’ hereafter.

[0072] In some aspects, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, pseudotypes, chimeras, and variants thereof; preferably the AAV vector is selected from the group consisting of AAV2, AAV5, AAV8, and AAV9.

[0073] In some aspects, the AAV vector comprises at least one transgene, and the transgene is optionally under control of a promoter.

[0074] In some aspects, the AAV vector comprises at least one transgene comprising the cDNA from a GBA gene, preferably from a human GBA gene, and the transgene is optionally under control of a promoter.

[0075] In some aspects, the invention also provides a pharmaceutical composition comprising an AAV vector particle according to the invention and at least one pharmaceutically acceptable vehicle.

[0076] In some aspects, the invention also relates to an AAV vector particle according to the invention or a pharmaceutical composition according to the invention, for use as a diagnostic agent and/or a medicament, preferably in gene therapy.

[0077] In other aspects the invention relates to the use of an AAV vector particle according to the invention or a pharmaceutical composition according to the invention, as a diagnostic agent and/or a medicament, preferably in gene therapy.

[0078] In other aspects, the invention provides a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein R₂ is selected from the group consisting of linear C_1-12 alkyl, branched C_3-12 alkyl, linear C_1-12 haloalkyl, branched C_3- ₁₂ haloalkyl and benzyl,; and R^L-NH- is a functional moiety as defined and described in classes and subclasses in the present invention. In some aspects, R₂ is methyl, ethyl or benzyl, preferably ethyl.

[0079] In some aspects, the functional moiety R^L-NH- comprises a group Z, one or more spacers L, and the compound of formula (III), or a pharmaceutically acceptable salt thereof, is represented by formula (Illa):

wherein R₂, Z and L are as defined and described in classes and subclasses in the present invention.

[0080] In some aspects, the compound of formula (Illa), or a pharmaceutically acceptable salt thereof, comprises one or more than one spacer L is selected from the group consisting of L₁, L₂ and L₃ and, is selected from the group consisting of formula (Illa₁), (IIIa₂), (IIIa₃), (IIIa₄), (IIIa₅), (IIIa₆), (IIIa₇), (IIIa₈), (IIIa₉), (Illa₁₀), (IlIa₁₁), (IlIa₁₂), (IlIa₁₃), (IIIa₁₄), (IlIa₁₅), (IIIa₁₆), (IIIa₁₇), (IlIa₁₈), (IIIa₁₉) and (IIIa₂₀):

wherein R₂, Z, L₁, L₂ and L₃ are as defined and described in classes and subclasses in the present invention.

[0081] In other aspects, the compound of formula (III) is a compound selected from the group consisting of formula (Illb) (IIIc), (IIId), (Ille), (I I If), (Illg), (Illh), (Illj), (Illk), (IIIm), (IIIn), (IIIp), (Illq), (Illr), (Ills), (lIlt), (IIIv), (IIIw) and (IIIx):

or a pharmaceutically acceptable salt thereof, wherein R_a, R_b and R_c, n, n’, m₁, m₂, m₃, m₄, Z, Ar, R₁ and R₂ are as defined and described in classes and subclasses disclosed in the present invention.

[0082] The compounds of formula (III) are useful to obtain the AAV vector particles comprising a moiety of formula (II), as defined and described in classes and subclasses in the present invention.

[0083] Accordingly, in some aspects, the invention also provides the use of a compound of formula (III), as defined and described in classes and subclasses in the present invention, to obtain a AAV vector particle comprising a moiety of formula (II), as defined and described in classes and subclasses in the present invention.

[0084] In particular, the compounds of formula (III) according to the present invention comprise a squarate ester moiety of formula (IV):

wherein R₂ is defined and described in classes and subclasses disclosed in the present invention, and wherein said squarate ester reacts, in suitable conditions, with the amino groups present in amino acid residue of the capsid of the AAV vector to form a squaramide linker of formula (I), as defined in the present disclosure.

[0085] In some aspects, the invention also provides a method of synthesizing an AAV vector particle comprising a moiety of formula (II), as defined and described in classes and subclasses in the present invention, wherein said method comprises the steps of incubating the AAV vector with a compound of formula (III), as defined and described in classes and subclasses of the present description, in conditions suitable for reacting a squarate moiety of the compound of formula (III) with at least one amino group of an amino acid residue of the capsid of the AAV vector so as to form a squaramide linker of formula (I). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0086] Various coupling chemistries that conjugate through an amino group (e.g., of a lysine sidechain) have been described. However, not all such coupling chemistries are compatible with and/or effective under particular conditions and/or with particular substrates.

[0087] The present disclosure relates to couplings that conjugate to amino acids of adeno-associated viruses (AAVs) using a squaramide linker of formula (I) as defined previously. The present disclosure appreciates that not all coupling chemistries are effective for such conjugation. For example, certain coupling chemistries require or are typically performed under conditions that may disrupt one or more structural or functional properties of the AAV (e.g., do not preserve AAV integrity). Alternatively or additionally, various coupling chemistries require or are typically performed under conditions that are not compatible with certain biochemical ligands, such as saccharides and/or (poly)peptides.

[0088] For example, some coupling strategies and conditions may favor self-coupling with another ligand molecule (inter- and/or intra-molecular coupling) rather than coupling with the AAV. In that connection, as previously indicated, in certain contexts, it has been observed that coupling chemistries comprising certain isothiocyanate groups are often too reactive and thus susceptible to significant self-coupling reactions.

[0089] Some coupling strategies include 2-step coupling reactions. However, in certain conditions, when only a first coupling reaction occurs, but not a second, then an AAV can be rendered immunogenic, for example due to non-natural chemical structures decorating the capsid surface. In that context, a one-step coupling chemistry has been described in, e.g., W02017/212019, which uses a certain isothiocyanate group that has been shown to be compatible in certain circumstances. However, as disclosed in WO2022/096681, a potential problem with use of such isothiocyanate groups for coupling to AAV is that, for example, such coupling reactions results in a lipophilic linker that may form an immunogenic hapten. [0090] WO2022/096681 discloses surface modified vectors obtained by 1-step reaction of a compound comprising a lactam (e.g., β-lactam) with an amino group present in an amino acid residue of the capsid proteins of the AAV which solved the issues referred to self-coupling reactions and to the formation of immunogenic hapten. However, due to the sensitizing potential of β-lactams, a cross contamination of such molecules into other products for human use has to be excluded on a ppb-level (1). Therefore areas where β- lactams are handled have to be completely and comprehensively separated from other areas, where products are handled that will be used for humane use. Therefore, already during development, β-lactams have to be completely separated from other development or production areas ((1) a) FDA, Guidance for Industry “Non-Penicillin Beta-Lactam Drugs: A CGMP Framework for Preventing Cross-Contamination, 2013; b) 21 CFR 211.176.

[0091] The present disclosure therefore recognizes a particular remaining need to provide suitable coupling chemistries that i) are compatible with and maintain the integrity of a multiplicity of AAV serotypes, ii) are 1-step, iii) minimize self-coupling of the ligand, iv) do not result in an immunogenic linker, v) provide a synthetic process which provides less constraints for industrial scale-up and vi) are more flexible in terms of biochemical ligands and AAV serotypes.

[0092] The squaramide moiety is a conformationally rigid cyclobutene ring derived from squaric acid (diketoclyclobutenediol) which benefits from unique physical and chemical properties which make it surprisingly useful for coupling a wide range of adeno- associated virus vectors to different type of ligands. Moreover, by selecting appropriate pH conditions the first and second substitution of the squarate can be controlled, allowing thus to provide a more selective substitution, resulting in a more flexible scaffold for coupling a diverse range of adeno-associated virus vectors to different type of ligands when compared to other solutions (linkers) known in the prior art.

[0093] In some aspects, the present invention relates to an adeno-associated virus (AAV) vector particle comprising a moiety of formula (II):

wherein N*, - — and R^L-NH- are as defined and described in classes and subclasses in the present invention.

[0094] In particular, the present invention relates to adeno-associated virus (AAV) vector particle of formula (II) as defined and described herein, resulting from reaction of a squarate ester of formula (III):

or a pharmaceutically acceptable salt thereof, with an amino group present within the capsid of the AAV vector (e.g., amino group of a lysine side chain), e.g., a modified AAV vector particle results from such reaction; wherein the group R₂ and the functional moiety R^L-NH- are as defined and described in classes and subclasses in the present invention.

[0095] For example, in some embodiments, the functional moiety R^L-NH- comprises a group Z, one or more spacers L, and the adeno-associated virus (AAV) vector particle comprises a moiety represented by formula (Ila), as disclosed herein, and results from the reaction of a compound of formula (Illa):

or a pharmaceutically acceptable salt thereof, with an amino group present within the capsid of the AAV vector; wherein R₂, Z and L are as defined and described in classes and subclasses in the present invention. [0096] In some embodiments, the functional moiety R^L-NH- comprises a group Z, and more than one spacers L, the moiety represented by formula (IIa) is selected from the group consisting of formula (IIa₁), (IIa₂) and (IIa₃), as disclosed herein, and the AAV vector particle comprising a moiety selected from the group consisting of formula (IIa₁), (IIa₂) and (IIa₃), results, respectively, from the reaction of a compound of formula (III) selected from the group consisting of formula (IIIa₁), (IIIa₂), (IIIa₃), (IIIa₄), (IIIa₅), (IIIa₆), (IIIa₇), (IIIa₈), (IIIa₉), (IIIa₁₀), (IIIa₁₁), (IIIa₁₂), (IIIa₁₃), (IIIa₁₄), (IIIa₁₅), (IIIa₁₆), (IIIa₁₇), (IIIa₁₈), (IIIa₁₉) and (IIIa₂₀):

,

or a pharmaceutically acceptable salt thereof, with an amino group present within the capsid of the AAV vector; wherein R₂, Z, L₁, L₂ and L₃ are as defined and described in classes and subclasses in the present invention.

[0097] In some particular embodiments, the adeno-associated virus (AAV) vector particle comprising a moiety of formula (II), as disclosed herein, results from the reaction of a compound of formula (III) selected from the group consisting of formula (Illb), (IIIc), (IIId), (Ille), (Illf), (Ille), (Illg), (Illh), (Illj), (Illk), (Illm), (Illn), (IIIp), (Illq), (Illr), (Ills), (IIlt), (IIIv), (IIIw) and (IIIx):

or a pharmaceutically acceptable salt thereof, with an amino group present within the capsid of the AAV vector; wherein R_a, R_b and R_c, n, n’, m₁, m₂, m₃, m₄, Z, Ar, R₁ and R₂ are as defined and described in classes and subclasses disclosed in the present invention. [0098] In some aspects, the present invention relates to methods of modifying AAV vectors, especially by modification of at least one amino acid residue of the capsid of the AAV. In some embodiments, the present invention provides methods of modifying an amino group of an amino acid residue of the capsid, preferably an amino group of a surface-exposed amino acid residue. As described herein, modification of an AAV vector was successfully achieved by covalently coupling a linker comprising a squarate ester as a reactive moiety. [0099] As a proof of concept, several compounds of formula (III) comprising a squarate ester moiety were prepared, in which the functional moiety R^L-NH- comprises a labelling agent, or a saccharide, or a peptide, for modifying amino groups of amino acid residues of the capsid of AAV vectors. It was evidenced that capsid proteins of several AAV serotypes, including AAV2 and AAV5, can be efficiently modified while maintaining the integrity of the AAV particles. It was further shown that these modified AAVs remain infectious, e.g., in the U87-MG glioblastoma cell line. AAV vectors [0100] AAV vectors suitable in the present invention may comprise or be derived from any natural or recombinant AAV serotype. [0101] 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). As used herein, AAV includes various naturally occurring and synthetic serotypes. [0102] In some embodiments, an AAV vector according to the present invention is selected from natural serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12; or pseudotypes, chimeras, and variants thereof.

[0103] In some embodiments, an AAV vector according to the present invention is of AAV phylogenetic Clade A, B, C, D, E, or F (see e.g., Gao et al., Clade of Adeno- Associated Viruses Are Widely Disseminated in Human Tissues. J. Virology. 2004). In some embodiments, an AAV vector according to the present invention is of AAV phylogenetic Clade E. In some embodiments, an AAV vector according to the present invention is of AAV phylogenetic Clade D. In some embodiments, an AAV vector according to the present invention is of AAV phylogenetic Clade F. In some embodiments, an AAV vector according to the present invention is of AAV phylogenetic Clade A. In some embodiments, an AAV vector according to the present invention is of AAV phylogenetic Clade C. In some embodiments, an AAV vector according to the present invention is of AAV phylogenetic Clade B. In some embodiments, an AAV vector according to the present invention is an AAV that does not belong to a classical phylogenetic Clade.

[0104] As used herein, the term “pseudotype” when referring to an AAV vector, or a “pseudotyped AAV vector”, refers to an AAV vector which comprises portions of an AAV genome, in particular the inverted terminal repeats (ITRs), of one AAV serotype packaged in the capsid of another AAV serotype. These pseudotypes are denoted using a slash or a hyphen, so that “AAV2/5” or “AAV2-5” indicates an AAV vector comprising a serotype 2 genome, packaged into a serotype 5 capsid.

[0105] In some embodiments, an AAV vector is transcapsidated. In some embodiments, transcapsidation approaches comprise transfection of combinations of AAV serotype helper plasmids to produce mosaic recombinant AAV capsid (see e.g., Rabinowitz et al. (2004), J. Virol. 78: 4421-4432). In some embodiments, polyploid (when utilizing more than two parental AAV helpers) or haploid (when only using two) approaches are utilized. In some embodiments, for example, AAV capsids can be made from VP1/VP2 of one serotype and VP3 donated from a unique serotype, or combinations thereof. In some embodiments, haploid AAVs have the potential to uniquely combine structural advantages of parental AAVs. In some embodiments, haploid AAVs have demonstrated 1) synergistic effects in transduction, 2) unexpected new tropisms, and 3) the ability to escape Nab (see e.g., Chai et. al. (2019), Viruses 11: 1138)

[0106] For example, in some embodiments, pseudotyped AAV vectors include, but are not limited to, AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/5, AAV2/6, AAV2/7, AAV2/8 and AAV2/9.

[0107] As used herein, the term “chimera” when referring to an AAV vector, or a “chimeric AAV vector”, refers to an AAV vector which comprises a capsid containing VP1, VP2 and VP3 proteins from at least two different AAV serotypes; or alternatively, which comprises VP1, VP2 and VP3 proteins, at least one of which comprises at least a portion from another AAV serotype.

[0108] Examples of chimeric AAV vectors include, but are not limited to, AAV-DJ, AAV2G9, AAV2i8, AAV2i8G9, AAV8G9, and AAV9il.

[0109] In some embodiments, an AAV vector according to the present invention is selected from the group comprising or consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV106.1/hu.37, AAV1 14.3/hu.4O, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43,

AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54,

AAV145.6/hu.55, AAV16.12/hu.l l, AAV16.3, AAV16.8/hu.lO, AAV161.1O/hu.6O, AAV161.6/hu.61, AAVl-7/rh.48, AAVl-8/rh.49, AAV2i8, AAV2i8G9,

AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.5O, AAV2-5/rh.51, AAV2.5T, AAV27.3, AAV29.3/bb.l, AAV29.5/bb.2, AAV2G9, AAV3B, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-1 l/rh.53, AAV3-3, AAV33.12/hu.l7, AAV33.4/hu.l5,

AAV33.8/hu.l6, AAV3-9/rh.52, AAV3a, AAV3b, AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2, AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/rh.64, AAV4-9/rh.54, AAV52.1/hu.2O, AAV52/hu.l9, AAV5-22/rh.58, AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27, AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6.1, AAV6.1.2, AAV6.2, AAV7m8, AAV7.2, AAV7.3/hu.7, AAV-8b, AAV8G9, AAV-8h, AAV9il, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVcy.5Rl, AAVcy.5R₂, AAVcy.5R3, AAVcy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.8, AAVhu.9, AAVhu.10, AAVhu.l 1, AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.l 5, AAVhu.l 6, AAVhu.l 7, AAVhu.l 8, AAVhu.l 9, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R₂, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R₂, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.53, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVpi.l, AAVpi.2, AAVpi.3, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh8R R533A mutant, AAVrh8R A586R mutant, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R₂, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44, AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.5O, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59, AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64Rl, AAVrh.64R₂, AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73, AAVrh.74, AAV-PHP.B, AAV-PHP.A, AAV-G2B-26, AAV-G2B-13, AAV-TH1.1-32, AAV-TH1.1-35, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.N/PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.B -ATP, AAV-PHP.B-ATT-T, AAV-PHP.B-DGT-T, AAV-PHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.B-SNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B-EGS, AAV-PHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.B-DST, AAV-PHP.B-STP, AAV-PHP.B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP, AAV-PHP.B-TMP, AAV-PHP.B-TTP, AAV-PHP.S/G2A12, AAV-G2A15/G2A3, AAV-G2B4, AAV-G2B5, PHP.S, AAAV, AAV A3.3, AAV A3.4, AAV A3.5, AAV A3.7, AAV CBr-7.3, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr-E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8„ AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-N4, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5,

AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H₂,

AAV CKd-H3, AAV CKd-H4, AAV CKd-H5, AAV CKd-H6, AAV CKd-N3,

AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4,

AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-M9, AAV CLv-R6, AAV CLv-1, AAV CLvl-1, AAV CLvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV CLvl-7, AAV CLvl-8, AAV CLvl-9, AAV CLv-2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El,

AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5,

AAV CLv-L6, AAV CLv-Ml, AAV CLv-Mll, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-Rl, AAV CLv-R₂,

AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R7, AAV CLv-R8,

AAV CLv-R9, AAV CSp-8.10, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV-LK08, AAV-LK15, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV SM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, AAV SM 10-8, AAV.VR-355, AAV-b, AAVC1, AAVC2, AAVC5, AAVCh.5, AAVCh.5Rl, AAV-DJ, AAV-DJ8, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3, AAVF3/HSC3, AAVF4/HSC4, AAVF5, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, AAVF9/HSC9, AAV-h, AAVH-l/hu. l, AAVH₂ , AAVH-5/hu.3, AAVH6, AAVhEl.l, AAVhErl.14, AAVhErl.16, AAVhErl.18, AAVhER1.23, AAVhErl.35, AAVhErl.36, AAVhErl.5, AAVhErl.7, AAVhErl.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC 12, AAV-PAEC11, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, Anc80, Anc80L65, Anc81, Anc82, Anc83, Anc84, Anc94, And 10, And 13, Ancl26, Ancl27, BAAV,BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprine AAV, Japanese AAV 10 serotype, UPENN AAV10, VOY101, and VOY201.

[0110] In some embodiments, “AAV vectors variants” include vectors which have been genetically modified, e.g., by substitution, deletion or addition of one or several amino acid residues in one or more of the capsid proteins VP1, VP2 and VP3. Examples of such variants include, but are not limited to, AAV vectors comprising at least one Y-to-F, K- to-R, T-to-A, S-to-A and/or T-to-V mutation in any one or several of their VP1, VP2 and/or VP3 capsid proteins.

[0111] Further examples of such variants include, but are not limited to, AAV1 with a Y73 IF mutation (or corresponding site in other AAV serotypes); AAV2 with one or more of Y272F, Y444F, T491V, Y500F, S662V and/or Y730F mutations (or corresponding sites in other AAV serotypes), such as AAV2 with Y444F mutation, AAV2 with Y444F+Y500F+Y730F mutations, AAV2 with Y272F+Y444F+Y500F+Y730F mutations, AAV2 with Y444F+ Y500F+Y730F+T491V mutations and AAV2 with Y272F+Y444F+Y500F+Y730F+T491V mutations; AAV3 with one or more of Y705F, Y731F and/or T492V mutations (or corresponding sites in other AAV serotypes); AAV5 with one or more of Y263F and/or Y719F mutations (or corresponding sites in other AAV serotypes); AAV6 with one or more of Y445F, T492V, S663V, Y705F and/or Y731F mutations (or corresponding sites in other AAV serotypes), such as AAV6 with Y445F mutation, AAV6 with Y705F+Y731F mutations, AAV6 with T492V mutation, AAV6 with Y705F+Y731F+T492V mutations, AAV6 with S663V mutation and AAV6 with S663V+T492V mutations; and AAV8 with one or more of Y447F, T494V and/or Y733F mutations (or corresponding sites in other AAV serotypes).

[0112] In some embodiments, an AAV vector according to the present invention is selected from the group consisting of AAV2, AAV5, AAV8, and AAV9.

In some embodiments, an AAV vector according to the present invention is AAV2.

In some embodiments, an AAV vector according to the present invention is AAV5.

In some embodiments, an AAV vector according to the present invention is AAV8.

In some embodiments, an AAV vector according to the present invention is AAV9.

In some embodiments, an AAV vector according to the present invention is AAV2/2.

In some embodiments, an AAV vector according to the present invention is AAV2/5.

In some embodiments, an AAV vector according to the present invention is AAV2/8.

In some embodiments, an AAV vector according to the present invention is AAV2/9.

[0113] In some embodiments, an AAV vector according to the present invention has a capsid of an AAV serotype selected from the group consisting of serotypes 2, 5, 8 and 9. In some embodiments, an AAV vector according to the present invention has a capsid of AAV serotype 2. In some embodiments, an AAV vector according to the present invention has a capsid of AAV serotype 5. In some embodiments, an AAV vector according to the present invention has a capsid of AAV serotype 8. In some embodiments, an AAV vector according to the present invention has a capsid of AAV serotype 9.

[0114] In some embodiments, an AAV vector can target a large variety of cells, tissues, and organs. In some embodiments, examples of cells targeted by AAV vectors encompass, but are not limited to, hepatocytes; cells of the retina; i.e. photoreceptors, retinal pigmented epithelium (RPE), Muller cells; muscle cells, i.e. myoblasts, satellite cells; cells of the central nervous system (CNS), i.e. neurons, glial cells; cells of the heart; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells; blood cells such as lymphocytes, monocytes, basophils, eosinophils, neutrophils, mast cells; hematopoietic cells including hematopoietic stem cells; cells of the inner ear (e.g., inner and/or outer hair cells, Hensen’s cells, Deiter’s cells, pillar cells, inner phalangeal cells, border cells, etc.); induced pluripotent stem cells (iPS) and the like. In some embodiments, examples of tissues and/or organs which can be targeted by AAV include eye, retina, liver, skeletal muscle, cardiac muscle, smooth muscle, ear, brain, spine, bone, connective tissue, heart, kidney, lung, lymph node, mammary gland, myelin, prostate, testes, thymus, thyroid, trachea, and the like. In some embodiments, preferred cell types are hepatocytes, retinal cells, muscle cells, cells of the CNS, cells of the PNS, and hematopoietic cells. In some embodiments, preferred tissue and/or organs are liver, muscle, heart, eye, and brain.

[0115] The tropism of AAVs can vary depending on their serotype. In some embodiments, for example, AAV2 can be used to transduce the central nervous system (CNS), kidney, and photoreceptor cells, while in some embodiments, for example, AAV8 is effective for transducing the CNS, heart, liver, photoreceptor cells, retinal pigment epithelium (RPE), and skeletal muscle.

[0116] In some embodiments, an 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 Examples section.

Recombinant AA V vectors

[0117] In some embodiments, an AAV vector modified according to the present invention may be a recombinant AAV (rAAV) vector. In general, wild-type (WT) AAVs have a single-stranded linear DNA genome about 5 kb long with two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The 5’ and 3’ ORFs encode replication, and capsid proteins, respectively. In general, an ITR contains 145 nucleotides and serves as an AAV genome replication origin and packaging signal. In recombinant AAV, viral ORFs are replaced by an exogenous gene expression cassette, while replication and capsid proteins are provided in trans. In some embodiments, an AAV vector modified according to the present invention may comprise a double- stranded, self-complementary DNA genome (scAAV) (see e.g., Buie et al., Self- complementary AAV Virus (scAAV) Safe and Long-term Gene Transfer in the Trabecular Meshwork of Living Rats and Monkeys. Invest Opthalmol Vis Sci. 2010).

[0118] Therefore, a “recombinant AAV vector” or “rAAV” herein refers to an AAV wherein an exogenous nucleic acid sequence (e.g., a payload, e.g., a transgene) 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 vector. For instance, said nucleic acid may comprise and/or may template any RNA or DNA sequence. In some embodiments, a nucleic acid may preferably comprise a DNA sequence. In some embodiments, rAAV vectors can be used as gene vectors for in vivo or in vitro applications.

[0119] For illustration, an exemplary rAAV vector modified according to the present invention may comprise an exogenous gene expression cassette replacing the viral ORFs and placed between two ITRs. In some embodiment, an exogenous gene expression cassette may comprise a promoter sequence, a sequence encoding a gene of interest, and a terminator sequence. In some embodiments, a promoter and a gene of interest are selected depending on a targeted tissue and/or organ and a known indication, e.g., for treatment and/or prevention of a disease state.

[0120] As an additional or alternative example, in some embodiments, a rAAV vector used in the present invention may comprise a DNA template for homologous recombination in cells. In some embodiments, a rAAV can be used in combination with gene editing tools, for promoting homologous recombination in targeted cells, in vivo, in vitro, and/or ex vivo. In some embodiments, a gene editing tools can be of any type, and encompass, without being limited to, CRISPR and its associated systems (Cas proteins, guide RNA), TALEN, Zinc Finger Nuclease, meganuclease, as well as RNA and/or DNA encoding said proteins. [0121] In some embodiments, an AAV vector modified according to the present invention comprises at least one transgene, selected in view of the intended use of the AAV vector.

[0122] The term “transgene”, as used herein, refers to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In some embodiments, a transgene confers a desired property to a cell into which it was introduced, or otherwise leads to a desired therapeutic or prophylactic outcome. In some embodiments, the transgene may be incorporated, either entirely or partially, in the host cell’s genome, such as, e.g., via corrective gene editing using a CRISPR-based method, TALEN-based method, ZFN-based method or the like, in presence of appropriate means. In some embodiments, a transgene may be transcribed into a molecule that mediates RNA interference (i.e., gene silencing), such as into a miRNA, siRNA, shRNA, piRNA, or the like.

[0123] In some embodiments, the at least one transgene comprises a cDNA encoding a protein or a fragment thereof.

[0124] As used herein, the term “cDNA” refers to complementary DNA and corresponds to a DNA molecule, usually synthesized from a single-stranded RNA (such as, e.g., a messenger RNA [mRNA] or a microRNA [miRNA]) template in a reaction catalyzed by a reverse transcriptase. In particular, when a cDNA is obtained from reverse transcription of a mRNA, it does not comprise an entire gene coding from a protein, but only the coding sequence of said protein (i.e., exons without introns).

[0125] In some embodiments, a fragment of a cDNA can comprise a part of said cDNA encoding the N-terminal part or the C-terminal part of a protein. In some embodiments, for example, such fragment could be useful in cases of large cDNAs which cannot readily be carried by a single AAV vector, and would thus require the use of more than one vector, e.g., dual AAV vectors.

[0126] In some embodiments, a fragment of a cDNA can comprise a part of said cDNA encoding a functional and/or structural portion of a protein. [0127] In some embodiments, a fragment of a cDNA can comprise a sequence encoding a functional and/or structural portion of an RNA molecule. In some embodiments, such an RNA molecule may be a ribosomal RNA, transfer RNA, small nuclear RNA, small nucleolar RNA, micro RNA, long non-coding RNA, short interfering RNA, guide RNA, and/or any functional RNA species.

[0128] In some particular embodiments, the cDNA is from the GBA gene, preferably from the human GBA gene. Exemplary sequences of the GBA gene may be found in WO2022/096681.

[0129] In some embodiments, at least one transgene is under the control of at least one element which enhances the transgene target specificity and/or expression. In some embodiments, examples of elements which enhance the transgene target specificity and/or expression include, but are not limited to, promoters, post-transcriptional regulatory elements (PREs), polyadenylation (poly A) signal sequences, translational regulatory elements, targets for control by endogenous RNA processing pathways, upstream enhancers (USEs), CMV enhancers, and introns.

[0130] In some embodiments, at least one transgene is under the control of at least one promoter.

[0131] A person skilled in the art may recognize that expression of transgenes in a target cell may require a specific promoter, including, but not limited to, a promoter that is species-specific, inducible, tissue-specific, temporally-specific, cell-specific, and/or cell cycle-specific.

[0132] In some embodiments, a promoter is a promoter having a tropism for a cell being targeted, i.e., a cell-specific promoter.

[0133] In some embodiments, a promoter drives expression of a transgene for a period of time in targeted tissues. Expression driven by a promoter may be for a period of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years,

10 years, or more than 10 years.

[0134] In some embodiments, a promoter is a weak promoter for sustained expression of a transgene.

[0135] In some embodiments, promoters may be naturally occurring or non-naturally occurring. In some embodiments, examples of promoters include, but are not limited to, viral promoters, plant promoters, and animal promoters (e.g., mammalian promoters). In some embodiments, a promoter may be a human promoter.

[0136] In some embodiments, a promoter may be truncated relative to a reference. In some embodiments, a promoter may be mutated relative to a reference.

[0137] In some embodiments, a promoter may be one that drives expression in multiple tissues. In some embodiments, such a promoter which drives or promotes expression in multiple tissues includes, but is not limited to, human elongation factor la-subunit (EFla), cytomegalovirus (CMV) immediate-early enhancer and/or promoter, chicken β- actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), and ubiquitin C (UBC).

[0138] In some embodiments, tissue- or cell-specific expression elements can be used to restrict expression of a transgene to certain cell types.

[0139] In some embodiments, a tissue and/or cell specific promoter may be a neuron specific promoter. Suitable examples of tissue- or cell-specific expression elements for neurons include, but are not limited to, neuron-specific enolase (NSE) promoter, platelet- derived growth factor (PDGF) promoter, platelet-derived growth factor B-chain (PDGF- β) promoter, synapsin (Syn) promoter, myelin basic protein (MBP) promoter, methyl- CpG binding protein 2 (MeCP2) promoter, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light (NFL) promoter, neurofilament heavy (NFH) promoter, β-globin minigene ηβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter and excitatory amino acid transporter 2 (EAAT2) promoter.

[0140] In some embodiments, a promoter is a ubiquitous promoter. In some embodiments, such a ubiquitous promoter can include, but is not limited to CMV, CBA (including its derivatives CAG, CBh, and the like), EF-la, PGK, UBC, GUSB (hGBp), and UCOE.

[0141] In some embodiments, a promoter is not tissue- or cell-specific.

[0142] In some embodiments, a promoter is an engineered promoter.

[0143] In some embodiments, a promoter is a promoter from a naturally-expressed protein.

[0144] In some preferred embodiments, a promoter is a CAG promoter (e.g., comprising a CMV immediate early enhancer and a chicken β-actin promoter).

Site of coupling on the AA V vector

[0145] In some embodiments of the invention, a provided AAV vector is one that is modified by covalent coupling of at least squarate ester to at least one amino acid of the AAV capsid (e.g., to at least one capsid protein of the AAV vector). A typical AAV capsid comprises three capsid proteins, named VP1, VP2 and VP3. In some embodiments, at least one squarate ester is covalently bound to at least one VP1 protein of an AAV vector. In some embodiments, at least one squarate ester is covalently bound to at least one VP2 protein of the AAV vector. In some embodiments, at least squarate ester moiety is covalently bound to at least one VP3 protein of the AAV vector.

[0146] In some embodiments, an AAV vector is modified by covalent coupling of at least one squarate ester to at least one surface-exposed amino acid residue of at least one capsid protein of the AAV vector. [0147] As used herein, the term “surface-exposed” refers to an amino acid residue with a side chain that is at least partially exposed at the outer surface of the AAV vector.

[0148] In some embodiments, at least one squarate ester is covalently bound to at least one amino group of a surface-exposed amino acid residue of the capsid of an AAV vector. In particular, the at least one squarate ester is a compound of formula (III), or a particular embodiment thereof selected from the group consisting of a compound of formula (Illa), (Illal), (IIIa2), (Illb) and (IIIc), as defined and described in classes and subclasses in the present invention.

[0149] By “amino group” it is herein referred to a primary amine group (-NH₂) or a secondary amine group (-NH-), or salts thereof; preferably the amino group is a primary amino group. In some preferred embodiments, the amino group is from a lysine residue, preferably from a surface-exposed lysine residue of the capsid of the AAV vector.

[0150] As used herein, “at least one amino group of an amino acid residue of the capsid” encompasses at least 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino groups of amino acid residue(s).

[0151] In some embodiments, the AAV vector of the invention comprises a plurality of (e.g., several) modified amino acid residues in its capsid. In some embodiments, a plurality of (e.g., several) amino acid residues of a same capsid protein are modified. In some embodiments, a plurality of (e.g., several) amino acid residues present in different capsid proteins are modified.

[0152] In some embodiments, the invention relates to an adeno-associated virus (AAV) vector particle comprising a moiety of formula (II):

wherein N* is a nitrogen atom of a primary amino group from a surface-exposed amino acid residue of a capsid polypeptide from the AAV vector;

— represents the point of attachment to the AAV vector’s capsid; and

[0153] R^L-NH- is a functional moiety comprising or consisting of a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins, or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties.

[0154] In some embodiments, the functional moiety R^L-NH- comprises a group Z and one or more spacers L, wherein

Z is H or comprises or consists in a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins, or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties; preferably Z is or comprises a peptide or a saccharide. In some embodiments, the peptide is or comprises a linear or a cyclic peptide, wherein said peptide may be a peptide featuring biological activity. In particular, the peptide is a blood brain barrier (BBB) shuttle peptide with an enhanced transduction activity across the blood brain barrier. In some preferred aspects, the BBB shuttle peptide is selected from the group consisting of a peptide THR or a peptide with a RGD motif, including a cyclic RGD peptide. In some embodiments, the saccharide selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and derivatives thereof, more preferably a saccharide selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, desosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, SI -fructose and Pl -fructose, preferably selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid and Pl -fructose, more preferably mannose; and L comprises or consists of one or more groups selected from the group consisting of an alkyl ether group, such as a polyethylene glycol (PEG) or a polypropylene glycol (PPG), preferably a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers; a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol; an arylene or a heteroarylene group Ar; being preferably Ar a phenylene or a pyridylene group optionally comprising one or more substitutions selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy; a C_1-6 alkylene group; a C_1- ₆ alkylene amine; a C_1-6 acyl group; an amino acid moiety, preferably an arginine moiety, or a β-alanine moiety; a polyamide such as a β-alanine polymer, preferably comprising 1 to 40 β-alanine monomers; a vinylic polymer such as pHPMA; a polyester, such as PLGA; and polymers of alkylene diamines; and combinations thereof.

[0155] In some particular embodiments L comprises one or more of a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers; a C_1-6 alkylene group; a C_1- 6 alkylene amine; a C_1-6 acyl group; a β-alanine polymer comprising 1 to 40 β-alanine monomers; a polyether of a branched C_3-12 polyol; an amino acid moiety, and an arylene or a heteroarylene group Ar.

[0156] In some aspects, the PEG and Ar groups are covalently linked by an amide moiety -N(R₁)C(O)-, or a bioisostere moiety thereof, wherein R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z-(OCH₂-CH₂)n-, Z-C(O)NH-(CH₂)q- (OCH₂-CH₂)n-, and Z-NHC(O)-(CH₂)q-(OCH₂-CH₂)n-, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂-CH₂)n- wherein n is selected from 1 to 40 and more preferably R₁ is H or Z-(OCH₂-CH₂)n- wherein n is selected from 1 to 40, even more preferably R₁ is H.

[0157] In some aspects, L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and one or more C_1-6 alkyl groups wherein said PEG and C_1-6 alkylene groups are covalently linked by an amide moiety or a bioisostere moiety thereof. [0158] In some aspects, L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and an arylene or a heteroarylene group Ar, preferably wherein said PEG and Ar groups are covalently linked by an amide moiety or a bioisostere moiety thereof.

[0159] In some aspects, L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, a β-alanine polymer comprising 1 to 40 β-alanine monomers and a C_1-6 acyl group. In other aspects, L comprises a β-alanine polymer comprising 1 to 40 β-alanine monomers and a C_1-6 alkylene amine. In other aspects, L comprises a β- alanine polymer comprising 1 to 40 β-alanine monomers and an arylene or a heteroarylene group Ar.

[0160] In other aspects, L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and one or more C_1-6 alkyl groups wherein said PEG and C_1-6 alkyl groups are covalently linked by an ether bond.

[0161] In other aspects L comprises a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, one or more C_1-6 alkylene groups, an arylene or a heteroarylene group Ar, and a poly ether of a branched C_3-12 polyol, wherein the PEG and the C_1-6 alkylene groups are covalently linked by an amide moiety or a bioisostere moiety thereof; wherein the polyether of a branched C_3-12 polyol and the C_1-6 alkyl group are covalently linked by an ether bond; and wherein the polyether of a branched C_3-12 polyol and the Ar group are covalently linked by an amide moiety or a bioisostere moiety thereof.

In some embodiments, the one or more spacer L is selected from the group consisting of L₁, L₂ and L₃, wherein L₁ is one or more groups selected from the group consisting of a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, or a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol and a β-alanine polymer comprising 1 to 40 β-alanine monomers, or a mixture thereof; L₂ comprises one or more arylene or a heteroarylene groups Ar, preferably L₂ comprises a phenylene or a pyridylene group; and L₃ is one or more groups selected from the group consisting of an amino acid moiety, preferably an arginine moiety, a β-alanine; C_1-6 alkylene amine group; a C_1-6 alkylene acyl group and a group C_1-6 alkylene; being said C_1-6 alkylene a linear C_1- 6 alkylene group or a branched C_3-6 alkylene group.

[0162] In some embodiments the moiety of formula (II) is selected from the group consisting of formula (lIb), (lIc), (lId), (lIe), (Ilf), (Ilg), (Ilh) (Ilj), (Ilk), (Ilm), (Iln), (IIp), (Ilq), (Ilr), (Ils), (lIt), (IIv), (IIw) and (IIx):

wherein

R_a, R_b and R_c are each independently H or a group R’ :

being at least one of R_a, R_b and R_c a group R’; n and n’ being each independently selected from 1 to 40, preferably n and n’ are each independently is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m₁ and, m2 being each independently selected from 0, 1 or 2; m₃, m₄ , m₅ and m₆ being each independently selected from 1 to 12, preferably 1 to 6, more preferably 1 or 2;

N* is a nitrogen atom of an amino group of an amino acid residue of the AAV vector’s capsid;

-- - -- represents the point of attachment to the capsid of the AAV vector;

Z is or comprises a linear or a cyclic peptide or a saccharide, wherein said peptide may be a peptide featuring biological activity, for example a blood brain barrier (BBB) shuttle peptide, preferably selected from the group consisting of a peptide THR or a peptide with a RGD motif, including a cyclic RGD peptide, and being the saccharide preferably selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and derivatives thereof, preferably a saccharide selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, desosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6- mannose, P6-glucose, sialic acid, SI -fructose and Pl -fructose, preferably selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid and Pl- fructose, more preferably mannose;

Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se, preferably a phenylene or pyridylene group, optionally comprising one or more substitutions selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy; and R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z-(OCH₂-CH₂)n-, Z-C(0)NH-(CH₂)q- (OCH₂-CH₂)n-, and Z-NHC(O)-(CH₂)q-(OCH₂-CH₂)n-, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂-CH₂)n-, wherein n is selected from 1 to 40 and more preferably R₁ H or Z-(OCH₂-CH₂)n-, wherein n is selected from 1 to 40, more preferably R₁ is H.

General Description and Definitions

[0163] The term “alkyl” refers to a monovalent or divalent, linear or branched, saturated hydrocarbon chain, comprising 1-8 carbon atoms (also named (C1-C8)alkyl), such as methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, tert-butyl- methyl, n-pentyl, n hexyl, n-heptyl, or n-octyl group. The term “alkylene group” corresponds to the bivalent group obtained by removal of a hydrogen atom from an alkyl group, as defined above herein, resulting in a moiety with two points of attachment.

[0164] The term “acyl” refers to a -C(O)R group, where R is an alkyl group as defined earlier or a phenyl group. An acyl group includes for example acetyl, ethyl carbonyl, or benzoyl group. [0165] The term “alkoxy” or “alkyloxy” refers to a -O-Alk group wherein Aik is an alkyl group as defined above. An alkoxy group includes for example methoxy, ethoxy, n- propyloxy, or tert-butyloxy group.

[0166] By “aryl group” it is herein referred to an aromatic monocyclic (i.e. phenyl) or bicyclic system (i.e. phenyl) comprising 4-12 carbon atoms, preferably 6 to 10, it being understood that in the case of a bicyclic system, one of the cycles is aromatic and the other cycle is aromatic or unsaturated. Aryl groups include for example phenyl, naphthyl, indenyl, or benzocyclobutenyl groups, optionally substituted by one or more groups optionally comprising one or more substitutions selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy. A preferred aryl group used herein is phenyl. The term “arylene group” corresponds to the bivalent group obtained by removal of a hydrogen atom from an aryl group, as defined above herein, resulting in a moiety with two points of attachment. A preferred arylene group used herein is phenylene optionally substituted by one or more groups optionally comprising one or more substitutions selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy.

[0167] By “heteroaryl group” it is herein referred to a 5 to 12 carbon-atom aromatic ring or ring system containing 1 to 2 rings which are fused together or linked covalently, typically containing 5 to 6 atoms on each ring; at least one of which is aromatic and in which one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen, sulfur or selenium atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quatemized. Such rings may be fused to an aryl ring. Non-limiting examples of such heteroaryl groups include: triazolyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatri azolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, imidazo[2,l-b][l,3]thiazolyl, thieno[3,2-b]furanyl, thieno[3,2-b]thiophenyl, thieno[2,3-d][l,3]thiazolyl, thieno[2,3-d]imidazolyl, tetrazolo[ 1,5 -a] pyridinyl, indolyl, indolizinyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, indazolyl, benzimidazolyl, 1,3-benzoxazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,3 -benzothiazolyl, 1,2-benzoisothiazolyl, 2,1 -benzoisothiazolyl, benzotriazolyl, 1,2,3-benzoxadiazolyl, 2,1,3-benzoxadiazolyl, 1,2,3-benzothiadiazolyl, 2,1,3-benzothiadiazolyl, thienopyridinyl, purinyl, imidazo[l,2-a]pyridinyl, 6-oxo-pyridazin-l(6H)-yl, 2-oxopyridin-l (2H)-yl, 6-oxo-pyrudazin-l(6H)-yl, 2-oxopyridin-l(2H)-yl, 1,3-benzodioxolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, optionally substituted by one or more groups selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy. A preferred heteroaryl group used herein is pyridyl. The term “heteroarylene group” corresponds to the bivalent group obtained by removal of a hydrogen atom from a heteroaryl group, as defined above herein, resulting in a moiety with two points of attachment. A preferred heteroarylene group used herein is pyridylene optionally substituted by one or more groups selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy.

[0168] The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, phosphorus, selenium, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, selenium, or silicon; the quatemized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H- pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl)).

[0169] The term “unsaturated”, as used herein, means that a moiety has one or more units of unsaturation.

[0170] The term “halogen” means F, Cl, Br, or I.

[0171] The term “arylalkyl” refers to a -Aik- Ar group, wherein Aik represents an alkyl group as defined earlier, and Ar represents an aryl group as defined earlier.

[0172] The term “heteroalkyl” refers to a linear or branched saturated hydrocarbon chain, comprising 1 to 5 carbon atoms and at least 1 or 2 heteroatoms, such as sulfur, nitrogen or oxygen atoms, in particular groups alkoxy, alkylamines, dialkylamines, thioethers, among others. Heteroalkyl groups, for example include -O(CH₂)_nOCH₃, - (CH₂)nOCH₃, -N(CH₂)n-N(CH₂CH₃)₂, -N(CH₂CH₃)₂, or -(CH₂)n-S-(CH₂)n-CH₃, where n is selected from 1 to 4, among others. [0173] The term “cycloalkyl” refers to a saturated monocyclic or polycyclic system, such as a fused or bridged bicyclic system, comprising 3-12 carbon atoms, such as the cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantly, decalinyl, or norbomyl groups.

[0174] The term “haloalkyl” means a linear or branched saturated hydrocarbon chain, comprising 1-6 carbon atoms and substituted with one or more, and notably 1-6 halogen atoms, such as the trifluoromethyl or 2,2,2-trifluoroethyl groups.

[0175] The term “O-R^a” refers to group in which the R group may be an alkyl, an aryl, a haloalkyl or an arylalkyl group, as defined earlier, is connected to the remainder of the molecule through an oxygen atom. O-cycloalkyl includes for example the O-cyclopentyl or O-cyclohexyl group.

[0176] As described herein, compounds may contain “optionally substituted” moi eties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety of compounds are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either

Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. [0177] Suitable substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; alkyl, acyl, aryl, heteroaryl, arylalkyl, heteroalkyl, cycloalkyl, alkoxy, haloalkyl, haloalkoxy, or a group O-R^a, wherein R^a and each of the substituents are as defined above herein, among others.

[0178] When the terms “moi eties of formula (II)” and “compounds of formula (III)” are used, said terms also include the possible pharmaceutically acceptable salts that said moieties and compounds may form. As used herein, the term “pharmaceutically acceptable salt” includes conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases as well as quaternary ammonium salts. More specific examples of suitable acid salts include hydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic, malic, steroic, tannic etc. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. For example, preferred salt forms include sodium salts of the compounds of formula (III) disclosed within the scope of the present description.

[0179] Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. For the purposes of the present specification, pharmaceutically acceptable salts also include zwitterionic pharmaceutically forms.

[0180] Many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". Solvates of the compounds of formula (III) are within the scope of the present invention. [0181] The term “isomer” refers to compounds of the invention which have identical molecular formulae as identified herein but which differ by nature or in the binding sequence of their atoms or in the layout of their atoms in space. Isomers which differ in the layout of their atoms in space are designated by “stereoisomers”. Stereosi omers which are not mirror images of each other, are designated as “diastereoisomers”, and stereoisomers which are non-superposable mirror images of each other are designated as “enantiomers” or “optical isomers”. “Stereoisomers” refer to racemates, enantiomers and diastereoisomers. A pair of diastereoisomers is designated as epimers. Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present disclosure. Unless otherwise stated, all tautomeric forms are within the scope of the disclosure.

[0182] The term “anomer” refers cyclic monosaccharides which are epimers and differ in the configuration of their C-l carbon atom if said monosaccharide is an aldose, and in the configuration of their C-2 carbon atom if they are ketoses, wherein said C-l or C-2 carbon atom is respectively named “anomeric carbon”.

[0183] The term “bioisostere”, when referred to a specific group or moiety, and in particular to the group amide included in the embodiments and aspects defined in the present invention, refers to other possible groups or moieties which are comparable in electronic and steric arrangement to said specific group, meaning that the bioisostere groups share some common biological properties in addition to their physicochemical analogy.

[0184] Additionally, unless otherwise stated, the present disclosure also includes compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present disclosure. In some embodiments, compounds of this disclosure comprise one or more deuterium atoms.

[0185] Combinations of substituents and variables envisioned by this disclosure are only those that result in the formation of stable compounds. The term “stable”, as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

Functional moiety R^L-NH-

[0186] The functional moiety R^L-NH- may be of any type and is typically selected depending on the biological effect which is sought when chemically modifying the capsid of the AAV vector.

[0187] In some embodiments, R^L-NH- comprises a cell -type specific ligand, a labelling agent, a steric shielding agent, a drug moiety or combinations thereof. In some embodiments, the functional moiety R^L-NH- may also comprise a (nano)-particle, including a magnetic (nano-) particle and a quantum dot. For instance, in some embodiments, R^L-NH- may comprise an iron, stain, silicium, gold or carbon (nano)- particle.

[0188] In some embodiments, R^L-NH- is a functional moiety comprising, or consisting of, a labeling agent, e.g. a fluorescent dye such as fluorescein, fluoroalanine, , rhodamine, boron-dipyrromethene (Bodipy®) dyes, and Alexa fluor®, or a radionuclide.

[0189] In some embodiments, R^L-NH- is a functional moiety comprising, or consisting of, a steric shielding agent, e.g. an agent able to mask certain epitopes of the capsid, thereby avoiding the binding of neutralizing antibodies. For instance, in some embodiments, R^L-NH- may comprise a polyethylene glycol (PEG), pHPMA, a peptide or a polysaccharide. In some embodiments, R^L-NH- comprises a polyethylene glycol (PEG), comprising from 1 to 40 ethylene glycol monomers, e.g. from 1 to 10, such as e.g. . -(OCH₂CH₂)- (referred to herein as “PEG1”), -(OCH₂CH₂)₂- (referred to herein as “PEG2”), -(OCH₂CH₂)₃- (referred to herein as “PEG3”), -(OCH₂CH₂)₄ (referred to herein as “PEG4”), or -(OCH₂CH₂)₅- (referred to herein as “PEG5”). In some particularly preferred embodiments R^L-NH- is a functional moiety comprising, or consisting of a saccharide or a peptide, being said saccharide and peptide as defined in the claims and embodiments of the present specification.

[0190] In some embodiments, the functional moiety R^L-NH- comprises a group Z and optionally one or more spacers L. In other embodiments, the functional moiety R^L-NH- consists of a group Z-NH- and does not comprise one or more spacers L.

Group Z

[0191] In some embodiments, Z is a functional moiety comprising, or consisting of, a cell-type specific ligand, namely a ligand enabling targeting of a specific type of cell. In some embodiments, such a ligand can enable modification of the tropism of the AAV vector, namely its capacity to selectively infect and/or transduce a given cell line, tissue, and/or organ. For instance, in some embodiments, Z can comprise or consist of a ligand which specifically binds to a membrane biological entity (e.g. a membrane receptor) of the targeted cell. In some embodiments, such a ligand can be, for instance, a saccharide, a hormone, including a steroid hormone, a peptide such as a peptide with a RGD motif, Angiopep-2 or muscle targeting peptides, a protein or a functionally active fragment thereof, a membrane receptor or a functionally active fragment thereof, CB 1 and CB2 ligands, an antibody including heavy-chain antibody, or functionally active fragments thereof such as Fab, Fab’, and VHH, a ScFv, a diabody, a spiegelmer, an aptamer including nucleic acid aptamer and peptide aptamer, a small chemical molecules known to bind to the targeted biological entity and the likes such as vitamins and drugs, and/or any suitable combination thereof.

[0192] As used herein, the term “cell-type specific ligand” refers to a compound (chemical or biological) that mediates specific binding and transduction of the target cell types and therefore can be used to selectively deliver transgenes into specific cell types in vivo, which increases the numbers of AAV vector particles reaching the targeted cells and tissues and decreases adverse effects in non-targeted cells and tissues. A cell-type specific ligand is an agent that exhibits high affinity for target cells and little to no affinity for non-target cells. For example, the target cell is of a particular tissue type, and the cell- type specific ligand specifically binds to a marker protein, surface antigen, receptor protein, that is expressed by cells of the target tissue.

[0193] By “functionally active fragment”, it is meant a fragment of, e.g., a protein, a membrane receptor or an antibody, which retains the functional activity of its full-length counterpart.

[0194] In some embodiments, Z comprises, or consists of, a cell-type specific ligand derived from a saccharide. Details on saccharides are provided hereafter.

[0195] In some embodiments, Z 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.

[0196] In some embodiments, Z comprises, or consists of, a cell-type specific ligand derived from vitamins such as folic acid.

[0197] In some embodiments Z is or comprises a linear or a cyclic peptide, wherein said peptide may be a peptide featuring biological activity. For example, the peptide may be peptide targeting transmembranal receptors, being said peptide targeting transmembranal receptors linked or not to cellular transcytose mechanisms allowing the crossing of a natural barrier, such as the blood brain barrier (BBB). In particular, the peptide is a blood brain barrier (BBB) shuttle peptide (or BBB-penetrating peptide) with an enhanced transduction activity across the blood brain barrier. In some preferred aspects, the BBB shuttle peptide is selected from the group consisting of a peptide THR or a peptide with a RGD motif, including a cyclic RGD peptide. In particular the peptide THR targets the transferrin receptor TfRl, and the RGD-based peptides target an integrin subclass. Expression of TfRl and integrins in different tissues is key for targeting CNS (via BBB) and muscle tissues. In a preferred embodiment Z comprises a peptide THR (SEQ ID No.: 1). [0198] In some embodiments, Z comprises, or consists of, a cell-type specific ligand derived from a muscle targeting peptide (MTP). In certain embodiments, Z is a cancer cell targeting peptide and comprises a peptide such as a peptide with RGD motif, including a cyclic RGD peptide.

[0199] In some embodiments, Z comprises, or consists of, a cell-type specific ligand derived from small molecules or hormones such as naproxen, ibuprofen, cholesterol, progesterone, or estradiol.

[0200] In some embodiments, Z comprises an antibody or antigen-binding portion thereof. In some such embodiments, an antibody may be or comprise, for example, a single chain antibody or variable domain, such as a camelid antibody, a heavy-chain antibody, a nanobody, a shark antibody, etc. In some embodiments, an antibody or antigen binding portion thereof may be or comprise a Fab, a Fab’, a VHH, a ScFv, a diabody, etc. In some particular embodiments, an antibody or antigen binding portion thereof may be characterized by having specific affinity for a particular cell-specific protein, membrane protein, and/or membrane protein receptor.

[0201] In some embodiments, Z comprises or consists of a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins, or fragments thereof, membrane receptors or fragments thereof, antibodies or fragments thereof, spiegelmers, nucleic acid or peptide aptamers, vitamins, and drugs.

[0202] In a specific embodiment, Z comprises or consists of a saccharide selected from the group consisting of monosaccharides, oligosaccharides and polysaccharides; preferably the saccharide is a monosaccharide, wherein said monosaccharide is preferably selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, desosamine, glucuronic acid, S6-galactose, S6-N- acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, SI -fructose and Pl -fructose, more preferably selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6- glucose, sialic acid and Pl -fructose, even more preferably mannose. [0203] In some particular embodiments, suitable examples of saccharides include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides, and derivatives thereof; or a saccharide substituted by a peptide.

[0204] As used herein, the term “derivatives” when referring to monosaccharides, oligosaccharides or polysaccharides, is meant to encompass saccharides containing one or more non-hydroxyl group(s). Examples of such non-hydroxyl groups include, but are not limited to, a hydrogen, an alkyl, an amino group (such as e.g. NH₂, an alkyl amino, a dialkyl amino), an N-acetylamino group and/or a thiol group.

[0205] In some embodiments, the non-hydroxyl group is a negatively charged group such as a phosphate, a phosphonate, a sulfate, a sulfonate and a carboxyl group.

[0206] “Monosaccharides”, also called “simple sugars”, are the simplest form of sugar and the most basic units of carbohydrates. Monosaccharides can be classified by the number of carbon atoms they contain, e.g., 3 (trioses), 4 (tetroses), 5 (pentoses), 6 (hexoses), 7 (heptoses), and so on.

[0207] Examples of monosaccharides include, but are not limited to, glycolaldehyde, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, and sedoheptulose.

[0208] Deoxymonosaccharides are common derivatives of monosaccharides encompassed in the present invention, i.e., monosaccharides that have had a hydroxyl group replaced with a hydrogen atom.

[0209] Examples of deoxymonosaccharides include, but are not limited to, deoxyribose, fucose, fuculose, rhamnose, quinovose, pneumose.

[0210] 2-amino-2-deoxymonosaccharides are also common derivatives of monosaccharides encompassed in the present invention, i.e., monosaccharides that have had a hydroxyl group replaced with an amino group. [0211] Examples of 2-amino-2-deoxymonosaccharides include, but are not limited to, glucosamine, galactosamine, and daunosamine, as well as their acetylated forms, including, but not limited to, N-acetylglucosamine, and N-acetylgalactosamine.

[0212] In some embodiments, the monosaccharide contains a negatively charged group such as a phosphate group, a sulfate group or a carboxyl group.

[0213] Examples of monosaccharides containing a phosphate group, include, but are not limited to, glucose-6-phosphate, mannose-6-phosphate and fructose- 1 -phosphate

[0214] Examples of monosaccharides containing a sulfate group, include, but are not limited to, galactose-6-sulfate (S6-galactose), N-acetylgalactosamine-6-sulfate (S6-N-acetylgalactosamine).

[0215] Examples of monosaccharides containing a carboxyl group, include, but are not limited to, glucuronic acid and sialic acid.

[0216] It is to be understood that the monosaccharides and derivatives thereof mentioned herein also encompass acyclic (open-chain) forms and cyclic forms.

[0217] It is also to be understood that the monosaccharides and derivatives thereof mentioned herein also encompass D-stereoi somers and L-stereoisomers, as well as mixtures of D- and L- stereoisomers (e.g., racemic mixtures).

[0218] It is also to be understood that the monosaccharides and derivatives thereof mentioned herein also encompass α-anomers and β-anomers, as well as racemic mixtures of α- and β-anomers.

[0219] “Oligosaccharides” are saccharide polymers comprising a small number (typically from two to ten) of monosaccharides.

[0220] In some embodiments, an oligosaccharide according to the present invention comprises at least two, three, four, five, six, seven, eight, nine or ten monosaccharides, e.g., selected from the monosaccharides disclosed hereinabove, including their derivatives. [0221] In some embodiments, such oligosaccharide(s) can be a homooligosaccharide (i.e., composed of units of the same monosaccharide, including their derivatives) or heterooligosaccharides (i.e., composed of units of at least two different monosaccharides, including their derivatives).

[0222] In some embodiments, examples of oligosaccharides include, but are not limited to, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, nonasaccharides, and decasaccharides.

[0223] In some embodiments, specific examples of disaccharides include, but are not limited to, cellobiose, chitobiose, gentiobiose, gentiobiulose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose, maltulose, mannobiose, melibiose, melibiulose, nigerose, palatinose, rutinose, rutinulose, sophorose, sucrose, trehalose, turanose, and xylobiose.

[0224] In some embodiments, specific examples of trisaccharides include, but are not limited to, kestose, maltotriose, maltotriulose, melezitose, nigerotriose, and raffinose.

[0225] In some embodiments, specific examples of tetrasaccharides include, but are not limited to, lychnose, maltotetraose, nigerotetraose, nystose, sesamose, and stachyose.

[0226] In some embodiments, specific examples of oligosaccharides include, but are not limited to, acarbose, fructooligosaccharide, galactooligosaccharide, isomaltooligosaccharide, and maltodextrin.

[0227] In some embodiments, oligosaccharides can be multi -antennary structures whereby some or all monosaccharides in the oligosaccharide are not linked to one another through O-glycosidic bonds but with branched linker structures. An example of a multi- antennary saccharide is tri-antennary N-acetylgalactosamine, which is a ligand for asialoglycoprotein receptor ASGPR (see e.g., Zhou et al., Development of Tri antennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins; ACS Cent. Sci. 2021). [0228] “Polysaccharides” are saccharide polymers comprising a large number (typically more than ten) of monosaccharides. They range in structure from linear to highly branched.

[0229] In some embodiments, a polysaccharide comprises more than ten monosaccharides (such as, e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more), e.g., selected from monosaccharides disclosed hereinabove, including their derivatives. In a similar way as described above for oligosaccharides, polysaccharides can be homopolysaccharides or heteropolysaccharides.

[0230] In some embodiments, examples of polysaccharides include, but are not limited to, beta-glucans, lentinan, sizofiran, zymosan, cellulose, hemicellulose, chitin, chitosan, dextrins, dextran, fructan, inulin, galactan, glucan, glycogen, levan [32— >6, lignin, mannan, pectin, starch, amylopectin, amylose, and xanthan gum.

[0231] In some embodiments, a saccharide or derivative thereof according to the present invention is a monosaccharide, preferably a hexose. In some embodiments, a preferential saccharide or derivative thereof according to the present invention is mannose, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, S6-galactose, S6-N-acetylgalactosamine, glucuronic acid, P6-galactose or Pl- galactose. In some embodiments, a preferential saccharide or derivative thereof according to the present invention is mannose, galactose, N-acetylglucosamine, or N-acetylgalactosamine.

[0232] In some embodiments, a saccharide or derivative thereof is mannose. In some embodiments, a saccharide or derivative thereof is galactose. In some embodiments, a saccharide or derivative thereof is N-acetylglucosamine. In some embodiments, a saccharide or derivative thereof is N-acetylgalactosamine.

[0233] In some embodiments, a saccharide or derivative thereof according to the present invention is a deoxymonosaccharide. In some preferential embodiments, a deoxymonosaccharide is preferably fucose.

[0234] In some embodiments, a saccharide or derivative thereof is a saccharide containing a non-hydroxyl group which is a dialkyl amino group. In some preferential embodiments, a saccharide containing a non-hydroxyl group which is a dialkyl amino group is a desosamine.

[0235] In some embodiments, a saccharide or derivative thereof is a saccharide containing a non-hydroxyl group which is a sulfate group. In some preferential embodiments, a saccharide containing a non-hydroxyl group which is sulfate group is S6-galactose, or S6-N-acetylgalactosamine.

[0236] In some embodiments, a saccharide or derivative thereof is a saccharide containing a non-hydroxyl group which is a phosphate group. In some preferential embodiments, a saccharide containing a non-hydroxyl group which is phosphate group is P6-glucose, P6- mannose, or Pl -fructose.

[0237] In some embodiments, a saccharide or derivative thereof is a saccharide containing a non-hydroxyl group which is a carboxyl group. In some preferential embodiments, a saccharide containing a non-hydroxyl group which is carboxyl group is glucuronic acid or sialic acid.

[0238] In some embodiments, the saccharide is selected from the group comprising, or consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, desosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6- mannose, P6-glucose, sialic acid, SI -fructose and Pl -fructose. In some preferred embodiments the saccharide is selected from the group consisting mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6- mannose, P6-glucose, sialic acid and Pl -fructose, more preferably mannose.

Spacer L

[0239] In some embodiments, the functional moiety R^L-NH- comprises a group Z and at least one spacer L. In particular, in some embodiments, one or more spacers L are present for linking the group Z to the squaramide linker of formula (I).

[0240] In some aspects, the functional moiety R^L-NH- comprises one or more groups Z and one or more spacers L. In some aspects, the functional moiety R^L-NH- comprises 1 to 3 groups Z, each of said groups Z linked to one or more spacers L present for linking each of the groups Z to the squaramide linker of formula (I).

[0241] In some embodiments, L may be any chemical chain which can comprise heteroatoms as well as cyclic moieties such as aryl and/or heteroaryl groups.

[0242] In some embodiments, L may comprise up to 1000 carbon atoms and even more. The length and the chemical nature of L may be optimized depending on the group Z which is intended to be coupled to the AAV vector and the biological effect which is sought.

[0243] In some embodiments, L 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.

[0244] In some embodiments, L connects the group Z to the squaramide linker of formula (I), as defined in the present disclosure, and preferably comprises up to 1000 carbon atoms and is preferably in the form of a chemical chain which optionally comprises heteroatoms (e.g. O, NH, S, Se or P) and/or cyclic moieties, such as aryl and/or heteroaryl groups.

[0245] In some embodiments, L may comprise one or more groups or moieties selected from alkyl (e.g., C_1-20, C_1-12, C_1-6 alkyl), aryl, heteroaryl, alkyl ether, polyether, polyester, acyl, alkyl amide, polyamide, a guanidine, or a combination thereof. As used herein, “combination” means that L may comprise several hydrocarbon chains, oligomer chains, polymeric chains (e.g. 2, 3, 4, 5 or 6) containing optionally one or more heteroatoms, aryl or heteroaryl groups, linked by any appropriate group, such as -O-, -S-, -NHC(O)-, - OC(O)-, -C(O)-O-C(O)-, -NH-, -NH-CO-NH-, -O-CO-, -NH-(CS)-NH-, -NH-CS- phosphodiester or phosphorothioate groups. The use of a variety of alkyls is contemplated, including, but not limited to, -(CH₂)_n-, wherein “n” is from about 2 to about 20 or more. In some embodiments, L comprises a C_2-20 straight or branched alkyl chain. [0246] In some embodiments, L is or comprises a polyether (e.g., polyethylene or polypropylene glycol). The use of a variety of ethers and polyethers is contemplated, including, but not limited to, -(OCH₂CH₂)_n-, wherein “n” is an integer from about 1 to about 40 or more. In some embodiments, L is or comprises a polyethylene glycol (“PEG”) of formula -(OCH₂CH₂)_n-, wherein “n” is an integer from 1-10, an integer from 1-6, and integer from 3-6, and integer from 3-5, or an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L is or comprises a polypropylene glycol, e.g., of formula -(OCH(CH₃)CH₂)_n-, wherein “n” is an integer from 1-10, an integer from 1-6, and integer from 3-6, and integer from 3-5, or an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0247] In some embodiments, L is or comprises an alkyl amide. The use of a variety of alkyl amides is contemplated, including, but not limited to, -(CH₂)_y-C(O)NH-(CH₂)_P- and -(OCH₂CH₂)_y-C(O)NH-(OCH₂CH₂)p- wherein “y” and “p” can be the same or different and “y” and “p” are from about 1 to about 20 or more. In some embodiments, L is or comprises an alkyl amide of formula -(CH₂)_y-C(O)NH-(CH₂)_P- or of formula - (OCH₂CH₂)y-C(O)NH-(OCH₂CH₂)_P-, wherein “y” and “p” are each independently selected from an integer from 1-10, an integer from 1-6, and integer from 3-6, and integer from 3-5, or an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The use of a variety of amides having the linking units of alkyl or ether bonds is contemplated, including, but not limited to, -R₄-C(O)NH-R₅-, wherein “R₄” and “R₅” are each independently selected from alkyls (e.g., C_1-20, C_1-12, C_1-6 alkyl), ethers, or polyethers (e.g., PEGs having a molecular weight between about 200 to 2,000 g/mol). In a preferred embodiment L comprises a polymer of β-alanine, preferably comprising 1 to 40 β-alanine monomers, more preferably Ito 10 β-alanine monomers.

[0248] In some embodiments, L may also comprise an alkylene diamine, e.g., -NH-(CH₂)r-NH-, where “r” is an integer from 2 to 20, for instance from 2 to 10, or an integer selected from 2, 3, 4, or 5. In some embodiments, L is a polymer of alkylene diamines (also known as polyamines), e.g., a compound of formula -NH-[(CH₂)_r-NH]t-, where “r” is as defined above and herein, 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. [0249] In some embodiments, L may also comprise polyamides obtained from vinylic monomers such as poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), (e.g., pHPMA having a molecular weight between about 200 and about 5000 g/mol).

[0250] In some embodiments, L may also comprise polyesters such as polycaprolactone (e.g., polycaprolactone having a molecular weight between about 200 and about 5000 g/mol) or poly(D,L-lactic-co-glycolic acid) (PLGA) (e.g., PLGA having a molecular weight between about 200 and about 5000 g/mol).

[0251] In some embodiments L may also comprise an acyl group, e.g., -(CH₂)r-C(O)-, where “r” is an integer from 2 to 20, for instance from 2 to 10, or an integer selected from 2, 3, 4, or 5.

[0252] In some embodiments, L may include one or more optionally substituted groups comprising, or consisting of, an arylene or a heteroarylene group, a saturated or unsaturated, linear or branched C₂-C₄₀ hydrocarbon chain, an alkylene amine containing group, an acyl containing group, an amino acid moeity, a polyethylene glycol, a polypropylene glycol, a polyether of a branched polyol, a β-alanine polymer, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof.

[0253] In some embodiments, L is or comprises a polyethylene glycol (PEG), comprising from 1 to 40 ethylene glycol monomers, e.g. from 2 to 10, such as e.g. - (OCH₂CH₂)₂- (referred to herein as “PEG2”), -(OCH₂CH₂)₃- (referred to herein as “PEG3”), -(OCH₂CH₂)₃- (referred to herein as “PEG3”), -(OCH₂CH₂)₄- (referred to herein as “PEG4”), or -(OCH₂CH₂)₅- (referred to herein as “PEG5”).

[0254] In some embodiments, L may comprise one or more arylene or a heteroarylene groups Ar. In some particular aspects, the arylene or a heteroarylene group Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se. In some embodiments the group Ar is substituted by an acyl or an amide moiety, or a bioisostere thereof. In some particular aspects the arylene or a heteroarylene group Ar is selected from the group consisting of phenylene and pyridylene. For example, in some embodiments, L comprises an optionally substituted phenylene moiety. For example, in some embodiments, L comprises an optionally substituted pyridylene moiety. In other embodiments said phenylene or pyridylene groups are substituted by one or more moieties selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy.

[0255] In some embodiments, L may comprise an alkylene, ether, polyether, alkylene amide, arylene group, heteroarylene group, an acyl group or a combination thereof. In a specific embodiment, L comprises a polyether, arylene group, heteroarylene group, acyl group or a combination thereof. In a particular embodiment, L comprises an arylene or a heteroarylene group Ar. Preferably said arylene or a heteroarylene group Ar is a 6- to 10- membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se. In some particular aspects the arylene or a heteroarylene group Ar is selected from the group consisting of phenylene and pyridylene optionally substituted by one or more moieties selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy.

[0256] In a specific embodiment L comprises a PEG. In another specific embodiment, L comprises a PEG and one or more aromatic groups, such as an arylene group and/or heteroarylene group Ar. In another specific embodiment, L comprises a PEG, one or more groups C_1-6 alkyl and one or more aromatic groups, such as an arylene group and/or heteroarylene group Ar. In a specific embodiment, L comprises a PEG and an aryl or a heteroaryl group Ar selected from the group consisting of phenylene and pyridylene optionally substituted by one or more moieties selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1-6 alkoxy. In another specific embodiment L comprises a PEG and an amino acid, preferably an arginine moiety. In another specific embodiment L comprises a PEG, a β-alanine polymer, and one or more aromatic groups, such as an arylene group and/or heteroarylene group Ar. In another specific embodiment L comprises a PEG, a β-alanine polymer, one or more groups C_1-6 alkyl, C_1-6 alkylamine or C_1-6 acyl; and an amino acid, preferably an arginine moi.

[0257] In a specific embodiment L comprises one or more polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, one or more C_1-6 alkylene groups, a polyether of a branched C_3-12 polyol, and one or more aromatic groups, such as an arylene group and/or heteroarylene group Ar.

[0258] In some embodiments, L consists of one or more groups selected from the group consisting of an arylene or a heteroarylene group, an optionally substituted group comprising saturated or unsaturated, linear or branched C₂-C₄₀ hydrocarbon chains, preferably one or more groups C_1-6 alkyl, C_1-6 alkylamine or C_1-6 acyl; a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, a polypropylene glycol (PPG) comprising 1 to 40 propylene glycol monomers, a polyether of a branched C_3-12 polyol, an arginine derivative, a β-alanine polymer comprising 1 to 40 β-alanine monomers, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof; and wherein L comprises at least one or more groups selected from the group consisting of an arylene or a heteroarylene group, a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and a β-alanine polymer comprising 1 to 40 β-alanine monomers.

R₁ and R₂

[0259] In some embodiments, wherein R₁ is selected from the group consisting of H, C_1- ₆ alkyl, C_1-6 haloalkyl, Z-(OCH₂-CH₂)n- Z-C(O)NH-(CH₂)q-(OCH₂-CH₂)n-, and Z- NHC(O)-(CH₂)q-(OCH₂-CH₂)n-, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention. In some embodiments R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂-CH₂)n-, wherein n is selected from 1 to 40. In a preferred embodiment R₁ is selected from the group consisting of H, C_1-6 alkyl and C_1-6 haloalkyl. Even more preferably R₁ is H.

[0260] In some embodiments, R₂ is selected from the group consisting of linear C_1-12 alkyl, branched C_3-12 alkyl, linear C_1-12 haloalkyl, branched C_3-12 haloalkyl, aryl, heteroaryl and benzyl. In some preferred embodiments R₂ is methyl, ethyl, trifluoromethyl, trifluoroethyl, phenyl, pyridyl or benzyl, more preferably ethyl. Arylene or a heteroarylene group Ar

[0261] In some embodiments, the group Ar is an arylene group, wherein said arylene group is as defined and described in classes and subclasses disclosed in the present invention. In other embodiments, the group Ar is an heteroarylene group, wherein said heteroaryl group is as defined and described in classes and subclasses disclosed in the present invention.

[0262] In some embodiments, said arylene or a heteroarylene group Ar is a 6- to 10- membered arylene group or a 5- or 12-membered heteroarylene group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se. In some particular embodiments, the arylene or a heteroarylene group Ar is selected from the group consisting of phenylene and pyridylene optionally substituted by one or more moieties selected from the group consisting of halogen, C_1-6 alkyl, C_1-6 haloalkyl, C_1-6 acyl and C_1- 6 alkoxy.

Formulae of AA V vectors

[0263] In some embodiments, the present invention relates to an AAV vector particle comprising a moiety of formula (II):

wherein said AAV vector particle comprises a squaramide linker of formula (I):

the group R^L and an amino acid residue of a capsid from the AAV vector are covalently linked to the squaramide linker of formula (I); N is a nitrogen atom from the amino moiety of a functional moiety R^L-NH- as defined in the present invention, and N* is a nitrogen atom of a primary amino group from a surface-exposed amino acid residue of a capsid polypeptide from the AAV vector.

[0264] In some embodiments, the present invention relates to an AAV vector particle comprising a moiety of formula (II), as defined in the present disclosure, in which the functional moiety R^L-NH- comprises a group Z, one or more spacers L, and the adeno- associated virus (AAV) vector particle is represented by formula (Ila):

wherein N*, Z and L, are as defined and described in classes and subclasses in the present invention.

[0265] In some embodiments, L comprises one or more optionally substituted groups selected from the group consisting of an arylene or an heteroarylene group Ar, a saturated or unsaturated, linear or branched C₂-C₄₀ hydrocarbon chain, preferably one or more groups C_1-6 alkylene, C_1-6 alkylamine or C_1-6 acyl, a polyethylene glycol, a polypropylene glycol, an amino acid moiety, a β-alanine polymer, a polyether of a branched C_3-12 polyol, pHPMA, PLGA, polymers of alkyl diamines and combinations thereof; preferably L is or comprises one or more groups selected from the group consisting of a polyethylene glycol, an aryl or an heteroaryl group Ar, a C_1-6 alkylene group, a C_1-6 alkylamine, a C_1-6 acyl group, an amino acid moiety, preferably an arginine moiety, a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and a β-alanine polymer comprising 1 to 40 β-alanine monomers. In some embodiments, L is or comprises a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers. In some preferred embodiments the polyethylene glycol (PEG) is PEG3, PEG4, or PEG5.

[0266] In some particular embodiments, the present invention relates to an AAV vector particle comprising a moiety of formula (II), as defined in the present disclosure, comprises more than one spacer L selected from the group consisting of L₁, L₂ and L₃, and said moiety of formula (II) is selected from the group consisting of formula (Ilai), (IIa2), (IIa3), (IIa4), (IIa5), (IIa6), (IIa7), (IIa8), (IIa9), (IIa10), (IIa11), (IIa12), (IIa13), (IIa14), (IIa₁₅), (IIa₁₆), (IIa₁₇), (IIa₁₈), (IIa₁₉) and (IIa₂₀):

wherein N*, Z, L₁, L₂ and L₃ are as defined and described in classes and subclasses disclosed in the present invention.

[0267] In some embodiments, L₁ is an optionally substituted group selected from the group consisting of polyethylene glycol, polypropylene glycol, pHPMA, PLGA, a β- alanine polymer, a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, polymers of alkyl diamines and combinations thereof; preferably L₁ is selected from the group consisting of a polyethylene glycol, a β-alanine polymer and a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol. In some embodiments, the polyethylene glycol (PEG), comprises 1 to 40 ethylene glycol monomers. In some preferred embodiments the polyethylene glycol (PEG) is PEG3, PEG4, or PEG5. In some embodiments the β-alanine polymer comprises 1 to 40 β-alanine monomers, preferably 1 to 10 β-alanine monomers.

[0268] In some embodiments, L₂ comprises one or more arylene or a heteroarylene groups Ar, as defined herein. In some preferred embodiments embodiments, L₂ comprises a phenylene group or a pyridylene group.

[0269] In some aspects, L₁ comprises one or more of a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, a β-alanine polymer comprising 1 to 40 β- alanine monomers, and a polyether of a branched C_3-12 polyol, preferably a branched C_3- ₆ polyol; L₂ comprises one or more arylene or a heteroarylene groups; and L₃ comprises a C_1-6 alkylene group, a C_1-6 alkylamine, a C_1-6 acyl group, or an amino acid moiety, preferably an arginine moiety.,

[0270] In some embodiments L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and L₁ and L₂ or L₁ and L₃ are covalently linked by an amide moiety, or a bioisostere moiety thereof, preferably an amide moiety -N(R₁)C(O)-, or a bioisostere moiety thereof, wherein R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z- (OCH₂-CH₂)n- Z-C(O)NH-(CH₂)q-(OCH₂-CH₂)n-, and Z-NHC(O)-(CH₂)q-(OCH₂- CH₂)n- wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; more preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z- (OCH₂-CH₂)n- wherein n is selected from 1 to 40, more preferably R₁ is H or Z-(OCH₂- CH₂)n- wherein n is selected from 1 to 40, and even more preferably R₁ is H.

[0271] In some aspects, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers; L₂ comprises one or more arylene or a heteroarylene groups; L₃ is a C_1-6 alkylene group; L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and L₁ and L₃ are covalently linked by an amide moiety -N(R₁)C(O)-, or a bioisostere moiety thereof, wherein R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z- (OCH₂-CH₂)n-, Z-C(O)NH-(CH₂)q-(OCH₂-CH₂)n-, and Z-NHC(O)-(CH₂)q-(OCH₂- CH₂)n- wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂- CH₂)n- wherein n is selected from 1 to 40, more preferably R₁ is H or Z-(OCH₂-CH₂)n- , wherein n is selected from 1 to 40, and even more preferably R₁ is H.

[0272] In some aspects, L₁ and L₂ are covalently linked by an amide moiety -N(R₁)C(O)-, or a bioisostere moiety thereof, wherein R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z-(OCH₂-CH₂)n- Z-C(O)NH-(CH₂)q- (OCH₂-CH₂)n-, and Z-NHC(O)-(CH₂)q-(OCH₂-CH₂)n-, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂-CH₂)n- wherein n is selected from 1 to 40 and more preferably R₁ is H or Z-(OCH₂-CH₂)n- wherein n is selected from 1 to 40, and even more preferably R₁ is H.

[0273] In some aspects, L₁ and L₃ are covalently linked by an amide moiety -N(R₁)C(O)-, or a bioisostere moiety thereof, wherein R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, Z-(OCH₂-CH₂)n- Z-C(O)NH-(CH₂)q- (OCH₂-CH₂)n- and Z-NHC(O)-(CH₂)q-(OCH₂-CH₂)n- wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in classes and subclasses disclosed in the present invention; preferably R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂-CH₂)n-, wherein n is selected from 1 to 40. more preferably R₁ is H or Z-(OCH₂-CH₂)n-, wherein n is selected from 1 to 40, and even more preferably R₁ is H.

[0274] In some embodiments, L₁ comprises one or more polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers and a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol; L₂ comprises one or more arylene or a heteroarylene groups Ar; and L₃ comprises one or more C_1-6 alkylene groups; the PEG and one C_1-6 alkylene group are covalently linked by an amide moiety or a bioisostere thereof, the polyether of a branched C_3-12 polyol and the and the C_1-6 alkylene group are covalently linked by an ether bond, the polyether of a branched C_3-12 polyol and an arylene or a heteroarylene group Ar are covalently linked by an amide moiety or a bioisostere thereof, preferably an amide moiety -N(R₁)C(O)-, or a bioisostere moiety thereof.

[0275] In some embodiments, L₁ comprises one or more of a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers and a β-alanine polymer comprising 1 to 40 β-alanine monomers; L₂ comprises one or more arylene or a heteroarylene groups Ar; and L₃ comprises one or more C_1-6 alkylene, C_1-6 alkylamine and C_1-6 acyl groups; and L₁ and L₂ or L₁ and L₃ are covalently linked by an amide moiety -N(R₁)C(O)-, or a bioisostere moiety thereof.

[0276] In some embodiments, L₁ comprises one or more of a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomer and a β-alanine polymer comprising 1 to 40 β-alanine monomers, L₃ comprises one or more C_1-6 alkylene groups, C_1-6 alkylamine groups, C_1-6 acyl groups and an amino acid moiety, preferably an arginine moiety.

[0277] In some preferred embodiments, n is 1, 2, 3, 4 or 5; n’ is selected from 2 to 8; mi and m2 are each independently 0, 1, or 2; m₃,m4, ms and me are each independently 1 to 3, preferably 2; Ar is a phenylene or a pyridylene group; and R₁ is H. [0278] Examples of bioisostere moieties of the amide moiety includes N-substituted and non- substituted moieties, for examples an amide -N(R₁)C(O)- and bioisoteres thereof that may be selected from -C(O)N(R₁)-, -N(R₃)C(O)N(R₁)-,

-N(R₁)C(O)N(R₃)-, -N(R₁)C(S)-, -C(S)N(R₁)-, -N(R₁)C(S)N(R₃)-, -N(R₃)C(S)N(R₁)-, -S(O)2-N(R₁)-, -N(R₁)-S(O)2- and a triazolyl group, among others, wherein R₃ and R₁ are each independently selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl, aryl, alkylaryl, Z-(OCH₂-CH₂)n- Z-C(O)NH-(CH₂)q-(OCH₂-CH₂)n-, and Z-NHC(O)- (CH₂)q-(OCH₂-CH₂)n-, wherein q is selected from 1 to 3, n is selected from 1 to 40, and R₃ may be the same or different from R₁.

[0279] In some embodiments, when L₂ comprises an arylene or a heteroarylene group Ar, and L₁ and the squaramide linker of formula (I), or L₁ and L₃, when present, are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para.

[0280] In some embodiments, when L₂ comprises an arylene or a heteroarylene group Ar and L₃ is a group C_1-6 alkylene, L₃ and the squaramide linker of formula (I) are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para.

[0281] In some embodiments, when L₂ comprises an arylene or a heteroarylene group Ar and L₃ is a group C_1-6 alkylene, one or more groups L₃ are covalently bonded to said arylene or a heteroarylene group Ar in positions orto, meta or para.

[0282] In some embodiments, L₁ or L₃ may be selected from alkyl (e.g., C_1-20, C_1-12, C_1- ₆ alkyl), ether, polyether, polyester, polyamide, alkyl amide, or a combination thereof. As used herein, “combination” means that L₁ or L₃ may comprise several hydrocarbon chains, oligomer chains or polymeric chains (e.g. 2, 3, 4, 5 or 6) linked by any appropriate group, such as -O-, -S-, -NHC(O)-, -OC(O)-, -C(O)-O-C(O)-, -NH-,

-NH-CO-NH-, -O-CO-, -NH-(CS)-NH-, -NH-CS- phosphodiester or phosphorothioate groups. The use of a variety of alkyls is contemplated, including, but not limited to, - (CH₂)_m-, wherein “m” is from about 2 to about 20 or more. In some embodiments, L₃ comprises a C_2-20 straight or branched alkyl chain. [0283] In some embodiments, L₁ or L₃ may also comprise polyesters such as poly caprolactone (e.g., poly caprolactone having a molecular weight between about 200 and about 5000 g/mol) or poly(D,L-lactic-co-glycolic acid) (PLGA) (e.g., PLGA having a molecular weight between about 200 and about 5000 g/mol).

[0284] In some embodiments, L₁ or L₃ may be selected from an optionally substituted group comprising, or consisting of, saturated or unsaturated, linear or branched C₂-C₄₀ hydrocarbon chains, polyethylene glycol, polypropylene glycol, a poly ether of a branched C_3-12 polyol, a β-alanine polymer, pHPMA, PLGA, polymers of alkylene diamines, amino acid moieties, such as an arginine or a β-alanine moiety, and combinations thereof. The use of a variety of alkyl amides is contemplated, including, but not limited to, -(CH₂) m₃- C(O)NH-(CH₂)m₄- and -(OCH₂CH₂)m₃-C(O)NH-(OCH₂CH₂)m₄-, wherein “m₃” and “m₄ " can be the same or different and “m₃” and “ m₄” are from about 1 to about 20 or more. In some embodiments, L₁ or L₃ is an alkyl amide of formula -(CH₂)m₃-C(O)NH- (CH₂) m₄- or -(OCH₂CH₂)m₃-C(O)NH-(OCH₂CH₂)m₄-, wherein “m₃” and “ m₄” are each independently selected from an integer from 1-10, an integer from 1-6, and integer from 3-6, and integer from 3-5, or an integer independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The use of a variety of amides having the linking units of alkyl or ether bonds is contemplated, including, but not limited to, -R₅-C(O)NH-R₆-, wherein “R₅” and “R₆” are each independently selected from alkyls (e.g., C_1-20, C_1-12, C_1-6 alkyl), ethers, or polyethers.

[0285] In some embodiments, L₁ may also comprise a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, an acyl group -C(O)-(CH₂)r, or an alkylene amine, e.g.,-NH-(CH₂)_r-, or an alkylene diamine, e.g., -NH-(CH₂)_r-NH- where “r” is an integer from 2 to 20, for instance from 2 to 10, or an integer selected from 2, 3, 4, or 5. In some embodiments, L₁ is a polymer of alkylene diamines (also known as polyamines), e.g., a compound of formula -NH-[(CH₂)_r-NH]_t-, where “r” is as defined above and herein, 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. In some embodiments, L₁ may also comprise polyamides such as β-alanine polymers with 1 to 40 β-alanine monomers, or polyamides obtained from amide vinylic monomers, such as poly(N-(2-hydroxypropyl)methacrylamide) (pHPMA), (e.g., pHPMA having a molecular weight between about 200 and about 5000 g/mol).

[0286] In some embodiments, L₃ is a group C_1-6 alkylene, preferably a -CH₂- or a -CH₂-CH₂- group; or a group C_1-6 alkylamine, preferably a -CH₂-CH₂-NH-; a group C_1-6 acyl, preferably a -CH₂-CH₂-C(O)- group; or an amino acid moiety, preferably an arginine or a β-alanine moiety.

[0287] In some particular embodiments, the AAV vector particle comprises a moiety of formula (II) selected from the group consisting of formula (lIb), (lIe), (lId), (lIe), (Ilf),

(Ilg), (IIh), (Ilj), (Ilk), (Ilm), (Iln), (lIp), (Ilq), (Ilr), (Ils), (lIt), (IIv), (IIw) and (IIx):

wherein R_a, R_b and R_c are each independently H or a group R’ :

and wherein n, n’, m₁, m₂, m₃, m₄, m₅, m₆, N*, Z, Ar and R₁ are as defined and described in classes and subclasses disclosed in the present invention.

[0288] In another preferred embodiment of the AAV vector particle has a moiety of formula (II) as defined herein, R^L comprises a group Z and one spacer L, wherein Z is a saccharide, L is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers and the moiety of formula (II) is represented by formula (lib), as defined herein.

[0289] In a preferred embodiment of the AAV vector particle has a moiety of formula (Ilal) comprising a group Z and one spacer L₁, wherein Z is a saccharide, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers and the moiety of formula (II) is represented by formula (lib), as defined herein.

[0290] In a preferred embodiment of the AAV vector particle has a moiety of formula (IIa2) or (IIa3) comprising a group Z and spacers L₁, L₂ and L₃, as defined herein, Z is a saccharide, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, L₂ is an arylene or a heteroarylene group Ar, L₃ is a group C_1-6 alkylene, L₁ and L₂ are covalently linked by an amide moiety -N(R₁)C(O)-, wherein R₁ is as defined herein and the moiety of formula (II) is represented by formula (lie).

[0291] In another preferred embodiment of the AAV vector particle has a moiety of formula (IIa4) or (IIa5) comprising a group Z and spacers L₁, L₂ and L₃, as defined herein, Z is a saccharide, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, L₂ is an arylene or a heteroarylene group Ar, L₃ is a group C_1-6 alkylene, L₁ and L₃ are covalently linked by an amide moiety -N(R₁)C(O)-, wherein R₁ is as defined herein and the moiety of formula (II) is represented by formula (IId).

[0292] In another preferred embodiment of the AAV vector particle has a moiety of formula (IIa6) comprising a group Z and spacers L₁ and L₃, as defined herein, Z is a peptide, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, L₃ is a group C_1-6 alkylene, L₁ and L₃ are covalently linked by an ether bond, and the moiety of formula (II) is represented by formula (lie) when the N-terminal end of the peptide Z and L₃ are covalently linked by an amide or a bioisostere moiety thereof, being the nitrogen atom of the amide the N-terminal end of the peptide Z; and the moiety of formula (II) is represented by formula (Ilf) when the C-terminal end of the peptide Z and L₃ are covalently linked by an amide or a bioisostere moiety thereof, being the carbonyl group of the amide the C-terminal end of the peptide Z, and wherein said amide may be N-substituted or non-substituted, for example an amide -N(R₁)C(O)-, being R₁ is as defined herein.

[0293] In another preferred embodiment of the AAV vector particle has a moiety of formula (IIa7) or (IIa8) or (IIa9) or (Ila10) comprising a group Z and spacers L₁, L₂ and one to three spacers L₃, as defined herein; wherein Z is a peptide, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, L₂ is an arylene or a heteroarylene group Ar, and L₃ is a group C_1-6 alkylene; wherein L₁ and a first spacer L₃ are covalently linked by an ether bond; L₁ and L₂ or L₁ and a second spacer L₃ are covalently linked by an amide moiety -N(R₁)C(O)-; and the moiety of formula (II) is represented by formula (Ilh) when the N-terminal end of the peptide Z and L₃ are covalently linked by an amide or a bioisostere moiety thereof, being the nitrogen atom of the amide the N-terminal end of the peptide Z; and the moiety of formula (II) is represented by formula (Ilg) when the C-terminal end of the peptide Z and L₃ are covalently linked by an amide or a bioisostere moiety thereof, being the carbonyl group of the amide the C-terminal end of the peptide Z; and wherein and wherein said amide may be N-substituted or non substituted, for example an amide -N(R₁)C(O)-, being R₁ is as defined herein.

[0294] In a preferred embodiment of the AAV vector particle has a moiety formula (Ilal 1) as defined herein, R^L-NH consists of a group Z-NH-, wherein Z is a peptide, and the nitrogen atom of the group Z-NH corresponds to the N-terminal group of the peptide, and the moiety of formula (II) is represented by formula (lib), as defined herein.

[0295] In another preferred embodiment of the AAV vector particle comprises a group Z and spacers L₁, L₂ and one to three spacers L₃, as defined herein; wherein Z is a peptide, L₁ is a β-alanine polymer, comprising 1 to 40 β-alanine monomers, L₂ is an arylene or a heteroarylene group Ar, and the L₃ spacers are a group C_1-6 alkyleneamine or a group C_1- ₆ acyl or a group C_1-6 alkylene; wherein L₁ and L₂ or L₁ and one spacer L₃ are covalently linked by an amide moiety -N(R₁)C(O)-; and the moiety of formula (II) is represented by formula (Ilj) when the N-terminal end of the peptide Z and L₁ are covalently linked by an amide or a bioisostere moiety thereof, being the nitrogen atom of the amide the N- terminal end of the peptide Z; and the moiety of formula (II) is represented by formula (Ilk) when the C-terminal end of the peptide Z and L₃ are covalently linked by an amide or a bioisostere moiety thereof, being the carbonyl group of the amide the C-terminal end of the peptide Z; and wherein said amide may be N-substituted or non- substituted, for example an amide -N(R₁)C(O)-, being R₁ is as defined herein.

[0296] In another preferred embodiment of the AAV vector particle comprises a group Z and spacers L₁ and L₃, as defined herein, Z is a peptide, L₁ is a β-alanine polymer, comprising 1 to 40 β-alanine monomers, L₃ is a group C_1-6 alkyleneamine or a group C_1- ₆ alkylene, L₁ and L₃ are covalently linked by an amide moiety, and the moiety of formula (II) is represented by formula (Ilm) when the C-terminal end of the peptide Z and L₁ are covalently linked by an amide moiety or a bioisostere moiety thereof, being the nitrogen atom of the amide the N-terminal end of the peptide Z; and the moiety of formula (II) is represented by formula (Iln) when the N-terminal end of the peptide Z and L₁ are covalently linked by an amide or a bioisostere moiety thereof; and wherein said amide may be N-substituted or non- substituted, for example an amide -N(R₁)C(O)-, being R₁ is as defined herein.

[0297] In another preferred embodiment of the AAV vector particle comprises a group Z, a spacer L₁ and spacers L₃, as defined herein, Z is a peptide, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, a first group L₃ is a group C_1-6 alkyleneamine or a group C_1-6 acyl, and a second spacer L₃ is an arginine moiety; wherein the group C_1-6 alkyleneamine or the group C_1-6 acyl and the spacer L₁ are covalently linked by an ether bond, L₁ and the squaramide linker are both covalently linked to the arginine moiety, and the moiety of formula (II) is represented by formula (IIp) when the N- terminal end of the peptide Z and a group C_1-6 acyl are covalently linked by an amide moiety or a bioisostere moiety thereof, being the nitrogen atom of the amide the N- terminal end of the peptide Z; and the moiety of formula (II) is represented by formula (Iq) when the C-terminal end of the peptide Z and a group C_1-6 alkyleneamine are covalently linked by an amide moiety or a bioisostere moiety thereof, being the carbonyl group of the amide the C-terminal end of the peptide Z; and wherein said amide may be N-substituted or non-substituted, for example an amide -N(R₁)C(O)-, being R₁ is as defined herein.

[0298] In another preferred embodiment of the AAV vector particle comprises a group Z, two spacers L₁ and two spacers L₃, as defined herein, Z is a peptide, a first L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, a second L₁ is a β-alanine polymer, comprising 1 to 40 β-alanine monomers; one spacer L₃ is a group C_1-6 alkyleneamine or a group C_1-6 acyl, and a second spacer L₃ is an arginine moiety; wherein the group C_1-6 alkyleneamine or the group C_1-6 acyl and the spacer L₁ are covalently linked by an ether bond, the PEG and the squaramide linker are both covalently linked to the arginine moiety, the β-alanine polymer is linked to the group C_1-6 alkyleneamine or the group C_1-6 acyl forming an amide moiety, and the moiety of formula (II) is represented by formula (Ilr) when the N-terminal end of the peptide Z and the β- alanine polymer are covalently linked by an amide moiety - or a bioisostere moiety thereof, being the nitrogen atom of the amide the N-terminal end of the peptide Z; and the moiety of formula (II) is represented by formula (Ils) when the C-terminal end of the peptide Z and the β-alanine polymer are covalently linked by an amide moiety or a bioisostere moiety thereof, being the carbonyl group of the amide the C-terminal end of the peptide Z; and wherein said amide may be N-substituted or non substituted, for example an amide -N(R₁)C(O)-, being R₁ is as defined herein..

[0299] In another preferred embodiment of the AAV vector particle comprises a group Z, two spacers L₁, one L₂ and one to three spacers L₃, as defined herein; wherein Z is a peptide, a first spacer L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers, a second spacer L₁ is a β-alanine polymer, comprising 1 to 40 β-alanine monomers; L₂ is an arylene or a heteroarylene group Ar; and each L₃ is a group C_1-6 alkylene or a group C_1-6 alkyleneamine or a group C_1-6 acyl; wherein the two spacers L₁ are both linked to a first spacer L₃ being a group C_1-6 alkyleneamine or a group C_1-6 acyl; the PEG and L₂ or the PEG and a second spacer L₃ being a group C_1-6 alkylene are covalently linked by an amide moiety -N(R₁)C(O)-; and the moiety of formula (II) is represented by formula (lit) when the N-terminal end of the peptide Z and the β-alanine polymer are covalently linked by an amide moiety or a bioisostere moiety thereof, being the nitrogen atom of the amide the N-terminal end of the peptide Z; and the moiety of formula (II) is represented by formula (IIv) when the C-terminal end of the peptide Z and the β-alanine polymer are covalently linked by an amide moiety or a bioisostere moiety thereof, being the carbonyl group of the amide the C-terminal end of the peptide Z; and wherein said amide may be N-substituted or non substituted, for example an amide - N(R₁)C(O)-, being R₁ is as defined herein.

[0300] In another preferred embodiment of the AAV vector particle comprises a group Z, one spacer L₂ and 0, 1 or 2 spacers L₃, as defined herein; wherein Z is a peptide, L₂ is an arylene or a heteroarylene group; L₃ is a group C_1-6 alkylene or a group C_1-6 alkyleneamine or a group C_1-6 acyl; and the moiety of formula (II) is represented by formula (IIw) when the N-terminal end of the peptide Z and L₂ or Z and L₃ are covalently linked by an amide moiety or a bioisostere moiety, being the nitrogen atom of the amide the N-terminal end of the peptide Z; and wherein said amide may be N-substituted or non- substituted, for example an amide -N(R₁)C(O)-, being R₁ is as defined herein.

[0301] In another preferred embodiment of the AAV vector particle has a moiety of formula (IIal2) or (IIal3) or (IIal4) or (IIal5) comprising three groups Z, being Z a saccharide or a peptide, and four spacers L₁, one spacer L₂ and three to five spacers L₃; wherein three spacers L₁ are polyethylene glycol (PEG) groups, comprising each independently 1 to 40 ethylene glycol monomers; one spacer L₁ is a polyether of a branched C_3-12 polyol; L₂ is an arylene or a heteroarylene group Ar; and each L₃ is a group C_1-6 alkylene; wherein the moiety of formula (II) is represented by formula (IIx), as defined herein.

[0302] It will be appreciated that, in the case wherein L (or L₁) comprises a PEG group directly linked to a saccharide Z, the terminal oxygen atom of the PEG group, when present on the side of Z, can be part of Z. This is the case for example when Z is a saccharide wherein the anomeric carbon bears the PEG linker. In an analogous manner, in the case Z is a peptide, the nitrogen atom of the amide Z-NR₃C(O)- belongs to the N- terminal end of the peptide, and the carbonyl group of the amide Z-C(O)NR₃- belongs to the C-terminal end of the peptide.

[0303] In an illustrative embodiment, the AAV vector particle of the invention is selected from:

[0304] For the purposes of the present disclosure, when the moiety of formula (II) comprises a saccharide, the stereochemistry of the anomeric carbon of said saccharides, when present, has not been represented in the drawings and figures of the AAV vector particles of the invention, or in the drawings and figures of the moieties of formula (II) or in the drawings and figures of the compounds of formula (III) disclosed in the present invention.

[0305] In an illustrative embodiment, the AAV vector of the invention comprises a moiety of formula (II) selected from:

[0306] In some embodiments, the AAV vector particle of the invention is selected from the group consisting of (l)-AAV, (2)-AAV and (3)-AAV. The same nomenclature is used for the other AAV serotypes and modifying groups.

[0307] In some embodiments, the AAV vector of the invention is (3)-AAV2 comprising at least one transgene comprising the cDNA from a GBA gene, preferably a human GBA gene, optionally the transgene is under control of a at least one regulatory element, preferably of a promoter as defined above, more preferably of a CAG promoter.

Compounds of formula (III) comprising squarate ester groups

[0308] In some embodiments, the invention further relates to a compound of formula (III), comprising a squarate ester, used to obtain the AAV vectors of the invention.

[0309] In some embodiments, the present invention provides a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein R₂ and R^L-NH- are defined and described in classes and subclasses in the present invention.

[0310] For example, in some embodiments, the functional moiety R^L-NH- comprises a group Z, one or more spacers L, and the compound of formula (III) is represented by formula (Illa):

wherein R₂, Z and L are as defined and described in classes and subclasses in the present invention. [0311] In some embodiments, the functional moiety R^L-NH- comprises a group Z, and one or more than one spacer L selected from the group consisting of L₁, L₂ and L₃ and, the compound of formula (Illa) is selected from the group consisting of formula (Illa₁), (IIIa₂), (IIIa₃), (IIIa₄), (Illa₅), (IIIa₆), (IIIa₇), (IIIa₈), (IIIa₉), (Illa₁₀), (IlIa₁₁), (IlIa₁₂), (Illa₁₃), (IIIa₁₄), (IlIa15), (IIIa₁₆), (IIIa₁₇), (IlIa₁₈), (IlIa₁₉) and (IIIa₂₀):

wherein R₂, Z, L₁, L₂ and L₃ are as defined and described in classes and subclasses in the present invention. In some aspects, L₁ is a polyethylene glycol (PEG), comprising 1 to 40 ethylene glycol monomers; L₂ comprises one or more arylene or a heteroarylene groups; L₃ is a C_1-6 alkylene group, L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and L₁ and L₂ or L₁ and L₃ are covalently linked by an amide moiety or a bioisostere moiety thereof.

[0312] In some particular embodiments, the compound of formula (III), as disclosed herein, is selected from the group consisting of formula (Illb), (IIIc), (IIId), (Ille), (II If), (Illg), (Illh), (Illj), (IIIk), (Illm), (IlIn), (IIIp), (Illq), (Illr), (Ills), (lIlt), (IIIv), (IIIw) and

(IIIx):

or a pharmaceutically acceptable salt thereof, wherein n, n’, m₁, m₂, m₃, m₄, Z, Ar, R_a, R_b, R_c, R₁ and R₂ are as defined and described in classes and subclasses disclosed in the present invention.

[0313] In some embodiments, the compound of formula (III) comprises a labelling agent (fluoresceine), or a saccharide (mannose, glucose, mannose phosphate) or a peptide (THR, SEQ ID No.: 1) and is selected from a compound of Table 2, or a salt thereof.

Method for obtaining the squaramide modified AAV vector particles comprising a moiety of formula (II)

[0314] In some embodiments, this invention further relates to methods of manufacturing of an AAV vector according to the invention. In some embodiments, a method of the present invention comprises incubating the AAV vector with a compound of formula (III), as defined and described in classes and subclasses of the present description, in conditions suitable for reacting a squarate moiety of the compound of formula (III) as defined and described in classes and subclasses disclosed in the present invention, with at least one amino group of an amino acid residue of the capsid of the AAV vector so as to form a squaramide linker of formula (I), as defined in the present invention.

[0315] In some embodiments, suitable conditions to obtain at least one moiety of formula (II) include suitable conditions to promote the formation of a covalent bond between an amino group of an amino acid residue of the capsid of the AAV vector and said squarate moiety without impairing the structural integrity of said AAV.

[0316] In some embodiments, an AAV vector is incubated with a compound of formula (III), and formulae (Illa₁), (IIIa₂), (IIIa₃), (IIIa₄), (Illa₅), (IIIa₆), (IIIa₇), (IIIa₈), (IIIa₉), (Illa₁₀), (IIla₁₁), (IlIa₁₂), (IIIa₁₃), (IIIa₁₄), (IIIa₁₅), (IIIa₁₆), (IIIa₁₇), (IlIa₁₈), (IIIa₁₉), (IIIa₂₀), (Illb), (IIIc), (IIId), (Ille), (IIIf), (Illg), (Illh), (Illj), (IIIk), (Illm), (IIIn), (IIIp), (Illq), (Illr), (Ills), (lIlt), (IIIv), (IIIw) or (IIIx) as described herein and in the section Examples.

[0317] In some embodiments, an incubation can be performed in an aqueous buffer having a pH ranging from 5.5 to 10, preferably from 7 to 10, e.g. from 9 to 10, such as 9.3. In some preferred embodiments, the pH is 9.3.

[0318] In some embodiments, an incubation buffer can be selected from TRIS buffer, borate buffer, Hepes buffer, acetate buffer, phosphate buffer e.g. PBS, or Dulbecco's phosphate-buffered saline (dPBS). In some preferred embodiments, the buffer is TRIS buffer. [0319] In some embodiments, an incubation can last from several minutes to several hours, for instance from 5 min to 6 hours, e.g. from 3 to 5 hours. In some preferential embodiments, the incubation is about 4 hours. In some embodiments, an incubation can last from several hours to several days, for instance from 6 to 72 hours, e.g. from 12 to 48 hours or from 16 to 24 hours. In some embodiments, the incubation is ended when a sufficient yield of coupling is achieved.

[0320] In some embodiments, the temperature of incubation is typically from 4 °C to 50 °C. In some preferential embodiments, 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 incubating solution can be stirred.

[0321] In some embodiments, the molar ratio of the compound of formula (III) to the AAV vector may be from 1.10⁵ to 1.10⁷, e.g. 1.10⁶ to 5.10⁶. In some preferential embodiments, there is a 3.10⁶ equivalents molar excess of the compound of formula (III).

[0322] In some embodiments, a method of the invention may comprise one or several additional steps prior to, or after the step of incubation as described above.

[0323] For instance, in some embodiments, a method of the invention may comprise a preliminary step of providing or producing an AAV vector to be modified.

[0324] In some embodiments, a 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 compound comprising a squarate group (e.g., a compound of formula (III)) at the end of the incubation step, e.g. by dialysis or tangential flow filtration, and/or a step of collecting the chemically modified AAV particles, and/or a step of purifying the AAV vector, and/or a step of recovering the AAV vector, and/or a step of formulating and/or packaging the AAV vector.

[0325] In some embodiments, a method of the invention may also comprise a preliminary step of providing or preparing the compound of formula (III) prior to the step of incubating the AAV vector with a compound of formula (III), as defined and described in classes and subclasses of the present description, in conditions suitable for reacting a squarate moiety of the compound of formula (III) with at least one amino group of an amino acid residue of the capsid of the AAV vector so as to form a squaramide linker of formula (I).

[0326] In particular, the compounds of formula (III), including all compounds of formula (III) disclosed in the present invention, selected from (Illa), (Illa₁), ( I IIa₂), (Illa₃), (IIIa4), (Illa₅), (IIIa₆), (IIIa₇), (IIIa₈), (IIIa₉), (Illa₁₀), (Illa₁₁), (Illa₁₂), (IIIa₁₃), (IIIa₁₄), (Illa₁₅), (Illa₁₆), (Illa₁₇), (IIIa₁₈), (IIIa₁₉), (IIIa₂₀), (IIIb), (IIIc), (Illd), (Ille), (Illf), (Illg), (Illh), (Illj), (Illk), (Illm), (IIIn), (IIIp), (Illq), (Illr), (Ills), (lIlt), (IIIv), (Illw)and (IIIx), can be prepared by several methods. The starting products are commercial products or products prepared according to known synthesis from commercial compounds or known to one skilled in the art.

[0327] In some examples the synthesis of a compound of formula (III) comprises reacting a functional moiety comprising an amino group R^L-NH₂, wherein R^L is as defined and described in classes and subclasses of the present description, with a diester of squaric acid of formula (V):

in suitable conditions to obtain a compound of formula (III); wherein R₂ is defined according to the classes and subclasses disclosed in the present invention.

[0328] In some examples, the synthesis of a compound of formula (III) comprises: providing a precursor compound of formula (VI):

wherein Z, L₁ are defined according to the classes and subclasses disclosed in the present invention; reacting said compound of formula (VI) with a diester of squaric acid of formula (V), as defined herein, in suitable conditions to obtain a compound of formula (Illa₁):

wherein R₂ is defined according to the classes and subclasses disclosed in the present invention.

[0329] The compounds of formula (VI) may be prepared, for example, by reduction of the azide function, by methods and conditions for azide reduction well-known in the art, starting from the corresponding azide precursor disclosed in document WO2022096681.

[0330] In other examples, the synthesis of a compound of formula (III) comprises: providing a precursor of formula (VIla) or a precursor of formula (VIlla):

wherein Z, L₁, L₃ and R₂ are defined according to the classes and subclasses disclosed in the present invention; reacting said compound of formula (VIla) or said compound of formula (VIlla) with a compound of formula (IX),

in suitable conditions to obtain, respectively, a compound of formula (Illa₄) or a compound of formula (Illa₅), wherein L₁ and L₃ are covalently linked by an amide moiety -N(R₁)C(O)-, and wherein R₁ is defined according to the classes and subclasses disclosed in the present invention.

[0331] Suitable conditions to react a compound of formula (VIla) or a compound of formula (VIlla) with a compound of formula (IX), as defined above herein are those well known in the art for peptidic coupling.

[0332] Intermediates compounds of formula (VIla) and (VIlla) may be prepared from respectively the corresponding amino acid precursor of formulas (VIla’) and (VIlla’):

with a compound of formula (V) in presence of a base and an alcoholic solvent, wherein L₂ and L₃ and the compound of formula (V) are defined according to the classes and subclasses disclosed in the present invention.

[0333] In other examples, the synthesis of a compound of formula (III) comprises: providing a precursor of formula (Vllb) or a precursor of formula (Vlllb):

wherein Z, L₂, L₃ and R₂ are defined according to the classes and subclasses disclosed in the present invention; reacting said compound of formula (Vllb) or said compound of formula (Vlllb) with a compound of formula (IX),

in suitable conditions to obtain, respectively, a compound of formula (Illa₂) or a compound of formula (Illa₃), wherein L₁ and L₂ are covalently linked by an amide moiety -N(R₁)C(O)-, and wherein R₁ is defined according to the classes and subclasses disclosed in the present invention.

[0334] Suitable conditions to react a compound of formula (Vllb) or a compound of formula (Vlllb) with a compound of formula (IX), as defined above herein are those well known in the art for peptidic coupling.

[0335] Intermediates compounds of formula (Vllb) and (Vlllb) may be prepared from respectively the corresponding amino acid precursor of formulas (Vllb’) and (Vlllb’):

[0336] In some embodiments of the compounds of formula (III) the group Z is a peptide which may be prepared by any of the methods known in the art (such as peptide synthesis on solid support, according to standard procedures), and coupled with an amine group or a carboxylic group of a spacer L, as described above herein, in suitable conditions (for example, on solid support, according to standard procedures) to obtain an amide moiety. Subsequently, after the release from the solid support of the group Z-L-NH₂, or the peptide Z-NH₂ (wherein the amine group is the N-terminal end of the peptide Z), the process of preparing the compounds of formula (III) comprises reacting, suitable conditions, for example in aqueous conditions, a compound of formula (V) with the primary amine group of Z-NH₂ or Z-L-NH₂ to obtain the compound of formula (III).

Uses of provided AAV vectors

[0337] In some embodiments, AAV vectors of the present invention can be used as a research tool. In some embodiments, AAV vectors of the present invention can be used as a medicament, for instance in gene therapy as vectors for the delivery of therapeutic nucleic acids such as DNA or RNA. In some embodiments, AAV vectors of the present invention can be used in a diagnostic means, e.g. as an imaging agent. In some embodiments, AAV vectors of the present invention can be used as a combination of both a therapeutic and diagnostic tool, e.g., theragnostic use.

Modifications of biological functionalities and/or properties of AA V vectors

[0338] In some embodiments, chemical modifications of the capsid of an AAV vector may modify one, or several, of its biological functionalities and/or properties. In some embodiments, biological functionalities and/or properties can depend on the nature of functional moiety R^L which is introduced to modify the AAV vector in the present invention. In some embodiments, one or more biological properties of a modified AAV vector can be altered compared to the unmodified AAV vector, such as: a modified selectivity of the AAV vector towards a specific organ, tissue, and/or cell type (e.g. an increased selectivity or a shifted selectivity from one tissue/organ/cell to another); and/or a modified immunoreactivity of the AAV vector, e.g. a decreased immunogenicity of the AAV vector and/or a decreased affinity for neutralizing antibodies, and/or said AAV vector triggers an altered humoral response when administered in vivo, e.g. does not generate AAV-directed neutralizing antibodies; and/or an increased infectivity efficiency of the AAV vector; and/or an increased transduction efficacy of the AAV vector towards a specific cell, tissue, and/or organ; and/or a reduced cellular toxicity when transducing cells in culture; and/or an induced cellular targeted mortality of cancer cells; and/or enabling the visualization/monitoring of the AAV vector upon in vivo administration or upon modification of cells in vitro; and/or enabling theragnostic applications; e.g. combining a therapeutic agent and a diagnostic agent.

[0339] In some embodiments, when the AAV vector is used as a medicament, e.g. as a gene vector for gene therapy, such modified properties may result in an improvement in the therapeutic index of the AAV vector. In some embodiments, an improvement in the therapeutic index of the AAV vector can result from decreases in the relative dose of AAV to administer to the subject in order to achieve the sought therapeutic effect, such a reduction in dosage can decrease the relative toxicity of the AAV therapeutic regime.

[0340] In some embodiments, an AAV vector of the present invention shows a preferential tropism for an organ or cell selected from liver, heart, brain, joints, retina, and/or skeletal muscle. In some embodiments, an AAV vector 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), and/or induced pluripotent stem cells (iPS). Uses and method for transducing cells

[0341] In some embodiments, the present invention relates to an AAV vector according to the present invention, for use in transducing a cell of a subject.

[0342] By “transducing a cell” it is herein referred to delivering a nucleic acid into a cell. The transduced nucleic acid of interest may be of any type and is selected depending on the sought effect. In some embodiments, when the AAV vector according to the present invention is used for transducing a cell, it comprises a transgene.

[0343] In some embodiments, for example, an AAV vector can comprise an exogenous gene expression cassette. In some embodiments, said cassette may comprise a promoter, a gene of interest, and a terminator. In some embodiments, as an additional or alternative example, an AAV vector of the invention may comprise a DNA template for homologous recombination in cells. In some embodiments, such an AAV vector can be used in combination with gene editing tools, for promoting homologous recombination in targeted cells. In some embodiments, the gene editing tools can be of any type, and encompass, without being limited to, CRISPR and its associated systems (including without limitation a Cas protein such as a Cas9 protein, or fusion protein thereof, a crRNA and tracrRNA, the latter two being either separate or linked together in a single gRNA), TALEN, Zinc Finger Nuclease, meganuclease, as well as RNA and DNA encoding said gene editing proteins and their associated systems.

[0344] In some embodiments, the present invention also relates to use of an AAV vector according to the present invention for transducing a cell of a subject.

[0345] In some embodiments, the present invention also relates to a method for transducing a cell of a subject, comprising administering an AAV vector according to the present invention to said subject.

[0346] In some embodiments, the present invention also relates to a method of delivering a transgene to a cell, the method comprising contacting a cell with an AAV vector particle of formula (II), as defined and described in classes and subclasses disclosed in the present, and a nucleic acid to be expressed in the contacted cell, in particular the transgene to be expressed in the contacted cell.

[0347] In some embodiments, the present invention also relates to a method for delivering a transgene into a cell of a subject, comprising administering an AAV vector according to the present invention comprising said transgene to said subject.

[0348] In some embodiments, the present invention further relates to an in vitro or ex vivo method for transducing a cell, comprising contacting said cell with an AAV vector according to the invention. In some embodiments, the cell may be from a subject (e.g., a patient). In some embodiments, after transduction, the cell may be transplanted to a subject in need thereof (e.g., the patient, and/or another subject).

[0349] In some embodiments, an AAV vector can be administered to a cell in vivo, ex vivo, or in vitro. In some embodiments, the cell may be derived from a mammal (e.g., humans, non-human primates, cows, mice, sheep, goats, pigs, rats, etc.) In some embodiments, the cell may be derived from a human. In some embodiments, the cell may be, but is not limited to, hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigmented cells, photoreceptors, chondrocytes, hematopoietic stem cells (HSC), or induced pluripotent stem cells (iPS).

[0350] In some embodiments, AAV vectors according to the present invention specifically transduce any or several of the following cells: neurons (such as, e.g., pyramidal neurons, Purkinje neurons, spindle neurons, medium spiny neurons, and/or interneurons [e.g., Golgi cells, Lugaro cells, basket cells, stellate cells, candelabrum cells, unipolar brush cells, granule cells, Renshaw cells, la inhibitory neurons, lb inhibitory neurons, parvalbumin-expressing interneurons, CCK-expressing interneurons, VIP-expressing interneurons, SOM-expressing interneurons, cholinergic interneurons, tyrosine hydroxylase-expressing interneurons, calretinin-expressing interneurons, or nitric oxide synthase-expressing interneurons]), oligodendrocytes, astrocytes, microglial cells, ependymal cells, radial glia cells and/or pituicytes. [0351] In some embodiments, AAV vectors according to the present invention do not specifically transduce one or more (or all) of the following cells: oligodendrocytes, astrocytes, microglial cells, ependymal cells, radial glia cells and/or pituicytes.

[0352] In some embodiments, an AAV vector according to the present invention can target a large variety of cells, tissues, and/or organs for treatment and/or prophylactic intervention. For example, in some embodiments, AAV vectors targets encompass, but are not limited to, hepatocytes; cells of the retina; i.e. photoreceptors, retinal pigmented epithelium (RPE), Muller cells; cells of the inner ear (e.g., inner and/or outer hair cells, Hensen’s cells, Deiter’s cells, pillar cells, inner phalangeal cells, border cells, etc.); muscle cells, i.e. myoblasts, satellite cells; cells of the central nervous system (CNS), i.e. neurons, glial cells; cells of the heart; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells; blood cells such as lymphocytes, monocytes, basophils, eosinophils, neutrophils, mast cells; 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, eye, retina, ear, liver, skeletal muscle, cardiac muscle, smooth muscle, brain, spine, bone, connective tissue, heart, kidney, lung, lymph node, mammary gland, myelin, prostate, testes, thymus, thyroid, trachea, and the like. In some embodiments, preferred cell types are hepatocytes, retinal cells, muscle cells, cells of the CNS, cells of the PNS and/or hematopoietic cells. In some embodiments, preferred tissues and/or organs are liver, muscle, heart, eye, and/or brain.

[0353] In some embodiments, an AAV described herein is considered to target cells of the CNS if it targets one or more cell types that include retinal cells; in some embodiments, targeting retinal cells is not considered to represent CNS targeting.

Use in gene therapy

[0354] In some embodiments, an AAV vector described herein may be particularly useful in gene therapy, e.g., to deliver a therapeutic nucleic acid of interest to a subject.

[0355] Accordingly, in some embodiments, the present invention also relates to an AAV vector according to the present invention, for use in gene therapy. [0356] In some embodiments, the present invention also relates to a method of gene therapy in a subject in need thereof, comprising administering an AAV vector according to the present invention to said subject.

[0357] In some embodiments, an AAV of the invention can be delivered by any appropriate route to the subject. In some embodiments, 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. In some embodiments, the route of administration is selected depending on the targeted tissue and/or organ, namely, depending on the tissue and/or organ in which transduction is sought.

[0358] In some embodiments, AAV vectors according to the present invention are to be administered by intraspinal and/or intracerebral administration.

[0359] In some embodiments, AAV vectors according to the present invention are to be administrated intraspinally. In some embodiments, intraspinal administration comprises or consists of intrathecal and epidural administration. In some embodiments, intraspinal administration comprises or consists of intrathecal administration.

[0360] In some embodiments, AAV vectors according to the present invention are to be administrated intracerebrally.

[0361] In some embodiments, AAV vectors according to the present invention are to be administered intracerebrally, wherein the intracerebral administration is at a site selected from the group comprising or consisting of: striatum (such as, e.g., putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, external globus pallidus and/or internal globus pallidus), thalamus, hypothalamus, epithalamus, subthalamus, parenchyma, cerebrum, medulla, deep cerebellar nuclei (such as, e.g., substantia nigra, dentate, emboliform, globose and/or fastigii nucleus), cerebrospinal fluid (CSF), meninges, dura mater, arachnoid mater, pia mater, subarachnoid cisterns (such as, e.g., cistema magna, pontine cistern, interpeduncular cistern, chiasmatic cistern, cistern of lateral cerebral fossa, superior cistern and/or cistern of lamina terminalis), subarachnoid space, cortex, septum, pons, and/or cerebellum.

[0362] In some embodiments, AAV vectors according to the present invention are to be administrated intrastriatally (i.e., in the striatum, such as, e.g., in the putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, external globus pallidus and/or internal globus pallidus), intrathalamically (i.e., in the thalamus), intraci stemally (i.e., in the subarachnoid cisterns, such as, e.g., in the cistema magna, pontine cistern, interpeduncular cistern, chiasmatic cistern, cistern of lateral cerebral fossa, superior cistern and/or cistern of lamina terminalis; preferably in the cisterna magna).

[0363] In some embodiments, conditions to be treated by administration of an AAV vector of the invention can be of any type. For example, in some embodiments, a condition to be treated may be selected from communicable diseases, and inherited as well as acquired genetic disorders. In some embodiments, genetic disorders of interest encompass but are not limited to genetic muscle disorders such as Duchenne Muscular Dystrophy, leukodystrophy, spinal muscular atrophy (SMA), hemophilia, sickle disease, and inherited retinal dystrophy. In some embodiments, AAV vectors of the present invention can also be used for treating disorders such as, but not limited to, cancers, arthritis, arthrosis, congenital and acquired cardiac diseases, Parkinson disease, Alzheimer’s disease, and infectious diseases (e.g., such as hepatitis C).

[0364] In some preferred embodiments, AAV vectors described herein can be particularly useful for preventing and/or treating an ophthalmic disease. Accordingly, in some embodiments, the present invention also relates to AAV vectors according to the present invention, for use in the prevention and/or treatment of an ophthalmic disease. In some embodiments, the present invention further relates to the use of AAV vectors according to the present invention, for the manufacture of a medicament for prevention and/or treatment of an ophthalmic disease. In some embodiments, the present invention also relates to a method of preventing and/or treating an ophthalmic disease in a subject in need thereof, comprising administering AAV vectors according to the present invention to said subject. [0365] In some preferred embodiments, an AAV vector described herein may also be particularly useful for preventing and/or treating a CNS disease. “Central nervous system” or “CNS” refers to both the brain and the spinal cord and contrasts with the “peripheral nervous system” or “PNS” which excludes the brain and the spinal cord. In some embodiments, the eye and in particular the retina is not considered to be part of the CNS. In some embodiments, the eye and in particular the retina can be considered to be part of the PNS. Accordingly, in some embodiments, the present invention also relates to modified AAV vectors according to the present invention, for use in the prevention or treatment of a CNS disease. In some embodiments, the present invention further relates to use of modified AAV vectors according to the present invention, for the manufacture of a medicament for the prevention or treatment of a CNS disease. In some embodiments, the present invention also relates to a method of preventing and/or treating a CNS disease in a subject in need thereof, comprising administering modified AAV vectors according to the present invention to said subject.

[0366] In some embodiments, a brain tissue may be or include the striatum, the thalamus, the substantia nigra, the parietal cortices, the hippocampus and/or the globus pallidus. In some embodiments, the CNS site may be in the striatum. In some embodiments, the CNS site may be in the thalamus. In some embodiments, the CNS site may be in the cistema magna.

[0367] As used herein, the terms “prevent”, “preventing” and “prevention” refer to prophylactic and preventative measures, wherein the object is to reduce the chances that a subject will develop a given disease over a given period of time. Such a reduction may be reflected, e.g., in a delayed onset of at least one symptom of the disease in the subject.

[0368] As used herein, the terms “treating” or “treatment” or “alleviation” refer to therapeutic treatment, excluding prophylactic or preventative measures; wherein the object is to slow down (lessen) a given disease. Those in need of treatment include those already with the disease as well those suspected to have the disease. A subject is successfully “treated” for a given disease if, after receiving a therapeutic amount of an AAV vector according to the present invention, said subject shows observable and/or measurable reduction in or absence of one or more of the following: one or more of the symptoms associated with the disease; reduced morbidity and mortality; and/or improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the targeted disease are readily measurable by routine procedures familiar to a physician.

[0369] As used herein, the term “subject” refers to a mammal, preferably a human. In some embodiments, a subject may be a “patient”, i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease. A “mammal” refers here to any mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammal is a primate, more preferably a human.

Composition

[0370] In some embodiments, the present invention further relates to a composition comprising AAV vectors according to the invention. In some embodiments, AAV vectors in the composition according to the present invention comprises at least one transgene.

[0371] In some embodiments, the composition is a pharmaceutical composition comprising an AAV vector according to the invention and at least one pharmaceutically acceptable vehicle.

[0372] The term “pharmaceutically acceptable”, when referring to vehicles, excipients, carriers, and/or preservatives, is meant to define molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a subject, preferably a human. For human administration, pharmaceutical compositions should meet sterility, pyrogenicity, and general safety and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.

[0373] In some embodiments, pharmaceutically acceptable vehicles, excipients, carriers and preservatives that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, proteins (such as, e.g., serum albumin, gelatin, immunoglobulins and the like), buffer substances (such as, e.g., phosphates, citrates or other organic acids, and the like), amino acids (such as, e.g., glycine, glutamine, asparagine, arginine, lysine and the like), antioxidants (such as, e.g., ascorbic acid and the like), chelating agents (such as, e.g., EDTA), sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate and the like), hydrophilic polymers (such as, e.g., polyvinylpyrrolidone, polyethylene- polyoxypropylene block polymers and the like), cellulose-based substances (such as, e.g., sodium carboxymethylcellulose), polyacrylates, waxes, nonionic surfactants (such as, e.g., Tween, pluronics, polyethylene glycol and the like), wool fat, and suitable combinations thereof.

[0374] In some embodiments, a pharmaceutical composition according to the present invention comprises vehicles which are pharmaceutically acceptable for a formulation intended for injection into a subject. In some embodiments, these may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0375] In some embodiments, a pharmaceutical composition according to the present invention comprise one or more agents that promote the entry of an AAV vector described herein into a mammalian cell, such as, e.g., natural and/or synthetic polymers, such as poloxamer, chitosan, cyclodextrins, dendrimers, poly(lactic-co-glycolic acid) polymers, and the like.

[0376] In some embodiments, AAV vectors comprising at least one transgene according to the present invention is comprised as part of a medicament. In some embodiments, the invention thus relates to a medicament comprising AAV vectors comprising at least one transgene according to the present invention. Transgene/disease combinations

[0377] As described above, AAV vectors according to the present invention may comprise at least one transgene, selected in view of the intended use of an AAV vector. Examples of transgenes that can be useful for treatment of ophthalmic diseases or CNS diseases are provided hereafter. In some embodiments, an eye is not considered to be a “CNS” site. In some embodiments, an eye can be considered a “PNS” site.

[0378] In some embodiments, ophthalmic diseases include inherited retinal diseases. In some embodiments, inherited retinal diseases include, but are not limited to, Leber’s congenital amaurosis, retinitis pigmentosa, retinitis punctata albescens, choroideremia, Stargardt disease, retinal dystrophies, choroidal dystrophies, cone dystrophies, cone-rod dystrophies, rod-cone dystrophies, macular dystrophies and macular degeneration, . In some embodiments, ophthalmic diseases include communicable diseases, such as infectious diseases (e.g., viral, bacterial, fungal, etc.). In some embodiments, an ophthalmic disease includes an injury. In some embodiments, an ophthalmic disease includes an auto-immune disease. In some embodiments, an ophthalmic disease includes a cancer.

[0379] In some embodiments, a CNS disease is a CNS infectious disease, a CNS degenerative disease, a CNS auto-immune disease, a CNS tumor disease, a cerebrovascular disease, a CNS injury, or a CNS structural defect.

[0380] In some embodiments, a CNS disease includes, but is not limited to, Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Friedreich’s ataxia, Canavan’s Disease, muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), Gaucher disease, adrenoleukodystrophy, Angelman syndrome, or epilepsy.

[0381] In some particular embodiments, a CNS disease is Parkinson’s disease or Gaucher disease. In some particular embodiments, a CNS disease is Parkinson’s disease. In some particular embodiments, a CNS disease is Gaucher disease.

[0382] In some particular embodiments, the gene involved in CNS diseases is the GBA gene, preferably the human GBA gene. [0383] In some embodiments, AAV vectors of the present invention are capable of effectively transducing certain areas of the brain, including the striatum, the thalamus, the substantia nigra, the parietal cortices, the hippocampus and/or the globus pallidus. Thus, in some embodiments, AAV vectors of the present invention are of great interest for targeting the striatum, the thalamus, the substantia nigra, the parietal cortices, the hippocampus and the globus pallidus, and/or for treating diseases affecting the striatum, the thalamus, the substantia nigra, the parietal cortices, the hippocampus and the globus pallidus.

[0384] In some embodiments, AAV vectors of the present invention are particularly suited for treating diseases of the striatum, the substantia nigra, the thalamus, the substantia nigra, the globus pallidus, the parietal cortices, and/or the hippocampus, such diseases include, but are not limited to, Huntington’s disease, Parkinson’s disease, multiple sclerosis atrophy, Lewis Body Dementia (LBD), progressive supranuclear palsy and Angelman syndrome.

[0385] In some embodiments, AAV vectors of the present invention are capable of effectively transducing neurons. Thus, in some embodiments, a CNS disease is a neurological disease or a disease affecting neurons.

[0386] In some embodiments, AAV vectors of the present invention are capable of effectively transducing neurons involved in the control of motor function. Thus, in some embodiments, a CNS disease is a disease affecting motor function. Non-limiting examples of diseases affecting motor function include, but are not limited to, Parkinson’s disease and Huntington’s disease.

[0387] In some particular embodiments, a CNS disease is Parkinson’s disease, and an AAV vector of the present invention has at least one transgene comprising the cDNA from the GBA gene, preferably from the human GBA gene.

[0388] In some embodiments, a CNS disease is muscular dystrophy, and an AAV vector of the present invention has at least one transgene, wherein the transgene comprises cDNA of a gene selected from the group comprising or consisting of the DMD gene. [0389] In some embodiments, a CNS disease is Gaucher disease, and an AAV vector of the present invention has at least one transgene, wherein the transgene comprises cDNA of the GBA gene.

[0390] One skilled in the art will recognize that a gene may have multiple transcriptional and/or translational isoforms, and that a transgene comprising a cDNA of a gene described herein encompasses the potential use of transcriptional variants and/or splice variants of a target gene.

Regimen

[0391] In some embodiments, modified AAV vectors according to the present invention are to be administered to a subject in need thereof in a therapeutically effective amount.

[0392] In some embodiments, modified AAV vectors according to the present invention are to be administrated at a dose ranging from about 10⁸ viral genomes (vg) to about 10¹⁵ vg, such as from about 10⁸ vg to about 10¹⁴ vg, from about 10⁸ vg to about 10¹³ vg, from about 10⁸ vg to about 10¹² vg, from about 10⁸ vg to about 10¹¹ vg, from about 10⁸ vg to about 10¹⁰ vg, from about 10⁸ vg to about 10⁹ vg, from about 10⁹ vg to about 10¹⁵ vg, from about 10⁹ vg to about 10¹⁴ vg, from about 10⁹ vg to about 10¹³ vg, from about 10⁹ vg to about 10¹² vg, from about 10⁹ vg to about 10¹¹ vg, from about 10⁹ vg to about 10¹⁰ vg, from about 10¹⁰ vg to about 10¹⁵ vg, from about 10¹⁰ vg to about 10¹⁴ vg, from about 10¹⁰ vg to about 10¹³ vg, from about 10¹⁰ vg to about 10¹² vg, from about 10¹⁰ vg to about 10¹¹ vg, from about 10¹¹ vg to about 10¹⁵ vg, from about 10¹¹ vg to about 10¹⁴ vg, from about 10¹¹ vg to about 10¹³ vg, from about 10¹¹ vg to about 10¹² vg, from about 10¹² vg to about 10¹⁵ vg, from about 10¹² vg to about 10¹⁴ vg, from about 10¹² vg to about 10¹³ vg, or from about 10¹³ vg to about 10¹⁵ vg.

[0393] The term “vector genome”, abbreviated as “vg”, refers to one or more polynucleotides comprising a set of the polynucleotide sequences of a vector, e.g., a viral vector. A vector genome may be encapsidated in a viral particle. Depending on the particular viral vector, a vector genome may comprise single-stranded DNA, double- stranded DNA, or single- stranded RNA, or double-stranded RNA. A vector genome may include endogenous sequences associated with a particular viral vector and/or any heterologous sequences inserted into a particular viral vector through recombinant techniques (e.g., a transgene). In some embodiments, the nucleic acid titer of a viral vector may be measured in terms of vg/mL. Methods suitable for measuring this titer are known in the art, and include, e.g., quantitative PCR. [0394] As used herein, the term “about”, when set in front of a numerical value, means that said numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Such small variations are, e.g., of ± 1%, ± 2%, ± 3%, ± 4%, ± 5%, ± 6%, ± 7%, ± 8%, ± 9%, ± 10% or more. [0395] In some embodiments, modified AAV vectors according to the present invention are to be administrated at a dose of about 1×10⁸ vg ± 0.5×10⁸, about 2×10⁸ vg ± 0.5×10⁸, about 2.75×10⁸ vg ± 0.5×10⁸, about 3×10⁸ vg ± 0.5×10⁸, about 4×10⁸ vg ± 0.5×10⁸, about 5×10⁸ vg ± 0.5×10⁸, about 6×10⁸ vg ± 0.5×10⁸, about 7×10⁸ vg ± 0.5×10⁸, about 8×10⁸ vg ± 0.5×10⁸, about 9×10⁸ vg ± 0.5×10⁸, about 1×10⁹ vg ± 0.5×10⁹, about 2×10⁹ vg ± 0.5×10⁹, about 3×10⁹ vg ± 0.5×10⁹, about 4×10⁹ vg ± 0.5×10⁹, about 5×10⁹ vg ± 0.5×10⁹, about 6×10⁹ vg ± 0.5×10⁹, about 7×10⁹ vg ± 0.5×10⁹, about 8×10⁹ vg ± 0.5×10⁹, about 9×10⁹ vg ± 0.5×10⁹, about 1×10¹⁰ vg ± 0.5×10¹⁰, about 2×10¹⁰ vg ± 0.5×10¹⁰, about 3×10¹⁰ vg ± 0.5×10¹⁰, about 4×10¹⁰ vg ± 0.5×10¹⁰, about 5×10¹⁰ vg ± 0.5×10¹⁰, about 6×10¹⁰ vg ± 0.5×10¹⁰, about 7×10¹⁰ vg ± 0.5×10¹⁰, about 8×10¹⁰ vg ± 0.5×10¹⁰, about 9×10¹⁰ vg ± 0.5×10¹⁰, about 1×10¹¹ vg ± 0.5×10¹¹, about 2×10¹¹ vg ± 0.5×10¹¹, about 3×10¹¹ vg ± 0.5×10¹¹, about 4×10¹¹ vg ± 0.5×10¹¹, about 5×10¹¹ vg ± 0.5×10¹¹, about 6×10¹¹ vg ± 0.5×10¹¹, about 7×10¹¹ vg ± 0.5×10¹¹, about 8×10¹¹ vg ± 0.5×10¹¹, about 9×10¹¹ vg ± 0.5×10¹¹, about 1×10¹² vg ± 0.5×10¹², about 2×10¹² vg ± 0.5×10¹², about 3×10¹² vg ± 0.5×10¹², about 4×10¹² vg ± 0.5×10¹², about 5×10¹² vg ± 0.5×10¹², about 6×10¹² vg ± 0.5×10¹², about 7×10¹² vg ± 0.5×10¹², about 8×10¹² vg ± 0.5×10¹², about 9×10¹² vg ± 0.5×10¹², about 1×10¹³ vg ± 0.5×10¹³, about 2×10¹³ vg ± 0.5×10¹³, about 3×10¹³ vg ± 0.5×10¹³, about 4×10¹³ vg ± 0.5×10¹³, about 5×10¹³ vg ± 0.5×10¹³, about 6×10¹³ vg ± 0.5×10¹³, about 7×10¹³ vg ± 0.5×10¹³, about 8×10¹³ vg ± 0.5×10¹³, about 9×10¹³ vg ± 0.5×10¹³, about 1×10¹⁴ vg ± 0.5×10¹⁴, about 2×10¹⁴ vg ± 0.5×10¹⁴, about 3×10¹⁴ vg ± 0.5×10¹⁴, about 4×10¹⁴ vg ± 0.5×10¹⁴, about 5×10¹⁴ vg ± 0.5×10¹⁴, about 6×10¹⁴ vg ± 0.5×10¹⁴, about 7×10¹⁴ vg ± 0.5×10¹⁴, about 8×10¹⁴ vg ± 0.5×10¹⁴, about 9×10¹⁴ vg ± 0.5×10¹⁴, about 1×10¹⁵ vg ± 0.5×10¹⁵, about 2×10¹⁵ vg ± 0.5×10¹⁵, about 3×10¹⁵ vg ± 0.5×10¹⁵, about 4×10¹⁵ vg ± 0.5×10¹⁵, about 5×10¹⁵ vg ± 0.5×10¹⁵, about 6×10¹⁵ vg ± 0.5×10¹⁵, about 7×10¹⁵ vg ± 0.5×10¹⁵, about 8×10¹⁵ vg ± 0.5×10¹⁵, or about 9×10¹⁵ vg ± 0.5×10¹⁵. [0396] In some embodiments, modified AAV vectors according to the present invention are to be administrated at a dose of about 1×10⁶ vg/kg ± 0.5×10⁶, about 2×10⁶ vg/kg ± 0.5×10⁶, about 3×10⁶ vg/kg ± 0.5×10⁶, about 4×10⁶ vg/kg ± 0.5×10⁶, about 5×10⁶ vg/kg ± 0.5×10⁶, about 6×10⁶ vg/kg ± 0.5×10⁶, about 7×10⁶ vg/kg ± 0.5×10⁶, about 8×10⁶ vg/kg ± 0.5×10⁶, about 9×10⁶ vg/kg ± 0.5×10⁶, about 1×10⁷ vg/kg ± 0.5×10⁷, about 2×10⁷ vg/kg ± 0.5×10⁷, about 3×10⁷ vg/kg ± 0.5×10⁷, about 4×10⁷ vg/kg ± 0.5×10⁷, about 5×10⁷ vg/kg ± 0.5×10⁷, about 6×10⁷ vg/kg ± 0.5×10⁷, about 7×10⁷ vg/kg ± 0.5×10⁷, about 8×10⁷ vg/kg ± 0.5×10⁷, about 9×10⁷ vg/kg ± 0.5×10⁷, about 1×10⁸ vg/kg ± 0.5×10⁸, about 2×10⁸ vg/kg ± 0.5×10⁸, about 3×10⁸ vg/kg ± 0.5×10⁸, about 4×10⁸ vg/kg ± 0.5×10⁸, about 5×10⁸ vg/kg ± 0.5×10⁸, about 6×10⁸ vg/kg ± 0.5×10⁸, about 7×10⁸ vg/kg ± 0.5×10⁸, about 8×10⁸ vg/kg ± 0.5×10⁸, about 9×10⁸ vg/kg ± 0.5×10⁸, about 1×10⁹ vg/kg ± 0.5×10⁹, about 2×10⁹ vg/kg ± 0.5×10⁹, about 3×10⁹ vg/kg ± 0.5×10⁹, about 4×10⁹ vg/kg ± 0.5×10⁹, about 5×10⁹ vg/kg ± 0.5×10⁹, about 6×10⁹ vg/kg ± 0.5×10⁹, about 7×10⁹ vg/kg ± 0.5×10⁹, about 8×10⁹ vg/kg ± 0.5×10⁹, about 9×10⁹ vg/kg ± 0.5×10⁹, about 1×10¹⁰ vg/kg ± 0.5×10¹⁰, about 2×10¹⁰ vg/kg ± 0.5×10¹⁰, about 3×10¹⁰ vg/kg ± 0.5×10¹⁰, about 4×10¹⁰ vg/kg ± 0.5×10¹⁰, about 5×10¹⁰ vg/kg ± 0.5×10¹⁰, about 6×10¹⁰ vg/kg ± 0.5×10¹⁰, about 7×10¹⁰ vg/kg ± 0.5×10¹⁰, about 8×10¹⁰ vg/kg ± 0.5×10¹⁰, about 9×10¹⁰ vg/kg ± 0.5×10¹⁰, about 1×10¹¹ vg/kg ± 0.5×10¹¹, about 2×10¹¹ vg/kg ± 0.5×10¹¹, about 3×10¹¹ vg/kg ± 0.5×10¹¹, about 4×10¹¹ vg/kg ± 0.5×10¹¹, about 5×10¹¹ vg/kg ± 0.5×10¹¹, about 6×10¹¹ vg/kg ± 0.5×10¹¹, about 7×10¹¹ vg/kg ± 0.5×10¹¹, about 8×10¹¹ vg/kg ± 0.5×10¹¹, about 9×10¹¹ vg/kg ± 0.5×10¹¹, about 1×10¹² vg/kg ± 0.5×10¹², about 2×10¹² vg/kg ± 0.5×10¹², about 3×10¹² vg/kg ± 0.5×10¹², about 4×10¹² vg/kg ± 0.5×10¹², about 5×10¹² vg/kg ± 0.5×10¹², about 6×10¹² vg/kg ± 0.5×10¹², about 7×10¹² vg/kg ± 0.5×10¹², about 8×10¹² vg/kg ± 0.5×10¹², about 9×10¹² vg/kg ± 0.5×10¹², about 1×10¹³ vg/kg ± 0.5×10¹³, about 2×10¹³ vg/kg ± 0.5×10¹³, about 3×10¹³ vg/kg ± 0.5×10¹³, about 4×10¹³ vg/kg ± 0.5×10¹³, about 5×10¹³ vg/kg ± 0.5×10¹³, about 6×10¹³ vg/kg ± 0.5×10¹³, about 7×10¹³ vg/kg ± 0.5×10¹³, about 8×10¹³ vg/kg ± 0.5×10¹³, about 9×10¹³ vg/kg ± 0.5×10¹³, or about 1×10¹⁴ vg/kg ± 0.5×10¹⁴. [0397] In some embodiments, the dose of modified AAV vectors required to achieve a desired effect or a therapeutic effect will vary based on several factors including, but not limited to, the specific route of administration, the level of gene, RNA or protein expression required to achieve a therapeutic effect, the specific disease being treated, and the stability of the gene, RNA, and/or protein product. A person skilled in the art can adjust dosing and/or determine a dose range to treat a particular subject and/or a particular disease based on the aforementioned factors, as well as other factors that are well known in the art. [0398] In some embodiments, the volume of modified AAV vectors administered to a subject will also depend, among other things, on the size of the subject, the dose of the AAV vector required to obtain therapeutic effect, the concentration of the AAV vector, and the proposed route of administration. [0399] In some embodiments, the rate of administration of AAV vectors delivered to a subject will also depend, among other things, on the size of the subject, the dose of the AAV vector required to obtain therapeutic effect, the concentration of the AAV vector, the volume of the AAV vector solution, and the proposed route of administration. For example, in some embodiments, for intracerebral administration, a rate of administration ranging from about 0.1 µL/min to about 1 µL/min or from about 1 µL/min to about 5 µL/min or from about 5 µL/min to about 10 µL/min can be used. [0400] In some embodiments, the rate of administration of AAV vectors administered to a subject is of about 0.1 µL/min ± 0.05 µL/min, about 0.2 µL/min ± 0.05 µL/min, about 0.3 µL/min ± 0.05 µL/min, about 0.4 µL/min ± 0.05 µL/min, about 0.5 µL/min ± 0.05 µL/min, about 0.6 µL/min ± 0.05 µL/min, about 0.7 µL/min ± 0.05 µL/min, about 0.8 µL/min ± 0.05 µL/min, about 0.9 µL/min ± 0.05 µL/min, 1 µL/min ± 0.5 µL/min, about 2 µL/min ± 0.5 µL/min, about 3 µL/min ± 0.5 µL/min, about 4 µL/min ± 0.5 µL/min, about 5 µL/min ± 0.5 µL/min, about 6 µL/min ± 0.5 µL/min, about 7 µL/min ± 0.5 µL/min, about 8 µL/min ± 0.5 µL/min, about 9 µL/min ± 0.5 µL/min, or about 10 µL/min ± 0.5 µL/min. [0401] In some embodiments, the total dose or total volume of AAV vectors may be administered continuously (i.e., wherein the total dose or total volume of modified AAV vectors is injected in a single shot or infusion); or discontinuously (i.e., wherein fractions of the total dose or total volume of AAV vectors are injected with intermittent periods between each shot, preferably with short intermittent periods such as periods of time of 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes between each shot or infusion). Kits [0402] The present invention also relates to kits and kits-of-parts, for: - transducing a cell of a subject; and/or - delivering a transgene to a subject; and/or - preventing and/or treating a disease in a subject. [0403] In some embodiments, the kits or kits-of-parts comprise one or more AAV vectors and/or compositions according to the present invention. [0404] In some embodiments, the kits or kits-of-parts further comprise a device for delivery of one or more AAV vectors and/or compositions according to the present invention. [0405] In some embodiments, the kits further include instructions for delivery of one or more AAV vectors and/ or compositions according to the present invention. In some embodiments, kits comprise instructions for preventing and/or treating a targeted disease, using the compositions, and/or methods described herein. [0406] In some embodiments, kits described herein may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and/or package inserts with instructions for performing any methods described herein. BRIEF DESCRIPTION OF THE FIGURES [0407] Figure 1: GFP staining of mouse brain slices at the striatal level after a single bilateral injection of AAV2-GFP or (3)-AAV2-GFP in the striatum. EXAMPLES [0408] The starting products used are commercial products or products prepared according to known synthesis from commercial compounds or known to one skilled in the art. [0409] The structures of the compounds described in the examples were determined according to the usual spectrophotometric techniques (nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC/MS) and purity was determined by high performance liquid chromatography (HPLC)). Synthesis intermediates and compounds of the invention are named according to the IUPAC (The International Union of Pure and Applied Chemistry) nomenclature and described in their neutral form. [0410] The following abbreviations have been used: ACN: acetonitrile BnBr: benzyl bromide Boc: tert-Butyloxycarbonyl CH₂Cl₂ or DCM: dichloromethane DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene DIC: N,N’-diisopropylcarbodiimide DIEA: N,N’-diisopropylethylamine DIPEA: N,N-diisopropylethylamine DMF : dimethylformamide DMSO : dimethylsulfoxide DTT: dithiothreitol EtOAc: ethyl acetate EtOH : ethanol FA : formic acid Fmoc: fluorenylmethoxycarbonyl HATU: Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium HCl: hydrochloric acid H₂O: water m-CPBA: meta-chloroperoxybenzoic acid MeOH: methanol MgSO₄: magnesium sulfate Na₂SO₄: sodium sulfate NaH: sodium hydride NaHCO₃: sodium bicarbonate NaN₃: sodium azide NEt₃ or TEA: tritethylamine NH₃: ammoniac PEG : polyethylene glycol Pd/C : palladium on carbon RT: room temperature tBu : tert-butyl TFA: trifluoroacetic acid TBAF: tetra-n-butylammonium fluoride Bu4NBr: tetrabutylammonium bromide TBDMSCl: tert-butyldimethylsilyl chloride TMSOTf : Trimethylsilyl trifluoromethanesulfonate TIPS: triisopropyl silane LC/MS: liquid chromatography-mass spectrometry HPLC: High Performance Liquid Chromatography UPLC : Ultra Performance Liquid Chromatography NMR: Nuclear Magnetic Resonance HPLC/MS method for purity determination for saccharide Z end-group Instruments : Shimadzu LCMS-2020 Single Quadrupole Liquid Chromatograph Mass Spectrometer, HPLC – Shimadzu Nexera-i LC-2040C 3D with DAD detector Column : Gemini‐NX 3 μm C18, 4.6*50 mm, or equivalent Column temperature : 30°C Eluent : A = H₂O, B = ACN Flow rate : 0.5 mL/min Gradient conditions :

UV/UPLC method for purity determination for peptide Z end-group : Instruments : UPLC Acquity HClass Column : BEHC18 (WATERS), 150*2.1 mm, or equivalent Column temperature : 60°C Eluent : A =H₂O (0.1 TFA), B = ACN (0.1% TFA) Flow rate : 0.5 mL/min Gradient conditions : 0 to 100% of B in 10 min Detection : 214 nm [0411] Preparation 1: Preparation of 4-(((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)methyl) benzoic acid

[0412] To a mixture of 4-(aminomethyl)benzoic acid (2.5 g) and DIPEA (4.3 mL) in EtOH was added 3,4-diethoxycyclobut-3-ene-1,2-dione (3.1 g). The reaction was stirred at room temperature for 3 h, next was diluted with H₂O, and treated with 1M HCl until the pH = 4. The resulting precipitate was filtered off, washed with H₂O, and dried. The crude material was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined, concentrated under reduced pressure to dryness to deliver desired intermediate (0.42 g, 43.7% yield) as a yellow solid. LC/MS (6 min): RT = 2.05 min, found [M+H]⁺ = 275.75.¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.95 (s, 1H), 9.40 – 8.99 (m, 1H), 8.01 – 7.84 (m, 2H), 7.42 (d, J = 8.0 Hz, 2H), 4.65 (ddd, J = 34.1, 26.5, 6.4 Hz, 4H), 1.49 – 1.18 (m, 3H) [0413] Preparation 2: Preparation of 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid

[0414] To a mixture of 4-aminobenzoic acid (5.0 g) and DIPEA (9.5 mL) in EtOH was added 3,4-diethoxycyclobut-3-ene-1,2-dione (6.8 g). The reaction mixture was stirred at room temperature for 3 h, and was next diluted with H₂O, and treated with 1M HCl until pH = 4. The resulting precipitate was filtered, washed with H₂O, and dried. A sample of the crude material was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined, concentrated under reduced pressure to deliver desired intermediate (0.249 g, 36.8% yield) as a yellow solid. LC/MS (6 min): RT = 2.1 min, found [M+H]⁺ = 261.75.¹H NMR (300 MHz, DMSO- d₆) δ (ppm): 12.80 (s, 1H), 11.00 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H), 4.80 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H). [0415] Preparation 3: Preparation of 4-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)ethyl)benzoic acid

[0416] To a mixture of 4-(2-aminoethyl)benzoic acid (500 mg) and DIPEA (0.79 mL) in EtOH was added 3,4-diethoxycyclobut-3-ene-1,2-dione (560 mg). The reaction mixture was stirred at room temperature overnight, next was diluted with H₂O, and treated with 1M HCl until the pH = 4. The resulting precipitate was filtered, washed with H₂O, and dried. The crude product was purified by Flash Column Chromatography using DCM/MeOH as eluent. The fractions containing the pure product were combined, concentrated under reduced pressure to dryness to deliver desired intermediate as a yellow solid (310 mg, 35% yield). LC/MS (6 min): RT = 2.1 min, found [M+H]⁺ 289.75, [M- H]- 287.70.¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 12.72 (s, 1H), 8.92 – 8.61 (m, 1H), 7.94 – 7.80 (m, 2H), 7.39 – 7.25 (m, 2H), 4.68 – 4.52 (m, 2H), 3.74 (d, J = 6.7 Hz, 1H), 3.54 (q, J = 6.6 Hz, 1H), 2.96 – 2.85 (m, 2H), 1.33 (q, J = 7.3 Hz, 3H) [0417] Preparation 4: Preparation of 3-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid

[0418] To a mixture of 3-aminobenzoic acid (1.0 g) and DIPEA (1.9 mL) in EtOH was added 3,4-diethoxycyclobut-3-ene-1,2-dione (1.4 g). The reaction mixture was stirred at room temperature overnight, next was diluted with H₂O, and treated with 1M HCl until the pH = 4. The resulting precipitate was filtered, washed with H₂O, and dried. The crude product was purified by Flash Column Chromatography using DCM/MeOH as eluent. The fractions containing the pure product were combined, concentrated under reduced pressure to dryness to deliver desired intermediate as a yellow solid (1.17 g, 61% yield). LC/MS (6 min): RT = 2.07 min, found [M+H]⁺ 261.70, [M-H]- 259.65. ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 13.05 (s, 1H), 10.89 (s, 1H), 7.98 (t, J = 1.9 Hz, 1H), 7.67 (dt, J = 7.6, 1.3 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 4.78 (q, J = 7.0 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H) [0419] Preparation 5: Preparation of 2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)phenyl)acetic acid

[0420] To a mixture of 4-aminophenylacetic acid (1.0 g) and DIPEA (1.7 mL) in EtOH was added 3,4-diethoxycyclobut-3-ene-1,2-dione (1.2 g). The reaction mixture was stirred at room temperature overnight, next was diluted with H₂O, and treated with 1M HCl until the pH = 4. The resulting precipitate was filtered, washed with H₂O, and dried. The crude product was purified by Flash Column Chromatography using DCM/MeOH as eluent. The fractions containing the pure product were combined, concentrated under reduced pressure to dryness to deliver desired intermediate as a yellow solid (1.3 g, 71% yield). LC/MS (6 min): RT = 2.3 min, found [M+H]⁺ 276.0, [M-H]- 274.15. ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 12.31 (s, 1H), 10.74 (s, 1H), 7.29 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H), 4.76 (q, J = 7.1 Hz, 2H), 3.53 (s, 2H), 1.41 (t, J = 7.1 Hz, 3H) [0421] Preparation 6: Preparation of 5-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)picolinic acid [0422] Step 1: Synthesis of 5-aminopicolinic acid

[0423] To a solution of 5-nitropicolinic acid 500 mg) in MeOH was added Pd/C under argon atmosphere. The inert gas was evacuated and backfilled with hydrogen (in total three times). The reaction mixture was stirred under hydrogen atmosphere (balloon) overnight. The resulting mixture was stirred at room temperature overnight. Then, it was filtered through a pad of Celite and concentrated under reduced pressure to deliver 5‐ aminopicolinic acid as a solid (425 mg, quantitative yield).¹H NMR (300 MHz, DMSO- d6) δ (ppm): 7.96 (d, J = 2.6 Hz, 1H), 7.74 (d, J = 8.5 Hz, 1H), 6.93 (dd, J = 8.5, 2.7 Hz, 1H), 6.17 – 5.99 (m, 2H). [0424] Step 2: 5-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)picolinic acid

[0425] To a mixture of 5‐aminopicolinic acid (422 mg) and TEA (0.56 mL) in EtOH was added 3,4-diethoxycyclobut-3-ene-1,2-dione (572 mg). The reaction was stirred at room temperature for 3 hours, next was diluted with H₂O, and treated with 1M HCl until the pH = 4. The resulting precipitate was filtered off, washed with H₂O, and dried to deliver desired intermediate as a yellow solid (609 mg, 76% yield). LCMS (6 min): RT = 2.0 min, found [M+H]⁺ 263.00 ; [M-H]- 261.15. ¹H NMR (300 MHz, DMSO-d6) δ (ppm): 11.16 (s, 1H), 10.48 (s, 1H), 8.76 – 8.50 (m, 2H), 8.13 – 8.00 (m, 3H), 7.92 (dd, J = 8.6, 2.7 Hz, 1H), 4.81 (q, J = 7.1 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H) EXAMPLE 1: PREPARATION OF COMPOUNDS OF FORMULA (III). Example 1.1 – Synthesis of compound (1): 3-ethoxy-4-((2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro- 2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)amino)cyclobut-3-ene-1,2-dione [0426] Step 1: Synthesis of (3S,4S,5S,6R)-2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol

[0427] To a solution of (3S,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (1.0 g), synthesized as described in the application WO2022096681, in MeOH, was added Pd/C (0.16 g) under argon atmosphere. The reaction mixture was stirred under hydrogen atmosphere overnight, filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to dryness. The crude product was used for the next step without further purification (0.74 g, yield 80% yield). LC/MS (6 min): RT = 0.5 min, found [M+H]⁺ = 312.10 [0428] Step 2: Synthesis of 3-ethoxy-4-((2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl) amino)cyclobut-3- ene-1,2-dione

[0429] To a solution of previous compound (150 mg) in EtOH was added under argon atmosphere TEA (0.09 mL) followed by 3,4-diethoxycyclobut-3-ene-1,2-dione (90 mg). The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure to dryness. The residue was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure to dryness. The resulting oil was dissolved in a small amount of H₂O, the solution was being frozen, and next freeze-dried to deliver the final material (0.111 g, 53% yield) as a colourless hard oil. LC/MS (12 min): RT = 6.28 min, found [M+H]⁺ = 436.1 ; [M-H]- = 434.0. HPLC purity: 95.6 % (200 nm), 96.6 % (270 nm) ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.92 – 8.50 (m, 1H), 4.80 – 4.59 (m, 5H), 4.54 (d, J = 5.8 Hz, 1H), 4.42 (t, J = 6.0 Hz, 1H), 3.77 – 3.35 (m, 18H), 1.37 (t, J = 7.0 Hz, 3H) Example 1.2 – Synthesis of compound (2): 4-(((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)methyl)-N-(2-(2-(2-(((3S,4S,5S,6R)- 3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy) ethoxy)ethoxy)ethyl)benzamide

[0430] To a mixture of intermediate 4-(((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)methyl) benzoic acid described in the preparation 1 (159 mg), HATU (220 mg) and DIPEA (0.25 mL) in DMF was added under argon atmosphere compound described in step 1 of example 1 (150 mg). The reaction mixture was stirred for 2 hours at room temperature, next the solvent was removed under reduced pressure. The residue was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure to dryness. The resulting oil was dissolved in a small amount of H₂O, the solution was being frozen, and next freeze-dried to deliver the final material (36 mg, 13.2% yield) as a white powder. LC/MS (12 min): RT = 7.28 min, [M+H]⁺ = 569.1 ; [M-H]- = 567.1 HPLC purity: 95.5 % (200 nm), 98.2 % (270 nm). ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): δ 9.39 – 8.96 (m, 1H), 8.50 (t, J = 5.5 Hz, 1H), 7.84 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 4.85 – 4.60 (m, 5H), 4.55 (d, J = 5.9 Hz, 2H), 4.43 (t, J = 5.9 Hz, 1H), 3.76 – 3.34 (m, 16H), 1.35 (dt, J = 14.2, 7.0 Hz, 3H) Example 1.3 – Synthesis of compound (3): 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl) benzamide

[0431] To a mixture of intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid described in preparation 2 (151 mg), HATU (220 mg) and DIPEA (0.25 mL) in DMF was added under argon atmosphere compound obtained in step 1 of example 1 (150 mg). The reaction mixture was stirred for 2 hours at room temperature. Next, the solvent was evaporated under reduced pressure. The residue was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure. The resulting oil was dissolved in a small amount of H₂O, the solution was being frozen, and next freeze-dried to deliver the final material (66 mg, 40.3% yield) as a white powder. LC/MS (12 min): RT = 4.79 min, [M+H]⁺ = 555.1 ; [M-H]- = 553.1. HPLC purity: 99.9 % (200 nm), 99.8 % (315 nm).¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 10.86 (s, 1H), 8.44 (t, J = 5.6 Hz, 1H), 7.84 (d, 2H), 7.43 (d, J = 8.5 Hz, 2H), 4.79 (q, J = 7.1 Hz, 2H), 4.70 (dd, J = 5.8, 4.6 Hz, 2H), 4.63 (d, J = 1.6 Hz, 1H), 4.54 (d, J = 5.8 Hz, 1H), 4.43 (t, J = 6.0 Hz, 1H), 3.76 – 3.35 (m, 18H), 1.44 (t, J = 7.1 Hz, 3H) Example 1.4 – Synthesis of compound (4): 3-ethoxy-4-((14-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H- pyran-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)amino)cyclobut-3-ene-1,2-dione [0432] Step 1: preparation of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1- iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0433] Mannose pentaacetate (5.0 g) was dissolved in dry DCM under argon atmosphere, morpholine (4.4 mL) was added, and the solution was stirred at room temperature overnight. The reaction mixture was then washed twice with 2M HCl (2x40 mL), water (50 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The resulting yellow oil was dissolved in dry DCM (100 mL) under argon atmosphere, the solution was cooled down to 0°C, and treated with trichloroacetonitrile. After being stirred for 1h at 0°C, DBU (0.38 mL) was added, the reaction mixture was stirred at 0°C for 1h, and next at room temperature for 1 hour. The solvents were removed under reduced pressure, and the brown oily residue was purified by silica gel column chromatography using hexane/ EtOAc (1:1) as eluent to yield the product (2.65 g, 42 % yield) as a yellowish oil. LC/MS no UV absorbance detected (lack of chromophore), no ionization detected (lack of ionophore).¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 10.15 (s, 1H), 6.22 (s, 1H), 5.36 – 5.19 (m, 3H), 4.26 – 4.04 (m, 3H), 2.16 (s, 3H), 2.07 – 1.96 (m, 10H) [0434] Step 2: Preparation of a mixture of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-((2,2- dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonadecan-19-yl)oxy)tetrahydro-2H- pyran-3,4,5-triyl triacetate and (2R,3R,4S,5S)-2-(acetoxymethyl)-6-((14-amino- 3,6,9,12-tetraoxatetradecyl)oxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate:

[0435] To a suspension of compound obtained in previous step (0.5 g) and molecular sieves 4 Å (to keep the reaction mixture anhydrous) in dry DCM under argon atmosphere was added commercially available N-Boc-PEG5-alcohol (0.514 g) at room temperature. The mixture was cooled down to -25°C, and TMSOTf (0.203 mL) was added. The suspension was stirred at -25°C for 1h, and then it was allowed to stir at room temperature overnight. After that time, the reaction was quenched with saturated NaHCO₃ solution, DCM (10 mL) was added, the phases were separated, and the organic phase was washed with water (15 mL), brine (15 mL), and dried over MgSO₄. The resulting solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was filtered through a silica gel pad to deliver 600 mg of the crude material containing both Boc- deprotected and Boc-protected products (a yellow oil). This crude material was used as such for the next step without further purification. Ionization shows a mixture of both Boc-deprotected (LC/MS (6 min): RT = 2.19 min, [M+H]⁺ = 568.35) and Boc- protected (LC/MS (6 min): RT = 3.16 min, [M+H]⁺ = 668.40). ¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 7.76 (t, J = 5.6 Hz, 1H), 5.29 – 5.05 (m, 3H), 4.57 (t, J = 5.4 Hz, 1H), 4.21 – 3.98 (m, 4H), 3.56 – 3.50 (m, 16H), 3.49 – 3.41 (m, 4H), 3.20 – 3.13 (m, 1H), 2.16 – 2.10 (m, 4H), 2.06 – 2.01 (m, 10H), 1.98 – 1.93 (m, 4H) [0436] Step 3: (3S,4S,5S,6R)-2-((14-amino-3,6,9,12-tetraoxatetradecyl)oxy)-6- (hydroxy methyl)tetrahydro-2H-pyran-3,4,5-triol

[0437] To a solution of previous compounds (0.6 g of crude material) in DCM was cooled down to 0°C and trifluoroacetic acid (3.44 mL) was added. The reaction mixture was stirred for 30 minutes at 0°C and next was allowed to stir at room temperature overnight. The solvent was removed under reduced pressure to obtain 580 mg of the crude product. The residue was purified by Reverse Phase flash column chromatography using ACN/H₂O as eluent to obtain 112 mg (31% yield) of pure compound. The obtained product was then dissolved in 7N NH3 in MeOH and stirred at room temperature overnight. The solvent was removed under reduced pressure to dryness. The crude product was used for the next step without further purification (85 mg, quantitative yield). LC/MS (6 min): RT = 1.30 min, [M+H]⁺ = 399.60 [0438] Step 4: Preparation of compound (4), 3-ethoxy-4-((14-(((3S,4S,5S,6R)-3,4,5- trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy)-3,6,9,12-tetraoxatetra decyl)amino)cyclobut-3-ene-1,2-dione

[0439] To a solution of previous compound (80 mg) in EtOH was added TEA (44 µL). After 5 minutes of stirring, 3,4-diethoxycyclobut-3-ene-1,2-dione (33 mg) was added and the reaction mixture was stirred at room temperature overnight. The solvents were evaporated under reduced pressure, and the residue was purified by preparative HPLC (ACN/water) to obtain 38 mg (34% yield) of desired compound. HPLC purity: 99.4% (261 nm).¹H NMR (300 MHz, DMSO-d₆) δ (ppm): 8.71 (d, J = 50.5 Hz, 1H), 4.71 – 4.69 (m, 1H), 4.67 (d, J = 3.0 Hz, 1H), 4.66 – 4.61 (m, 2H), 4.54 (d, J = 5.8 Hz, 1H), 4.42 (t, J = 6.0 Hz, 1H), 3.72 – 3.47 (m, 20H), 3.47 – 3.42 (m, 2H), 3.41 – 3.32 (m, 3H), 1.37 (t, J = 7.1 Hz, 3H). Example 1.5 : Synthesis of compound (5): 3-((3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)amino)-4-ethoxy cyclobut-3-ene-1,2-dione

[0440] A mixture of commercially available of 5-aminofluorescein (50 mg) and 3,4- diethoxycyclobut-3-ene-1,2-dione (25 mg) in anhydrous EtOH under argon atmosphere was cooled down to 0°C. Then, DIPEA (28 µL) was added to the solution, and the reaction mixture was allowed to stir at room temperature overnight. Next, 1M HCl was added dropwise to bring the pH to 3. The resulting mixture was extracted with DCM (3x15 mL). The combined organic layers were dried over Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure to obtain 46 mg of the crude product. [0441] The reaction was repeated using similar conditions, only replacing DIPEA with TEA (22 µL). The crude product (52 mg) was combined with the previous batch and the material was purified by preparative HPLC to obtain in total 57 mg of desired compound as an orange solid. LC/MS (6 min): RT = 2.82 min, [M+H]⁺ = 471.55. HPLC purity: 99.92 % (222 nm), 99.96 % (303 nm).¹H NMR (300 MHz, DMSO-d6) δ 10.17 (s, 1H), 7.96 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 8.4, 2.2 Hz, 1H), 7.31 – 7.21 (m, 1H), 6.70 – 6.48 (m, 6H), 4.81 (q, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H). Example 1.6 - Synthesis of compound (6): 4-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)ethyl)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy) ethoxy)ethyl)benzamide

[0442] To a mixture of intermediate 4-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)ethyl)benzoic acid described in preparation 3 (167 mg), HATU (219 mg) and DIPEA (0.25 mL) in DMF was added under argon atmosphere compound obtained in step 1 of example 1 (150 mg). The reaction mixture was stirred overnight at room temperature. Next, the solvent was evaporated under reduced pressure. The residue was purified twice by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure. After freeze-drying process, the final compound was obtained as a white powder (20 mg, 7% yield). LC/MS (12 min): RT = 4.8 min, found [M-H]- = 581.2. HPLC purity: 99.6 % (200 nm), 99.7 % (260 nm).¹H NMR (400 MHz, DMSO-d₆) δ (ppm): 8.87 (s, 0.5H), 8.68 (s, 0.5H), 8.45 (d, J = 6.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.34 – 7.22 (m, 2H), 4.71 (dd, J = 7.4, 4.8 Hz, 2H), 4.63 (d, J = 1.6 Hz, 2H), 4.59 (d, J = 7.0 Hz, 1H), 4.55 (d, J = 5.9 Hz, 1H), 4.43 (t, J = 6.0 Hz, 1H), 3.66 (dt, J = 11.1, 5.8 Hz, 2H), 3.61 – 3.36 (m, 18H), 2.89 (s, 2H), 1.35 (q, J = 7.4 Hz, 3H).

Example 1.7 - Synthesis of compound (7): 3-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)benzamide

[0443] To a mixture of intermediate 3-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid described in preparation 4 (251 mg), HATU (366 mg) and DIPEA (0.42 mL) in DMF was added under argon atmosphere compound obtained in step 1 of example 1 (250 mg). The reaction mixture was stirred overnight at room temperature. Next, the solvent was evaporated under reduced pressure. The residue was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure. After freeze-drying process, the final compound was obtained as a white powder (71 mg, 33% yield). LC/MS (12 min): RT = 4.9 min, found [M+H]⁺ = 555.1 ; [M-H]- = 553.1. HPLC purity: 99.9 % (200 nm), 99.9 % (315 nm).¹H NMR (400 MHz, DMSO-d6) δ (ppm): 10.87 (s, 1H), 8.47 (t, J = 5.6 Hz, 1H), 7.80 (t, J = 1.9 Hz, 1H), 7.57 (dt, J = 7.6, 1.4 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 4.77 (q, J = 7.1 Hz, 2H), 4.70 (dd, J = 7.9, 4.8 Hz, 2H), 4.62 (d, J = 1.6 Hz, 1H), 4.54 (d, J = 5.9 Hz, 1H), 4.42 (t, J = 5.8 Hz, 1H), 3.70 – 3.61 (m, 2H), 3.58 – 3.32 (m, 16H), 1.41 (t, J = 7.1 Hz, 3H) Example 1.8 - Synthesis of compound (8): 2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)phenyl)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy) ethoxy)ethyl)acetamide

[0444] To a mixture of intermediate 2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)phenyl)acetic acid described in preparation 5 (159 mg), HATU (219 mg) and DIPEA (0.25 mL) in DMF was added under argon atmosphere compound obtained in step 1 of example 1 (150 mg). The reaction mixture was stirred overnight at room temperature. Next, the solvent was evaporated under reduced pressure. The residue was purified twice by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure. After freeze-drying process, the final compound was obtained as a white powder (21 mg, 10% yield). LC/MS (12 min): RT = 4.8 min, found [M-H]- = 567.1. HPLC purity: 99.6 % (200 nm), 99.7 % (300 nm).¹H NMR (400 MHz, DMSO-d6) δ (ppm): 10.62 (s, 1H), 8.10 (t, J = 5.7 Hz, 1H), 7.28 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 8.6 Hz, 2H), 4.82 – 4.72 (m, 3H), 4.74 – 4.69 (m, 2H), 4.64 (d, J = 1.7 Hz, 1H), 4.55 (d, J = 5.9 Hz, 1H), 4.44 (t, J = 5.9 Hz, 1H), 3.73 – 3.62 (m, 2H), 3.62 – 3.35 (m, 16H), 3.21 (q, J = 5.8 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H) Example 1.9 - Synthesis of compound (9): 5-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl) picolinamide

[0445] To a mixture of intermediate 5-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)picolinic acid described in preparation 6 (328 mg), HATU (454 mg) and DIPEA (0.52 mL) in DMF was added under argon atmosphere compound obtained in step 1 of example 1 (310 mg). The reaction mixture was stirred 3 hours at room temperature. Next, the solvent was evaporated under reduced pressure. The residue was purified by preparative Reverse Phase HPLC using ACN-FA/H₂O-FA as eluent. The fractions containing the pure product were combined and ACN was partially evaporated under reduced pressure. The aqueous solution was frozen, and next freeze-dried to deliver the final compound as a yellow solid foam (36 mg, 6% yield). LC/MS (12 min): RT = 4.59 min, found [M+H]⁺ = 556.0 ; [M-H]- = 554.0. HPLC purity: 95.7% (200nm), 95.6% (323nm).¹H NMR (300 MHz, DMSO-d6) δ (ppm): 11.11 (s, 1H), 8.66 (dd, J = 2.6, 0.8 Hz, 1H), 8.61 (t, J = 5.8 Hz, 1H), 8.02 (dd, J = 8.5, 0.7 Hz, 1H), 7.92 (dd, J = 8.5, 2.6 Hz, 1H), 4.80 (q, J = 7.1 Hz, 2H), 4.68 (s, 2H), 4.63 (d, J = 1.6 Hz, 1H), 4.47 (d, J = 32.8 Hz, 2H), 3.67 (td, J = 9.7, 8.0, 4.9 Hz, 2H), 3.61 – 3.52 (m, 8H), 3.48 (dd, J = 11.7, 5.8 Hz, 8H), 1.44 (t, J = 7.1 Hz, 3H) Example 1.10 - Synthesis of compound (10): 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)-N-(2-(2-(2-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro- 2H-pyran-2-yl)oxy) ethoxy)ethoxy)ethyl)benzamide [0446] Step 1: synthesis of (2R,3R,4S,5R)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1- iminoethoxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0447] To a solution of α-D(+)-glucose pentaacetate (10 g) was added morpholine (8.8 mL), and the mixture was stirred at room temperature overnight. The reaction mixture was next washed with 2M HCl (2x200 ml), water (200 mL), and dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting yellow oil was dissolved in dry DCM under argon atmosphere, the solution was cooled down to 0 °C, and treated with trichloroacetonitrile (25.7 mL). After being stirred for 1 hour at 0 °C, DBU (0.76 mL) was added, the reaction mixture was stirred at 0 °C for 1 hour, and next at room temperature overnight. The solvents were removed under reduced pressure, and the residue was purified by column chromatography eluting with hexane and EtOAc mixture to deliver the desired product as a yellow oil (10.3 g, 82% yield).¹H NMR (300 MHz, DMSO-d6) δ (ppm): 9.99 (s, 1H), 6.43 (s, 1H), 5.41 (t, J = 9.9 Hz, 1H), 5.19 – 5.05 (m, 2H), 4.25 – 4.07 (m, 3H), 2.01 – 1.94 (m, 12H) [0448] Step 2: synthesis of (2R,3R,4S,5R)-2-(acetoxymethyl)-6-(2-(2-(2-azidoethoxy) ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0449] To a suspension of compound obtained at previous step (10.3 g) and molecular sieves in dry DCM was added 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (6.9 g), synthesized as described in the application WO2022096681, at room temperature under argon atmosphere. The mixture was cooled down to -25^oC, and TMSOTf (4.5 mL) was added. The reaction mixture was stirred at -25°C for 1 hour, and then was allowed to stir at room temperature overnight. The reaction was quenched with saturated NaHCO₃ solution, DCM was added, the phases were separated, and the organic phase was washed with water, brine, and dried over Na₂SO₄. The resulting suspension was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography eluting with hexane and EtOAc mixture to give the desired product as a yellow oil (3.8 g, 29% yield).¹H NMR (300 MHz, DMSO-d6) δ (ppm): 5.25 (t, J = 9.4 Hz, 1H), 4.90 (t, J = 9.7 Hz, 1H), 4.85 – 4.72 (m, 2H), 4.21 – 4.12 (m, 1H), 4.05 – 3.99 (m, 1H), 3.99 – 3.92 (m, 1H), 3.84 – 3.75 (m, 1H), 3.61 – 3.52 (m, 9H), 3.40 – 3.37 (m, 2H), 2.02 (s, 3H), 2.00 – 1.96 (m, 6H), 1.94 (s, 3H) [0450] Step 3: synthesis of (3R,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0451] Compound obtained at previous step (3.8 g) was dissolved in 7N NH3 in MeOH and stirred at room temperature overnight. Next, the solvent was evaporated under reduced pressure, and the crude desired product was used for the next step without further purification (3.0 g, quantitative yield).¹H NMR (300 MHz, DMSO-d6) δ (ppm): 4.99 – 4.84 (m, 2H), 4.57 – 4.40 (m, 1H), 4.15 (d, J = 7.8 Hz, 1H), 3.92 – 3.81 (m, 1H), 3.70 – 3.22 (m, 18H) [0452] Step 4: synthesis of (3R,4S,5S,6R)-2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0453] To a solution of compound obtained at previous step (500 mg) in MeOH was added Pd/C under argon atmosphere. The inert gas was evacuated and backfilled with hydrogen (in total three times). The reaction mixture was stirred under hydrogen atmosphere (balloon) overnight, filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to dryness. The crude product was used for the next step without further purification (460 mg, quantitative yield). LC/MS (6 min): RT = 0.5 min, found [M+H]⁺ = 312.05. [0454] Step 5: 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2- (((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy) ethoxy)ethoxy)ethyl)benzamide

[0455] To a mixture of intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid described in preparation 2 (256 mg), HATU (372 mg) and DIPEA (0.43 mL) in DMF was added under argon atmosphere compound obtained in previous step (254 mg). The reaction mixture was stirred for 2 hours at room temperature. The solvent was evaporated under reduced pressure. The residue was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure. The resulting oil was dissolved in a small amount of H₂O, the solution was being frozen, and next freeze- dried to deliver the final product as a white solid foam (74 mg, 16% yield). LC/MS (12 min): RT = 4.79 min, found [M+H]⁺ = 555.1 ; [M-H]- = 553.1. HPLC purity: 99.8% (200nm), 99.8% (315nm).¹H NMR (300 MHz, DMSO-d6) δ (ppm): 10.91 (s, 1H), 8.44 (t, J = 5.5 Hz, 1H), 7.90 – 7.79 (m, 2H), 7.44 (d, J = 8.5 Hz, 2H), 4.96 (d, J = 4.9 Hz, 1H), 4.89 (m, 2H), 4.79 (m, 2H), 4.48 (t, J = 5.9 Hz, 1H), 4.15 (d, J = 7.7 Hz, 1H), 3.92 – 3.78 (m, 1H), 3.71 – 3.61 (m, 1H), 3.61 – 3.49 (m, 9H), 3.43 (m, 3H), 3.08 (m, 3H), 2.95 (m, 1H), 1.42 (t, 3H) Example 1.11 - Synthesis of compound (11): 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)-N-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H- pyran-2-yl)oxy)ethyl)benzamide [0456] Step 1: synthesis of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2- bromoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0457] To a stirred solution of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1- iminoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (1.0 g) obtained in step 1 of example 1.4 in dry DCM was added 2-bromoethanol (0.115 mL) followed by boron trifluoride diethyl etherate (0.01 mL). The reaction mixture was stirred at room temperature overnight. Triethylamine (0.2 mL) was added, and the resulting mixture was concentrated under reduced pressure. The crude material was purified by flash chromatography with hexane/EtOAc, as eluent to give the desired product as a white solid (710 mg, 76% yield).¹H NMR (300 MHz, Chloroform-d) δ (ppm): 5.38 – 5.24 (m, 3H), 4.87 (d, J = 1.7 Hz, 1H), 4.27 (dd, J = 12.6, 5.9 Hz, 1H), 4.17 – 4.09 (m, 2H), 4.03 – 3.83 (m, 2H), 3.52 (dd, J = 6.3, 5.7 Hz, 2H), 2.16 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H). [0458] Step 2: synthesis of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2- azidoethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate

[0459] To a stirred solution of compound obtained at previous step (700 mg) in dry DMF was added NaN₃ (800 mg) and the mixture was stirred at 60°C overnight. The solvent was removed under reduced pressure, and the crude material was treated with EtOAc, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography with hexane/EtOAc as eluent to afford desired product as a colorless oil (600 mg, 93% yield). ¹H NMR (400 MHz, Chloroform-d) δ (ppm): 5.36 (dd, J = 10.1, 3.3 Hz, 1H), 5.31 (d, J = 9.8 Hz, 1H), 5.28 (dd, J = 3.4, 1.8 Hz, 1H), 4.87 (d, J = 1.8 Hz, 1H), 4.29 (dd, J = 12.3, 5.3 Hz, 1H), 4.16 – 4.10 (m, 2H), 4.05 (ddd, J = 9.8, 5.3, 2.4 Hz, 1H), 3.87 (ddd, J = 10.6, 6.8, 3.8 Hz, 1H), 3.67 (ddd, J = 10.6, 6.0, 3.6 Hz, 1H), 3.54 – 3.39 (m, 2H), 2.16 (s, 3H), 2.11 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H) [0460] Step 3: synthesis of (3S,4S,5S,6R)-2-(2-azidoethoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0461] Compound obtained at previous step (600 mg) was dissolved in 7N NH₃ in MeOH and stirred at room temperature overnight. The solvent was removed under reduced pressure to dryness, and the crude product was used for the next step without further purification (358 mg, quantitative yield). ¹H NMR (400 MHz, DMSO-d6) δ (ppm): 4.75 (t, J = 5.0 Hz, 2H), 4.67 (d, J = 1.6 Hz, 1H), 4.59 (d, J = 5.9 Hz, 1H), 4.46 (t, J = 6.0 Hz, 1H), 3.78 (ddd, J = 11.0, 6.4, 3.5 Hz, 1H), 3.70 – 3.64 (m, 1H), 3.62 (ddd, J = 4.7, 3.4, 1.6 Hz, 1H), 3.55 (ddd, J = 11.0, 6.5, 3.3 Hz, 1H), 3.51 – 3.35 (m, 6H). [0462] Step 4: synthesis of (3S,4S,5S,6R)-2-(2-aminoethoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0463] To a solution of compound obtained at previous step (358 mg) in MeOH was added 10% Pd/C under gentle flow of argon. The inert gas was evacuated and backfilled with hydrogen (in total three times). The reaction mixture was stirred under hydrogen atmosphere (balloon) overnight. The resulting mixture was filtered through a pad of Celite, washed with MeOH, and concentrated under reduced pressure to afford the product as a colorless oil. The crude product was used for the next step without further purification (320 mg, quantitative yield). ¹H NMR (300 MHz, Deuterium Oxide) δ (ppm): 4.89 (d, J = 1.8 Hz, 1H), 3.98 (dd, J = 3.4, 1.8 Hz, 1H), 3.91 (dd, J = 12.0, 1.5 Hz, 1H), 3.87 – 3.72 (m, 3H), 3.70 – 3.62 (m, 2H), 3.55 (ddd, J = 10.4, 6.0, 4.4 Hz, 1H), 2.93 – 2.78 (m, 2H) [0464] Step 5: synthesis of 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2- (((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)ethyl)benzamide

[0465] To a stirred solution of intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid described in preparation 2 (211 mg) in DMF was added DIPEA (0.35 mL). The mixture was stirred for 5 minutes, then HATU (307 mg) was added, followed by a solution in DMF of compound obtained at previous step (150 mg). The mixture was stirred at room temperature for 2h. The solvents were removed under reduced pressure and the crude material was purified by reversed phase flash column chromatography to afford desired product as a white powder (97 mg, 31% yield). LC/MS (12 min): RT = 7.65 min, found [M-H]- = 465.0. HPLC purity: 98.5% (200nm), 98.9% (315nm).¹H NMR (400 MHz, Deuterium Oxide) δ (ppm): 7.79 – 7.74 (m, 2H), 7.41 (d, J = 8.5 Hz, 2H), 4.91 (d, J = 1.7 Hz, 1H), 4.84 – 4.76 (m, 2H), 3.96 (dd, J = 3.5, 1.7 Hz, 1H), 3.90 (ddd, J = 10.8, 7.1, 4.1 Hz, 1H), 3.83 – 3.77 (m, 2H), 3.77 – 3.55 (m, 6H), 1.48 (t, J = 7.1 Hz, 3H) Example 1.12 - Synthesis of compound (12): ((2R,3S,4S,5S)-6-(2-(2-(2-(4-((2-ethoxy- 3,4-dioxocyclobut-1-en-1-yl)amino)benzamido) ethoxy)ethoxy)ethoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)methyldihydrogen phosphate [0466] Step 1: synthesis of (3S,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6- (((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triol

[0467] To a solution of (3S,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6- (hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (1.0 g), synthesized as described in the application WO2022096681, in DMF were added TBDMSCl (490 mg) and imidazole (400 mg) under Ar. The reaction mixture was stirred at RT overnight. Then, the reaction mixture was diluted with EtOAc (20 mL) and washed with water, and brine. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product obtained as an oil (1.1 g, 83% yield) was used for the next step without further purification. LC/MS (6 min): RT = 3.25 min, found [M+H]⁺ 452.25. ¹H NMR (300 MHz, Chloroform-d) δ (ppm): 4.84 – 4.69 (m, 1H), 3.87 (dd, J = 3.3, 1.6 Hz, 1H), 3.81 – 3.65 (m, 5H), 3.61 – 3.47 (m, 10H), 3.30 (t, J = 5.0 Hz, 2H), 0.80 (s, 9H), 0.00 (d, J = 2.7 Hz, 6H) [0468] Step 2: synthesis of (((2R,3R,4S,5S)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)- 3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methoxy)(tert-butyl)dimethylsilane

[0469] Compound obtained at previous step (1.0 g) was dissolved in anhydrous DMF under argon atmosphere, and the solution was cooled down to 0°C. NaH 60% (440 mg) was slowly added and the mixture was stirred at 0°C for 10 min. BnBr (1.31 mL) was added and the reaction mixture was stirred at 0°C for 10 min, then at room temperature overnight. The reaction was quenched with methanol, and the mixture was concentrated to dryness in vacuo. The crude material was diluted with EtOAc, washed with water, and brine. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduce pressure. The product was purified by column chromatography using hexane/EtOAc as eluent to provide desired product as an oil (1.2 g, 76% yield). ¹H NMR (300 MHz, Chloroform-d) δ (ppm): 7.57 – 6.85 (m, 16H), 4.93 – 4.80 (m, 2H), 4.75 – 4.51 (m, 5H), 3.95 – 3.83 (m, 2H), 3.78 (dd, J = 4.8, 2.8 Hz, 3H), 3.74 – 3.68 (m, 1H), 3.63 – 3.47 (m, 10H), 3.27 (t, J = 5.1 Hz, 2H), 0.83 (s, 9H), 0.01 (d, J = 3.4 Hz, 6H) [0470] Step 3: synthesis of ((2R,3R,4S,5S)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)- 3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methanol

[0471] Compound obtained at previous step (1.1 g) was dissolved in THF under argon atmosphere, and the mixture was cooled down to 0°C. A solution of TBAF – 1M in THF (2.3 mL) was slowly added and the reaction mixture was stirred at 0°C for 10 min, then at room temperature for 3h. The reaction was quenched with water, and the resulting mixture was extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by column chromatography using DCM/MeOH as eluent to provide desired product as an oil (750 mg, 81% yield). LC/MS (6 min): RT = 4.33 min, found [M+Na]⁺ = 630.5 ; [M+H₂O]⁺ = 625.5. ¹H NMR (300 MHz, Chloroform-d) δ (ppm): 7.63 – 7.06 (m, 15H), 4.92 – 4.76 (m, 2H), 4.75 – 4.53 (m, 5H), 3.88 (dd, J = 5.3, 2.5 Hz, 2H), 3.81 – 3.46 (m, 14H), 3.26 (t, J = 5.0 Hz, 2H) [0472] Step 4: synthesis of ((2R,3R,4S,5S)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)- 3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methyl dibenzyl phosphate

[0473] To a suspension of compound obtained at previous step (550 mg) and 4Å molecular sieves in anhydrous DCM was added under argon atmosphere a solution of tetrazole – 0.45M in ACN (6.2 mL). The mixture was stirred at room temperature for 2h, then dibenzyl diisopropylphosphoramidite (0.64 mL) was added under argon atmosphere. The reaction mixture was stirred at room temperature overnight, next was cooled down to 0°C, and m-CPBA (470 mg) was added. The resulting mixture was allowed to stir at room temperature for 1h, and the reaction was quenched with water. The suspension was filtered through a pad of Celite, the filtrate was washed with water, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The product was purified by column chromatography using hexane/EtOAc and DCM/MeOH as eluent to provide desired product as an oil (320 mg, 41% yield). LC/MS (6 min): RT = 4.92 min, found [M+H]⁺ = 868.75.¹H NMR (300 MHz, DMSO-d6) δ (ppm): 7.66 – 7.14 (m, 25H), 5.00 (ddd, J = 9.6, 6.1, 2.1 Hz, 4H), 4.78 (d, J = 10.9 Hz, 1H), 4.64 (d, J = 8.7 Hz, 3H), 4.53 (d, J = 11.5 Hz, 2H), 4.19 (dd, J = 6.7, 3.2 Hz, 2H), 3.90 (d, J = 1.9 Hz, 1H), 3.83 – 3.61 (m, 4H), 3.58 – 3.47 (m, 10H), 3.28 (t, J = 4.9 Hz, 2H).³¹P NMR (300 MHz, DMSO-d6) δ (ppm) : -1.0 (s) [0474] Step 5: synthesis of ((2R,3R,4S,5S)-6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)- 3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methyl dibenzyl phosphate

[0475] To a suspension of compound obtained at previous step (200 mg) in a mixture of THF/H₂O was added PPh₃ (91 mg). The reaction mixture was stirred overnight at RT, then extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was purified by column chromatography to provide desired product as an oil (117 mg, 60% yield). ¹H NMR (300 MHz, DMSO-d6) δ (ppm): 7.66 – 7.14 (m, 25H), 5.00 (ddd, J = 9.6, 6.1, 2.1 Hz, 4H), 4.78 (d, J = 10.9 Hz, 1H), 4.64 (d, J = 8.7 Hz, 3H), 4.53 (d, J = 11.5 Hz, 2H), 4.19 (dd, J = 6.7, 3.2 Hz, 2H), 3.90 (d, J = 1.9 Hz, 1H), 3.83 – 3.61 (m, 4H), 3.58 – 3.47 (m, 10H), 3.28 (t, J = 4.9 Hz, 2H), 2.70 (m, 2H). ³¹P NMR (300 MHz, DMSO-d6) δ (ppm) : -1.0 (s) [0476] Step 6: synthesis of dibenzyl (((2R,3R,4S,5S)-3,4,5-tris(benzyloxy)-6-(2-(2-(2- (4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzamido)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)methyl) phosphate

[0477] To a mixture of intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1- yl)amino)benzoic acid described in preparation 2 (30 mg), HATU (43 mg) and DIPEA (0.05 mL) in DMF was added under argon atmosphere compound obtained at previous (80 mg). The reaction mixture was stirred for 2 hours at room temperature. Next, the solvent was evaporated under reduced pressure. The residue was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure. The resulting oil was dissolved in a small amount of H₂O, the solution was being frozen, and next freeze-dried to deliver the final material (50 mg, 49% yield) as a white powder. LC/MS (6 min): RT = 4.25 min, found [M+H]⁺ = 1085.6. [0478] Step 7: synthesis of ((2R,3S,4S,5S)-6-(2-(2-(2-(4-((2-ethoxy-3,4-dioxocyclobut- 1-en-1-yl)amino)benzamido)ethoxy)ethoxy)ethoxy)-3,4,5-trihydroxytetrahydro-2H- pyran-2-yl) methyl dihydrogen phosphate

[0479] To a solution of compound obtained at previous step (37 mg) in THF was added Pd/C 10% under argon atmosphere. The reaction mixture was stirred under hydrogen atmosphere (a balloon) until starting material was consumed, then filtered through a pad of Celite. The filtrate was evaporated to dryness under reduced pressure. The residue was purified by preparative Reverse Phase HPLC using ACN/H₂O as eluent. The fractions containing the pure product were combined and concentrated under reduced pressure. The resulting oil was dissolved in a small amount of H₂O, the solution was being frozen, and next freeze-dried to deliver the final material (1.5 mg, 7% yield) as a white powder. LC/MS (6 min): RT = 1.61 min, found [M-H]- = 633.10. HPLC purity: 92.1% (200nm), 93.1% (315nm). Example 1.13 - Synthesis of compounds of formula (III) comprising a peptide group Z.

[0480] (&) In bold shown the group -NH- of R^L-NH- / (#) -NH- from R^L-NH- not shown (amino group of threonine indicated in bold T). The peptide group THRPPMWSPVWP is the peptide THR and corresponds to SEQ ID No: 1. [0481] 1.13.1. General procedure for peptide synthesis: Peptide synthesis: [0482] All peptides were synthesized on solid support [Rink amide resin for compounds (14), (17), (15), (18), (19), (20) and (22); 2-chloro chloroTrityl resin for compounds (13), (16), (21) and (23) at a scale of 0.25 mmol on a GYROS PROTEIN Symphony X peptide synthesizer. The syntheses were performed according to a standard protocol in Fmoc/tBu strategy using as activator the DIC/ Oxyma Pure couple and a piperidine solution in DMF for deprotection of the Fmoc protecting group. Deprotection: [0483] The cleavage from the resin and the deprotection of the side chains were carried out by treatment with a mixture TFA/H₂O/TIPS/DTT (90: 5: 2.5: 2.5) for 90 min. Bu₄NBr (20 eq.) was added to the mixture 15 min before the deprotection stop to prevent methionine oxidation. After filtration, the resin was washed with TFA, the filtrate was concentrated, the peptide was precipitated in diethyl ether, isolated by centrifugation, and freeze-dried in order to eliminate the maximum of residual TFA. 1.13.2. Grafting: General procedure for 3,4-diethoxycyclobut-3-ene-1,2-dione grafting To a solution of peptide (0.1 mmol) in distilled water (0.5 mL)were added DIEA (until a pH between 7 and 7.5 is reached) and a solution of 3,4-diethoxycyclobut-3-ene-1,2-dione (1.2 eq., 0.12 mmol) in EtOH (0.5 mL). The reaction was stirred during 2h at room temperature. 1.13.3. Purification and salt exchange : [0484] The raw peptides were purified directly from the reaction mixture using a reverse phase preparative HPLC system (Waters Delta Prep 4000) using a reverse phase column (Vydac Denali prep C-18, 10 μm, 120 Å, 50 300 mm) and a suitable gradient of CAN+TFA 0.1%/ H₂O+TFA 0.1% as eluent. The fractions containing the purified target peptide were identified by UV measurement (UV/Visible Waters 2489 detector) at 214 nm and the selected fractions were then combined and freeze-dried. [0485] The exchange of trifluoroacetate salt into acetate was carried out during purification via a proprietary buffer. [0486] All peptides were synthesized according to the general procedure on solid support described above, then by an adapted grafting procedure in solution, followed by a purification/salt exchange sequence. 1.13.4. Synthesis of compound (13): [0487] ESI-MS (m/z): [M+H]⁺ calcd for C92H125N22O21S: 1907.20, found: 1906.37. [M+2H]²⁺: 954.10, found: 953.75, [M+3H]³⁺: 636.40, found: 636.48. UPLC purity: 91.6 % (214 nm), RT = 5.20 min 1.13.5. Synthesis of compound (14): [0488] ESI-MS (m/z): [M+H]⁺ calcd for C84H110N21O18S: 1733.99, found: 1733.47. [M+2H]²⁺: 867.49, found: 866.68, [M+3H]³⁺: 578.66, found: 578.45. UPLC purity: 95.6 % (214 nm), RT = 4.52 min 1.13.6. Synthesis of compound (15): [0489] ESI-MS (m/z): [M+H]⁺ calcd for C91H123N22O21S: 1893.17, found: 1892.56. [M+2H]²⁺: 947,08, found: 946.41, [M+3H]³⁺: 631.72, found: 631.58. UPLC purity: 95.6 % (214 nm), RT = 4.55 1.13.7. Synthesis of compound (16): [0490] ESI-MS (m/z): [M+H]⁺ calcd for C85H120N21O20S: 1788.07, found: 1787.37. [M+2H]²⁺: 894,53, found: 893.69, [M+3H]³⁺: 596.69, found: 596.34. UPLC purity: 95.6 % (214 nm), RT = 4.61 min 1.13.8. Synthesis of compound (17): [0491] ESI-MS (m/z): [M+H]⁺ calcd for C77H105N20O17S: 1614.86, found: 1614.44. [M+2H]²⁺: 807.93, found: 807.09, [M+3H]³⁺: 538.95, found: 538.86. UPLC purity: 95.0 % (214 nm), RT = 4.38 min 1.13.9. Synthesis of compound (18): [0492] ESI-MS (m/z): [M+H]⁺ calcd for C84H118N21O20S: 1774.05, found: 1773.12, [M+2H]²⁺: 887.02, found: 886.95, [M+3H]³⁺: 591.68, found: 591.86. UPLC purity: 95.5 % (214 nm), RT = 4.06 min 1.13.10. Synthesis of compound (19): [0493] ESI-MS (m/z): [M+H]⁺ calcd for C105H151N22O28S: 2201.54, found: off- spectrum, [M+2H]²⁺: 1101.27, found: 1101.23, [M+3H]³⁺: 734.51, found: 734.51. UPLC purity: 96.6 % (214 nm), RT = 5.25 min 1.13.11. Synthesis of compound (20): [0494] ESI-MS (m/z): [M+H]⁺ calcd for C93H125N24O21S: 1946.22, found: 1946.37, [M+2H]²⁺: 974.11, found: 973.96, [M+3H]³⁺: 649.74, found: 649.54. UPLC purity: 97.8 % (214 nm), RT = 4.94 min 1.13.12. Synthesis of compound (21): [0495] ESI-MS (m/z): [M+H]⁺ calcd for C87H122N23O20S: 1842.13, found: 1841.35, [M+2H]²⁺: 921.56, found: 920.83, [M+3H]³⁺: 614.71, found: 614.51. UPLC purity: 95.1 % (214 nm), RT = 4.77 min 1.13.13: Synthesis of compound (22) ESI-MS (m/z): [M+H]⁺ calcd for C108H150N29O26S: 2302.62, found: off-spectrum, [M+2H]²⁺: 1151.81, found: 1151.56, [M+3H]³⁺: 768.20, found: 767.91. UPLC purity: 95.0 % (214 nm), RT = 4.63 min 1.13.14: Synthesis of compound (23) ESI-MS (m/z): [M+H]⁺ calcd for C102H147N28O25S: 2197.52; found : off-spectrum, [M+2H]²⁺: 1099.26, found: 1099.12; [M+3H]³⁺: 733.17, found: 732.88; [M+4H]⁴⁺: 550.13, found: 549.95. UPLC purity: 95.0 % (214 nm), RT = 4.42 min EXAMPLE 2: SYNTHESIS AND COUPLING OF AAVs. [0496] Conjugated AAVs were generated by coupling the squarate moieties of the invention to at least one AAV surface-exposed primary amine as described below. II.1. Production and purification of AAVs. [0497] AAVs were produced and purified according to well-known techniques in the art. II.2. Production and purification of chemically-conjugated AAVs. Materials [0498] Compounds (1)-(11), (13)-(17) and (19)-(23) were obtained as detailed above in Example 1. [0499] The following AAVs, obtained as detailed in example II.1, were used: - AAV2-eGFP: 1.0E13 vg/mL in DPBS + Ca²⁺, Mg²⁺, 0.001% Pluronic F68 at pH 7.4; - AAV5-eGFP: 1.0E13 vg/mL in DPBS + Ca²⁺, Mg²⁺, 0.001% Pluronic F68 at pH 7.4; - AAV9-eGFP: 1.0E13 vg/mL in DPBS + Ca²⁺, Mg²⁺, 0.001% Pluronic F68, at pH 7.4. Table 3 - Other material and reagents

Methods [0500] The coupling of the squarate linkers on AAV2-eGFP, AAV5-eGFP or AAV9- eGFP capsids (2E11 or 1E12 vg) was carried out with a solution of TRIS buffer pH = 9.3 containing compounds (1)-(11), (13)-(17) and (19)-(23) a molar ratio of 1E6 or 3E6 equivalents and incubated during 4h, 16h, 48h or 72h at 20°C. At the end of the incubation period, unbound linkers were removed using PD MidiTrap G-25 columns. The columns were first equilibrated with 5x4 ml of formulation buffer (DPBS, Ca2+, Mg2+, 0.001% F68). The coupling reactions mixtures were then loaded onto one column each, and the samples were allowed to enter the bed. Elution of the rAAV vectors was performed with 1.5 ml formulation buffer. 5 fractions at 0.3 ml were collected dropwise in 1.5 ml PP- tubes. Fractions 2-5 were pooled and qPCR or ddPCR titer was determined for each pooled fraction. Pooled fractions were then sterile filtrated using Acrodisc PP, PES, 0.2 μM 1.3 cm2. II.3. Characterization of chemically-conjugated AAVs II.3.a. Titration of Vector Genones (vg) [0501] For all coupling reactions quantitative real time PCR (qPCR) titers were determined using a LightCycler 480 (Roche) or droplet digital PCR (ddPCR) were determined using a QX200 (Biorad) for samples taken after the formulation/filtration step. II.3.b. Analysis of coupling by SDS-PAGE and Lectin WB [0502] The purity and integrity of the obtained conjugated AAV vectors was evaluated by silver staining of SDS-PAGE gels. The efficacy of the coupling of the mannose moieties on the AAVs was further studied by western blot analysis using concanavalin- HRP lectin (ConA) staining which binds selectively to mannose. Successful coupling should result in a shift of the VP proteins towards higher molecular masses, and specific ConA staining for mannose conjugated AAV. Results [0503] The results are summarized in Table 4 below

ND: not determined; NA: not applicable. [0504] Conclusion: Mobility shifts, mass shift and/or ConA staining were observed for all modified AAVs, providing evidences of effective coupling of squarate linkers on AAVs. The rate of conjugation can also be modulated based on the design of linkers, offering a versatile approach to achieve varying ligand loading on AAVs. [0505] Parallel assessment of ligand stability in either buffer showed that over 90% of the ligand presented an intact structure after 24h of incubation. II.3.c. Infectivity assay (U87-MG glioblastoma cells) [0506] It was tested if infectivity of the conjugated AAVs can be observed. For this purpose, U87-MG cells were transduced at MOI 1E5 with (1)-AAV2, (2)-AAV2, or (3)- AAV2 vectors and were analyzed 72h after transduction via monitoring of transduced (eGFP-positive) and non-transduced cell population by fluorescence microscopy. [0507] Conclusion: mannose conjugated AAV2-eGFP particles were infectious in U87- MG cell line. II.3.d. Infectivity assay in HEK-TfR1(+)cells [0508] It was tested whether infectivity of the conjugated AAVs can be observed. For this purpose, HEK-293 cells overexpressing the human transferrin receptor (HEK- TfR1(+)) were transduced at an MOI of 1E4 with (13)-AAV9, (16)-AAV9, or (23)-AAV9 vectors. Twenty-four (24) hours post-infection, transduction was measured by quantitative PCR to determine the number of vector genome copiesper cell. [0509] Results: Ten (10) copies per cell were measured for (13)-AAV9 and (16)-AAV9 vectors. Twenty-eight (28) copies per cell were measured for the (23)-AAV9 vector. [0510] Conclusion: AAV9 vectors conjugated with peptide-based ligands were infectious in HEK-TfR1(+) cells. EXAMPLE 3: EVALUATION OF THE TRANSDUCTION PROPERTIES OF TWO AAV VECTORS, INCLUDING A CONJUGATED VECTOR, IN THE MOUSE BRAIN [0511] The objective of the study was to investigate the transduction properties of two recombinant AAV2 vectors expressing GFP (AAV2 and (3)-AAV2) in the mouse brain following a single bilateral intrastriatal injection. Materials Animals [0512] Six (6) adult male C57BL/6 mice (Mus musculus), purchased from Charles River Laboratories. Test items [0513] “AAV2” is a recombinant AAV2 vector comprising an unmodified capsid and carrying a CAG-eGFP expression cassette. [0514] “(3)-AAV2” is a recombinant AAV2 vector comprising a modified capsid with surface-bound mannose linkers and carrying a CAG-eGFP expression cassette. Methods Test items [0515] Compound of formula (III) was covalently attached to primary amino group from amino acids exposed at the surface of the capsid after a 4-hr co-incubation with the AAV2 vectors in Tris buffer pH 9.3 at 20°C. Formulation and elimination of free molecules that did not bind to the AAV capsid were performed by Tangential Flow Filtration of the mix against buffered saline sterile solution (BSSS) + 0.001% poloxamer. Conjugated AAV solution was 0.22µm-filtered. Study design [0516] Six (6) mice underwent stereotactic surgery and were randomly injected with the test items into the striatum, according to Table .

Surgical procedures [0517] Anesthesia was maintained during surgery with a concentration of 1.5-2 % isoflurane. For pain management, Buprenorphine 0.04 mg/kg was administered s.c. Each animal was placed in a stereotaxic apparatus. A midline incision of the scalp was made. Holes were drilled above the target regions, using Bregma as a reference point. A 33G Hamilton syringe was used to inject the test items. The injection rate of the test item solution was 0.25 μL/minute. After injection, wounds were closed with a surgical suture. Ex Vivo Analysis Euthanasia and tissue processing [0518] Six weeks after the stereotactic injections, all animals were euthanized by intraperitoneal (i.p.) injection of 600 mg/kg pentobarbital. After transcardial perfusion with 0.9% saline and 4% paraformaldehyde (PFA) in phosphate buffer, whole brains were removed and fixed for 3 h at room temperature. They were transferred to 15 % sucrose/PBS for few days. Then, they were transferred to cryomolds, embedded in OCT medium, frozen in dry ice-cooled isopentane and coronally cryosectioned at 10 μm thickness on a cryotome. Sections were mounted on glass slides and stored at -20°C. GFP Immunofluorescence [0519] All steps were executed in Dulbecco’s phosphate buffered saline pH 7.2-7.8 (DPBS) at room temperature unless noted otherwise. Cryosections were air-dried and washed. Unspecific binding sites were blocked, and sections were washed. Sections were incubated with primary antibodies in 1% normal donkey serum/DPBS overnight at 4°C: Goat anti-GFP polyclonal antibody (Abcam, ab5450; 1:5000). Sections were washed and incubated with the secondary antibody: Donkey anti-goat IgG (H+L), Alexa Fluor 488 (Abcam, ab150129), 1:500. Sections were washed and incubated with DAPI (AppliChem, Cat. No 1001). Sections were washed, covered and coverslipped. [0520] Whole slide scans of all stained sections were acquired on a Zeiss automatic microscope AxioScan Z1 with high aperture lenses, equipped with a Zeiss Axiocam 506 mono and a Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 3.3 software. Results [0521] Representative images of G1 and G2 groups at the striatal level are shown in Erreur ! Source du renvoi introuvable.. These results demonstrate that (3)-AAV2 is fully functional and transduced the region of interest, the striatum, after an intrastriatal injection. Moreover, GFP expression is increased after injection of (3)-AAV2 compared to AAV2.

Claims

CLAIMS 1. An adeno-associated virus (AAV) vector particle comprising a moiety of formula

wherein N* is a nitrogen atom of a primary amino group from a surface-exposed amino acid residue of a capsid polypeptide from the AAV vector; ---- represents the point of attachment to the AAV vector’s capsid; and R^L-NH- is a functional moiety including a nitrogen containing group -NH- and a group R^L, and wherein said functional moiety R^L-NH- comprises a steric shielding agent, a labelling agent, a cell-type specific ligand, a drug moiety or a combination thereof.

2. The AAV vector particle according to claim 1, wherein the functional moiety R^L- NH- comprises a group Z and one or more spacers L, and the adeno-associated virus (AAV) vector particle is represented by formula (IIa):

wherein Z is H or a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins, or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties, and L comprises one or more groups selected from the group consisting of an arylene or a heteroarylene group, an optionally substituted group comprising saturated or unsaturated, linear or branched, C₁-C₄₀ hydrocarbon chains, preferably one or more groups C_1-6 alkyl, C_1-6 alkylamine or C_1-6 acyl, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; an acyl group, an amino acid moiety, a polyether of a branched polyol, a β-alanine polymer, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof.

3. The AAV vector particle according to claim 2, wherein L comprises more than one spacers groups selected from the group consisting of L₁, L₂ and L₃, and said moiety of formula (IIa) is selected from the group consisting of formula (IIa₁), (IIa₂), (IIa₃), (IIa₄), (IIa₅), (IIa₆), (IIa₇), (IIa₈), (IIa₉), (IIa₁₀), (IIa₁₁), (IIa₁₂), (IIa₁₃), (IIa₁₄), (IIa₁₅), (IIa₁₆), (IIa₁₇), (IIa₁₈), (IIa₁₉) and (IIa₂₀):

wherein L₁ is one or more groups selected from the group consisting of a polyethylene glycol (PEG), comprising 1 to 40, preferably 1 to 20, ethylene glycol monomers, a polyether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and a β-alanine polymer comprising 1 to 40, preferably 1 to10, β-alanine monomers, or a mixture thereof; L₂ comprises one or more arylene or a heteroarylene groups; L₃ is one or more groups selected from an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, a C_1-6 alkylene group, being said C_1-6 alkylene a linear C_1-6 alkylene group or a branched C_3-6 alkylene group; a C_1-6 alkylenamine group and C_1-6 acyl group; wherein L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and wherein, L₁ and L₂, or L₁ and L₃ are covalently linked by an amide moiety or a bioisostere moiety thereof; or wherein L₁ and L₃ covalently linked by an ether bond.

4. The AAV vector particle according to any of claims 2 or 3, or a pharmaceutically acceptable salt thereof, wherein Z comprises a linear or a cyclic peptide or a saccharide, preferably wherein said peptide features biological activity, and being the saccharide selected from the group consisting of monosaccharides, oligosaccharides and polysaccharides.

5. The AAV vector particle according to any of claims 2 to 4, wherein the peptide is a blood brain barrier (BBB) shuttle peptide, preferably a BBB shuttle peptide selected from the group consisting of a peptide THR or a peptide with a RGD motif, including a cyclic RGD peptide; and wherein the saccharide is selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose trehalose, desosamine, glucuronic acid, S6-galactose, S6-N- acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose and P1- fructose.

6. The AAV vector particle according to any of claims 1 to 5, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12; or pseudotypes, chimeras, and variants thereof.

7. The AAV vector particle according to any of claims 1 to 6, wherein the moiety of formula (II) is selected from the group consisting of formula (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (IIm), (IIn), (IIp), (IIq), (IIr), (IIs), (IIt), (IIv), (IIw) and (IIx):

(IIc),

being at least one of R_a, R_b and R_c a group R’, and wherein Z is a peptide or a saccharide, Ar is arylene group or a heteroarylene group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se; R₁ is selected from the group consisting of H, C_1-6 alkyl, C_1-6 haloalkyl and Z-(OCH₂-CH₂)n-, Z- C(O)NH-(CH₂)q-(OCH₂-CH₂)n- and Z-NHC(O)-(CH₂)q-(OCH₂-CH₂)n-; preferably R1 is H; m₁ and m₂ are each independently 0, 1 or 2, m₃, m₄, m₅ and m₆ are each independently selected from 1 to 6, preferably 1 or 2, and n and n’ are each independently selected from 1 to 40, preferably 1 to 20, and q is selected from 1 to 3.

8. A pharmaceutical composition comprising an AAV vector particle according to any of claims 1 to 7, and at least one pharmaceutically acceptable vehicle.

9. An AAV vector particle according to any of claims 1 to 7, or a pharmaceutical composition according to claim 8, for use as a medicament.

10. An AAV vector particle according to any of claims 1 to 7, or a pharmaceutical composition according to claim 8, for use in gene therapy.

11. A method of delivering a nucleic acid to a cell, the method comprising contacting a cell with an AAV vector particle according to any one of claims 1 to 7 and a nucleic acid to be expressed in the contacted cell.

12. A compound of formula (Illa):

wherein

Z is H or a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties, and

L consists of one or more groups selected from the group consisting of an arylene or a heteroarylene group, an optionally substituted group comprising saturated or unsaturated, linear or branched C₂-C₄₀ hydrocarbon chains, a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, a polypropylene glycol (PPG) comprising 1 to 40 propylene glycol monomers, a polyether of a branched C_3-12 polyol, an arginine derivative, a β-alanine polymer comprising 1 to 40 β-alanine monomers, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof; and wherein L comprises at least one or more groups selected from the group consisting of an arylene or a heteroarylene group, a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and a β-alanine polymer comprising 1 to 40 β-alanine monomers.

13. The compound of claim 12, wherein L comprises more than one spacers groups selected from the group consisting of L₁, L₂, and L₃, and said compound of formula (III) is selected from the group consisting of formula (Illa₁), (IIIa₂), (Illa₃), (IIIa₄), (Illa₅), (IIIa₆), (IIIa₇), (IIIa₈), (IIIa₉), (Illa₁₀), (Illa₁₁), (IIIa₁₂), (IIIa₁₃), (IIIa₁₄), (Illa₁₅), (IIIa₁₆), (IIIa₁₇), (IIIa₁₈), (IIIa₁₉) and (IIIa₂₀):

wherein L₁ is one or more groups selected from the group consisting of a polyethylene glycol (PEG), comprising 1 to 40, preferably 1 to 20, ethylene glycol monomers, a poly ether of a branched C_3-12 polyol, preferably a branched C_3-6 polyol, and a β-alanine polymer comprising 1 to 40, preferably 1 to10, β-alanine monomers, or a mixture thereof; L₂ comprises one or more arylene or a heteroarylene groups; L₃ is one or more groups selected from the group consisting of an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, a C_1-6 alkylene group, being said C_1-6 alkylene a linear C_1-6 alkylene group or a branched C_3-6 alkylene group; a C_1-6 alkylenamine group and C_1-6 acyl group, being said C_1-6 alkylene a linear C_1-6 alkylene group or a branched C_3-6 alkylene group; wherein L₃ is covalently linked to L₂ by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and wherein L₁ and L₂ or L₁ and L₃ are covalently linked by an amide moiety or a bioisostere moiety thereof, or wherein L₁ and L₃ covalently linked by an ether bond.

14. A method for manufacturing an AAV vector particle according to any one of claims 1 to 7, wherein said method comprises reacting a compound of formula (III) according to any of claims 12 or 13 with an amino group present within the capsid of the AAV vector.