Squaramide-modified viral vectors

Abstract

Viral capsid comprising a viral capsid protein covalently linked to a functional group are described, wherein the functional group comprises a crosslinked group resulting from a click-chemistry reaction between a first and a second group forming a click-chemistry reactive pair; the viral capsid protein is covalently attached to a squaramide moiety of formula (SQ): (SQ) wherein N* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein; ---- represents the point of attachment to the viral capsid protein; and N is a nitrogen atom of the functional group.

Description

SQUARAMIDE-MODIFIED VIRAL VECTORSFIELD OF INVENTION

[0001] The present invention relates to viral vectors, in particular adeno-associated virus (AAV) vectors, modified by the covalent coupling of a squarate ester to a primary amino group of a natural amino acid residue of the capsid, wherein the formed squaramide group is covalently linked to a functional group. In some embodiments, provided viral vectors are useful in delivering a nucleic acid to a cell, especially for treating a disease.BACKGROUND OF INVENTION

[0002] The delivery of nucleic acid sequences into cells can be carried-out by various methods, such as using a viral vector which is genetically engineered to deliver the sequences of interest. Examples of types of viral vectors used for those purposes are adeno-associated virus, enveloped viruses, such as herpes simplex virus, lentiviruses and retroviruses. In particular, recombinant adeno-associated virus (AAV) vectors are widely used for that purpose because of their ability to transduce both dividing and non-dividing cells, their relatively broad tropism, their long-term persistence as episomal DNA in transduced cells, and a high safety profile, in particular because wild-type AAV is not associated with any human diseases. These characteristics make them appealing for applications in therapeutic applications, such as gene therapy.

[0003] Clinical trials of gene therapy using AAV vectors were conducted or are ongoing for several types of diseases. Nevertheless, many current AAV vectors are insufficient at transducing certain cell types and tissues and / or show unselective distribution. Moreover, the ability of the immune system to recognize and mount an immune response against current AAV vectors limits the clinical use of such vectors, particularly in patients with pre-existing immunity against AAV or where vectors are to be administered multiple times.

[0004] In particular, it was evidenced that many current AAV vectors have poor transduction efficiency in desired target cells or tissues, necessitating high doses of vectors. These high doses not only pose challenges for vector production, but alsoincrease the risk of immune response and toxicity. Consequently, more infectious AAV vectors would be highly desirable.

[0005] Another limitation of AAV vectors is their broad tropism, which results in transgene expression in both target and non-target cells, thus lacking specificity. This can lead to reduced efficacy and increased toxicity. Consequently, there is a need for AAV vectors with improved transduction of specific cells for more targeted expression.

[0006] AAV infects cells through various capsid / receptor interactions that ultimately results in internalization of the virus capsid by the infected cells and transduction. These capsid / receptor interactions can be manipulated to enhance the desired tropism of AAV vectors, by creating novel interactions with a specific receptor or marker, either alone or in combination with modifications decreasing or eliminating the natural tropism of the virus.

[0007] The most common approach to manipulate ligand / receptor interactions is to genetically modify the capsid proteins, and thus the viral capsid surface. Viral capsid proteins are genetically engineered to express a targeting ligand. However, this approach requires positioning the targeting ligand within a tolerable location that does not interfere with capsid assembly during production while driving productive interaction with the targeted receptor. This implies size constraints on the ligand and / or limits the range of usable molecules. Moreover, the number of ligands is fixed by the number of modified capsid units, reducing flexibility. Finally, ligands inserted directly into the virus capsid are often not modular and must be redesigned for each serotype.

[0008] Alternatively or additionally, the capsid protein is modified prior assembly to present an “anchor” or “signature”, which is conjugated post-production to a targeting ligand. Modification of the AAV capsid proteins is generally achieved by (1) a single site genetic substitution on the capsid, (2) genetic insertion of a peptide tag, or (3) insertion of non-canonical amino acids in the capsid proteins (e.g., azido-lysine supplemented into the cell media). For example, Erickson SB et al. (Bioconjug Chem. 2024 Jan 17;35( l):64-71. doi: 10.1021 / acs.bioconjchem.3c00411) propose site-specific incorporation of bioorthogonal noncanonical amino acids (ncAA), such as azidomodified lysines, into the AAV capsid proteins for cRGD-mediated retargeting. WO2021222899A1 proposes replacing at least one capsid protein with a mutated protein at one or more amino acid residues to incorporate other natural amino acids (for examplecysteine) or unnatural amino acids (for example bearing an azido group for biorthogonal conjugation) relative to the naturally occurring amino acids of the wild-type viral capsid protein. Puzzo et al. (Mol Ther Nucleic Acids. 2023 Jan 21 ;31:383-397. doi: 10.1016 / j.omtn.2023.01.007) propose modifying viral capsid proteins by a single site substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry. WO2024129990A2 also proposes the use of variant AAV capsid proteins including an azide-bearing unnatural amino acid substitution. Although the introduction of a “scaffold” or “signature” in the capsid protein pre-assembly is advantageous because of the flexibility and modular nature of the targeting ligand used, this adds significant complexity in the preparation of the AAV vectors and often impacts manufacturing productivity. Moreover, genetically modifying the capsid to introduce the “scaffold” or “signature” suffers the same limitations as genetically inserting the ligand into the capsid in terms of size and location of the “scaffold” or “signature” to maintain the structure of the capsid, and / or in the number of the “scaffold” or “signature” tags that can be introduced per capsid unit.

[0009] Another strategy is to perform a chemical modification on the viral capsid postassembly, without modification of the capsid proteins prior production. For example, W02017 / 212019 provides surface modified AAV vectors obtained by covalently coupling a ligand bearing a certain isothiocyanate linker 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. On the other hand, WO2022 / 096681 provides surface modified vectors obtained by reaction of a compound comprising a lactam (e.g., P-lactam) linker with an amino group present in a natural amino acid residue of the capsid proteins of the AAV.

[0010] But not all these coupling chemistries are compatible with and / or effective under particular conditions and / or with particular types of ligands.

[0011] Accordingly, there remains a need for new methods for improving the desired properties of AAV vectors (already genetically modified or not), not requiring modifications of the vector prior assembly for subsequent conjugation, while remaining adaptable for nucleic acid delivery into a variety of target cells.SUMMARY

[0012] The present invention relates to the provision of modified viral capsids comprising functional groups covalently attached to a viral capsid protein for gene delivery and gene therapy. In particular, the present invention provides viral vector particles, preferably AAV vector particles, with a viral capsid comprising functional groups covalently attached to a viral capsid protein.

[0013] Thus, one aspect of the present invention relates to a viral capsid, preferably an AAV capsid, comprising a viral capsid protein covalently linked to a functional group, wherein the functional group comprises a crosslinked group resulting from a clickchemistry reaction between a first and a second group forming a click-chemistry reactive pair; the viral capsid protein is covalently attached to a squaramide moiety of formula (SQ):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; andN is a nitrogen atom of the functional group.

[0014] The viral capsid is not particularly limited and can be selected from nonenveloped viruses, such as adenoviruses and parvoviruses including bocavirus and AAVs, or from enveloped viruses, such as herpes simplex virus, lentiviruses and retroviruses. In particular, the viral capsid is a viral capsid of an AAV.

[0015] In some embodiments, the click-chemistry reaction is selected from the group consisting of a strained-promoted alkyne-azide click-chemistry (SPAAC) reaction, astrain-promoted alkyne-nitrone cycloaddition (SPANC); and an inverse electron-demand Diels-Alder (IEDDA) reaction.

[0016] In some embodiments, the click-chemistry reactive pair is selected from the group consisting of i. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine; viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyne; wherein each of the groups azide, strained alkyne, tetrazine, olefine, alkyne, 1,2,3- triazine, amidine, guanidine and 1,2,4-triazine are as defined in the claims and embodiments of the present invention.

[0017] In some particular embodiments, said viral capsid comprises a viral capsid protein covalently linked to a moiety of formula (I):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; andZ is H or is a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof;each Si and S2 comprise independently a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branched polyol, a P- alanine polymer, pHPMA, PLGA, a sarcosine polymer and combinations thereof; andR is a crosslinked group resulting from a click-chemistry reaction between a first and a second group forming a click-chemistry reactive pair, wherein the click-chemistry reaction and the first and the second group forming a click-chemistry reactive pair are as defined in the embodiments and claims of the present invention.

[0018] In some embodiments, the viral capsid comprising a moiety of formula (I) results from a click chemistry reaction between a compound of formula (II) and a viral capsid covalently linked to a moiety of formula (III):wherein the crosslinked group R of the moiety of formula (I) results from a click chemistry reaction between R3 and R4, wherein R3 and R4 are, respectively, either the first group or the second group forming a click-chemistry reactive pair, and wherein the first and the second group forming a click-chemistry reactive pair, Z, Si, S2, N, N* and - — , are as defined in the embodiments and claims of the present invention.

[0019] Yet another aspect of the present invention relates to a viral vector particle comprising a nucleic acid and the viral capsid as defined in the embodiments and claims of the present invention, preferably wherein the viral capsid is an AAV capsid of an AAV vector selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO and AAVrh74; or pseudotypes, chimeras, and variants thereof.

[0020] Another aspect of the present invention relates to a viral capsid comprising a viral capsid protein covalently linked to a moiety of formula (III):wherein R4 is, either the first group or the second group forming a click-chemistry reactive pair; preferably R4 is selected from the group consisting of an azide, a strained alkyne, a tetrazine, a cyclic olefine, an alkyne, a 1,2,3-triazine, an amidine, and a 1,2,4- triazine; and whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; and Si comprises a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branched polyol, a P-alanine polymer, pHPMA, PLGA, a sarcosine polymer, and combinations thereof; being said viral capsid covalently linked to a moiety of formula (III) an intermediate in the manufacturing a viral capsid covalently linked to a moiety of formula (I) as defined in the embodiments and claims of the present invention.

[0021] Another aspect of the present invention relates to the use of a pair of compounds for manufacturing a viral vector particle, preferably an AAV vector particle, wherein said pair of compounds consists of a compound of formula (II):and a compound of formula (IV), or a pharmaceutically acceptable salt thereof:whereinRi is selected from the group consisting of a linear Ci-12 alkyl, a branched C3-12 alkyl, a linear Ci-12 haloalkyl, a branched C3-12 haloalkyl, benzyl, phenyl and pyridyl;Z is H or is a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof; each Si and S2 comprise independently a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branched polyol, a P- alanine polymer, pHPMA, PLGA, a sarcosine polymer, and combinations thereof; andR3 and R4 are, respectively, either the first group or the second group of a click-chemistry reactive pair, said click-chemistry reactive pair selected from the group consisting of i. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine; viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyne; wherein each of the groups azide, strained alkyne, tetrazine, olefine, alkyne, 1,2,3- triazine, amidine, guanidine and 1,2,4-triazine are as defined in the claims and embodiments of the present invention.

[0022] Another aspect of the present invention relates to a method for manufacturing a viral vector particle, preferably an AAV vector particle, said method comprising: reacting a viral particle, preferably an AAV vector, with a compound of formula (IV):in conditions suitable for reacting a squarate moiety of the compound of formula (IV) with at least one amino group from a surface-exposed natural amino acid residue of a viral capsid protein from the viral vector so as to form a viral vector particle, preferably an AAV vector particle comprising a moiety of formula (III):reacting the viral vector particle, preferably the AAV vector particle comprising a moiety of formula (III) with a compound of formula (II):in conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react and obtain a viral vector particle, preferably an AAV vector particle comprising a moiety of formula (I):whereinRi is selected from the group consisting of a linear Ci-12 alkyl, a branched C3-12 alkyl, a linear Ci-12 haloalkyl, a branched C3-12 haloalkyl, benzyl, phenyl and pyridyl;R is a group resulting from a click-chemistry reaction between a first and a second group of a click-chemistry reactive pair, wherein R3 and R4 are, respectively, either the first group or the second group of said click-chemistry reactive pair, preferably R3 and R4 are, respectively, either the first group or the second group of said click-chemistry reactive pair selected from the group consisting ofi. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine; viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyne; wherein each of the groups azide, strained alkyne, tetrazine, olefine, alkyne, 1,2,3- triazine, amidine, guanidine and 1,2,4-triazine are as defined in the claims and embodiments of the present invention;N* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein;Z is H or is a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof; and each Si and S2 comprise independently a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branched polyol, a P- alanine polymer, pHPMA, PLGA, a sarcosine polymer and combinations thereof.

[0023] Another aspect of the present invention relates to a pharmaceutical composition comprising a viral vector particle, preferably an AAV vector particle, as defined in the embodiments and claims of the present invention, and at least one pharmaceutically acceptable vehicle.

[0024] Another aspect of the present invention relates to a viral vector particle, preferably an AAV vector particle, or to a pharmaceutical composition, as defined in the embodiments and claims of the present invention, for use as a medicament.

[0025] Yet another aspect of the present invention relates to a viral vector particle, preferably an AAV vector particle, or to a pharmaceutical composition, as defined in the embodiments and claims of the present invention, for use in gene therapy.

[0026] Another aspect of the present invention relates to a viral vector particle, preferably an AAV vector particle, or to a pharmaceutical composition, as defined in the embodiments and claims of the present invention, for use in a method of delivering a nucleic acid to a cell, the method comprising contacting a cell with said viral vector particle comprising a nucleic acid to be expressed in the contacted cell.

[0027] Yet another aspect of the invention relates to a non-therapeutic method of delivering a nucleic acid to a cell, the method comprising contacting a cell with a viral vector particle, preferably an AAV vector particle, as defined in the embodiments and claims of the present invention, comprising a nucleic acid to be expressed in the contacted cell.

[0028] Preferably the viral vector particle is a parvovirus particle, more preferably a bocavirus or an AAV particle, even more preferably an AAV particle.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTSGeneral definitions

[0029] The term “alkyl” refers to a monovalent or divalent, linear or branched, saturated hydrocarbon chain, comprising 1-12 carbon atoms (also named Ci-12 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” 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.

[0030] 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, ethylcarbonyl, or benzoyl group.

[0031] The term “alkoxy” or “alkyloxy” refers to a -O-R group wherein the R group may be an alkyl, an aryl, a haloalkyl or an arylalkyl group, as defined herein, is connectedto the remainder of the molecule through an oxygen atom. O-cycloalkyl includes for example the O-cyclopentyl or O-cyclohexyl group.

[0032] 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, Ci-6 alkyl, Ci-6 haloalkyl, Ci-6 acyl and Ci-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, Ci-6 alkyl, Ci-6 haloalkyl, Ci-6 acyl and Ci-6 alkoxy.

[0033] 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, thiatriazolyl, 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, tetrazolof l,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, Ci-6 alkyl, Ci-6 haloalkyl, Ci-6 acyl and Ci-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, Ci-6 alkyl, Ci-6 haloalkyl, Ci-6 acyl and Ci-6 alkoxy.

[0034] 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)).

[0035] The term “unsaturated”, as used herein, means that a moiety has one or more units of unsaturation.

[0036] The term “halogen” means F, Cl, Br, or I.

[0037] The term “arylalkyl” refers to a -Aik- Ar group or to an -Ar- Aik group, wherein Aik represents an alkyl group as defined earlier, and Ar represents an aryl group as defined earlier.

[0038] 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.

[0039] 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 thecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantly, decalinyl, or norbomyl groups.

[0040] 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.

[0041] The term “azide” refers to a linear, a branched or a cyclic hydrocarbon chain (saturated, comprising unsaturations or aromatic groups) containing optionally one or more heteroatoms, which is substituted with a group -N3 (i.e. -N=N+=N"). Examples of azides, according to the present invention, are those which are adapted to react with a strained alkyne in a strain-promoted alkyne-azide click chemistry (SPAAC) reaction. In some embodiments, the azide. In some embodiments the azide is preferably an aryl azide, preferably a phenyl azide. When the azide is a phenyl azide?said azide may be in orto, meta or para position.

[0042] The term “nitrone” refers to a linear, a branched or a cyclic hydrocarbon chain (saturated, comprising unsaturations or aromatic groups) containing optionally one or more heteroatoms, which is substituted with a group -CRa=+NRb-O'wherein Raand Rbmay be H or another group as long as the resulting nitrone retains the desired reactivity in a strain-promoted alkyne-nitrone click chemistry (SPANC) reaction.

[0043] The term “alkyne” refers to a linear, a branched or a cyclic hydrocarbon chain, containing optionally one or more heteroatoms, which comprises a carbon-carbon triple bond -C=C- featuring a linear geometry. Examples of alkynes, according to the present invention, are those which are adapted to react with a tetrazine or with a 1,2,4-triazine in an inverse electron demand Diels-Alder click-chemistry reaction. In some embodiments, the alkyne is selected from the group consisting of:

[0044] On the other hand, the term “strained alkyne” refers to a linear, a branched or a cyclic hydrocarbon chain, containing optionally one or more heteroatoms, which comprises a carbon-carbon triple bond -C=C- featuring a bond angle, a geometry distorted from the ideal linear geometry of regular alkynes, and imparts to the compound or group with a higher reactivity, lowering the amount of energy required to react with dienes, 1,3- dipoles, and other molecular systems. Examples de strained alkynes are, in particular, those which are adapted to react with an azide in a SPAAC reaction, or are adapted to react with a nitrone in a SPANC reaction, or are adapted to react with a tetrazine or with a 1,2,4-triazine in an IEDDA reaction. In some embodiments, the strained alkyne is selected from the group consisting of dibenzoazacyclooctyne (DBCO), dibenzylcyclooctyne (DIBO), bicyclononyne (BCN), cyclooctyne (OCT), monofluorated cyclooctyne (MOFO), difluorooctyne (DIFO), dimethoxy azacyclooctyne (DIMAC), carboxymethylmonobenzocyclooctyne (COMBO), biarylazacyclooctynone (BARAC), 2,3,6,7-tetramethoxy-dibenzylcyclooctyne (TMDIBO), sulfonylated dibenzylcyclooctyne (S-DIBO), pyrrolocyclooctyne (PYRROC) and 3, 3,6,6- tetramethylthiaheptyne (TMTH). In some embodiments the strained alkyne is DIB AC or BCN. Particularly, the strained alkyne may comprise one moiety selected from the group consisting of:

[0045] In a preferred embodiment the strained-alkyne is:

[0046] The term “tetrazine” refers to a linear, a branched or a cyclic hydrocarbon chain (saturated, comprising unsaturations or aromatic groups) containing optionally one or more heteroatoms, substituted with a 6-membered aromatic ring containing 4 nitrogen atoms in the form of 1,2,4,5-tetrazines. Examples of tetrazines are those which are adapted to react with olefines (cyclic olefines), alkynes and strained alkynes in an IEDDAreaction. In some embodiments, the tetrazine is a 1,2, 4, 5 tetrazine comprising an alkyl or an electron withdrawing group, more preferably the tetrazine is an aryl tetrazine comprising an alkyl or an electron withdrawing group, even more preferably the tetrazinewherein R2 is selected from the group consisting of H,C1-12 alkyl, and an electron withdrawing group. In some embodiments, R2 is selected from the group consisting of H, Ci-12 alkyl, Ci-12 haloalkyl, aryl, heteroaryl, -CN, -C(0)NH2, - S(O)2Raand -C(O)Ra, wherein Rais an Ci-12 alkyl group. In a preferred embodiment R2 is selected from the group consisting of H and Ci-12 alkyl, more preferably selected from the group consisting of H and methyl.

[0047] The term “olefine” refers for the purposes of the click chemistry reactions disclosed herein to a cyclic hydrocarbon chain containing optionally one or more heteroatoms, comprising a double bond -C=C- Accordingly, for the purposes of the present disclosure the terms “olefine” and “cyclic olefine” refer both to a cyclic hydrocarbon chain containing optionally one or more heteroatoms, comprising a double bond -C=C-. For the purposes of the present invention, preferred olefines are cyclic hydrocarbon groups, comprising an electron donating group, as defined herein, as a substituent. Examples of olefines are those which are adapted to react with tetrazines or 1,2,4-triazine in an IEDDA reaction. In some embodiments, the olefine is selected from the group consisting of cyclopropene, transcyclooctene, noroborene and N-acylazetine. In some embodiments the olefine is selected from the group consisting of methylcyclopropene, transcyclooctene and noroborene, more preferably cyclopropene and transcyclooctene; even more preferably the olefine is selected from the group consisting

[0048] The term “1,2,3-triazine” refers to a linear, a branched or a cyclic hydrocarbon chain (saturated, comprising unsaturations or aromatic groups) comprising optionally oneor more heteroatoms, substituted with 6-membered aromatic ring with 3 nitrogen atoms in positions 1, 2 and 3. Examples of 1,2,3-triazines are those which are adapted to react with guanidines or amidines in an IEDDA reaction. In some embodiments the 1,2,3- triazine is an aryl 1,2,3-triazine:

[0049] The term “1,2,4-triazine” refers to a linear, a branched or a cyclic hydrocarbon chain (saturated, comprising unsaturations or aromatic groups) comprising optionally one or more heteroatoms, substituted with 6-membered aromatic ring with 3 nitrogen atoms in positions 1, 2 and 4. Examples of 1,2,4-triazines are those which are adapted to react with olefines (cyclic olefines), alkynes and strained alkynes in an IEDDA reaction. In some embodiments the 1,2,4-triazine is an aryl 1,2,4-triazine:

[0050] The term “amidine” refers to a group. Examples of amidines are those which are adapted to react with 1,2,3-triazines in an IEDDA reaction.

[0051] The term “guanidine” refers to a group. Examples of guanidines are those which are adapted to react with 1,2,3-triazines in an IEDDA reaction

[0052] The term “electron withdrawing group” refers to an atom or a group of atoms that draws electron density from neighboring atoms towards itself, usually by resonance or inductive effects. Examples of electron withdrawing groups are -C(O)OR, where R is an alkyl group, -CN, -C(O)NRR’, wherein R and R’ are each independently, either H and alkyl, -S(O2)R, where R is an alkyl group, an haloalkyl group, -C(O)R, where R is an alkyl group, aryl groups and heteroaryl groups, among others. On the other hand, the term“electron donor group” refers to an atom or a group of atoms that releases electron density to neighboring atoms from itself, usually by resonance or inductive effects. Examples of electron donor groups are alkyl (linear or branched), alcohols, amines, ethers, alkoxy groups, among others.

[0053] As described herein, compounds may contain “optionally substituted” moieties. 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 thatQRasuitable 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.

[0054] 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’, wherein R’ and each of the substituents are as defined above herein, among others.

[0055] As used herein, the term “pharmaceutically acceptable salt” includes conventional salts formed from pharmaceutically acceptable inorganic or organic acidsor 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, salt forms of the compounds of formula (IV) disclosed herein are within the scope of the present description.

[0056] 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.

[0057] 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".

[0058] 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”. Stereoisomers 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, regio isomers 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, Z and E conformational isomers, or orto, meta and para regio-isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, regio and geometric (or conformational) mixtures of the present compounds are within the scope of the present disclosure. In particular, the products of a click chemistry reaction may include regio isomers, for example, when the strained alkyne triple bond does not comprise symmetry axis. Unless otherwise stated, all isomeric forms of the click chemistry crosslinked group are within the scope of the disclosure.

[0059] 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”.

[0060] 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. Examples of bioisostere moieties include, for example, bioisostere moieties of the squarate groups, such as dithiosquarates and dithionosquarates, preferably dithionosquarates, or bioisostere moieties of the squaramide group, such as dithionosquaramides, as discussed by Taylor et al., J. Am. Chem. Soc. 2023 November 22; 145(46):25056-25060, or bioisostere moieties of an amide moiety which may be selected from -C(O)N(R)-, -N(R’)C(O)N(R)-, -N(R’)C(O)N(R)-, -N(R)C(S)-, -C(S)N(R)-, -N(R)C(S)N(R’)-, -N(R)C(S)N(R’)-, -S(O)2-N(R)-, -N(R)-S(O)2-, and a triazolyl group, among others, wherein R and R’ are each independently selected from the group consisting of H, Ci-6 alkyl, Ci-6 haloalkyl, aryl, alkylaryl, alkylether, alkylamide, among others.

[0061] 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 a13C- or14C-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 inaccordance with the present disclosure. In some embodiments, compounds of this disclosure comprise one or more deuterium atoms.

[0062] 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).Viral capsid comprising a functional group covalently linked to a squaramide of formula (SQ)

[0063] Various coupling chemistries that conjugate a viral capsid, for example a viral capsid from an AAV, through an amino group (e.g., of a lysine sidechain) of a viral capsid protein have been described.

[0064] Some coupling strategies include 2 step coupling reactions, with the modification of the capsid prior to assembly of the capsid, to introduce an “anchor” or “signature”, which is conjugated post-production to a targeting ligand. However, this adds significant complexity in the preparation of the viral vectors and often reduces efficiency of production.

[0065] In that context, a one-step post capsid assembly coupling chemistry have 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. WO2022 / 096681 discloses surface modified vectors obtained by 1-step reaction of a compound comprising a lactam (e.g., P-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.

[0066] In addition, as previously mentioned, not all such one-step, post capsid assembly coupling chemistries are compatible with and / or effective under particular conditions and / or with particular substrates or ligands.

[0067] For example, when directly conjugating the viral capsid with a ligand, certain coupling chemistries require or are typically performed under conditions (pH, temperature, etc.) that may disrupt one or more structural or functional properties of the viral capsid (e.g., do not preserve AAV capsid integrity) or of the ligand (e.g. mAb, scFv, Fab region, etc.). Alternatively or additionally, various coupling chemistries require or are typically performed under conditions that are not compatible for the coupling of certain biochemical ligands, in particular ligands comprising residues which will affect the bioconjugation reaction between AAV and ligand. The present disclosure therefore recognizes a particular remaining need to expand and improve post-assembly viral capsid coupling methods, to provide solutions that i) are compatible with and maintain the integrity of a broad variety of AAV serotypes, ii) are compatible with and maintain the integrity of ligands; 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) is compatible with a larger variety of ligands (such as large peptides, mAb, Fab region, for example) and AAV serotypes.

[0068] 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 direct chemical modification of the viral capsid, and without need for any preliminary mutation of the capsid proteins. Moreover, by using a click chemistry reactive pair, a 2-step coupling of a wide variety of ligands is possible, reducing in that manner the risk of disrupting one or more structural or functional properties of the AAV and of the ligand, without the need of introducing an “anchor” or “signature”, which is conjugated post-production to a targeting ligand, and at the same time, avoiding cross-reactivity between reactive groups of some type of ligands which would compete in the bioconjugation reaction with the AAV.

[0069] The conjugation based on squaramide linkers, especially in a 2 steps method as described and claimed in the present invention, represents a well-controlled process of modification of natural amino acids only at the surface of the viral capsids, withoutunconjugated ligands, under optimal conditions (that are both suitable to viral capsids and end groups such as peptides and proteins) avoiding a potential cross reactivity between the squaramide linkers and these particular end groups.

[0070] Thus, the present invention relates to a viral capsid comprising a viral capsid protein covalently linked to a functional group, wherein the functional group comprises a crosslinked group resulting from a click-chemistry reaction between a first and a second group forming a click-chemistry reactive pair; the viral capsid protein is covalently attached to a squaramide moiety of formula (SQ):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; andN is a nitrogen atom of the functional group; wherein the functional group, the click chemistry reaction, the click chemistry pair, the first and second groups forming said click-chemistry reactive pair, the surface-exposed natural amino acid residue, the viral capsid protein and the viral capsid, are as defined in the claims and embodiments of the present invention.

[0071] In particular, the viral capsid may be the viral capsid from a non-enveloped virus, preferably a parvovirus particle, such as a bocavirus and an AAV vector particle. In some embodiments, an AAV vector modified according to the present invention comprises at least a nucleic acid, in particular, at least a transgene which is selected in view of the intended use of the AAV vector.

[0072] Thus one aspect of the invention refers to an AAV vector particle comprising a viral capsid protein covalently linked to a functional group, wherein the functional group comprises a crosslinked group resulting from a click-chemistry reaction between a first and a second group forming a click-chemistry reactive pair; the viral capsid protein is covalently attached to a squaramide moiety of formula (SQ):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; andN is a nitrogen atom of the functional group; wherein the functional group, the click chemistry reaction, the click chemistry pair, the first and second groups forming said click-chemistry reactive pair, the surface-exposed natural amino acid residue, the viral capsid protein and the viral capsid, are as defined in the claims and embodiments of the present invention.Viral capsid comprising a moiety of formula (I)

[0073] In some embodiments of the invention, the functional group of the viral capsid, as disclosed in the claims and embodiments of the present invention, comprises an end group Z, and two spacers groups Si and S2 covalently linked by a crosslinked group R, resulting from a click-chemistry reaction between a first and a second group forming a click-chemistry reactive pair are as defined in the embodiments and claims of the present invention, being a first of the spacers groups Si covalently linked to the squaramidemoiety of formula (SQ) and the end group Z covalently linked to the second of the spacers groups S2.

[0074] Thus, in some embodiments, the viral capsid comprising a squaramide moiety of formula (SQ), as defined in the claims and embodiments of the present invention, comprises a viral capsid protein covalently linked to a moiety of formula (I):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; and wherein Z, Si and S2 and R are as defined in the claims and embodiments of the present invention, in particular:Z is H or is a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof; each Si and S2 comprise independently a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branched polyol, a [3- alanine polymer, pHPMA, PLGA, a sarcosine polymer and combinations thereof; andR is a crosslinked group resulting from a click-chemistry reaction between a first and a second group forming a click-chemistry reactive pair, wherein the click-chemistry reaction and the first and the second group forming a click-chemistry reactive pair are as defined in the embodiments and claims of the present invention.

[0075] In some embodiments, the squaramide moiety (SQ) is covalently attached to a group of the functional group which is different from the crosslinked group resulting from a click chemistry reaction.

[0001] In particular, the viral capsid comprising a viral capsid protein covalently linked to a moiety of formula (I) may be the viral capsid from an AAV vector particle. In some embodiments, an AAV vector comprising a viral capsid protein covalently linked to a moiety of formula (I), as defined in the claims and embodiments of the present invention, comprises at least a nucleic acid, in particular, at least a transgene which is selected in view of the intended use of the AAV vector.Click chemistry reaction and click chemistry pairs - R3 and R4

[0076] The term “click chemistry reactions” refers a set of efficient and selective chemical reaction, referring specifically the term “bioorthogonal reaction” to click chemistry reactions that can occur within living systems without interfering with natural biological processes, and refer to highly selective chemical processes which are: biocompatible covalent bonds, ideally in a water based environment, at room temperature, with quick kinetics, chemo- selective and resulting in low amounts of (non-toxic) byproducts. Accordingly, a click chemistry reaction according to the present invention is: (1) biocompatible, (2) highly selective, (3) features quick kinetics and (4) may be carried out in aqueous media so that both ingredients and products are stable in said aqueous conditions (Vida Terzic, PhD thesis, Universite Paris-Saclay, 31-08-2016).

[0077] The term biocompatible reaction refers to a reaction which may be carried out at physiological conditions and compatible with a living. The term selective refers to the fact that the reaction ingredients react in a specific manner among each other and irreversibly, without intervention of biomolecules present in the reaction medium (enzymes, biological nucleophiles such as thiol or amine groups, etc.) (Vida Terzic, PhD thesis, Universite Paris-Saclay, 31-08-2016; https: / / theses.fr / 2016SACLS190).

[0078] In some embodiments, the click-chemistry reaction is selected from the group consisting of a strained-promoted alkyne-azide click-chemistry (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC); and an inverse electron-demand Diels-Alder (IEDDA) reaction.

[0079] In some embodiments, the click-chemistry reactive pair is selected from the group consisting ofi. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine); viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyne; wherein each of the groups azide, strained alkyne, tetrazine, olefine, alkyne, 1,2,3- triazine, amidine, guanidine and 1,2,4-triazine are as defined in the claims and embodiments of the present invention.

[0080] In some embodiments R3 and R4 are, respectively, either the first group or the second group of a click-chemistry reactive pair, said click-chemistry reactive pair selected from the group consisting of i. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine; viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyne; wherein each of the groups azide, strained alkyne, tetrazine, olefine, alkyne, 1,2,3- triazine, amidine, guanidine and 1,2,4-triazine are as defined in the claims and embodiments of the present invention.

[0081] A strained-promoted alkyne- azide click-chemistry (SPAAC) reaction is a [3+2] cycloaddition between an azide and a strained-alkyne, i.e. a compound comprising a triple bond featuring a bond angle, a geometry distorted from the ideal linear geometry of regular alkynes. Accordingly, when the click chemistry reaction is a SPAAC reaction, theclick chemistry pair is an azide and a strained alkyne. A SPAAC reaction between a strained alkyne and an azide may be illustrated schematically herein below:Strained alkyne

[0082] In some embodiments the click chemistry reaction is a SPAAC reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either an azide -N3 or a strained alkyne selected from the group consisting of:and the resulting crosslinked group, in particular the crosslinked group R, is, respectively:including any regio isomer and stereoisomer thereof.

[0083] In some embodiments the click chemistry reaction is a SPAAC reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either an aryl azide or a strained alkyne selected from the group consisting of:and the resulting crosslinked group, in particular the crosslinked group R, is, respectively:

[0084] A strained promoted alkyne-nitrone cycloaddition (SPANC) is also a [3+2] cycloaddition between a nitrone and a strained triple bond of a strained alkyne featuring a bond angle, a geometry distorted from the ideal linear geometry of regular alkynes. Accordingly, when the click chemistry reaction is a SPAAC reaction, the click chemistry pair is a nitrone and a strained alkyne. A SPANC reaction between a strained alkyne and a nitrone may be illustrated schematically herein below:NitroneStrained alkyne

[0085] An inverse electron-demand Diels-Alder (IEDDA) reaction is a [4+2] cycloaddition between an electron rich dienophile and electron poor diene resulting in the formation of a 6-membered ring. In some embodiments, when the click chemistry reaction is an IEDDA reaction, the click chemistry pair is selected from the group consisting of a tetrazine and a cyclic olefine, a tetrazine and an alkyne, a tetrazine and a strained alkyne, a 1,2,3-triazine and an amidine, a 1,2,3-triazine and a guanidine, a 1,2,4- triazine and a cyclic olefine, a 1,2,4-triazine and an alkyne and a 1,2,4-triazine and a strained alkyne.

[0086] An IEDDA reaction between a tetrazine and a cyclic olefine may be illustrated schematically herein below:OlefineTetrazine

[0087] In some embodiments the click chemistry reaction is an IEDDA reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either a tetrazine:or an olefine selected from the group consisting of:and the resulting crosslinked group, in particular the crosslinked group R, is, respectively:wherein R2 is as defined in the claims and embodiments of the present invention.

[0088] An IEDDA reaction between a tetrazine and an alkyne or a strained alkyne may be illustrated schematically herein below:Alkyne or Strainedalkyne Tetrazine

[0089] In some embodiments the click chemistry reaction is an IEDDA reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either a tetrazine:or an alkyne selected from the group consisting of:and the resulting crosslinked group, in particular the crosslinked group R, is, respectively:including any regio isomers thereof, and wherein R2 is as defined in the claims and embodiments of the present invention.

[0090] In some embodiments the click chemistry reaction is an IEDDA reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either a tetrazine:and the resulting crosslinked group, in particular the crosslinked group R, is:including any regio isomers and stereoisomers thereof, and wherein R2 is as defined in the claims and embodiments of the present invention.

[0091] An IEDDA reaction between a 1,2,3-triazine and an amidine may be illustrated schematically herein below:

[0092] In some embodiments the click chemistry reaction is an IEDDA reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either an amidine:and the resulting crosslinked group, in particular the crosslinked group R, is:, including any regio isomers thereof.

[0093] An IEDDA reaction between a 1,2,3-triazine and a guanidine may be illustrated schematically herein below:

[0094] In some embodiments the click chemistry reaction is an IEDDA reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either a guanidine:and the resulting crosslinked group, in particular the crosslinked group R, is:, including any regio isomers thereof.

[0095] An IEDDA reaction between a 1,2,4-triazine and a cyclic olefine may be illustrated schematically herein below:Olefine 1,2,4-triazine

[0096] In some embodiments the click chemistry reaction is an IEDDA reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either a 1,2,4-triazine:and the resulting crosslinked group, in particular the crosslinked group R, is:, including any regio isomers and stereoisomers thereof.

[0097] An IEDDA reaction between a 1,2,4-triazine and an alkyne or a strained alkyne may be illustrated schematically herein below:Alkyne or Strained1,2,4-triazine alkyne

[0098] In some embodiments the click chemistry reaction is an IEDDA reaction, and the first and second group of the click-chemistry reactive pair, in particular of the click chemistry pair R3 and R4, are either a 1,2,4-triazine:or a strained alkyne selected from the group consisting of:and the resulting crosslinked group, in particular the crosslinked group R, is, respectively:, including any regio isomers and stereoisomers thereof.

[0099] The click chemistry reaction results in a crosslinked group, in particular a crosslinked group R, which is the result of the reaction between a first and a second group forming a click-chemistry reactive pair, in particular between R3 and R4. According to the present invention, said crosslinked group includes any possible isomers, regio isomers or stereoisomers (enantiomers or diastereomers), resulting from the click chemistry reaction of the click-chemistry reactive pair.End group Z

[0100] The end group Z may be H or a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof.

[0101] As used herein, the term “cell-type targeting ligand” refers to a compound (chemical or biological) that mediates binding and transduction of the target cell types, or increases transduction by different mechanisms, such as increased cell entry, and therefore can be used to increase efficiency and / or specificity of gene transfer into the targeted cell types. For example, the target cell is of a particular tissue type, and the celltype specific ligand binds to a marker protein, surface antigen, receptor protein, that is expressed by cells of the target tissue

[0102] As used herein, the term “receptor targeting ligand” refers to a compound (chemical or biological) that is able to bind to a specific receptor and direct (or target) the AAV to this receptor and / or drive subsequent AAV-receptor internalization, increasing efficiency in AAV transport and / or transduction to / of the targeted cells or tissues.

[0103] In some embodiments, Z comprises or consists of a cell-type targeting ligand, namely a ligand enabling targeting of a specific type of cell, or a receptor targeting ligand, namely a ligand enabling targeting a specific receptor. In some embodiments, such a ligand can enable modification of transduction efficiency of the AAV vector, namely its capacity to get transported, access and / or transduce a given cell line, tissue, and / or organ. 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 receptor mediating transport through natural barriers. 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.

[0104] In some embodiments, such a ligand can be, as non-limiting examples, a saccharide, in particular saccharides with amino substitutions and / or charged saccharides, a hormone, including a steroid hormone, a peptide, such as a cyclic peptide with a RGD motif, Angiopep-2 or muscle targeting peptides, a protein a glycoprotein or a fragment thereof, a membrane receptor or fragments thereof, an antibody including heavy-chainantibody, or functionally active fragments thereof such as Fab, Fab’, VHH, scFV, a nanobody, a diabody, a spiegelmer, a nucleic acid or peptide aptamer, a DARPIN, a small chemical molecules known to bind to the targeted biological entity such as vitamins, and drugs, and / or any suitable combination thereof.

[0105] In some embodiments Z can comprise a polypeptide, preferably wherein said polypeptide features biological activity; preferably the polypeptide is an antibody, an antibody fragment or an antigen-binding fragment, preferably a single-chain variable fragment (scFv) or a VHH (nanobodies).

[0106] 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.

[0107] In some embodiments, Z comprises, or consists of, a cell-type targeting ligand derived from certain types of saccharides, such as amino-substituted saccharides and saccharides with charged groups. Details on saccharides are provided hereafter.

[0108] In some embodiments, Z comprises, or consists of, a cell-type targeting ligand or a receptor targeting ligand derived from proteins such as a cytokine, transferrin, a growth factor such as Epidermal Growth Factor (EGF), and basic Fibroblast Growth Factor FGF.

[0109] 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 a 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 transport activity across the blood brain barrier. In some preferred aspects, the BBB peptide targets the transferrin receptor TfRl. In some preferred aspects, the peptide is a peptide with a RGD motif, including a cyclic RGD peptide. 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.

[0110] In some embodiments, Z comprises, or consists of, a cell-type targeting ligand or a receptor targeting ligand derived from a muscle targeting peptide. 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.

[0111] In some embodiments, Z is or comprises a peptide, such as a linear or cyclic peptide, a protein, a glycoprotein or a fragment thereof, a membrane receptor or fragments thereof, an antibody, Fab, Fab’, VHH, scFV, a nanobody, a diabody, a spiegelmer, a nucleic acid or peptide aptamer, a DARPIN; preferably Z is or comprises a cyclic peptide with a RGD motif, Angiopep-2, BBB or muscle targeting peptides, an antibody, Fab, Fab’, VHH and scFV.

[0112] In some embodiments, Z comprises, or consists of, a cell-type targeting ligand or a receptor targeting ligand derived from small molecules or hormones such as naproxen, ibuprofen, cholesterol, progesterone, or estradiol.

[0113] 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 embodiments Z may be an antibody fragment obtained from camelids (such as llamas and alpacas), in particular a VHH (Variable Heavy domain of Heavy chain) or a nanobody. In some particular embodiments, an antibody or antigen binding portion thereof may be characterized by having specific affinity and / or specificity for a particular cell- specific protein, membrane protein, and / or membrane protein receptor.

[0114] In some embodiments, Z comprises or consists of a cell-type specific ligand or a receptor specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins or fragments thereof, lectins, membrane receptors or fragments thereof, antibodies or fragments thereof, spiegelmers, DARPIN, nucleic acid or peptide aptamers, vitamins, immunosuppressive molecules, and drugs and any combination thereof. Also contemplated are gene editing nucleases suchas Cas9, such as IgG / IgM proteases, as well as cytokines and protein based and small molecule adjuvants for vaccination or cancer therapy.

[0115] In specific embodiments, Z comprises or consists of steric shielding agents for avoiding interactions with immune components (e.g. neutralizing antibodies and / or complement system), such as, but not limited to, synthetic polymers.

[0116] 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 an osamine, such as glucosamine, galactosamine and mannosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6- mannose, P6-glucose, sialic acid, Sl-fructose and Pl-fructose, more preferably selected from the group consisting of glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid and Pl-fructose. In some embodiments the oligosaccharide and polysaccharide comprise one or more monosaccharides selected from the group consisting of an osamine, such as glucosamine, galactosamine and mannosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, Sl-fructose and Pl-fructose, more preferably selected from the group consisting of glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6- mannose, P6-glucose, sialic acid and Pl-fructose.

[0117] Examples saccharides include saccharides comprising non-hydroxyl groups such as, an amino group (such as e.g. NH2, an alkyl amino, a dialkyl amino), an N- acetylamino group and / or a thiol group.

[0118] 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.

[0119] “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.

[0120] 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.

[0121] 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.

[0122] In some embodiments, the monosaccharide contains a negatively charged group such as a phosphate group, a sulfate group or a carboxyl group.

[0123] Examples of monosaccharides containing a phosphate group, include, but are not limited to, glucose-6-phosphate, mannose-6-phosphate and fructose- 1 -phosphate

[0124] Examples of monosaccharides containing a sulfate group, include, but are not limited to, galactose-6-sulfate (S6-galactose), N-acetylgalactosamine-6-sulfate (S 6-N- acetylgalacto s amine) .

[0125] Examples of monosaccharides containing a carboxyl group, include, but are not limited to, glucuronic acid and sialic acid.

[0126] It is to be understood that the monosaccharides and derivatives thereof mentioned herein also encompass acyclic (open-chain) forms and cyclic forms.

[0127] It is also to be understood that the monosaccharides and derivatives thereof mentioned herein also encompass D- stereoisomers and L-stereoisomers, as well as mixtures of D- and L- stereoisomers (e.g., racemic mixtures).

[0128] It is also to be understood that the monosaccharides and derivatives thereof mentioned herein also encompass a-anomers and P-anomers, as well as racemic mixtures of a- and P-anomers.

[0129] In some embodiments, the oligosaccharide and the polysaccharide or a derivative thereof according to the present invention comprises a monosaccharide selected from the group consisting of an osamine, such as glucosamine, galactosamine and mannosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6- mannose, P6-glucose, sialic acid, Sl-fructose and Pl-fructose, more preferably selectedfrom the group consisting of glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid and Pl-fructose.

[0130] “Oligosaccharides” are saccharide polymers comprising a small number (typically from two to ten) of monosaccharides.

[0131] 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.

[0132] 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).

[0133] In some embodiments, examples of oligosaccharides include, but are not limited to, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, nonasaccharides, and decasaccharides.

[0134] 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 Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins; ACS Cent. Sci. 2021).

[0135] “Polysaccharides” are saccharide polymers comprising a large number (typically more than ten) of monosaccharides. They range in structure from linear to highly branched.

[0136] 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 asimilar way as described above for oligosaccharides, polysaccharides can be homopolysaccharides or heteropolysaccharides.

[0137] In some embodiments, the saccharide (i.e., monosaccharide, oligosaccharide and polysaccharide) or derivative thereof is a saccharide containing a non-hydroxyl group which is an amine group. In some preferential embodiments, the saccharide containing a non-hydroxyl group which is an amine group comprises or consists of a glucosamine, galactosamine or mannosamine.

[0138] In some embodiments, a saccharide (i.e., monosaccharide, oligosaccharide and polysaccharide) 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 comprises or consists of S6-galactose, or S6- JV-acetylgalactosamine.

[0139] 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 comprises or consists of P6-glucose, P6- mannose, or Pl -fructose.

[0140] 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 comprises or consists of glucuronic acid or sialic acid.

[0141] In some embodiments, the saccharide consists or comprises a saccharide containing a non-hydroxyl group selected from the group comprising, or consisting of glucosamine, galactosamine mannosamine, glucuronic acid, S6-galactose, S6-N- acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, Sl-fructose and Pl-fructose, more preferably selected from the group consisting of glucuronic acid, S6-galactose, S6- N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid and Pl-fructose.Spacers Si and S2

[0142] In some embodiments, each of the spacers Si and S2 may be, independently, any chemical chain which can comprise heteroatoms as well as cyclic moieties such as aryl and / or heteroaryl groups.

[0143] In some embodiments, each of the spacers Si and S2 may comprise, independently, up to 1000 carbon atoms and even more. The length and the chemical nature of each of the spacers Si and S2 may be designed depending on the end group Z and the crosslinked group R to which said spacers are covalently attached to, and also depending the biological effect which is sought.

[0144] In some embodiments, each of the spacers Si and S2 is, independently, 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.

[0145] In some embodiments, each of the spacers Si and S2 may, independently, comprise 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.

[0146] In some embodiments, each of the spacers Si and S2 may, independently, comprise one or more groups or moieties selected from alkyl (e.g., Ci-20, Ci-12, C1-6 alkyl), aryl, heteroaryl, alkyl ether, polyether, polyester, acyl, alkyl amide, polyamide, a guanidine, or a combination thereof. As used herein, “combination” means that each of the spacers Si and S2 may, independently, 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, -(CEh / n-, wherein “n” is from about 2 to about 20 or more. In some embodiments, each of the spacers Si and S2 may, independently, comprise a C2-20 straight or branched alkyl chain.

[0147] In some embodiments, each of the spacers Si and S2, independently, may be or may comprise 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, each of the spacers Si and S2, independently, may be or may comprise a polyethylene glycol (“PEG”) of formula -(OCH2CH2)n-, wherein “n” is an integer from 1 to 24, 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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24. In some embodiments, each of the spacers Si and S2, independently, may be or may comprise a polypropylene glycol, e.g., of formula - (OCH(CH3)CH2)n-, wherein “n” is an integer from 1 to 24, 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, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24.

[0148] In some embodiments, each of the spacers Si and S2, independently, may be or may comprise 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, each of the spacers Si and S2, independently, may be or may comprise 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-Rs-, wherein “R4” and “R5” are each independently selected from alkyls (e.g., Ci-20, Ci-12, C1-6 alkyl), ethers, or polyethers (e.g., PEGs having a molecular weight between about 200 to 2,000 g / mol). In a preferred embodiment each of the spacers Si and S2, independently, may comprise a polymer of P-alanine, preferably comprising 1 to 40 P-alanine monomers, more preferably Ito 24 P-alanine monomers, for example 1 to 10 P-alanine monomers.

[0149] In some embodiments, each of the spacers Si and S2, independently, may be or may comprise a sarcosine monomer (also known as N-methyl glycine). In a preferredembodiment each of the spacers Si and S2, independently, may comprise a polymer of sarcosine, preferably comprising 1 to 40 sarcosine monomers, more preferably comprising 1 to 24 sarcosine monomers, for example 1 to 10 sarcosine monomers.

[0150] In some embodiments, each of the spacers Si and S2, independently, 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 each of the spacers Si and S2, independently, may be 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.

[0151] In some embodiments, each of the spacers Si and S2, independently, 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).

[0152] In some embodiments, each of the spacers Si and S2, independently, 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).

[0153] In some embodiments each of the spacers Si and S2, independently, 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.

[0154] In some embodiments, each of the spacers Si and S2, independently, 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 P-alanine polymer, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof.

[0155] In some embodiments, each of the spacers Si and S2, independently, may be or may comprise a polyethylene glycol (PEG), comprising from 1 to 40 ethylene glycol monomers, e.g. from 2 to 24, 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”),-(OCH2CH2)5- (referred to herein as “PEG5”), -(OCH2CH2)IO- (referred to herein as “PEG10”), or -(OCH2CH2)24- (referred to herein as “PEG24”).

[0156] In some embodiments, each of the spacers Si and S2, independently, 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, each of the spacers Si and S2, independently, may comprise an optionally substituted phenylene moiety. For example, in some embodiments, each of the spacers Si and S2, independently, may comprise 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, C1-6 alkyl, C1-6 haloalkyl, C1-6 acyl and C1-6 alkoxy.

[0157] In some embodiments, each of the spacers Si and S2, independently, may comprise an alkylene, ether, polyether, alkylene amide, arylene group, heteroarylene group, an acyl group or a combination thereof. In a specific embodiment, each of the spacers Si and S2, independently, may comprise 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 ormore moieties selected from the group consisting of halogen, Ci-6 alkyl, Ci-6 haloalkyl, Ci-6 acyl and Ci-6 alkoxy.

[0158] In a specific embodiment each of the spacers Si and S2, independently, may comprise a PEG. In another specific embodiment, each of the spacers Si and S2, independently, may comprise a PEG and one or more aromatic groups, such as an arylene group and / or heteroarylene group Ar. In another specific embodiment, each of the spacers Si and S2, independently, may comprise a PEG, one or more groups C1-6 alkyl and one or more aromatic groups, such as an arylene group and / or heteroarylene group Ar. In a specific embodiment, each of the spacers Si and S2, independently, may comprise 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, C1-6 alkyl, C1-6 haloalkyl, C1-6 acyl and C1-6 alkoxy. In another specific embodiment each of the spacers Si and S2, independently, may comprise a PEG and an amino acid, preferably an arginine moiety. In a specific embodiment each of the spacers Si and S2, independently, may comprise a PEG. In another specific embodiment, each of the spacers Si and S2, independently, may comprise a PEG and one or more sarcosine polymers. In another specific embodiment each of the spacers Si and S2, independently, may comprise a PEG, a P-alanine polymer, a sarcosine polymer and one or more aromatic groups, such as an arylene group and / or hetero arylene group Ar. In another specific embodiment each of the spacers Si and S2, independently, may comprise a PEG, a P-alanine polymer, one or more groups C1-6 alkyl, C1-6 alkylamine or C1-6 acyl; and an amino acid, preferably an arginine moiety.

[0159] In a specific embodiment each of the spacers Si and S2, independently, may comprise one or more polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, one or more C1-6 alkylene groups, a polyether of a branched C3-12 polyol, and one or more aromatic groups, such as an arylene group and / or heteroarylene group Ar.

[0160] In some embodiments, each of the spacers Si and S2, independently, 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 C1-6 alkyl, C1-6 alkylamine or C1-6 acyl; a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycolmonomers, a polypropylene glycol (PPG) comprising 1 to 40 propylene glycol monomers, a polyether of a branched C3-12 polyol, an arginine derivative, a P-alanine polymer comprising 1 to 40 P-alanine monomers, pHPMA, PLGA, polymers of alkylene diamines, a sarcosine polymer and combinations thereof; and wherein each of the spacers Si and S2, independently, 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 a P-alanine polymer comprising 1 to 40 P- alanine monomers, and a sarcosine polymer comprising 1 to 40 sarcosine monomers.Ri and R2

[0161] In some embodiments, Ri is selected from the group consisting of linear Ci-12 alkyl, branched C3-12 alkyl, linear Ci-12 haloalkyl, branched C3-12 haloalkyl, aryl, heteroaryl and benzyl. In some preferred embodiments Ri is methyl, ethyl, trifluoromethyl, trifluoroethyl, phenyl, pyridyl or benzyl, more preferably ethyl.

[0162] In some embodiments, R2 is selected from the group consisting of H, alkyl, and an electron withdrawing group, as defined in the claims and embodiments of the present invention. In some embodiments R2 is selected from the group consisting of H, alkyl, haloalkyl, aryl, heteroaryl, -CN, -C(O)NH2, -S(O)2Ra, -C(O)Ra, -C(O)ORa, wherein Ra is an alkyl group. Preferably the alkyl is selected from the group consisting of linear Cn 12 alkyl and branched C3-12 alkyl, more preferably the alkyl is methyl. Preferably the haloalkyl is selected from the group consisting of linear Ci-12 haloalkyl and branched C3- 12 haloalkyl.Viral capsids

[0163] In embodiments of the present disclosure, the viral capsid is not particularly limited. In some embodiments, the viral capsid is selected from non-enveloped viruses, such as an adenovirus or a parvovirus, for instance a bocavirus or an AAV. In some embodiments, the viral capsid is selected from an enveloped virus, such as herpes simplex virus, lentivirus and retrovirus. Embodiments include naturally and non-naturally occurring capsids, and include any genetic, biologic or chemical alteration or variation thereof. In some embodiments, the viral capsid is a viral capsid of an adeno-associated virus, an enveloped virus, such as herpes simplex virus, lentiviruses and retroviruses,preferably the viral capsid is a parvovirus, for instance a bocavirus or an AAV, more preferably an AAV capsid.

[0164] AAV is composed of a 4.7kb single-stranded DNA genome inside an icosahedral capsid. The genome contains three open reading frames (ORF) flanked by internal terminal repetitions (ITR) that function as the origin of viral replication and the packaging signal. The ORF rep encodes four structural nonproteins that play a role in viral replication, transcription, site-specific integration and virus assembly. The ORF cap encodes three structural proteins, VP1, VP2 and VP3, that combine to form the viral capsid.

[0165] There are several naturally occurring serotypes and more than 100 known variants of AAV, each of which differs in relation to the amino acid sequence, particularly within the hypervariable regions of the capsid proteins and so in relation to its properties of targeting and gene supply.

[0166] For the purpose of the disclosure presented here, the terminology “AAV” includes, without limitation, the virus itself and its derivatives. Unless otherwise stated, the terminology refers to all serotypes, and to both replication and recombinant competent forms. As used herein, AAV includes any naturally occurring and synthetic serotypes.

[0167] The term AAV includes all identified natural serotypes. In some embodiments, AAV capsids according to the present invention are those of natural serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrhlO and AAVrh74.

[0168] Also, the term AAV includes any engineered serotypes generated by nonnatural methods, such as, but not limited to amino acid mutagenesis, peptide insertion, deletions in one or more of the capsid proteins VP1, VP2 and VP3, capsid shuffling from various serotypes, VP ancestral reconstruction or in silico design.

[0169] Engineered serotypes include any combination of capsid proteins from natural and modified serotypes that can assemble and produce a new AAV virus that is not known to exist in nature.

[0170] In some embodiments, the AAV serotypes are further modified to reduce or remove natural tropism, e.g. to reduce galactose binding, integrin binding and / or heparin sulfate proteoglycan binding.

[0171] In some embodiments, an AAV capsid according to the present invention is selected from AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. In some embodiments, an AAV capsid according to the present invention is from AAV1. In some embodiments, an AAV capsid according to the present invention is from AAV2. In some embodiments, an AAV capsid according to the present invention is from AAV5. In some embodiments, an AAV capsid according to the present invention is from AAV6.

[0172] In some embodiments, an AAV capsid according to the present invention is from AAV8. In some embodiments, an AAV capsid according to the present invention is from AAV9. In some embodiments, the AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9 capsids are optionally further engineered to reduce or modify natural mammalian cell surface target binding.Site of coupling on the viral capsid

[0173] In some embodiments of the invention, a provided viral capsid is a viral capsid from a viral vector, preferably parvoviruses including bocavirus and AAVs, more preferably an AAV vector, and a viral capsid protein of the viral vector, preferably of the AAV vector (e.g., at least one capsid protein of the AAV vector) is covalently attached to a squaramide moiety of formula (SQ):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein.

[0174] 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.

[0175] In some embodiments, a viral vector, preferably an AAV vector, is modified by covalent coupling of at least one squarate ester to at least one surface-exposed natural amino acid residue of at least one capsid protein of the viral vector, preferably of the AAV vector.

[0176] As used herein, the term “surface-exposed” refers to a natural amino acid residue with a side chain that is at least partially exposed at the outer surface of the viral vector, in particular of the AAV vector.

[0177] In some embodiments, at least one squarate ester is covalently bound to at least one amino group of a surface-exposed natural amino acid residue of the capsid of a viral vector, preferably of an AAV vector.

[0178] 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 viral vector, preferably of the AAV vector.

[0179] As used herein, “at least one amino group of a natural 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 (a) natural amino acid residue(s).

[0180] In some embodiments, the viral vector, preferably 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.Viral capsid comprising a moiety of formula (III)

[0181] In some embodiments, the viral capsid comprising a moiety of formula (I), as defined in the claims and embodiments of the present invention, results from a click chemistry reaction between a compound of formula (II) and a viral capsid protein covalently linked to a moiety of formula (III):wherein the crosslinked group R of the moiety of formula (I) results from a click chemistry reaction between R3 and R4, wherein R3 and R4 are, respectively, either the first group or the second group forming a click-chemistry reactive pair, and wherein the first and the second group forming a click-chemistry reactive pair, Z, Si, S2, N, N* and - — , are as defined in the embodiments and claims of the present invention.

[0182] Thus, the present invention also refers to the provision of a viral capsid comprising a viral capsid protein covalently linked to a moiety of formula (III):wherein R4, N*, — and Si are as defined in the embodiments and claims of the present invention; and being said viral capsid covalently linked to a moiety of formula (III) an intermediate in the manufacturing a viral capsid covalently linked to a moiety of formula (I) as defined in the embodiments and claims of the present invention.

[0183] In particular, the viral capsid comprising a viral capsid protein covalently linked to a moiety of formula (III), as defined in the claims and embodiments of the present invention, may be the viral capsid from a viral vector particle, preferably an AAV vector particle. In some embodiments, a viral vector comprising a viral capsid protein covalently linked to a moiety of formula (III), as defined in the claims and embodiments of the present invention, comprises at least a nucleic acid, in particular, at least a transgene which is selected in view of the intended use of the viral vector. Preferably, an AAV vector comprising a viral capsid protein covalently linked to a moiety of formula (III), as defined in the claims and embodiments of the present invention, comprises at least a nucleic acid, in particular, at least a transgene which is selected in view of the intended use of the AAV vector.Recombinant vectors

[0184] In some embodiments, the viral capsid comprising a modified viral capsid protein according to the present invention may be the viral capsid from a recombinant viral vector, preferably recombinant parvoviruses including recombinant bocavirus and recombinant AAVs, more preferably a recombinant AAV vector. For the purposes of the present invention, the term “vector” refers to a viral vector and, more in particular, to the preferred AAV viral vectors according to the disclosure

[0185] A “recombinant viral vector” herein refers to a viral particle that comprises an exogenous polynucleotide (i.e., a polynucleotide that is not a wild-type viral genome, for example, a transgene that must be supplied to a target cell). Thus, a “recombinant AAV vector” or “rAAV” herein refers to an AAV particle that comprises an exogenous polynucleotide (i.e., a polynucleotide that is not a wild-type AAV genome, for example, a transgene that must be supplied to a target cell). In general, the exogeneous polynucleotide is flanked by at least one, and usually by two, ITR of the AAV.

[0186] The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides. The term polynucleotide, as used here, refers interchangeably to single and double- stranded molecules. The polynucleotide packaged inside the AAV vectors in the present invention can be any kind of polynucleotide usefully transduced into cells by AAV vectors. In some embodiments, the polynucleotide packaged in the AAV vector is an expressible polynucleotide, which encodes at least one protein. In some embodiments, the polynucleotide encodes at least one transgene.

[0187] As an additional or alternative example, in some embodiments, the polynucleotide serves as a DNA template for homologous recombination in cells.

[0188] The term “transgene”, as used herein, refers to an expressing polynucleotide that performs a function of some kind in the cell. For example, a transgene may contain an ORF that encodes a transgene product. Non-limiting examples of transgene products include a protein, an intracellular or a secreted antibody, a peptide toxin, a pharmacology molecule, CRISPR based-editors or CRIS PR-based epigenetic tools. Another example of a transgene product is an RNA, for example, a functional RNA product, an aptamer, an interfering RNA, a ribosomal RNA, a transporter RNA, a non-coding RNA, a guide RNA for nucleases, which are transcribed but not translated. In some embodiments, a transgeneconfers 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.Viral vector particles comprising a moiety of formula (I)

[0189] In some embodiments, the viral capsid is a viral capsid of a non-enveloped virus, for instance, a parvovirus, such as a bocavirus and an adeno-associated virus (AAV), an enveloped virus, such as and herpes simplex virus, a lentivirus and retrovirus. Preferably, the viral capsid is a parvovirus, for instance a bocavirus or an AAV, more preferably an AAV capsid. Thus, one aspect of the invention refers to a viral vector particle selected from a non-enveloped virus, for instance, a parvovirus, such as a bocavirus and an adeno-associated virus (AAV),, an enveloped virus, such as herpes simplex virus, lentiviruses and retroviruses, and preferably to an AAV vector particle, comprising a viral capsid protein covalently linked to a moiety of formula (I):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; and wherein Z, Si and S2 and R are as defined in the claims and embodiments of the present invention

[0190] In some embodiments the viral vector particle, preferably the AAV viral vector particle, comprises a viral capsid, as defined in the present claims and embodiments, and a nucleic acid encapsulated therein.

[0191] 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). Insome 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.

[0192] 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.

[0193] 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)

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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 1, 2, 5, 6, 8 and 9. In some embodiments, an AAV vector according to the present invention has a capsid of AAV serotype 1. 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 6. 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.

[0198] 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.Method for obtaining the AAV vector particles

[0199] In some embodiments, this invention further relates to methods of manufacturing of a viral vector, wherein the viral capsid of the viral vector is selected from non-enveloped virus, for instance a parvovirus, such as a bocavirus and an adeno-associated virus, an enveloped virus, such as herpes simplex virus, lentiviruses and retroviruses, preferably an AAV vector according to the invention. Thus, in some embodiments the present invention relates to a method for manufacturing a viral vector particle, preferably an AAV vector particle comprising: reacting a viral vector, preferably an AAV vector with a compound of formula (IV):in conditions suitable for reacting a squarate moiety of the compound of formula (IV) with at least one amino group from a surface-exposed natural amino acid residue of a viral capsid protein from the viral vector, preferably from the AAV vector so as to form a viral vector particle, preferably an AAV vector particle comprising a moiety of formula (III):reacting the viral vector particle, preferably the AAV vector comprising a moiety of formula (III) with a compound of formula (II):in conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react and obtain an AAV vector particle comprising a moiety of formula (I):wherein Z, Si, S2, R, Ri, R3, R4, N* and — are defined according to the claims and embodiments of the present invention.

[0200] In some embodiments, a method of the present invention comprises incubating the viral vector, preferably the AAV vector, with a compound of formula (IV) 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 (IV) as defined and described in classes and subclasses disclosed in the present invention, with at least one amino group of a natural amino acid residue of the capsid of the viral vector so as to form a moiety of formula (III), as defined in the present invention.

[0201] In some embodiments, suitable conditions to obtain at least one moiety of formula (III) include suitable conditions to promote the formation of a covalent bond between an amino group of a natural amino acid residue of the capsid of the viral vector, preferably of the AAV vector, and said squarate moiety without impairing the structural integrity of said viral vector.

[0202] In some embodiments, an incubation can be performed in an aqueous buffer, optionally containing a cosolvent, for example DMSO, an alcohol, a polyol, among others solvents soluble or miscible with water which do not affect the stability of the compounds and viral vectors herein disclosed, 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.

[0203] 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.

[0204] In some embodiments, the incubation may be carried out including the addition of a cosolvent which is compatible with viral vectors, such as DMSO, an alcohol, a polyol, among others. In some embodiments the incubation is carried out in the presence of a nonionic surfactant, such as Pluronic®.

[0205] 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.

[0206] 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.

[0207] In some embodiments, the molar ratio of the compound of formula (III) to the viral vector, preferably the AAV vector, may be from 1.102to 5.107, from 1.105to 3.106. In some preferential embodiments, there is between 1.105to 3.106equivalents molar excess of the compound of formula (III).

[0208] 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.

[0209] For instance, in some embodiments, a method of the invention may comprise a preliminary step of providing or producing a viral vector, preferably an AAV vector to be modified.

[0210] 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 (IV) at the end of the incubation step, e.g. by dialysis or tangential flow filtration, and / or a step of collecting the chemically modified viral vector particles, preferably the chemically modified AAV vector particles comprising a moiety of formula (III), and / or a step of purifying the viral vector particles, preferably the AAV vector particles comprising a moiety of formula (III), and / or a step of recovering the viral vector particles, preferably the AAV vector particles comprising a moiety of formula (III), and / or a step of formulating and / or packaging the viral vector particles, preferably the AAV vector particles comprising a moiety of formula (III).

[0211] Further, the method of the invention comprises reacting the viral vector, preferably the AAV vector comprising a moiety of formula (III) with a compound of formula (II):in conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react and obtain a viral vector particle, preferably an AAV vector particle comprising a moiety of formula (I):wherein Z, Si, S2, R, Ri, R3, R4, N* and — are defined according to the claims and embodiments of the present invention.

[0212] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting a strained- promoted alkyne-azide click-chemistry (SPAAC) reaction, between an azide and a strained alkyne, wherein R3 and R4 are, respectively, either an azide or a strained alkyne as defined in the claims and embodiments of the present invention.

[0213] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting a strain- promoted alkyne-nitrone cycloaddition (SPANC) reaction, between a nitrone and a strained alkyne, wherein R3 and R4 are, respectively, either a nitrone or a strained alkyne as defined in the claims and embodiments of the present invention.

[0214] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels-Alder (IEDDA) reaction, between a tetrazine and a cyclic olefine, wherein R3 and R4 are, respectively, either a tetrazine or a cyclic olefine as defined in the claims and embodiments of the present invention.

[0215] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels-Alder (IEDDA) reaction, between a tetrazine and an alkyne, wherein R3 and R4 are, respectively, either a tetrazine or an alkyne as defined in the claims and embodiments of the present invention.

[0216] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels-Alder (IEDDA) reaction, between a tetrazine and a strained alkyne, wherein R3 and R4 are, respectively, either a tetrazine or a strained alkyne as defined in the claims and embodiments of the present invention.

[0217] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels- Alder (IEDDA) reaction, between a 1,2,3-triazine and an amidine, wherein R3 and R4 are, respectively, either a 1,2,3-triazine or an amidine as defined in the claims and embodiments of the present invention.

[0218] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels- Alder (IEDDA) reaction, between a 1,2,3-triazine and a guanidine, wherein R3 and R4 are, respectively, either a 1,2,3-triazine or a guanidine as defined in the claims and embodiments of the present invention.

[0219] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels- Alder (IEDDA) reaction, between a 1,2,4-triazine and a cyclic olefine, wherein R3 and R4 are, respectively, either a 1,2,4-triazine or a cyclic olefine as defined in the claims and embodiments of the present invention.

[0220] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels- Alder (IEDDA) reaction, between a 1,2,4-triazine and an alkyne, wherein R3 and R4 are, respectively, either a 1,2,4-triazine or an alkyne as defined in the claims and embodiments of the present invention.

[0221] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react are conditions suitable for conducting an inverse electron-demand Diels- Alder (IEDDA) reaction, between a 1,2,4-triazine and a strained alkyne, wherein R3 and R4 are, respectively, either a 1,2,4-triazine or a strained alkyne as defined in the claims and embodiments of the present invention.

[0222] In some embodiments the conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react include a pH between 6 and 8.5, preferably 6.5and 8, more preferably between 7 and 7.5; and a aqueous buffer, such as a phosphate buffered saline (PBS) solution, optionally also comprising a cosolvent compatible with viral vectors, such as DMSO. In some embodiments the reaction to form a click-chemistry pair is carried out in the presence of a nonionic surfactant, such as Pluronic®.

[0223] In some embodiments 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 S2 covalently linked to a group R3, as described above herein, in suitable conditions (for example, on solid support, according to standard procedures).

[0224] Examples of compounds of formula (IV) are exemplified in Table 1, and of resulting AAV viral capsids comprising a moiety of formula (III) are exemplified in Table 2:Table 1: Compounds of formula (IV)Table 2: Examples of AAV viral capsids comprising a moiety of formula (III)

[0225] Examples of compounds of formula (II) are exemplified in Table 3, and examples of AAV viral capsids comprising a moiety of formula (I) resulting from the click chemistry reaction of the compounds of formula (II) of Table 3 and the AAV viral capsids comprising the moieties of formula (III) of Table 2 are exemplified schematically in Table 4 and Fig. 3:Table 3: Examples of compounds of formula (II)Table 4: Examples of AAV viral capsids comprising a moiety of formula (I)Uses of the provided viral vectors

[0226] In some embodiments, the viral vectors, preferably the rAAV vectors can be used as gene vectors for in vivo or in vitro applications. In some embodiments, the viral vectors, preferably the AAV vectors of the present invention can be used as a research tool. In some embodiments, the viral vectors, preferably the 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. For instance, the disclosure refers to the viral vectors, preferably the AAV vectors of the present invention for use as a medicament, or to the use of the viral vectors, preferably the AAV vectors of the present invention for the manufacturing of a medicament, or to a method for the prevention or treatment of a disease comprising administering an effective amount of the viral vectors, preferably the AAV vectors of the present invention, to a subject in need thereof. In some embodiments, the viral vectors, preferably the AAV vectors of the present invention canbe used in a diagnostic means, e.g. as an imaging agent. In some embodiments, the viral vectors, preferably the 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 viral vectors

[0227] In some embodiments, chemical modifications of the capsid of a viral vector, preferably 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 group which is introduced to modify the viral vector, preferably the AAV vector in the present invention. In some embodiments, one or more biological properties of a modified viral vector, preferably a modified AAV vector can be altered compared to the unmodified vector, such as: a modified tropism of the vector for specific organ, tissue, and / or cell type (e.g. an increased selectivity or a shifted selectivity from one tissue / organ / cell to another) compared to the capsid that has not been surface modified; and / or a modified immunoreactivity of the vector, e.g. a decreased immunogenicity of the vector and / or a decreased affinity for neutralizing antibodies or complement systems, and / or said vector triggers an altered immune response when administered in vivo, e.g. generate less AAV-directed innate and adaptive immune responses compared to the capsid that has not been surface modified; and / or an increased infectivity rate of the vector (i.e., greater efficiency or similar efficiency at lower doses) compared to the capsid that has not been modified and / or an improved transduction of 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 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.

[0228] In some embodiments, when the viral vector, preferably 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 viral vector, preferably of the AAV vector. In some embodiments, an improvement in the therapeutic index can result from decreases in the relative dose of viral vector, in particular the relative dose of AAV vector, 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 therapeutic regime.

[0229] In some embodiments, an AAV vector of the present invention shows a preferential tropism for an organ or cell selected from CNS, liver, lung, heart, , 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

[0230] In some embodiments, the present invention relates to a viral vector, preferably to an AAV vector according to the present invention or a pharmaceutical composition comprising the same, for use in transducing a cell of a subject. In some embodiments, the present invention relates to a viral vector, preferably to an AAV vector according to the present invention or a pharmaceutical composition comprising the same, for use in a method of delivering a nucleic acid to a cell for the treatment of a disease. In other embodiments, the present invention relates to a non-therapeutic method of delivering a nucleic acid to a cell, the method comprising contacting a cell with a viral vector, preferably with an AAV vector particle according to the present invention comprising a nucleic acid to be expressed in the contacted cell.

[0231] 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.

[0232] . In some embodiments, such a 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.

[0233] In some embodiments, the present invention also relates to use of a viral vector, preferably an AAV vector according to the present invention for transducing a cell of a subject.

[0234] In some embodiments, the present invention also relates to a method for transducing a cell of a subject, comprising administering a viral vector, preferably an AAV vector according to the present invention to said subject.

[0235] 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 a viral vector, preferably an AAV vector particle of formula (I), 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.

[0236] In some embodiments, the present invention also relates to a method for delivering a transgene into a cell of a subject, comprising administering a viral vector, preferably an AAV vector according to the present invention comprising said transgene to said subject.

[0237] 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 a viral vector, preferably 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).

[0238] In some embodiments, a viral vector, preferably 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 someembodiments, 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).

[0239] In some embodiments, a viral vector, preferably 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, viral vector targets, preferably AAV vectors targets encompass, but are not limited to, hepatocytes; cells of the retina; muscle cells; cells of the central nervous system (CNS); cells of the heart; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells; blood 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 a viral vector, preferably an AAV vector, 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.Use in gene therapy

[0240] Viral vectors, and in particular AAV vectors hold great promise in human gene therapy and have been used for the treatment of several diseases due to its ability to provide long-term gene expression and lack of pathogenicity.

[0241] In some embodiments, a viral vector, and preferably an AAV vector described herein may be particularly useful in gene therapy, e.g., to deliver a therapeutic transgene of interest to a subject.

[0242] Accordingly, in some embodiments, the present invention also relates to a viral vector, and preferably to an AAV vector according to the present invention, for use in gene therapy.

[0243] In some embodiments, the present invention also relates to the use of a viral vector, and preferably an AAV vector according to the present invention for manufacturing a medicament for gene therapy, or to a method of gene therapy in a subjectin need thereof, comprising administering a viral vector, and preferably an AAV vector according to the present invention to said subject.

[0244] In some embodiments, a viral vector, and preferably an AAV of the invention can be delivered by any appropriate route to the subject. The administration can be systemic, local, or systemic combined with local; systemic includes parenteral and oral, and local includes local and loco-regional. In some embodiments, appropriate administration routes encompass, without being limited to, inhalational, topical, intratissue (e.g. intramuscular, intracardiac, intrahepatic, intrarenal), conjunctival (e.g. intraretinal, subretinal), mucosal (e.g. buccal, nasal), intra- articular, intravitreal, intracranial, intravascular (e.g. intravenous (IV) or intraarterial), intraventricular, intracistemal, intraperitoneal, and intralymphatic routes. In some preferred embodiments, the administration is parenteral, preferably intravascular such as intravenous (IV) or intraarterial. The parenteral administration is advantageously by injection or perfusion. 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.

[0245] In some embodiments, a viral vector, and preferably an AAV vector according to the present invention may be useful for in a method for preventing or treating a disease, disorder, or condition of any type, i.e. in the manufacturing of a medicament for preventing or treating a disease, disorder, or condition of any type. For example, such viral vectors, and preferably such AAV vectors may be useful in treating a disease, disorder, or condition characterized by missing or aberrant protein or polypeptide activity. In some embodiments, the AAV vector includes a transgene encoding the missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent transduction may lead to expression of the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide. Alternatively, or in addition, AAV vector may be useful to transduce cells and transform them in a biofactory for therapeutic proteins, such as, but not limited to, secreted antibodies.

[0246] Unlimited examples of diseases, disorders, and / or conditions for which a viral vector, and preferably an AAV vector according to the present invention may be administered include infectious diseases such as SCID or hepatitis C (as both vaccines and therapeutics), , cancers, proliferative diseases, genetic diseases, autoimmunediseases, inflammatory diseases, pain, diabetes, neurodegenerative diseases (e.g., such as Alzheimer’s disease, Parkinson’s disease), neurodevelopmental diseases (e.g. such as epilepsia), cardiovascular diseases, metabolic diseases, respiratory disorders, muscle diseases, kidney disorders, sensory disorders such as ocular diseases, inherited retinal diseases and inner ear disorders. C).

[0247] In some embodiments, a viral vector, and preferably an AAV vector of the present invention can be used for treating muscle diseases. Muscle diseases include various inherited (genetic) and acquired diseases or disorders affecting the structure or function of muscle including skeletal and / or cardiac muscle.

[0248] In some embodiments, the muscle disease is a neuromuscular genetic disorder such as the muscular dystrophies (MD) which are a group of inherited genetic conditions that gradually cause the muscles to weaken, leading to an increasing level of disability. Neuromuscular genetic disorders include in particular: muscular dystrophies (MD), congenital myopathies, myotonic syndromes, ion channel muscle diseases, hereditary cardiomyopathies, congenital myasthenic syndromes, motor neuron diseases, hereditary motor and sensory neuropathies and other neuromuscular disorders.

[0249] In some embodiments, a viral vector, and preferably an AAV vector of the present invention can also be used for treating enzyme-deficiency diseases such as lysosomal storage diseases (LSDs) including any disorder resulting from a defect in lysosome function. Currently, approximately 50 lysosomal storage disorders have been identified, the most well-known of which include Tay-Sachs, Gaucher, and Niemann- Pick disease.

[0250] In some embodiments, viral vectors, and preferably AAV vectors of the present invention can also be used for treating Central Nervous System (CNS) diseases. CNS diseases include various inherited (genetic) and acquired diseases or disorders affecting the structure or function of brain or one of its parts and structures. CNS diseases include, but are not limited to Huntington's disease, epilepsy, Parkinson's disease, Alzheimer's disease and frontotemporal dementia (FTD).

[0251] 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.

[0252] 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.

[0253] 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

[0254] In some embodiments, the present invention further relates to a composition comprising viral vectors, preferably AAV vectors according to the invention. In some embodiments, the viral vectors, preferably the AAV vectors in the composition according to the present invention comprises at least one transgene.

[0255] In some embodiments, the composition is a pharmaceutical composition comprising a viral vector, preferably an AAV vector according to the invention and at least one pharmaceutically acceptable vehicle.

[0256] 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 whenadministered 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.

[0257] 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, polyethylenepolyoxypropylene block polymers and the like), cellulose-based substances (such as, e.g., sodium carboxymethylcellulose), poly acrylates, waxes, nonionic surfactants (such as, e.g., Tween, pluronics, polyethylene glycol and the like), wool fat, sugars (saccharides) such as sucrose, sugar alcohols such as mannitol, and suitable combinations thereof.

[0258] 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, sodium, potassium, magnesium or calcium citrate 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.

[0259] In some embodiments, a pharmaceutical composition according to the present invention comprise one or more agents that promote the entry of a viral vector, in particular 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.

[0260] In some embodiments, viral vectors and preferably 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 viral vectors, and preferably AAV vectors comprising at least one transgene according to the present invention.Regimen

[0261] In some embodiments, modified viral vectors, preferably the modified AAV vectors according to the present invention are to be administered in an effective amount. The dose of viral vectors, in particular of the 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 transgene product expression required to achieve a therapeutic effect, the specific disease being treated, the stability of the gene, RNA, and / or protein product, the age of the patient and the weight of the patient. 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.

[0262] In some embodiments, modified viral vectors, preferably the modified AAV vectors according to the present invention are to be administrated at a dose ranging from about 107viral genomes (vg) to about 1018vg, such as from about 108vg to about 1014Overall, systemically administrated viral vectors require higher dosages than those delivered to targeted sites. Usually, systemically delivered viral vectors, in particular AAVs are dosed at 1014-1016vg, whereas targeted delivered AAVs are dosed at 10n-1013vg-

[0263] 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, doublestranded 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 recombinanttechniques (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.

[0264] 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.

[0265] In some embodiments, the volume of modified viral vectors, in particular of the modified AAV vectors administered to a subject will also depend, among other things, on the age and 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.

[0266] In some embodiments, the rate of administration of viral vectors, in particular of the modified AAV vectors delivered to a subject will also depend, among other things, on the age and size of the subject, the dose of the viral vector, in particular of the AAV vector required to obtain therapeutic effect, the concentration of the viral vector, in particular of the AAV vector, the volume of the viral vector, in particular of the AAV vector solution, and the proposed route of administration. In some embodiments, the total dose or total volume of the viral vectors, in particular of the AAV vectors, may be administered in a single shot or infusion; or the viral vectors, in particular the AAV vectors are injected with intermittent periods between each shot.Kits

[0267] 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.

[0268] In some embodiments, the kits or kits-of-parts comprise one or more viral vectors, preferably one or more AAV vectors and / or compositions according to the present invention.

[0269] In some embodiments, the kits or kits-of-parts further comprise a device for delivery of one or more viral vectors, preferably one or more AAV vectors and / or compositions according to the present invention.

[0270] In some embodiments, the kits further include instructions for delivery of one or more viral vectors, preferably 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.

[0271] 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

[0272] FIG.l: 1.1012vg of AAV2-eGFP, were added to a solution of the compound of formula (IV), (1) (3E6 eq) in TBS (pH 9.3), and incubated for 16h at 20°C resulting in the AAV viral particle comprising a moiety of formula (III) (AAV2-1). This (AAV2-1) was then incubated with compounds of formula (II), (B) and (E), at a 1:1 (v / v) ratio for 2 hours to obtain, respectively, the viral particles comprising a moiety of formula (I), AAV2-1B and AAV2-1E. 1010vg of each coupling were analyzed by SDS-PAGE and silver staining or dot blot analysis: Fig. 1 are SDS-PAGE gels with silver staining evidencing AAV capsid integrity. VP1, VP2 and VP3 are the three proteins constituting the AAV capsid of (first and forth columns numbering from left to right) the unmodified AAV2, of (second and fifth columns numbering from left to right) the modified AAV2 comprising a moiety of formula (III), AAV2-1, after bioconjugation with the compound of formula (IV), (1), and (third and sixth column numbering from left to right) the modified AAV2 comprising a moiety of formula (I), AAV2-1B and AAV2-1E, after click chemistry reaction with, respectively, a compound of formula (II), (B) or (E). Capsid protein molecular weight is indicated at the right of the images according to a protein ladder. Fig. IB are dot blot analysis using Streptavidin-HRP antibody to detect biotin coupled to AAV capsids of (left) the unmodified AAV2, of (center) the modified AAV2comprising a moiety of formula (III), AAV2-1, after bioconjugation with the compound of formula (IV), (1), and of (right) the modified AAV2 comprising a moiety of formula (I), AAV2-1B, after click chemistry reaction with compound of formula (II), (B). Fig. 1C are dot blot analysis of (left) the unmodified AAV2, of (right) the modified AAV2 comprising a moiety of formula (III), AAV2-1, after bioconjugation with the compound of formula (IV), (1), and of (right) the modified AAV2 comprising a moiety of formula (I), AAV2-1E, after click chemistry reaction with compound of formula (II), (E), using biotinylated avb3 integrin, followed by Streptavidin-HRP, to detect peptide ligands coupled to their linker. Fig. ID are mass photometry analysis by SamuxMP (Refeyn) evidencing the mass shift of the non-modified AAV in comparison with conjugated AAV comprising a moiety of formula (III), AAV2-1 (upper figure) and comprising a moiety of formula (I), AAV-1B (lower figure), after conducting a SPAAC click chemistry reaction with compound of formula (II), (B).

[0273] FIG. 2: 1.1012vg of AAV2-eGFP, were added to a solution of the compound of formula (IV) (3) (3E6 eq) in TBS (pH 9.3) and incubated for 16h at 20°C resulting in the AAV viral particle comprising a moiety of formula (III) (AAV2-3). The AAV viral particle comprising a moiety of formula (III), (AAV2-3) was then incubated with compounds of formula (II), (B) and (E) at a 1:1 (v / v) ratio for 2 hours to obtain, respectively, the viral particles comprising a moiety of formula (I), AAV2-3B and AAV2- 3E. IO10vg of each coupling were analyzed by SDS-PAGE and silver staining or dot blot analysis: Fig. 2A are SDS-PAGE gels with silver staining evidencing AAV capsid integrity. VP1, VP2 and VP3 are the three proteins constituting the AAV capsid of (first and forth columns numbering from left to right) the unmodified AAV2, of (second and fifth columns numbering from left to right) the modified AAV2 comprising a moiety of formula (III), AAV2-3, after bioconjugation with the compound of formula (IV), (3), and of (third and sixth columns numbering from left to right) the modified AAV2 comprising a moiety of formula (I), AAV2-3B and AAV2-3E, after click chemistry reaction with, respectively, a compound of formula (II), (B) or (E). Capsid protein molecular weight is indicated at the right of the images according to a protein ladder. Fig. 2B are dot blot analysis of (left) the unmodified AAV2, of (center) the modified AAV2 comprising a moiety of formula (III), AAV2-3, after bioconjugation with the compound of formula (IV), (3), and of (right) the modified AAV2 comprising a moiety of formula (I), AAV2-3B, after click chemistry reaction with compound of formula (II), (B) using Streptavidin- HRP antibody to detect biotin coupled to AAV capsids. Fig. 2C are dot blot analysis of (left) the unmodified AAV2, of (center) the modified AAV2 comprising a moiety of formula (III), AAV2-3, after bioconjugation with the compound of formula (IV), (3), and of (right) the modified AAV2 comprising a moiety of formula (I), AAV2-3E, after click chemistry reaction with compound of formula (II), (E), using biotinylated avb3 integrin, followed by Streptavidin-HRP, to detect peptide ligands coupled to their linker. Fig. 2D are mass photometry analysis by SamuxMP (Refeyn) evidencing the mass shift of the non-modified AAV in comparison with conjugated AAV comprising a moiety of formula (III), AAV2-3.

[0274] FIG. 3: Chemical structure of compounds of formula (II) included in table 3.

[0275] FIG. 4: 4.1011vg of AAV2-CAG-eGFP, were added to a solution of the compound of formula (IV), (13) (3 mM) or (11) (0.3 mM) in TBS (pH 9.3), and incubated for 2h at 20°C resulting in AAV viral particles comprising a moiety of formula (III), respectively, (AAV2-11) or (AAV2-13). AAV were then incubated with the compound of formula (II), (K) or (L) (100 pM), at a 1 : 1 (v / v) ratio for 2 hours to obtain, respectively, the viral particles comprising a moiety of formula (I), AAV2-11K and AAV2-13L. 1010vg of each coupling were analyzed by SDS-PAGE and silver staining or 5.109vg of each coupling by dot blot: Fig. 4A are SDS-PAGE gels with silver staining evidencing AAV capsid integrity. VP1, VP2 and VP3 are the three proteins constituting the AAV capsid of the unmodified AAV2 (first column of each gel), of the modified AAV2 comprising a moiety of formula (III), V2-11 and AAV2-13, after bioconjugation with, respectively, the compound of formula (IV), (11) or (13), (second column of each gel) and the modified AAV2 comprising a moiety of formula (I), AAV2-11K and AAV2-13L, after click chemistry reaction with, respectively, a compound of formula (II), (K) or (L) (last column of each gel). Capsid protein’s molecular weights are indicated according to a protein ladder. Fig. 4B are dot blot analysis of AAV capsids of the unmodified AAV2 (left), of the modified AAV2 comprising a moiety of formula (III), AAV2-11 and AAV2-13, after bioconjugation with, respectively, the compound of formula (IV), (11) or (13), (center), and of the modified AAV2 comprising a moiety of formula (I), AAV2-11K and AAV2- 13L, after click chemistry reaction with, respectively, a compound of formula (II), (K) or (L) (right) using Streptavidin-HRP antibody to detect coupled biotin. Fig. 4C are massphotometry analysis by SamuxMP (Refeyn) evidencing the mass shift of the nonmodified AAV2 in comparison with conjugated AAV2 comprising a moiety of formula (I), AAV2-11K and AAV2-13L, after click chemistry reaction with, respectively, a compound of formula (II), (K) or (L).FIG. 5: 3.1012vg of AAV2-4pmut-CAG-nanoLUC, were added to a solution of the compound of formula (IV), (3) (3 mM) in TBS (pH 9.3), and incubated for 2h at 20°C resulting in AAV viral particles comprising a moiety of formula (III) (AAV2-4pmut-3). AAV were then incubated with the compound of formula (II), (N) or (P) (25 pM), at a l:l(v / v) ratio for 2 hours to obtain, respectively, the viral particles comprising a moiety of formula (I), AAV2-4pmut-3N and AAV2-4pmut-3P. IQ10vg of each coupling were analyzed by SDS-PAGE and silver staining or 5.109vg of each coupling by dot blot: Fig. 5A is SDS-PAGE gel with silver staining evidencing AAV capsid integrity. VP1, VP2 and VP3 are the three proteins constituting the AAV capsid of the unmodified AAV2- 4pmut (first and last columns of the gel), of the modified AAV2-4pmut comprising a moiety of formula (III), AAV2-4pmut-3, after bioconjugation with, respectively, the compound of formula (IV), (third column of the gel) and the modified AAV2-4pmut comprising a moiety of formula (I), V2-4pmut-3N and V2-4pmut-3P, after click chemistry reaction with, respectively, a compound of formula (II), (N) or (P) (second and fourth columns of the gel). Capsid protein’s molecular weights are indicated according to a protein ladder. Fig. 5B are dot blot analysis of AAV capsids of the unmodified AAV2- 4pmut (first column), of the modified AAV2-4pmut comprising a moiety of formula (III), AAV2-4pmut-3 after bioconjugation with, respectively, the compound of formula (IV), (3) (second column) and the modified AAV2-4pmut comprising a moiety of formula (I), AAV2-4pmut-3N and AAV2-4pmut-3P, after click chemistry reaction with, respectively, a compound of formula (II), (N) or (P) (last column) using TFR1 -Biotin protein, followed by Streptavidin-HRP, to detect coupled VHH ligands. Fig. 5C is the result of the transduction efficiency assay of the unmodified AAV2-4pmut (first column), the modified AAV2-4pmut comprising a moiety of formula (III), V2-4pmut-3 after bioconjugation with the compound of formula (IV), (3) (second column) and the modified AAV2-4pmut comprising a moiety of formula (I), AAV2-4pmut-3N and AAV2-4pmut- 3P, after click chemistry reaction with, respectively, a compound of formula (II), (N) or (P) on HEK cell-line surexpressing TFR1 at a MOI of 300. Data represents the values ofluciferase bioluminescence after 24 h normalized by the number of cells and expressed as a fold change of the unmodified AAV2-4pmut.EXAMPLES

[0276] 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.

[0277] 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 or ultra performance liquid chromatography (HPLC or UPLC)).

[0278] The following abbreviations have been used:ACN: acetonitrileBoc: tert-ButyloxycarbonylCa.: circaCH2Q2 or DCM: dichloromethaneCV : column volumeDIPEA: A,A-diisopropylethylamineDMF : dimethylformamideDMSO : dimethylsulfoxideEtOAc: ethyl acetateEtOH : ethanolHATU: Hexafluorophosphate Azabenzo triazole Tetramethyl UroniumHex.: hexaneHFIP : hexafluoroisopropanolHO Ac: acetic acidH2O: waterMeOH: methanol min.: minute mL: milliliterNa2SO4: sodium sulfateNEt or TEA: triethylamineNH3: ammoniacPbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonylPyBOP : benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluoropho sphateRT: room temperatureTFA: trifluoro acetic acidTIPS: triisopropyl silaneTLC: thin-layer chromatographyLC / MS: liquid chromatography-mass spectrometryHPLC: High Performance Liquid ChromatographyUPLC : Ultra Performance Liquid ChromatographyNMR: Nuclear Magnetic Resonance

[0279] HPLC / MS method for purity determination :

[0280] Instruments: Shimadzu LCMS-2020 Single Quadrupole Liquid ChromatographMass Spectrometer, HPLC - Shimadzu Nexera-i LC-2040C 3D with DAD detector

[0281] Column: Gemini-NX 3 p C18, 4.6 x 50 mm), or equivalent

[0282] HPLC conditions:Wavelength range: 200 nm ± 4.0 nm, 267 nm ± 4.0 nm,Flow: 0.5 ml / minColumn temperature: 30 °C Injection volume: 0.3 pLAnalysis time: 14 min

[0283] Elution: gradientTime [min] Mobile phase A [%] Mobile phase B [%] Flow [ml / min]0.0 95 5 0.52.0 95 5 0.59.5 20 80 0.510.5 20 80 0.512.0 95 5 0.514.0 95 5 0.5

[0284] Re versed-phase HPLC / MS analyses were carried out with a Waters Alliance 2795 HPLC equipped with an autosampler, an inline membrane degasser, a column oven 10 (T° = 45 °C), a UV detector, and a ZQ quadrupole mass detector working in ionization electrospray mode. Analyzed compounds (0.1 to 0.3 mg) were solubilizedin a minimum amount of DMSO completed with acetonitrile (total volume: 1 mL). Standard analytical parameters: flow rate: 1 mL / min, Vinj.: 5pL. Acidic conditions: Waters XSelect CSH C18 column (3.5pm, 2. lx 50 mm). Gradient: (H2O + 0.04% v / v HCO2H (10mM)) / ACN from 95 / 5 to 0 / 100 in 2.5 min. Alkaline conditions: Waters XSelect CSH C18 column (3.5pm, 2. lx 50 mm). Gradient: (H2O + 0.06% v / v NH3 (aq.) (10mM)) / ACN from 95 / 5 to 0 / 100 in 2.5 min.

[0285] Acidic Reverse-Layer UPLC-MSSystem: Waters Aquity UPLCDetectors: TUV (E10TUV106N), Sedex 100 LT-ELSD, and SQ detector(LBA751)Column Oven temperature: 45 °CColumn: Waters XSelect CSH C18 2.5 pm, 2.1 x 50 mm (SKU: 186006101)Mobile Phase A: 10 mM FA in H2OMobile Phase B: MeCNGradient 2.5 min: 1 ml.min'1, 5% B for 5 min, 5% to 98% B over 1.17 min, 98% B for 0.25 min, 98% to 5% B over 0.23 min and 5% B for 0.35 min.Gradient 4 min: 1 ml.min'1, 5% B for 5 min, 5% to 98% B over 2.65 min, 98% B for 0.27 min, 98% to 5% B over 0.25 min and 5% B for 0.33 min.

[0286] Basic Reverse-Layer HPLC-MSSystem: Waters Aliance 2795 HPLC UPLCDetectors: Waters PDA 2996, Sedex 100 LT-ELSD, and Waters MicroMass ZqColumn Oven Temperature: 45 °CColumn: Waters XSelect CSH C18 2.5 pm, 2.1 x 50 mm (SKU: 186006101)Mobile Phase A: 10 mM NH3 in H2OMobile Phase B: MeCNGradient 4.0 min: 1 ml.min'1, 5% to 95% B over 2.50 min, 95% B for 0.50 min, 95% to 5% B over 0.22 min and 5% B for 0.78 min.

[0287] 'H NMR :JH NMR spectra (400 MHz) were recorded with a Bruker ULTRASHIELD 400 spectrometer. Processing and analyses of the spectra were performed with MestReNova.

[0288] Preparation 1: Preparation of 4-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino)benzoic acid

[0289] 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 IM HC1 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. 'H NMR (300 MHz, DMSO- d6) 5 (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)EXAMPLE 1: PREPARATION OF COMPOUNDS OF FORMULA (IV).

[0290] The following compounds of formula (IV), compounds (1) to (13) were prepared to be subsequently conjugated to an AAV vector particle viral capsid for obtaining an AAV vector particle comprising a moiety of formula (III):

[0291] Compound (1): 4-azido-V-(2-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino)ethyl) benzamide

[0292] Compound (2): 4-azido-V-(2-(2-(2-(4-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino) benzamido)ethoxy)ethoxy)ethyl)benzamide

[0293] Compound (3): A^-(l-amino-6-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino)-l-imino-7-oxo-ll,14-dioxa-2,8-diazahexadecan-16-yl)-4-azidobenzamide, acetic acid salt

[0294] Compound (4): N6-diazo-N2-(4-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino)benzoyl)-L-lysine

[0295] Compound (5): N6-(4-azidobenzoyl)-N2-(4-((2-ethoxy-3,4-dioxocyclobut-l- en- l-yl)amino)benzoyl)-L- lysine

[0296] Compound (6): N-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12) ,4(9), 5, 7, 13, 15 -hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] ethoxy] ethoxy] ethyl] - 4-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino] benzamide

[0297] Compound (7): (2S)-N-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12) ,4(9), 5, 7, 13, 15 -hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] ethoxy] ethoxy] ethyl] - 2-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino]-5-guanidino-pentanamide

[0298] Compound (8): tert-butyl (2S)-6-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca- 1(16), 4, 6, 8, 12,14-hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] -2- [ [4- [(2-ethoxy-3 ,4- dioxo-cyclobuten-l-yl)amino]benzoyl] amino]hexanoic acid

[0299] Compound (9): (3S)-4-[2-[[(lR,8S)-9-bicyclo[6.1.0]non-4- ynyl] methoxycarbonylamino] ethylamino]-3-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten-l- yl)amino]benzoyl] amino] -4-oxo-butanoic acid

[0300] Compound (10): (3S)-3-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten-l- yl)amino]benzoyl]amino]-4-oxo-4-[2-(2-tricyclo[10.4.0.04,9]hexadeca- l(16),4,6,8,12,14-hexaen-10-ynyloxycarbonylamino) ethylamino]butanoic acid

[0301] Compound (11): (S)-14-(4-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino)benzamido)-l-(4-(6-methyl- 1,2,4, 5-tetrazin-3-yl)phenoxy)- 13-oxo-3, 6,9- trioxa- 12-azahexadecan- 16-oic acid

[0302] Compound (12): (2-methylcycloprop-2-en-l-yl)methyl (2-(2-(2-(4-((2- ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)benzamido)ethoxy)ethoxy)ethyl)carbamate

[0303] Compound (13): 4-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)-N-(l-(2- methylcycloprop-2-en- l-yl)-4-oxo-2,8, 1 l-trioxa-5-azatridecan- 13-yl)benzamideExample 1.1 - Synthesis of compound (1): 4-azido-N-(2-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino) ethyl)benzamide Step 1: Synthesis of tert-butyl (2-(4-azidobenzamido)ethyl)carbamate

[0304] To a solution of 4- azidobenzoic acid (500 mg) in DMF at room temperature were added HATU (1750 mg) and DIPEA (1.6 mL). The mixture was stirred for 10 min, and 1-Boc-ethylenediamine (589 mg) was added. The reaction mixture was stirred for 2 hours at room temperature and was poured onto an ice. The product was extracted with EtOAc (3x30 mL). The combined organic layers were washed with ca. 10% solution of HO Ac (3x10 mL) to remove unreacted amine, and next with brine (1x30 mL). The organic layer was next concentrated under reduced pressure to get a yellowish solid. This material was dissolved in a mixture of acetone- water (ca.l: l), and the solution was partially concentrated under reduced pressure. The resulting suspension was filtered off and dried to get the desired product as a yellowish solid (715 mg, 76.4% yield). LC / MS (6 min): RT = 3.1 min, found [M+H]+= 306.05. 'H NMR (400 MHz, DMSO-de) 5 (ppm): 8.46 (t, 1H), 7.90 - 7.87 (m, 2H), 7.22 - 7.18 (m, 2H), 6.91 (t, 1H), 3.26-3.30 (q, 2H), 3.07-3.12 (q, 2H), 1.37 (s, 9H)Step 2: Synthesis of A-(2-aminoethyl)-4-azidobenzamide

[0305] To a solution of previous compound (700 mg) in DCM was added TFA (0.88 mL) at room temperature. The reaction mixture was stirred over the night at room temperature and was next concentrated under reduced pressure to get a yellow oil. The material was purified by Reverse Phase Flash Column Chromatography (acetonitrile / water) to get the title product as a TFA salt in the form of a yellow oil (674 mg, 92.0% yield). LC / MS (6 min): RT = 1.75 min, found [M+H]+= 206.05 (free amine). 'H NMR (400 MHz, DMSO-de) 5 (ppm): 8.66 (t, 1H), 7.95-7.88 (m, 5H), 7.27 - 7.23 (m, 2H), 3.54 - 3.51 (m, 2H), 3.04-2.98 (q, 2H)Step 3: Synthesis of 4-azido-A-(2-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)ethyl) benzamide

[0306] To a solution of previous compound (650 mg) as TFA salt in a mixture of EtOH / DIPEA (9.75 mL / 1.77 mL) at room temperature was added 3,4-diethoxycyclobut- 3-ene- 1,2-dione (0.9 mL). The reaction mixture was stirred overnight at room temperature. The precipitate was filtered off, washed with a mixture of EtOH / DCM and dried. The crude product was triturated with EtOAc at room temperature for several hours. The obtained product was filtered off, washed with EtOAc, and dried to get the target compound (471 mg, 70.2% yield) as a yellowish solid. LC / MS (6 min): RT = 2.7 min, found [M+H]+= 330.0. HPLC purity: 99.5 % (200 nm), 99.8 % (267 nm). 'H NMR (400 MHz, DMSO-de) 5 (ppm): 8.78 (br s, 1H), 8.61-8.64 (m, 1H), 7.89 - 7.85 (m, 2H), 7.22-7.19 (m, 2H), 4.66-4.42 (2xq, 2H), 3.64-3.61 (t, 1H), 3.42 (s, 3H), 1.38-1.10 (2xt, 3H)Example 1.2 - Synthesis of compound (2): 4-azido-A-(2-(2-(2-(4-((2-ethoxy-3,4- dioxocyclobut- 1 -en- 1 -yl)amino)benzamido)ethoxy) ethoxy)ethyl)benzamideStep 1: Synthesis of tert-butyl (2-(2-(2-(4-azidobenzamido)ethoxy)ethoxy)ethyl) carbamate

[0307] To a solution of 4- azidobenzoic acid (500 mg) in DMF at room temperature were added HATU (1750 mg) and DIPEA (1.60 mL). The mixture was stirred for 10 min, and BocNH-PEG2-NH2 (0.73 mL) was added. The reaction mixture was stirred for 2 hours at room temperature and was poured onto an ice. The product was extracted with a mixture of EtO Ac-Hex (1:1, 3x30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to get an orange oil. This material was purified by Flash Column Chromatography on silica gel using DCM-MeOH (100:0-95:5) as eluent to get the desired product as an orange oil (1.0 g, 82.9% yield). LC / MS (6 min): RT = 3.2 min, found [M+H]+= 394.15. 'H NMR (300 MHz, DMSO-dfi) 5 (ppm): 8.52 (t, 1H), 7.92 - 7.88 (m, 2H), 7.22 - 7.18 (m, 2H), 6.75 (t, 1H), 3.55 - 3.48 (m, 6H), 3.43 - 3.37 (m, 4H), 3.05 (q, 2H), 1.37 (s, 9H)Step 2: Synthesis of A-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-4-azidobenzamide

[0308] To a solution of previous compound (1.0 g) in DCM at room temperature was added TFA (2.34 mL). The reaction mixture was stirred overnight at room temperature and was next concentrated under reduced pressure to get a yellow oil. The material was purified by Reverse Phase Flash Column Chromatography (acetonitrile / water) to get the target product ( 1.34 g, yield > 100% yield) as a TFA salt in the form of a solidified orange oil. This material was used further without an additional purification. LC / MS (6 min): RT = 2.0 min, found [M+H]+= 294.0 (free amine). 'H NMR (300 MHz, DMSO-dg) 5 (ppm): 8.54 (t, 1H), 7.91 - 7.88 (m, 2H), 7.79 (s, 3H), 7.22 - 7.19 (m, 2H), 3.60 - 3.57 (m, 8H), 3.56 - 3.40 (m, 2H), 2.96 (h, 2H)Step 3: Synthesis of 4-azido-A-(2-(2-(2-(4-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino)benzamido)ethoxy) ethoxy)ethyl)benzamide

[0309] To a solution of previous compound (450 mg) as TFA salt in DMF at room temperature was added DIPEA (1.54 mL). The mixture was stirred for 15 minutes and was treated with 4-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)benzoic acid (289 mg) described in the preparation 1 followed by HATU (630 mg). The reaction mixture was stirred at room temperature overnight and was poured onto an ice. The product was extracted with EtOAc (3x50 mL), the combined organic layers were dried, and concentrated under reduced pressure. The residue was purified by Reverse Phase Flash Column Chromatography (water / acetonitrile 100 / 0 to 60 / 40). The appropriate fractions with the product were partially concentrated under reduced pressure, and the product was extracted with EtOAc (3x50 mL). The removal of the solvent under reduced pressure delivered the pure product (209 mg, 35.2% yield) as a yellow solid. LC / MS (6 min): RT = 2.85 min, found [M+H]+= 537.3. HPLC purity: 97.6 % (200 nm), 97.8 % (316 nm). 'H NMR (300 MHz, DMSO-d6) 5 (ppm): 10.92 (s, 1H), 8.52 (t, 1H), 8.43 (t, 1H), 7.91 - 7.81 (m, 4H), 7.43 (d, 2H), 7.20 - 7.16 (m, 2H), 4.79 (q, 2H), 3.55 - 3.51 (m, 8H), 3.41 (m, 4H), 1.43 (t, 3H)Example 1.3 - Synthesis of compound (3):N-( 1 -amino-6-((2-ethoxy-3,4-dioxocyclobut- 1 -en- 1 -yl)amino)- 1 -imino-7 -oxo- 11,14- dioxa-2,8-diazahexadecan-16-yl)-4-azidobenzamide acetic acid saltStep 1: Synthesis of A2-(tert-butoxycarbonyl)-A“-((2,2,4,6,7-pentamethyl-2,3- dihydrobenzofuran- 5 -yl) sulfonyl) arginine

[0310] To a suspension of commercially available H-Arg(Pbf)-OH (5 g) in ACN was added DIPEA (6.13 mL) at room temperature. The mixture was stirred for 10 min, and Boc anhydride (5.12 g) was added. The reaction mixture was stirred overnight at room temperature, and next was concentrated under reduced pressure. The residue was purified by Reverse Phase Flash Column Chromatography (acetonitrile / water, ACN 5% to 60%). All fractions containing the product were combined, partially concentrated under reduced pressure to remove acetonitrile, extracted with EtOAc (3x100 mL), and concentrated in vacuo to dryness to deliver the desired product (5.5 g, 89.1% yield) as a colorless oil. LC / MS (6 min): RT = 3.4 min, found [M+H]+= 527.3. 'H NMR (300 MHz, DMSO- d6) 8 (ppm): 7.05 (d, 1H), 6.69-6.39 (2xbr s, 3H), 3.84-3.79 (td, 1H), 3.01 (q, 2H), 2.97 (s, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.01 (s, 3H), 1.67 - 1.44 (m, 4H), 1.49-1.34 (m, 15H).Step 2: Synthesis of tert-butyl (l-(4-azidophenyl)-18-imino-l,12-dioxo-18-((2,2,4,6,7- pentamethyl-2,3-dihydrobenzofuran)-5-sulfonamido)-5,8-dioxa-2, 11,17- triazaoctadecan- 13-yl)carbamate

[0311] To a solution of Boc-Arg(Pbf)-OH (310 mg) obtained previously and N-{2-[2- (2-aminoethoxy)ethoxy]ethyl}-4-azidobenzamide TFA salt (240 mg) described in step 2 of example 1.2 in DMF was added DIPEA (0.41 mL) at room temperature. The mixture was stirred 5 minutes, and HATU (340 mg) was added. The reaction mixture was stirred overnight at room temperature and was poured onto an ice. The product was extracted with EtOAc (3x 30 mL), the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to get an orange oil. This material was purified by Reverse Phase Flash Column Chromatography (acetonitrile / water, ACN 20% to 60%). All fractions containing the product were combined, partially concentrated under reduced pressure to remove acetonitrile, and the product was extracted with EtOAc (3x30 mL), concentrated in vacuo to deliver 350 mg of the product (350 mg, 74.2% yield) as a white solid foam. LC / MS (6 min): RT = 3.65 min, found [M+H]+= 802.65. 'H NMR(300 MHz, DMSO-rffi) 5 (ppm): 8.53 (t, 1H), 7.92 - 7.84 (m, 2H), 7.82 (t, 1H), 7.22 - 7.17 (m, 2H), 6.79 and 6.65 (d and br s, 2H), 6.41 (br s, 2H), 3.86 (d, 1H), 3.52 - 3.50 (m, 6H), 3.42 - 3.40 (m, 4H), 3.19 (q, 2H), 3.01 (d, 2H), 2.96 (s, 2H), 2.48 (s, 3H), 2.42 (s, 3H), 2.01 (s, 3H), 1.58-1.54 (m, 1H), 1.41-1.39 (m, 17H)Step 3: Synthesis of 4-azido-A-(l,6-diamino-l-imino-7-oxo-l l,14-dioxa-2,8- diazahexadecan- 16-yl)benzamide

[0312] A solution of compound obtained previously in a mixture of TFA / TIS / water (3.23 mL / 0.09 mL / 0.09 mL) was stirred 4 hours at room temperature. Next, the mixture was poured into diethyl ether (50 mL) resulting in a pinkish precipitation. The solid material was filtered off and washed with diethyl ether. As this material was a highly hygroscopic, the solid was dissolved in a mixture of water-ethanol and concentrated under reduced pressure to dryness to get the TFA salt form of product (259 mg, 90.2% yield) as a pale brown oil. This material was used for the next step without further purification.LC / MS (6 min): RT = 1.75 min, found [M+H]+= 450.2 (free amine).Step 4: Synthesis of A-(l-amino-6-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)-l- imino-7-oxo-l l,14-dioxa-2,8-diazahexadecan-16-yl)-4-azidobenzamide, acetic acid salt

[0313] A suspension of previous compound obtained as TFA salt (180 mg), 3,4- diethoxycyclobut-3-ene- 1,2-dione (0.04 mL) and DIPEA 0.14 mL) in a mixture of EtOH / water were stirred at room temperature overnight. To the reaction mixture was next added HOAc (5 eq), stirred for 5 minutes, and the solvents were removed under reducedpressure. The residue was purified by Reverse Phase Flash Column Chromatography (acetonitrile / water, ACN 0% to 10%). The fractions containing the product were combined, treated with HOAc (5 eq), and concentrated under reduced pressure to get a colourless heavy oil. This material was dissolved in a small portion of water, the sample was frozen, and next freeze-dried to get the acetate salt form of the desired product as off- white amorphous powder (128 mg, 76.2% yield). The product turned into yellowish crystals after several hours at room temperature. LC / MS (6 min): RT = 2.25 min, found [M+H]+= 574.35 (free amine). HPLC purity: 99.2 % (200 nm), 97.4 % (270 nm). 'H NMR (300 MHz, DMSO-d6) 5 (ppm): 8.86 - 8.74 (m, 1H), 8.53 (t, 1H), 8.17 - 8.12 (m, 1H), 7.92 - 7.87 (m, 2H), 7.50 (br s, 1H), 7.23 - 7.18 (m, 2H), 7.06 (br s, 4H), 4.71 - 4.58 (m, 2H), 4.54 and 4.08 (m, 1H), 3.54 - 3.51 (m, 6H), 3.43 - 3.40 (m, 4H), 3.23 - 3.21 (m, 2H), 3.10 - 3.06 (m, 2H), 1.79 (m, 1H), 1.63 (m, 1H), 1.48 (m, 2H), 1.41 - 1.31 (m, 3H)Example 1.4 - Synthesis of compound (4):

[0314] N6-diazo-N2-(4-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)benzoyl)-L- lysine

[0315] Step 1: Synthesis of tert-butyl N2-(((9H-fhioren-9-yl)methoxy)carbonyl)-N6- diazo-L-lysinate

[0316] To a stirred solution of Fmoc-Lys(N3)-OH (800 mg) and tert-butyl-2,2,2- trichloroacetimidate (1.1 mL) in anhydrous tetrahydrofuran (19 mL) under an argon atmosphere was added boron trifluoride diethyl etherate (50 pL) at ambient temperature. The reaction was stirred 2 h, after which an additional portion of tert-butyl-2,2,2- trichloroacetimidate (370 pL) was added. Stirring was continued at ambient temperature for a further 2 h. The reaction mixture was poured over saturated aqueous NaHCCh (100 mL), and the aqueous layer was extracted with EtOAc (100 mL). The organic layer was dried over anhydrous Na2SO4, fdtered, and concentrated under reduced pressure. Thecrude product was purified by column chromatography on silica gel (0% to 50% ethyl acetate in cyclohexane) to afford the titled compound as a colorless oil (770 mg, 84% yield). 'H NMR (400 MHz, Chloroform-d) 5 (ppm): 7.77 (d, J =7.5 Hz, 2H), 7.60 (d, J =7.5 Hz, 2H), 7.40 (t, J =7.4 Hz, 2H), 7.32 (td, J =7.5, 1.2 Hz, 2H), 5.34 (d, J =8.2 Hz, 1H), 4.40 (d, J =7.1 Hz, 2H), 4.34 - 4.25 (m, 1H), 4.23 (t, J =7.0 Hz, 1H), 3.28 (t, J =6.8 Hz, 2H), 1.86 (ddt, 7 = 15.4, 10.9, 5.5 Hz, 1H), 1.75 - 1.59 (m, 3H), 1.48 (s, 11H). UPLC- MS (Acid, 4.0 min) Rt = 1.86 min, m / z (ES+) = 451.2 [M+H]+

[0317] Step 2: tert-butyl N6-diazo-L-lysinate

[0318] To a solution of compound obtained previously (770 mg) in N,N- dimethylformamide (6.8 mL) was added piperidine (1.7 mL) at ambient temperature. The reaction was stirred for 1.5 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (0% to 10% (7N NH3 in MeOH) in DCM) to afford the titled compound as a colorless liquid (347 mg, 89% yield). 'H NMR (400 MHz, DMSO-t e) 5 (ppm): 3.31 (t, J =6.8 Hz, 2H), 3.14 (dd, J =6.8, 5.6 Hz, 1H), 1.62 (s, 2H), 1.58 - 1.48 (m, 3H), 1.41 (s, 12H). HPLC-MS (Basic, 4 min) Rt = 2.14 min, m / z (ES+) = 229.2 [M+H]+, 173.2 [M-tBu+H]+

[0319] Step 3: (2S)-6-azido-2-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino] benzoyl] amino] hexanoic acid

[0320] To a solution of compound obtained previously (240 mg), 4-[(2-ethoxy-3,4- dioxo-cyclobuten-l-yl)amino]benzoic acid obtained in preparation 1 (300 mg) and O- (7 -azabenzo triazol- 1 -yl)-A, N, N', A^'-tctramcthyluron ium hexafluorophosphate (HATU) (400 mg) in anhydrous A,A-dimethylformamide (4.8 mL, 0.20 M) was added N- diisopropylethylamine (DIPEA) (500 pL). The reaction was stirred for 2 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (25% to 100% MeOH in DCM) to afford an inseparable mixture of the precursor protected compound and its substituted analogue, in which the ethoxy moiety of the squarate was replaced by an additional lysine residue, as a colorless liquid. To a solution of the crude mixture containing the precursor protected compound (451 mg) in dichloromethane (5 mL) was added trifluoroacetic acid (5 mL) at ambient temperature. The reaction was stirred for 1 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (0% to 15% MeOH + 1% AcOH in DCM) to afford the titled compound as a white solid (219 mg, 53% yield). 'H NMR (400 MHz, DMSO- d6) 6 (ppm): 12.55 (s, 1H), 10.93 (s, 1H), 8.50 (d, J =7.8 Hz, 1H), 7.97 - 7.79 (m, 2H), 7.45 (d, J =8.6 Hz, 2H), 4.79 (q, J =7.1 Hz, 2H), 4.36 (ddd, J =9.5, 7.7, 5.1 Hz, 1H), 3.33 (t, J =6.8 Hz, 2H), 1.81 (dddd, J = 17.7, 13.5, 10.0, 5.5 Hz, 2H), 1.66 - 1.50 (m, 2H), 1.43 (t, J =7.1 Hz, 5H). UPLC-MS (Acid, 2.5 min) Rt = 1.37 min, m / z (ES+) = 416.1 [M+H]+Example 1.5 - Synthesis of compound (5):

[0321] N6-(4-azidobenzoyl)-N2-(4-((2-ethoxy-3,4-dioxocyclobut- 1 -en- 1 -yl)amino) benzoyl)-L-lysine

[0322] Step 1: tert-butyl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(4- azidobenzoyl)-L-lysinate

[0323] To a solution of tert-butyl (2S)-6-amino-2-(9H-fluoren-9- ylmethoxycarbonylamino)hexanoate hydrochloride (500 mg), 4- azidobenzoic acid (212 mg), and 2-(lH-benzo[d][l,2,3]triazol-l-yl)-l,l,3,3-tetramethylisouronium tetrafluoroborate (TBTU) (418 mg) in anhydrous A-di methylformamide (5 mL) was added A,A-diisopropylethylamine (DIPEA, 0.76 mL) at room temperature. The reaction mixture was stirred for 16 h, after which O-(7-azabcnzotriazol- l -yl)- A',A'- tetramethyluronium hexafluorophosphate (HATU, 412 mg) was added. The mixture was stirred at room temperature for an additional 23 h, followed by the addition of triethylamine (760 pL). Stirring was continued for 30 min at room temperature. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (15% to 75% ethyl acetate in cyclohexane) to afford the titled compound as an off-white sticky oil (354 mg, 55% yield). 'H NMR (400 MHz, Chloroform-d) 5 (ppm): 7.75 (dd, J =8.7, 2.2 Hz, 4H), 7.56 (dd, J =7.5, 3.8 Hz, 2H), 7.39 (td, J =7.5, 2.8 Hz, 2H), 7.30 (tdd, J =7.5, 3.2, 1.2 Hz, 2H), 6.95 (d, J =8.2 Hz, 2H), 6.30 (t, J =4.3 Hz, 1H), 5.46 (d, J =8.1 Hz, 1H), 4.42 - 4.30 (m, 2H), 4.30 - 4.23 (m, 1H), 4.19 (t, J =7.2 Hz, 1H), 3.45 (q, J =6.6 Hz, 2H), 1.94 - 1.81 (m, 1H), 1.77 - 1.58 (m, 5H), 1.47 (s, 11H). 'H NMR (400 MHz, DMSO-de) 6 (ppm): 8.45 (t, J =5.6 Hz, 1H), 7.97 - 7.83 (m, 4H), 7.71 (d, J =7.5 Hz, 2H), 7.64 (d, J =7.8 Hz, 1H), 7.41 (t, J =7.4 Hz, 2H), 7.31 (td, J =7.5, 1.2 Hz, 2H), 7.22 - 7.11 (m, 2H), 4.35 - 4.24 (m, 2H), 4.21 (q, J =6.7 Hz, 1H), 3.87 (td, J =8.5, 5.2 Hz, 1H), 3.25 (q, J =6.6 Hz, 2H), 1.66 (ddd, 7 = 17.3, 14.0, 8.0 Hz, 2H), 1.52 (p, J =7.0 Hz, 2H), 1.37 (s, 11H) ; UPLC- MS (Acid, 2.5 min) Rt = 1.81 min, m / z (ES+) = 570.3 [M+H]+

[0324] Step 2: tert-butyl N6-(4-azidobenzoyl)-L-lysinate

[0325] To a solution of [4-[[(5S)-6-tert-butoxy-5-(9H-fluoren-9- ylmethoxycarbonylamino)-6-oxo-hexyl]carbamoyl]phenyl]-diazonio-azanide (354 mg) in dichloromethane (6.0 mL) was added piperidine (237 pL) at room temperature. The reaction was stirred for 18 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (0% to 10% MeOH in DCM) to afford the titled compound as a yellow sticky oil (186 mg, 90% yield). 'H NMR (400 MHz, DMSO-de) 5 (ppm): 8.43 (t, J =5.7 Hz, 1H), 7.98 - 7.77 (m, 2H), 7.25 - 7.12 (m, 2H), 3.23 (q, J =6.7 Hz, 2H), 3.13 (dd, J =7.0, 5.7 Hz, 1H), 1.70 - 1.39 (m, 6H), 1.38 (s, 9H), 1.36 - 1.30 (m, 2H). UPLC-MS (Acid, 2.5 min) Rt = 1.14 min, m / z (ES+) = 348.2 [M+H]+

[0326] Step 3: tert-butyl N6-(4-azidobenzoyl)-N2-(4-((2-ethoxy-3,4-dioxocyclobut-l- en- 1-yl) amino)benzoyl)-L-lysinate

[0327] To a solution of compound obtained previously (181 mg), HATU (207 mg), and 4-[(2-ethoxy-3,4-dioxocyclobuten-l-yl)amino]benzoic acid obtained in preparation 1 (155 mg) in anhydrous A,A-dimethylformamide (2.47 mL) was added DIPEA (258 pL) at room temperature. The reaction mixture was stirred for 45 min. The reaction was diluted with EtOAc (25 mL) and washed successively with saturated aqueous NaHCCh (25 mL), 1 M HCl / brine (1:1, 25 mL), and brine (25 mL). The organic layer was driedover anhydrous Na2SO4, filtered, and concentrated under reduced pressure. Purification of the crude product by column chromatography on silica gel (0% to 10% MeOH in DCM) afforded the titled compound as a colorless sticky oil (203 mg, 70% yield). 'H NMR (400 MHz, DMSO-de) 8 (ppm): 10.93 (s, 1H), 8.45 (d, J =2.3 Hz, 1H), 8.43 (s, 1H), 7.93 - 7.78 (m, 4H), 7.43 (d, J =8.3 Hz, 2H), 7.22 - 7.09 (m, 2H), 4.79 (q, J =7.1 Hz, 2H), 4.27 (q, J =7.5 Hz, 1H), 3.29 - 3.22 (m, 2H), 1.86 - 1.71 (m, 2H), 1.55 (h, J =6.5 Hz, 2H), 1.43 (t, J =7.1 Hz, 4H), 1.39 (s, 10H). UPLC-MS (Acid, 2.5 min) Rt = 1.54 min, m / z (ES+) = 591.3 [M+H]+

[0328] Step 4: N6-(4-azidobenzoyl)-N2-(4-((2-ethoxy-3,4-dioxocyclobut-l-en-l- yl)amino)benzoyl)-L-lysine

[0329] To a solution of compound obtained previously (184 mg) in dichloromethane (1.5 mL) was added trifluoroacetic acid (1.5 mL) at room temperature. The reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure to afford titled compound as a white solid (162 mg, 93% yield). 'H NMR (400 MHz, DMSO-t e) 6 (ppm): 10.93 (s, 1H), 8.45 (dd, J =6.6, 4.9 Hz, 2H), 7.96 - 7.77 (m, 4H), 7.43 (d, J =8.3 Hz, 2H), 7.26 - 7.05 (m, 2H), 4.79 (q, J =7.1 Hz, 2H), 4.35 (ddd, J =9.3, 7.5, 5.0 Hz, 1H), 3.25 (q, J =6.5 Hz, 2H), 1.95 - 1.70 (m, 2H), 1.62 - 1.47 (m, 2H), 1.43 (t, J =7.1 Hz, 5H). UPLC-MS (Acid, 4.0 min) Rt = 1.85 min, m / z (ES+) = 535.2 [M+H]+Example 1.6 - Synthesis of compound (6):

[0330] N-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-l(12),4(9),5,7,13,15- hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] ethoxy] ethoxy] ethyl] -4- [(2-ethoxy-3 ,4- dioxo-cyclobuten- 1 -yl)amino] benzamide

[0331] Step 1: tert-butyl N-[2-[2-[2-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten-l- yl)amino]benzoyl] amino]ethoxy]ethoxy]ethyl]carbamate

[0332] To a solution of 4-[(2-ethoxy-3,4-dioxocyclobuten-l-yl)amino]benzoic acid obtained in preparation 1 (70% pure, 541 mg) and N-Boc- 2,2'-(ethylenedioxy)diethylamine (300 mg) in N,N-dimethylformamide (6.1 mL) at room temperature, were added DIPEA (0.42 mL) and TBTU (465 mg). The mixture was stirred for 16h. The volatiles were removed under reduced pressure. The residue was partitioned between DCM / MeOH (9 / 1) and 0.5N HC1. The biphasic yellow suspension was filtered over fritted glass and filter cake was washed with DCM and water. The layers of the filtrate were separated. The organic extract was dried (Na2SO4), filtered and concentrated under reduced pressure.; The residue was purified by by column chromatography on silica gel (DCM / MeOH = 99 / 1 to 80 / 20) to afford an impure residue. It was further purified by Flash Chromatography to afford titled compound as a yellow solid (390 mg, 66% yield). 'H NMR (400 MHz, DMSO-de) d (ppm): 10.91 (s, 1H), 8.42 (t, J= 5.6 Hz, 1H), 7.92 - 7.77 (m, 2H), 7.44 (d, J= 8.1 Hz, 2H), 6.74 (t, J= 5.9 Hz, 1H), 4.79 (q, J = 7.0 Hz, 2H), 3.60 - 3.48 (m, 6H), 3.45 - 3.35 (m, 4H), 3.05 (q, J = 6.0 Hz, 2H), 1.44 (t, J= 7.0 Hz, 3H), 1.37 (s, 9H). MS (ESI+): [M+H]+492.3

[0333] Step 2: N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]-4-[(2-ethoxy-3,4-dioxo- cyclobuten-l-yl)amino]benzamide, TFA salt

[0334] To a solution of compound obtained previously (75 mg) in DCM (0.6 mL) at RT, was added trifluoroacetic acid (294 pL). The mixture was stirred for 2h. The volatiles were removed under reduced pressure and the residue was azeotroped with toluene (2x1 mL) to afford titled compound as a yellow sticky oil (113 mg, quantitative yield). 'H NMR (400 MHz, DMSO-de) d (ppm): 8.44 (t, J= 5.6 Hz, 1H), 7.97 - 7.56 (m, 5H), 7.45(d, J= 8.3 Hz, 2H), 4.80 (q, J = 7.1 Hz, 2H), 3.65 - 3.50 (m, 8H), 3.43 (q, J = 5.9 Hz, 2H), 2.97 (q, J = 5.5 Hz, 2H), 1.44 (t, J= 7.1 Hz, 3H). MS (ESI+): [M+H]+392.1

[0335] Step 3: N-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12) ,4(9), 5, 7, 13, 15 -hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] ethoxy] ethoxy] ethyl] - 4-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino]benzamide

[0336] To a solution of DBCO-acid (CAS n°: 1353016-70-2; 56 mg) in DMF (2 mL) at RT, were added DIPEA (0.11 mL) and TBTU (59 mg). The mixture was stirred at RT for 30 min and a solution of compound obtained at previous step in DMF (1.1 mL) was added dropwise. The mixture was stirred for 30 min. The reaction mixture was partitioned between EtOAc and 0.5N HC1. The layers were separated and the aqueous phase was extracted with EtOAc. The combined organic extracts were washed (0.5N HC1, water, brine), dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by FC (DCM / MeOH = 99 / 1 to 90 / 10) to afford titled compound as a light yellow solid (80 mg, 66% yield). 'H NMR (400 MHz, DMSO-de) d (ppm): 10.91 (s, 1H), 8.42 (t, J = 5.5 Hz, 1H), 7.88 - 7.79 (m, 2H), 7.74 (t, J = 5.5 Hz, 1H), 7.68 (dd, J = 6.9, 2.2 Hz, 1H), 7.62 (d, J = 6.8 Hz, 1H), 7.56 - 7.40 (m, 5H), 7.40 - 7.25 (m, 3H), 5.03 (d, J = 14.1 Hz, 1H), 4.79 (q, J = 7.1 Hz, 2H), 3.61 (d, J = 14.0 Hz, 1H), 3.57 - 3.44 (m, 6H), 3.44 - 3.35 (m, 2H), 3.35 - 3.25 (m, 2H), 3.13 - 3.04 (m, 2H), 2.63 - 2.50 (m, 1H), 2.29 - 2.18 (m, 1H), 2.06 - 1.94 (m, 1H), 1.83 - 1.70 (m, 1H), 1.43 (t, J = 7.1 Hz, 3H). MS (ESI+): [M+H]+679.4Example 1.7 - Synthesis of compound (7):

[0337] (2S)-N-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-l(12),4(9),5,7,13,15- hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] ethoxy] ethoxy] ethyl] -2- [(2-ethoxy-3 ,4- dioxo-cyclobuten-l-yl)amino]-5-guanidino-pentanamide[(2,2,4,6,7-pentamethyl-3H-benzofuran-5-yl)sulfonyl]carbamimidoyl]amino]pentanoic acid

[0339] A solution of H-Arg(Pbf)-OH (2.04 g), 3, 4-diethoxy-3-cyclobutene- 1,2-dione(775 mg) and DIPEA (0.95 mL) in EtOH (15.2 mL) was stirred at RT for 16h. The volatiles were removed under reduced pressure. The residue was partitioned between EtOAc and 0.5N HC1. The layers were separated, and the aqueous phase was extracted with EtOAc (2 x). The combined organic extracts were washed (0.5N HC1, brine), dried (Na2SO4), filtered and concentrated under reduced pressure to afford title compound as a white foam (2.5 g, 100% yield). 'H NMR (400 MHz, DMSO-de) S (ppm): 13.09 (br s, 1H), 9.10 - 8.75 (m, 1H), 7.30 - 6.10 (br m, 3H), 4.75 - 4.60 (m, 2H), 4.58 - 4.45 & 4.15 - 4.05 (m, 1H), 3.12 - 3.00 (m, 2H), 2.97 (s, 2H), 2.48 (s, 3H), 2.43 (s, 3H), 2.01 (s, 3H), 1.95 - 1.78 (m, 1H), 1.75 - 1.60 (m, 1H), 1.56 - 1.26 (m, 11H). MS (ESI+): [M+H]+551.3

[0340] Step 2: tert-butyl N-[2-[2-[2-[[(2S)-2-[(2-ethoxy-3,4-dioxo-cyclobuten-l- yl)amino]-5-[[N-[(2,2,4,6,7-pentamethyl-3H-benzofuran-5- yl) sulfonyl] carbamimidoyl] amino]pentanoyl] amino] ethoxy] ethoxy] ethyl] carbamate

[0341] To a solution of compound obtained in previous step (1.26 g) in DMF (9.2 mL) at RT, were added successively and quickly N-Boc- 2,2'-(ethylenedioxy)diethylamine (682 mg), DIPEA (0.80 mL) and TBTU (882 mg). The mixture was stirred for Ih. The reaction mixture was partitioned between EtOAc and 0.5N HC1. The layers were separated, and the aqueous phase was extracted with EtOAc (2x). The combined organic extracts were washed (0.5N HC1, brine), dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (cHex / (EtOAc / EtOH = 3 / 1) = 90 / 10 to 0 / 100) to afford titled compound as a white foam (1.45g, 82% yield) as a white foam. 'H NMR (400 MHz, DMSO-de) d (ppm): 8.83 & 8.71 (d, J = 8.5 Hz, IH), 8.10 (d, J = 18.5 Hz, IH), 7.14 - 5.91 (m, 4H), 4.72 - 4.58 (m, 2H), 4.58 - 4.45 & 4.10 - 4.00 (m, IH), 3.48 (s, 4H), 3.45 - 3.32 (m, 4H), 3.21 (br s, 2H), 3.10 - 3.00 (m, 4H), 2.96 (s, 2H), 2.47 (s, 3H), 2.42 (s, 3H), 2.00 (s, 3H), 1.80 - 1.69 (m, IH), 1.68 - 1.50 (m, IH), 1.50 - 1.30 (m, 20H). MS (ESI+): [M+H]+781.5

[0342] Step 3: (2S)-N-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12) ,4(9), 5, 7, 13, 15 -hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] ethoxy] ethoxy] ethyl] - 2-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino]-5-[[N-[(2,2,4,6,7-pentamethyl-3H- benzofuran-5-yl)sulfonyl]carbamimidoyl] amino]pentanamide

[0343] To a solution of compound obtained previously (780 mg) in DCM (2.6 mL) at RT, was added HC1 (4M solution in dioxane) (1.25 mL). The mixture was stirred for 2 h. The volatiles were removed under reduced pressure. The residue was dissolved in DMF (5 mL) and added dropwise to a pre-stirred (15 min) solution of DBCO-acid (366 mg), DIPEA (696 pL) and TBTU (385 mg) in DMF (5 mL) at RT. The resulting mixture was stirred for 1 h. The reaction mixture was partitioned between EtOAc and 0.5N HC1. The layers were separated, and the aqueous phase was extracted with EtOAc (2 x). The combined organic extracts were washed (0.5N HC1, brine), dried (Na2SO4), filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (DCM / MeOH = 99 / 1 to 90 / 10) to afford titled compound as a white foam (892mg, 92% yield). 'H NMR (400 MHz, DMSO-de) d (ppm): 8.84 & 8.71 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 19.9 Hz, 1H), 7.80 - 7.60 (m, 3H), 7.55 - 7.25 (m, 6H), 7.00 - 6.20 (br m, 3H), 5.03 (d, J = 14.0 Hz, 1H), 4.75 - 4.65 (m, 2H), 4.57 - 4.47 & 4.10 - 4.00 (m, 1H), 3.61 (d, J = 13.9 Hz, 1H), 3.50 - 3.42 (m, 4H), 3.42 - 3.35 (m, 2H), 3.35 - 3.25 (m, 2H), 3.25- 3.15 (m, 2H), 3.15 - 3.00 (m, 4H), 2.96 (s, 2H), 2.65 - 2.50 (m, 1H), 2.48 (s, 3H), 2.42 (s, 3H), 2.30 - 2.20 (m, 1H), 2.05 - 1.95 (m, 1H), 2.00 (s, 3H), 1.82 - 1.70 (m, 2H), 1.66- 1.52 (m, 1H), 1.41 (s, 11H). MS (ESI+): [M+H]+968.6

[0344] Step 4 : (2S)-N-[2-[2-[2-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-1(12) ,4(9), 5, 7, 13, 15 -hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] ethoxy] ethoxy] ethyl] - 2-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino]-5-guanidino-pentanamide

[0345] To the compound obtained previously (882 mg) at RT, was added a 5% m- cresol solution in trifluoroacetic acid (12.3 mL). The mixture was stirred for Ih. The volatiles were removed under reduced pressure and the residue was azeotroped with toluene (10 mL). Et2<D (75 mL) was added, and the mixture was sonicated to afford a beige suspension. It was filtered over fritted glass and the filter cake was washed with Et2<D (2x20 mL) and collected. The residue was purified by Reverse Phase Flash Column Chromatography (water+0.1%AcOH / acetonitrile 95 / 5 to 2 / 98). The fractions of interest were combined and lyophilized to afford title compound as a white foam (200mg, 31% yield). Product is contaminated with 19mol% m-cresol. 'H NMR (400 MHz, DMSO-t / e) <5 (ppm): 8.85 & 8.74 (d, J= 8.5 Hz, IH), 8.13 (d, J= 21.2 Hz, IH), 7.80 - 7.60 (m, 3H), 7.56 - 7.43 (m, 4H), 7.42 - 7.27 (m, 3H), 7.25 - 6.75 (br s, 3H), 5.03 (d, J = 14.0 Hz, IH), 4.72 - 4.57 (m, 2H), 4.57 - 4.48 & 4.12 - 4.03 (m, IH), 3.62 (d, J = 14.0 Hz, IH), 3.50 - 3.42 (m, 4H), 3.42 - 3.36 (m, 2H), 3.32 - 3.27 (m, 2H), 3.25 - 3.17 (m, 2H), 3.14 - 3.04 (m, 4H), 2.63 - 2.50 (m, IH), 2.29 - 2.18 (m, IH), 2.06 - 1.95 (m, IH), 1.86 - 1.72 (m, 2H), 1.70 - 1.56 (m, IH), 1.56 - 1.42 (m, 2H), 1.42 - 1.30 (m, 3H). MS (ESI+): [M+H]+716.3Example 1.8 - Synthesis of compound (8):

[0346] tert-butyl (2S)-6-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca-l(16),4,6,8,12,14- hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] -2- [ [4- [(2-ethoxy-3 ,4-dioxo-cyclobuten- 1 - yl)amino]benzoyl] amino]hexanoic acid

[0347] Preparation of (2,5-dioxopyrrolidin-l-yl) 4-(2- azatricyclo[10.4.0.04,9]hexadeca-l(16),4,6,8,12,14-hexaen-10-yn-2-yl)-4-oxo- butanoate (SI)

[0348] To a solution of DBCO-acid (150 mg) and A-hydroxysuccinimide (68 mg) in anhydrous dichloromethane (1.7 mL), EDCI (113 mg) was added at room temperature. The reaction was stirred for 1 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (25% to 100% EtOAc in cyclohexane) to afford the titled compound as an off-white solid (176 mg, 89% yield). 'H NMR (400 MHz, Chloroform-d) 5 (ppm): 7.78 - 7.62 (m, 1H), 7.45 - 7.34 (m, 5H), 7.31 (td, J =7.5, 1.3 Hz, 1H), 7.27 - 7.24 (m, 2H), 5.18 (d, J =13.9 Hz, 1H), 3.68 (d, J =13.8 Hz, 1H), 2.97 (dt, J = 17.3, 7.6 Hz, 1H), 2.89 - 2.80 (m, 1H), 2.78 (s, 4H), 2.64 (ddd, J = 17.4, 7.6, 5.4 Hz, 1H), 2.08 (ddd, J = 16.8, 7.8, 5.4 Hz, 1H). UPLC-MS (Acid, 2.5 min) Rt = 1.57 min, m / z (ES+) = 403.1 [M+H]+

[0349] Step 1: tert-butyl (2S)-6-(tert-butoxycarbonylamino)-2-[[4-[(2-ethoxy-3,4- dioxo-cyclobuten-l-yl)amino]benzoyl]amino]hexanoate

[0350] To a solution of commercially available tert-butyl (2S)-2-amino-6-(tert- butoxycarbonylamino)hexanoate (447 mg), HATU (561 mg) and 4-[(2-ethoxy-3,4- dioxo-cyclobuten-l-yl)amino]benzoic acid (421 mg) obtained in preparation 1 in anhydrous A, A-dimcthylformamidc (6.7 mL) was added AA-diisopropylethylamine (DIPEA) (702 pL). The reaction was stirred for 1 h. The reaction mixture wasconcentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (25% to 100% EtOAc in cyclohexane) to afford the titled compound as a colourless sticky oil (598 mg, 80% yield). 'H NMR (400 MHz, DMSO-de) 8 (ppm): 10.92 (s, 1H), 8.45 (d, J =7.4 Hz, 1H), 7.98 - 7.81 (m, 2H), 7.45 (d, J =8.4 Hz, 2H), 6.76 (t, J =5.8 Hz, 1H), 4.79 (q, J =7.1 Hz, 2H), 4.25 (q, J =7.5 Hz, 1H), 3.08 - 2.78 (m, 2H), 1.74 (d, J =6.1 Hz, 2H), 1.42 (d, J =7.3 Hz, 4H), 1.38 (d, J =20.2 Hz, 21H). UPLC-MS (Acid, 2.5 min) Rt = 1.58 min, m / z (ES+) = 546.3 [M+H]+

[0351] Step 2: tert-butyl (2S)-6-amino-2-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten- 1- yl)amino]benzoyl] amino]hexanoic acid 2,2,2-trifluoroacetic acid

[0352] To a solution of compound obtained in previous step (598 mg) in dichloromethane (5 mL) was added trifluoroacetic acid (5.0 mL) at room temperature. The reaction was stirred for 2.5 h. The solvent was removed under reduced pressure to afford title compound as a white solid (642 mg, quantitative yield) that was used as it for the next step without further purification. 'H NMR (400 MHz, DMSO-t / e) 6 (ppm): 12.34 (s, 1H), 10.95 (s, 1H), 8.50 (d, J =7.7 Hz, 1H), 7.94 - 7.86 (m, 2H), 7.74 (s, 3H), 7.46 (d, J =8.4 Hz, 2H), 4.79 (q, J =7.1 Hz, 2H), 4.36 (ddd, J =9.6, 7.7, 5.0 Hz, 1H), 2.79 (q, J =6.7, 6.0 Hz, 2H), 1.91 - 1.70 (m, 2H), 1.58 (p, J =6.5 Hz, 2H), 1.43 (t, J =7.0 Hz, 5H). UPLC-MS (Acid, 2.5 min) Rt = 0.22 / 0.99 min, m / z (ES+) = 390.2 [M+H]+

[0353] Step 3: tert-butyl (2S)-6-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca- 1(16), 4, 6, 8, 12,14-hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] -2- [ [4- [(2-ethoxy-3 ,4- dioxo-cyclobuten-l-yl)amino] benzoyl] amino]hexanoic acid N-ethyl-N-isopropyl- propan-2-amine.

[0354] To a solution of activated DBCO (SI) (149 mg) and compound obtained at previous step (186 mg) in anhydrous A,A-dimethylformamide (3.7 mL) was added DIPEA (193 pL) at room temperature. The reaction was stirred for 2 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (0% to 15% MeOH+l%AcOH in DCM) to afford the titled compound as a colorless sticky oil (98 mg, 33% yield). 'H NMR (400 MHz, DMSO-de) 8 (ppm): 10.93 (s, 1H), 8.38 (d, J =7.6 Hz, 1H), 7.87 (dd, J =8.7, 2.1 Hz, 2H), 7.71 - 7.64 (m, 2H), 7.62 (dd, J =7.3, 1.6 Hz, 1H), 7.52 - 7.40 (m, 5H), 7.36 (qd, J =7.4, 1.6 Hz, 2H), 7.29 (td, J =7.5, 1.6 Hz, 1H), 5.02 (d, J =14.0 Hz, 1H), 4.79 (q, J =7.1 Hz, 2H), 4.28 (td, J =8.0, 4.9 Hz, 1H), 3.60 (d, J = 13.9 Hz, 1H), 3.44 (s, 1H), 3.40 - 3.23 (m, 2H), 2.92 (q, J =6.3 Hz, 2H), 2.55 (dd, 7 = 16.1, 8.0 Hz, 1H), 2.21 (dt, J = 15.2, 7.6 Hz, 1H), 1.97 (ddd, 7 = 14.7, 8.0, 5.7 Hz, 1H), 1.75 (tq, 7 = 15.5, 5.3, 4.6 Hz, 3H), 1.43 (t, 7 =7.1 Hz, 3H), 1.38 - 1.22 (m, 4H), 1.22 - 1.03 (m, 4H). UPLC-MS (Acid, 4.0 min) Rt = 1.55 min, m / z (ES+) = 677.3 [M+H]+

[0355] Step 4: tert-butyl (2S)-6-[[4-(2-azatricyclo[10.4.0.04,9]hexadeca- 1(16), 4, 6, 8, 12,14-hexaen- 10-yn-2-yl)-4-oxo-butanoyl] amino] -2- [ [4- [(2-ethoxy-3 ,4- dioxo-cyclobuten-l-yl)amino] benzoyl] amino]hexanoic acid

[0356] The A,A-diisopropylethylamine counterion can be quantitively exchange for a proton by passing a solution of compound obtained at previous step in ethanol with few drops of dichloromethane through a Dowex-H+column to afford the desired compound as an off-white solid after solvent removal. 'H NMR (400 MHz, DMSO-de) 5 (ppm): 10.92 (s, 1H), 8.43 (d, J =7.6 Hz, 1H), 7.92 - 7.83 (m, 2H), 7.70 - 7.63 (m, 2H), 7.62 (dd, J =7.2, 1.6 Hz, 1H), 7.53 - 7.40 (m, 5H), 7.36 (qd, J =7.4, 1.6 Hz, 2H), 7.29 (td, J =1A, 1.7 Hz, 1H), 5.03 (d, J =14.0 Hz, 1H), 4.79 (q, J =7.1 Hz, 2H), 4.30 (td, J =8.4, 5.4 Hz, 1H), 3.60 (d, J = 14.0 Hz, 1H), 2.92 (d, J =5.9 Hz, 2H), 2.56 (dt, J = 16.1, 7.7 Hz, 1H), 2.21 (dt, J = 15.3, 7.6 Hz, 1H), 1.97 (ddd, 7 = 15.2, 8.0, 5.7 Hz, 1H), 1.86 - 1.65 (m, 3H), 1.43 (t, J =7.1 Hz, 3H), 1.30 (tq, J =12.4, 7.0, 6.5 Hz, 4H). UPLC-MS (Acid, 4.0 min) Rt = 1.57 min, m / z (ES+) = 677.3 [M+H]+Example 1.9 - Synthesis of compound (9):

[0357] (3S)-4-[2-[[(lR,8S)-9-bicyclo[6.1.0]non-4-ynyl]methoxycarbonylamino] ethylamino] -3-[[4- [(2-ethoxy-3,4-dioxo-cyclobuten- 1 -yl)amino]benzoyl] amino] -4-oxo- butanoic acid

[0358] Step 1: tert-butyl (3S)-3-(benzyloxycarbonylamino)-4-[2-(tert- butoxycarbonylamino) ethylamino] -4-oxo-butanoate

[0359] To a solution of Z-Asp(OtBu)-OH (2.0 g) and HATU (2.6 g) in anhydrous N, N- dimethylformamide (12 mL) was added A-Boc-ethylenediamine (1.1 mL) followed by DIPEA (3.2 mL). The reaction was stirred at room temperature for 2 h. The reaction was diluted in EtOAc:MTBE (100 mL, 1:1) and washed with NaOH 1 M (100 mL), 1 M HCkbrine (1:1, 100 mL), 10% w / v aq. citric acid (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4, fdtered and evaporated to afford the titled compound as an off-white solid (2.78 g, 96% yield) that was used as it for the next step without further purification. 'H NMR (400 MHz, DMSO-t / e) 5 (ppm): 7.96 (t, J =5.6 Hz, 1H), 7.49 (d, J =8.4 Hz, 1H), 7.42 - 7.19 (m, 5H), 6.75 (t, J =5.6 Hz, 1H), 5.07 (d, J = 12.6 Hz, 1H), 4.99 (d, J = 12.6 Hz, 1H), 4.32 (td, J =8.7, 5.2 Hz, 1H), 3.08 (dt, J =26.8,6.2 Hz, 2H), 2.98 (p, J =6.5, 5.5 Hz, 2H), 2.72 - 2.60 (m, 1H), 2.42 (dd, J = 15.9, 8.9 Hz, 1H), 1.37 (s, 9H), 1.36 (s, 9H). UPLC-MS (Acid, 2.5 min) Rt = 1.60 min, m / z (ES+) =466.2 [M+H]+

[0360] Step 2: (3S)-3-amino-4-[2-(tert-butoxycarbonylamino)ethylamino]-4-oxo- butanoate

[0361] A solution of compound obtained previously (2.64 g) dissolved in ethanol (28 mL) and ethyl acetate (28 mL) was degassed with 3 vacuum / argon cycles, before 10% palladium on carbon 50% wet (0.6 g) was added. The reaction was again degassed with 3 vacuum / argon cycles and then put under hydrogen with 3 vacuum / H2 cycles. The reaction was stirred at room temperature for 16 h. The reaction mixture was filtered over celite and rinsed with a 1 : 1 mixture of EtOH and EtOAc. The solvent was removed under reduced pressure to afford the titled compound as a green oil (1.9 g, quantitative yield) that was used as it for the next step without further purification. 'H NMR (400 MHz, DMSO-t / e) 8 (ppm): 7.94 (t, J =5.7 Hz, 1H), 6.78 (t, J =5.7 Hz, 1H), 3.51 - 3.40 (m, 1H), 3.08 (h, J =7.2 Hz, 2H), 2.98 (q, J =6.2 Hz, 2H), 2.62 - 2.47 (m, 2H), 2.30 (dd, J = 15.6,7.7 Hz, 2H), 1.39 (s, 9H), 1.37 (s, 9H). UPLC-MS (Acid, 4.0 min) Rt = 1.99 min, m / z (ES+) = 332.2 [M+H]+

[0362] Step 3: (3S)-4-[2-(tert-butoxycarbonylamino)ethylamino]-3-[[4-[(2-ethoxy- 3,4-dioxo-cyclo buten-l-yl)amino]benzoyl] amino] -4-oxo-butanoate

[0363] To a solution of compound obtained previously (1.59 g) and DIPEA (2.5 mL) dissolved in anhydrous N, A-dimethylformamide (24 mL) was added at room temperature HATU (2.0 g), quickly followed by 4-[(2-ethoxy-3,4-dioxo-cyclobuten-l- yl)amino]benzoic acid obtained in preparation 1 (1.50 g). The reaction was stirred for 15 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by silica gel column chromatography (6% to 50% (2:8, MeOH:DCM) in DCM) to afford the titled compound as a white solid (1.21 g, 44% yield). 'H NMR (400 MHz, DMSO-de) 8 (ppm): 10.92 (s, 1H), 8.52 (d, J =8.2 Hz, 1H), 7.98 (t, J =5.6 Hz, 1H), 7.93 - 7.78 (m, 2H), 7.44 (d, J =8.3 Hz, 2H), 6.75 (t, J =5.6 Hz, 1H), 4.79 (q, J =7.0 Hz, 3H), 3.08 (dq, J =19.4, 6.9 Hz, 2H), 2.98 (q, J =6.2 Hz, 2H), 2.82 - 2.72 (m, 1H), 2.58 (dd, J =15.8, 9.1 Hz, 1H), 1.43 (t, J =7.1 Hz, 3H), 1.36 (s, 9H), 1.35 (s, 9H). UPLC-MS (Acid, 4.0 min) Rt = 1.49 min, m / z (ES+) = 575.3 [M+H]+

[0364] Step 4: (3S)-4-[2-[[(lR,8S)-9-bicyclo[6.1.0]non-4-ynyl]methoxycarbonyl amino] ethylamino] -3- [ [4- [(2-ethoxy-3 ,4-dioxo-cyclobuten- 1 -yl)amino]benzoyl] amino] - 4-oxo-butanoic acid

[0365] To a solution of compound obtained previously (200 mg) in dichloromethane (1.7 mL) was added trifluoroacetic acid (1.7 mL) at room temperature. The reaction was stirred for 2 h, after which the solvent was removed under reduced pressure, and theresulting crude residue was co-evaporated with toluene to afford crude deprotected intermediate.

[0366] Crude deprotected intermediate was dissolved in A-dimcthylformamidc (1.7 mL), and endo-BCN-NHS carbonate (CAS n° 1426827-79-3; 101 mg) was added, directly followed by tri ethylamine (438 pL). The reaction mixture was stirred at room temperature for 1.5 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by reverse-phase column chromatography (5% to 37% MeCN in water + 10 mM Formic Acid) to afford titled compound as a white solid (90.9 mg, 44% yield). 'H NMR (400 MHz, DMSO-de) 8 (ppm): 12.14 (s, 1H), 10.97 (s, 1H), 8.56 (d, J =7.7 Hz, 1H), 7.96 (t, J =5.4 Hz, 1H), 7.92 - 7.79 (m, 2H), 7.44 (d, J =8.4 Hz, 2H), 7.06 (t, J =5.6 Hz, 1H), 4.79 (q, J =7.1 Hz, 2H), 4.71 (td, J =8.0, 5.4 Hz, 1H), 3.97 (d, J =8.0 Hz, 2H), 3.11 (q, J =5.9, 5.4 Hz, 2H), 3.07 - 2.97 (m, 2H), 2.75 (dd, J = 16.2, 5.4 Hz, 1H), 2.62 (dd, J =16.3, 8.4 Hz, 1H), 2.32 - 2.03 (m, 6H), 1.49 (s, 2H), 1.43 (t, J =7.1 Hz, 3H), 1.23 (p, J =8.3 Hz, 1H), 0.84 (t, J =9.9 Hz, 2H). UPLC-MS (Acid, 4.0 min) Rt = 1.89 min, m / z (ES+) = 595.3 [M+H]+Example 1.10 - Synthesis of compound (10):

[0367] (3S)-3-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino]benzoyl]amino]-4- oxo-4-[2-(2-tricyclo[10.4.0.04,9]hexadeca-l(16),4,6,8,12,14-hexaen-10- ynyloxycarbonylamino) ethylamino]butanoic acid

[0368] To a solution of compound obtained in step 3 of example 1.9 (150 mg) in dichloromethane (1.3 mL) was added trifluoroacetic acid (1.3 mL) at room temperature. The reaction was stirred for 2 h, after which the solvent was removed under reduced pressure, and the resulting crude residue was co-evaporated with toluene to afford the corresponding crude deprotected intermediate.

[0369] The deprotected intermediate was dissolved in A,A-dimethylformamide (1.3 mL), and commercially available l l,12-didehydro-5,6-dihydrodibenzo[a,e]cycloocten-5- yl carbonic acid 4-nitrophenyl ester (CAS n°: 1027338-09-5; 101 mg) was added, directly followed by tri ethylamine (182 pL). The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by reverse-phase column chromatography (45% to 55% MeCN in water + 10 mM Formic Acid). Mixed fractions were subjected to a second purification by reverse-phase column chromatography (40% to 50% MeCN in water + 10 mM Formic Acid). Pure fractions were combined and concentrated under reduced pressure to afford titled compound as a white solid (57 mg, 32% yield). 'H NMR (400 MHz, DMSO-de) 8 (ppm): 12.21 (s, 1H), 10.91 (s, 1H), 8.56 (d, J =7.8 Hz, 1H), 8.07 - 7.97 (m, 1H), 7.87 (d, J =8.6 Hz, 2H), 7.53 (d, J =7.2 Hz, 2H), 7.49 - 7.34 (m, 9H), 5.27 (s, 1H), 4.76 (dq, J = 15.2, 7.8, 7.4 Hz, 3H), 3.16 (td, J =12.5, 11.7, 5.6 Hz, 3H), 3.05 (q, J =6.3 Hz, 2H), 2.83 - 2.72 (m, 2H), 2.65 (dd, J =16.3, 8.6 Hz, 1H), 1.42 (t, J =7.1 Hz, 3H). UPLC-MS (Acid, 4.0 min) Rt = 2.21 min, m / z (ES+) = 665.3 [M+H]+Example 1.11 - Synthesis of compound (11):

[0370] (S)-14-(4-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)benzamido)-l-(4-(6- methyl- 1,2, 4, 5-tetrazin-3-yl)phenoxy)- 13-oxo-3, 6, 9-trioxa-12-azahexadecan- 16-oic acid

[0371] Step 1: tert-butyl N-[2-[2-[2-[2-[4-(6-methyl-l,2,4,5-tetrazin-3- yl)phenoxy ] ethoxy] ethoxy] ethoxy] ethyl] c arb amate

[0372] To a solution of 4-(6-Methyl-l,2,4,5-tetrazin-3-yl)phenol (300 mg) and tert- Butyl (2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethyl)carbamate (766 mg) in anhydrous acetonitrile (10 mL) was added potassium carbonate (991 mg) at room temperature. The reaction was stirred at 70 °C for 5 h. The reaction mixture was diluted in EtOAc (50 mL) and washed with EtOAc (50 mL), water (50 mL) and brine (2 x 50 mL). The organic layer was dried over anhydrous Na2SO4, fdtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (12% to 100% EtOAcin cyclohexane) to afford the titled compound as a bright pink oil (518 mg, 67% yield). 'H NMR (400 MHz, Chloroform-d) 5 (ppm): 8.64 - 8.39 (m, 2H), 7.15 - 7.01 (m, 2H), 5.05 (d, J =7.3 Hz, 1H), 4.23 (dd, J =5.7, 3.9 Hz, 2H), 3.96 - 3.85 (m, 2H), 3.78 - 3.71 (m, 2H), 3.70 - 3.66 (m, 2H), 3.64 (ddd, J =5.7, 3.1, 1.2 Hz, 2H), 3.60 (ddd, J =5.9, 3.2, 1.2 Hz, 2H), 3.52 (t, J =5.2 Hz, 2H), 3.29 (q, J =5.4 Hz, 2H), 3.03 (s, 3H), 1.42 (s, 9H). UPLC-MS (Acid, 4.0 min) Rt = 2.46 min, m / z (ES-) = 462.3 [M-H]’

[0373] Step 2: (3S)-3-(9H-fluoren-9-ylmethoxycarbonylamino)-4-[2-[2-[2-[2-[4-(6- methyl- 1,2,4, 5-tetrazin-3-yl)phenoxy]ethoxy]ethoxy]ethoxy]ethylamino]-4-oxo- butanoate

[0374] To a solution of previously obtained compound (441 mg) in dichloromethane (2.4 mL) was added 4M hydrogen chloride in dioxane (2.4 mL) at room temperature. The reaction was stirred for 1 h, after which solvent was evaporated under reduce pressure to afford the corresponding deprotected crude intermediate.

[0375] To the deprotected crude intermediate dissolved in anhydrous N,N- dimethylformamide (9.5 mL) were added at room temperature Fmoc-Asp(OtBu)-OH (431 mg), HATU (398 mg), followed by DIPEA (830 pL). The reaction was stirred for 1 h. The reaction mixture was dilute in EtOAc (50 mL) and washed with saturated aqueous NaHCCh (50 mL), brine (50 mL), 1 M HCTBrine (2:8, 50 mL), and brine (50 mL). The organic layer was dried over Na2SO4, fdtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (5% to 75% EtOAc:EtOH (3:1) in cyclohexane) to afford the titled compound as a bright pink solid (735.2 mg, quantitative yield). 'H NMR (400 MHz, Chloroform-d) 5 (ppm): 8.64 - 8.42 (m, 2H), 7.75 (dt, J =7.5, 0.9 Hz, 2H), 7.58 (d, J =7.5 Hz, 2H), 7.44 - 7.36 (m, 2H), 7.30 (tt, J =7.5, 1.0 Hz, 2H), 7.11 - 7.04 (m, 2H), 6.84 (s, 1H), 5.97 (d, J =8.6 Hz, 1H), 4.51 (s, 1H), 4.41 (d, J =7.3 Hz, 2H), 4.25 - 4.15 (m, 3H), 3.87 (dd, J =5.6, 4.0 Hz, 2H), 3.77 - 3.67 (m, 2H), 3.68 - 3.63 (m, 2H), 3.63 - 3.57 (m, 4H), 3.54 (t, J =5.1 Hz, 2H), 3.51 -3.37 (m, 2H), 3.05 (s, 3H), 2.92 - 2.80 (m, 1H), 2.63 (dd, J =16.9, 6.4 Hz, 1H), 1.44 (s, 9H). UPLC-MS (Acid, 4.0 min) Rt = 2.92 min, m / z (ES+) = 757.5 [M+H]+

[0376] Step 3: tert-butyl (3S)-3-amino-4-[2-[2-[2-[2-[4-(6-methyl-l,2,4,5-tetrazin-3- yl)phenoxy]ethoxy]ethoxy]ethoxy]ethylamino]-4-oxo-butanoate

[0377] To a solution of compound obtained previously (654 mg) in dichloromethane (6 mL) was added piperidine (1.7 mL) at room temperature. The reaction was stirred for 1 h. The reaction mixture was concentrated under reduced pressure, and the resulting crude residue was purified by column chromatography on silica gel (0% to 10% (7N NH3 in MeOH) in DCM) to afford the titled compound as a pink oil (389 mg, 81% yield). 'H NMR (400 MHz, Chloroform-d) 5 (ppm): 8.53 (d, J =9.0 Hz, 2H), 7.65 (s, 1H), 7.09 (d, J =9.0 Hz, 2H), 4.33 - 4.19 (m, 2H), 3.96 - 3.87 (m, 2H), 3.79 - 3.72 (m, 2H), 3.72 - 3.68 (m, 2H), 3.68 - 3.60 (m, 5H), 3.56 (dd, J =5.9, 4.9 Hz, 2H), 3.45 (p, J =5.2 Hz, 2H), 3.05 (s, 3H), 2.83 (dd, J =16.6, 3.8 Hz, 1H), 2.48 (dd, J = 16.5, 8.6 Hz, 1H), 1.44 (s, 9H). HPLC-MS (Basic, 4.0 min) Rt = 2.22 min, m / z (ES+) = 535.3 [M+H]+

[0378] Step 4: tert-butyl (3S)-3-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten-l- yl)amino]benzoyl] amino] -4- [2- [2- [2- [2- [4-(6-methyl- 1 ,2,4,5-tetrazin-3- yl)phenoxy]ethoxy]ethoxy]ethoxy]ethylamino]-4-oxo-butanoate

[0379] To a solution of 4-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino]benzoic acid obtained in preparation 1 (230 mg), compound obtained in the previous step (370 mg) and HATU (279 mg) in anhydrous A,A-dimethylformamide (4.6 mL) was added at room temperature DIPEA (466 pL). The reaction was stirred for 25 min. The reaction mixturewas diluted in EtOAc (50 mL) and washed with 1 M HCkBrine (2:8, 50 mL), saturated aqueous NaHCOs (50 mL), and brine (50 mL). The organic layer was dried over ISfeSCL, filtered and evaporated under reduced pressure The crude product was purified by column chromatography on silica gel (25% to 89% EtOAc:EtOH (3:1) in Cyclohexane) to afford the titled compound as a bright red oil (364 mg, 70% yield). 'H NMR (400 MHz, Chloroform-d) 5 (ppm): 8.48 (d, J =9.0 Hz, 2H), 7.82 (d, J =8.4 Hz, 2H), 7.67 (d, J =7.9 Hz, 1H), 7.40 (d, J =8.3 Hz, 2H), 7.12 - 7.01 (m, 3H), 4.95 (td, J =6.9, 4.6 Hz, 1H), 4.89 (q, J =7.1 Hz, 2H), 4.23 (dd, J =5.7, 3.9 Hz, 2H), 3.92 - 3.84 (m, 2H), 3.73 (dd, J =5.8, 3.3 Hz, 2H), 3.66 (dd, J =5.8, 3.4 Hz, 2H), 3.60 (q, J =1.3 Hz, 4H), 3.54 (t, J =5.2 Hz, 2H), 3.45 (q, J =5.3 Hz, 2H), 3.05 (s, 3H), 2.93 (dd, J =16.6, 4.7 Hz, 1H), 2.67 (dd, J = 16.5, 6.6 Hz, 1H), 1.51 (t, J =7.1 Hz, 3H), 1.45 (s, 9H). HPLC-MS (Basic, 4.0 min) Rt = 2.13 min, m / z (ES+) = 778.5 [M+H]+

[0380] Step 5: (3S)-3-[[4-[(2-ethoxy-3,4-dioxo-cyclobuten-l- yl)amino]benzoyl] amino] -4- [2- [2- [2- [2- [4-(6-methyl- 1 ,2,4,5-tetrazin-3- yl)phenoxy] ethoxy] ethoxy] ethoxy] ethyl amino] -4-oxo-butanoic acid

[0381] To compound obtained the previous step (350 mg) in dichloromethane (2.2 mL) was added trifluoroacetic acid (2.2 mL) at room temperature. The reaction was stirred for 1.5 h, after which toluene (1 mL) was added. The solvent was evaporated under reduced pressure, and the residue obtained was resuspended in a mixture of acetonitrile and water, frozen and freeze-dried to afford the titled compound as a dark pink solid (243 mg, 71% yield). 'H NMR (400 MHz, DMSO-t e) 8 ‘ppm): 10.91 (s, 1H), 8.53 (d, J =7.9 Hz, 1H), 8.44 - 8.36 (m, 2H), 7.96 - 7.78 (m, 3H), 7.54 - 7.36 (m, 2H), 7.22 - 7.17 (m, 2H), 4.78 (q, J =6.9 Hz, 3H), 4.29 - 4.16 (m, 2H), 3.84 - 3.72 (m, 2H), 3.59 (td, J =4.1, 1.1 Hz, 2H), 3.55 - 3.51 (m, 2H), 3.49 (s, 4H), 3.40 (t, J =6.0 Hz, 2H), 3.21 (p, J =6.1 Hz, 2H), 2.96 (s, 3H), 2.77 (dd, J =16.4, 5.4 Hz, 1H), 2.63 (dd, J = 16.4, 8.6 Hz, 1H), 1.42 (t, J =7.1 Hz, 3H). HPLC-MS (Basic, 4.0 min) Rt = 1.46 min, m / z (ES+) = 722.4 [M+H]+Example 1.12 - Synthesis of compound (12):

[0382] (2-methylcycloprop-2-en-l-yl)methyl (2-(2-(2-(4-((2-ethoxy-3,4- dioxocyclobut- 1 -en- 1 -yl)amino)benzamido)ethoxy)ethoxy)ethyl)carbamate

[0383] Step 1: (2-methylcycloprop-2-en-l-yl)methyl (4-nitrophenyl) carbonate

[0384] To a solution of 2-methyl-3-trimethylsilyl-cycloprop-2-en-l-yl]methanol (Chem. Commun., 2019, 55, 1092 - 1095) (203 mg) in THF (6.5 mL) at room temperature, was added tetrabutylammonium fluoride (IM solution in THF) (1.56 mL). The mixture was stirred for 2 h. The reaction mixture was cooled down to 0°C and diluted with pyridine (5.2 mL). 4-Nitrophenylchloroformate (707 mg) was added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was partitioned between 0.5N HC1 and EtOAc. The layers were separated, and the aqueous phase was extracted with EtOAc (2x). The combined organic extracts were washed (IN HC1 until pH < 3, brine), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (cHex / EtOAc = 97 / 3 to 70 / 30) to afford titled compound (63mg, 19% yield) as a colorless oil. 1H NMR (400 MHz, Chloroform- d) 5 (ppm): 8.33 - 8.27 (m, 2H), 7.44 - 7.37 (m, 2H), 6.64 (s, 1H), 4.30 - 4.20 (m, 1H), 4.16 (dd, J = 10.9, 5.5 Hz, 1H), 2.20 (d, J = 1.2 Hz, 3H), 1.80 (td, J = 5.3, 1.6 Hz, 1H)

[0385] Step 2: (2-methylcycloprop-2-en-l-yl)methyl N-[2-[2-[2-[[4-[(2-ethoxy-3,4- dioxo-cyclobuten-l-yl)amino]benzoyl]amino]ethoxy]ethoxy]ethyl]carbamate

[0386] To a solution of compound obtained in step 2 of example 1.6 (78 mg) in DMF (1 mL) at room temperature, were successively added a solution of compound obtained previously (61 mg) in THF (1 mL) and DIPEA (89 pL). The mixture was stirred at RT for 2h. The reaction mixture was partitioned between EtOAc and 0.5N HC1. The layers were separated, and the aqueous phase was extracted with EtOAc (2x). The combined organic extracts were washed (0.5N HC1, water, brine), dried over Na2SO4, filtered andconcentrated under reduced pressure. The residue was purified by flash chromatography (DCM / MeOH = 99 / 1 to 90 / 10) and then preparative TLC (DCM / MeOH = 93 / 7) to afford title compound as a light yellow solid (41mg, 38% yield). 'H NMR (400 MHz, DMSO- d6) <5 (ppm): 10.91 (s, 1H), 8.42 (t, J= 5.6 Hz, 1H), 7.99 - 7.75 (m, 2H), 7.43 (d, J= 8.3 Hz, 2H), 7.03 (s, 1H), 6.85 (s, 1H), 4.79 (q, J= 7.1 Hz, 2H), 3.86 - 3.69 (m, 2H), 3.59 - 3.46 (m, 6H), 3.45 - 3.35 (m, 4H), 3.10 (q, J= 5.9 Hz, 2H), 2.10 (d, J= 1.2 Hz, 3H), 1.50 (dt, J= 5.3, 2.6 Hz, 1H), 1.43 (t, J= 7.1 Hz, 3H). MS (ESI+): [M+H]+502.2Example 1.13 - Synthesis of compound (13):4-((2-ethoxy-3,4-dioxocyclobut-l-en-l-yl)amino)-N-(l-(2-methylcycloprop-2-en-l-yl)- 4-oxo-2,8,l l-trioxa-5-azatridecan-13-yl)benzamide

[0387] Step 1: tert-butyl 2- [(2-methylcycloprop-2-en-l-yl)methoxy] acetate

[0388] To a solution of 2-methyl-3-trimethylsilyl-cycloprop-2-en-l-yl]methanol (133 mg) and t-butyl bromoacetate (0.3 mL) in DCM (4.3 mL) at room temperature, were added tetra-N-butylammonium hydrogen sulfate (58 mg) and NaOH 32% (4.4 mL). The mixture was vigorously stirred for 6 h. The reaction mixture was partitioned between water and MTBE. The layers were separated, and the aqueous phase was extracted with MTBE (2x). The combined organic extracts were washed (water, brine), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (cHex / EtOAc = 100 / 0 to 80 / 20) to afford the desired cyclopropenylsilane (159 mg) contaminated with t-butyl bromoacetate. It was dissolved in THF (2.9 mL) at room temperature and tetrabutylammonium fluoride (IM solution in THF) (1.2 mL). The mixture was stirred for 1 h. The reaction mixture was concentrated under reduced pressure and directly purified by flash chromatography (cHex / EtOAc = 100 / 0 to 80 / 20) to afford titled compound (62 mg, 37% over two steps) as a colorless oil. 'H NMR (400 MHz, Chloroform-d) <5 (ppm): 6.64 (s, 1H), 3.98 (s, 2H), 3.51 - 3.36 (m, 2H), 2.17 (d, J = 1.1 Hz, 3H), 1.69 (td, J= 5.1, 1.6 Hz, 1H), 1.50 (s, 9H)

[0389] Step 2: 2- [(2-methylcycloprop-2-en-l-yl)methoxy] acetic acid

[0390] To a solution of compound obtained previously (60 mg) in THF / MeOH / water (2 / 2 / 1, 3.75 mL) at room temperature, was added NaOH 2N (0.75 mL). The mixture was stirred for h. The volatiles were removed under reduced pressure. DCM was added, followed by 2N HC1 until pH < 2. The layers were separated, and the aqueous phase was extracted with DCM (2x). The combined organic extracts were dried over Na2SO4, filtered and concentrated under reduced pressure to afford titlted compound as a colorless oil (42 mg, 98% yield). 'H NMR (400 MHz, DMSO-de) d (ppm): 12.50 (s, 1H), 6.89 (s, 1H), 3.95 (s, 2H), 3.36 (dd, J= 10.0, 4.9 Hz, 1H), 3.25 (dd, J = 10.0, 5.3 Hz, 1H), 2.12 (d, J= 1.1 Hz, 3H), 1.49 (td, J= 5.1, 1.6 Hz, 1H)

[0391] Step 3: 4-[(2-ethoxy-3,4-dioxo-cyclobuten-l-yl)amino]-N-[2-[2-[2-[[2-[(2- methylcycloprop-2-en- 1 -yl)methoxy] acetyl] amino] ethoxy] ethoxy] ethyl]benzamide

[0392] To a solution of compound obtained previously (39 mg) in DMF ( 1 mL) at room temperature, were successively added DIPEA (128 pL) and TBTU (83 mg). The mixture was stirred for 30 min and a solution of compound obtained in step 2 of example 1.6 (72 mg) in DMF (2.7 mL). The mixture was stirred for 30 min. The reaction mixture was partitioned between EtOAc and 0.5N HC1. The layers were separated, and the aqueous phase was extracted with EtOAc (2x). The combined organic extracts were washed (0.5N HC1, water, brine), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (DCM / MeOH = 99 / 1 to 90 / 10) and then preparative TLC (DCM / MeOH = 95 / 5) to afford titled compound (61 mg, 64% yield) as a light yellow solid. 'H NMR (400 MHz, DMSO-t / e) S (ppm): 10.90 (s, 1H), 8.42 (t, J = 5.6 Hz, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.53 (s, 1H), 7.43 (d, J = 8.3 Hz, 2H), 6.90 (s, 1H), 4.78 (q, J= 7.1 Hz, 2H), 3.81 (s, 2H), 3.57 - 3.50 (m, 6H), 3.45 - 3.20 (m, 8H), 2.11 (d, J= 1.2 Hz, 3H), 1.50 (td, J= 5.1, 1.5 Hz, 1H), 1.43 (t, J = 7.0 Hz, 3H). MS (ESI+): [M+H]+516.3EXAMPLE 2: PREPARATION OF COMPOUNDS OF FORMULA (II).

[0393] Several compounds of formula (II) were obtained commercially (indicated for each supplier or CAS number):Compound A: Endo-BCN-PEG4-biotin (BP-40780; Broadpharm),Step 1: Peptide synthesis

[0394] Linear peptidic sequence RKfDG (SEQ ID NO: 1) was synthesized on solid support [2-chloroTrityl resin] on an automated CS Bio peptide synthesizer, each amino acid bearing the adapted protecting group for its side chain. The synthesis was performed according to a standard protocol in Fmoc / tBu strategy using as activator for example HATU and a piperidine solution in DMF for deprotection of the Fmoc protecting group.Step 2 : Solid support cleavage

[0395] After drying in vacuo, the peptidyl resin is exposed to 20% HFIP in DCM solution several times to lead to the sequence cleaved from the resin. The filtrate was concentrated in vacuo, solubilized in ACN / H2O followed by lyophilization.Step 3 : Backbone cyclisation

[0396] The protected peptide was dissolved in DMF and treated with DIPEA in the presence of PyBOP leading to the cyclization of the backbone intermediate. Once the conversion was complete, the reaction mixture was concentrated in vacuo, dissolved in ACN / H2O and submitted to a new freeze-dry cycle.Step 4 : Side chain deprotection

[0397] The side chain protecting group of the peptide was cleaved using a mixture of TFA / H2O / TIPS during 2 hours. Isolation of the cyclic backbone peptide was performed by precipitation using Et2O / Pentane and the material was washed several times with this mixture to remove protecting groups and maximum of residual TFA. The material was then solubilized in ACN / H2O followed by freeze-drying step.Step 5 : Linker conjugation

[0398] The peptidic material was dissolved in DMSO and pre-treated with DIPEA to neutralize potential contamination of residual TFA. The NHS ester of DBCO-PEG2-NHS (CAS n° 2585653-12-7) in solution in DMSO was added and the pH of the reaction mixture was adjusted to pH=8 by addition of DIPEA. Once a quantitative conversion to the desired conjugate was determined by LC-MS, the reaction mixture was concentrated in vacuo, dissolved in ACN / H2O and then lyophilized.Step 6: Peptide purification

[0399] The cyclized crude material was dissolved in an appropriate co-mixture of ACN / Milli-Q H2O and injected on a Cl 8 preparative HPLC column using acidic eluents (A : MilliQ-H2O+0.1% AcOH ; B : ACN+0.05% AcOH). ESI-MS m / z) [M+H]+calcd for C53H67N11O12: 1050.2, found: 1051.8. [M+2H]2+: 526.1, found: 526.23. UPLC purity: 95.1 % (215 nm), RT = 1.57 minSynthesis of compound F: Modified DBCO-PEG4-AngioPep2Step 1: Peptide synthesis

[0400] The linear peptidic sequence TFFYG GSRGK RNNFK TEEY (SEQ ID No: 2) was carried out on a GPT Prelude X peptide synthesiser on a 0.15 mmol scale using TG R RAM resin with a loading of 0.2 mmol / g. Oxyma / DIC chemistry was applied for all couplings using 8 eq. of building block and reagents. Fmoc deprotection was carried out with piperidine (20% v / v) in DMF. A capping step with acetic anhydride was carried out after each coupling.Step 2: Mercaptopropionic acid coupling and cleave from solid support

[0401] S-trityl-3-mercaptopropionic acid was coupled with standard Oxyma / DIC chemistry on the GPT Prelude X peptide synthesiser under single couple extended conditions (80 °C, 15 min), after which the bulk resin was cleaved in TFA:TIS:water:EDT (4 hours) leading to the crude modified peptide.Step 3 : Intermediate peptide purification

[0402] The crude material was purified by reverse phase HPLC to >90% purity prior to conjugation to the bromoacetamido-PEG4-amido-DBCO. A Phenomenex Luna C18 250x21.2 mm column was used with solvent system A: water + 0.1% TFA, B: MeCN + 0.1% TFA. The peptide was purified using a gradient of 10-45% B over 12 CV. Fractions containing the right intermediate were pooled and lyophilised leading to the peptide with 92.5% purity.Step 4: Conjugation to bromoaceto-PEG4-amido-DBCO

[0403] A conjugation was performed between the purified material and bromoaceto- PEG4-amido-DBCO / DMF (CAS n°: 2852742-74-4). The mixture was stirred in 10% MeCN / O.OlM Phosphate buffer (20 mL, pH 7.4) for 16 h. The peptide once conjugated to the DBCO precipitated to a white solid in solution.Step 5 : Purification and acetate exchange

[0404] The crude peptide was purified by reverse phase HPLC with a Phenomenex Luna C18 250x10.0 mm column using solvent system A: water + 0.1% TFA, B: MeCN + 0.1% TFA and a suitable gradient of 25-50% B over 12 CV. Fractions containing the right compound were pooled and lyophilised to yield targeted peptide as TFA salt.

[0405] The exchange of trifluoroacetate salt into acetate was carried out on-column acetate exchange via a proprietary buffer. ESI-MS (m / z): [M+H]+ calcd for C138H191N33O38S: 2953.30, found: 2951.44. [M+2H]2+: 1477.15, found: 1476.71. [M+3H]3+: 985.10, found: 984.83. [M+4H]4+: 739.07, found: 738.62. HPLC purity: 97.2 % (214 nm), RT = 22.32 minCompound G: Azido-PEG3-Biotin (CAS n° : 875770-34-6)Synthesis of compound H: Azido-PEG4-AngioPep2Step 1: Peptide synthesis

[0406] As the main peptide chain for compound H was the same as for compound F, identical synthesis strategy was carried out.Step 2: Peptide conjugation and cleave from solid support

[0407] Azido-PEG4-NHS ester (CAS n°: 1807534-82-2) and DIPEA were dissolved in DMF and added to pre-swollen resin. The reaction mixture was stirred until disappearance of the starting material, with additional reagent if necessary. The bulk resin was cleaved in 5 volumes TFA:TIS: water leading to the crude peptide.Step 3 : Purification and acetate exchange

[0408] The crude peptide was purified by reverse phase HPLC with a Phenomenex Luna C18 250x21.2 mm column using solvent system A: water + 0.1% TFA, B: MeCN + 0.1% TFA and a suitable gradient of 10-50% B over 10 CV. Fractions containing the right compound were pooled and lyophilised to yield targeted peptide as TFA salt of 90.2 purity.

[0409] The exchange of trifluoroacetate salt into acetate was carried out on-column acetate exchange via a proprietary buffer. ESI-MS (m / z): [M+H]+ calcd for C115H169N33O35: 2574.81, found: 2573.32. [M+2H]2+: 1287.90, found: 1287.64. [M+3H]3+: 858.93, found: 858.78. [M+4H]4+: 644.45, found: 644.34. HPLC purity: 97.2 % (214 nm), RT = 19.95 minSynthesis of compound J: Azido-PEG2-cRGDfK

[0410] The linear peptide RKfDG (SEQ ID NO: 1) was synthesized on solid support (2- chloro chloroTrityl resin) at a scale of 0.25 mmol on a GYROS PROTEIN Symphony X peptide synthesizer, in a similar way to compound 7B1 described in WO2025051994A1. The syntheses were performed according to a standard protocol in Fmoc / tBu strategy using as activator HATU and a piperidine solution in DMF for deprotection of the Fmoc protecting group.Step 1: Peptide DfKRG synthesis and spacer introduction on solid support

[0411] The peptidyl resin Fmoc-Asp(Otbu)-DPhe-Lys(Alloc)-Arg(Pbf)-Gly-resin was synthesized, followed by the deprotection of the Alloc group by using 0.35 eq. of Palladium (0) and 24 eq. of phenylsilane. Then the reagent azido-PEG2-NHS ester (CAS n° 1312309-64-0) and DIPEA were dissolved in DMF and added to the pre-swollen resin.The reaction mixture was stirred until disappearance of starting material then N-terminal Fmoc deprotection was performed in appropriate conditions.Step 2: Cleavage from the solid support

[0412] The cleavage from the resin was performed under selective mild conditions by the treatment of HFIP / DCM (20:80) for Ih at RT, the resin was removed by filtration and the filtrate was evaporated to dryness under vacuum. Crude peptide was precipitated by the addition of cold diethylether. Protected peptide was thus obtained by filtration.Step 3 : Head to tail cyclization

[0413] Protected peptide H-Asp(OtBu)-DPhe-Lys(Boc-Spacer)-Arg(Pbf)-Gly-OH was solubilized in DMF (at 10 mM). To this solution were successively added DIEA (3 eq.) and BOP (1.1 eq.). After stirring at room temperature overnight, the solution containing the protected cyclic peptide was concentrated under vacuum, precipitated by added Et2O and dried under vacuum.Step 4:_Acido labile protecting group removal

[0414] Protected cyclic peptide was treated with TFA / H2O / TIPS (92.5 / 5 / 2.5, v / v / v)) solution at room temperature for 2 hours. Isolation of the cyclic backbone peptide was performed by precipitation using Et2O / Pentane and the material was washed several times with this mixture to remove protecting groups and maximum of residual TFA. The material was then solubilized in ACN / H2O followed by freeze-drying step.Step 5 : Purification and salt exchange

[0415] The cyclized crude material was 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 pm, 120 A, 50 300 mm) and a suitable gradient of ACN+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.

[0416] The exchange of trifluoroacetate salt into acetate was carried out during purification via a proprietary buffer. ESI-MS (m / z): [M+H]+ calcd for C34H52N12O10: 789.86, found: 789.39. [M+2H]2+: 395.43, found: 395.37. UPLC purity: 95.1 % (214 nm), RT = 3.78 minCompound K: TCO-PEG3-Biotin (HY- 136050, MedChemExpress)Compound L: Methyltetrazine-PEG3 -biotin (CAS n° : 1835759-81-3)Synthesis of compound M: Methyl tetrazine-PEG4-Angiopep2Step 1: Peptide synthesis

[0417] As the main peptide chain for compound M was the same as for compounds F andH, identical synthesis strategy was carried out.Step 2: Peptide conjugation and cleave from solid support

[0418] Methyltetrazine-PEG4-NHS (CAS n° 1802907-92-1) and DIPEA were dissolved in DMF and added to the pre-swollen resin. The reaction mixture was stirred until disappearance of the starting material, with additional reagent if necessary. The bulk resin was cleaved in 5 volumes TFA:TIS:EDT leading to the crude peptide.Step 3 : Purification and acetate exchange

[0419] The crude peptide was purified by reverse phase HPLC with a Phenomenex Luna C18 (250x21.2 mm) column using solvent system A: water + 0.1% TFA, B: MeCN + 0.1% TFA and a suitable gradient of 10-50% B over 10 CV. Fractions containing the right compound were pooled and lyophilised to yield targeted peptide as TFA salt.

[0420] The exchange of trifluoroacetate salt into acetate was carried out on-column acetate exchange via a proprietary buffer. ESI-MS (m / z): [M+H]+ calcd for C124H176N34O36: 2719.97, found: 2719.34. [M+2H]2+: 1360.48, found: 1360.16. [M+3H]3+: 907.32, found: 907.13. [M+4H]4+: 680.60, found: 680.60. HPLC purity: 90.4 % (214 nm), RT = 22.74 min.SynthesisStep 1: Cloning of the expression construct

[0421] After gene synthesis, the corresponding VHH sequence was cloned into the E. coli expression vector pTac, including an N-terminal H14n2-bdSUMO tag and a C- terminal ectopic cysteine for site-specific labeling. Plasmids were purified using mini prep and sequenced using Sanger sequencing.Step 2 : Protein expression and purification from E. coli cells

[0422] SHuffle® Express E. coli cells were transformed with the plasmid of interest and cultivated in TB media in shaker flasks overnight at 23°C. Media was centrifuged to harvest cells that were lysed using a high-salt buffer. The H14n2-bdSUMO tagged VHH was purified by affinity using IMAC resin. The affinity tag was cleaved during IMAC purification using bdSENPl protease taking the N-terminal SG tagged VHH of interest down. A Polishing step using size exclusion chromatography (SEC) with Superdex 75 in phosphate buffer pH 6.0 was carried out to obtain >95% purity.Step 3: DBCO labelling

[0423] The protein (3.65 mg / mL) was labelled on its C-terminal cysteine with DBCO maleimide (1.5 eq.) in phosphate buffer pH 6.0 for Ih at 4°C. The excess of DBCO reactant was removed by size exclusion chromatography (Superdex™ 75 Increase 10 / 300 GL, Cytiva) and the VHH of interest eluted in PBS, concentrated at 1 mg / mL and lyophilized.Synthesis of compound P: V02.19.6 (L138) - VHH02-DBCOStep 1: Cloning of the expression construct

[0424] After gene synthesis, the corresponding VHH sequence was cloned into the E. coli expression vector pTac, including an N-terminal H14n2-bdSUMO tag and a C-terminal ectopic cysteine for site-specific labeling. Plasmids were purified using mini prep and sequenced using Sanger sequencing.Step 2 : Protein expression and purification from E. coli cells

[0425] SHuffle® Express E. coli cells were transformed with the plasmid of interest and cultivated in TB media in shaker flasks overnight at 23°C. Media was centrifuged to harvest cells that were lysed using a high-salt buffer. The H14n2-bdSUMO tagged VHH was purified by affinity using IMAC resin. The affinity tag was cleaved during IMAC purification using bdSENPl protease taking the N-terminal SG tagged VHH of interest down. A Polishing step using size exclusion chromatography (SEC) with Superdex 75 in phosphate buffer pH 6.0 was carried out to obtain >95% purity.Step 3: DBCO labelling

[0426] The protein (3.65 mg / mL) was labelled on its C-terminal cysteine with Maleimide-PEG4-DBCO (1.5 eq.) in phosphate buffer pH 6.0 for Ih at 4°C. The excess of DBCO reactant was removed by size exclusion chromatography (Superdex™ 75 Increase 10 / 300 GL, Cytiva) and the VHH of interest eluted in PBS, concentrated at 1 mg / mL and lyophilized.Synthesis of compound Q and R : V01.1.8 (L137) - VHHOl-azido and V02.1.8 (L139) -VHH02-azidoStep 1: Cloning of the expression construct

[0427] After gene synthesis, the corresponding VHH sequence was cloned into the E. coli expression vector pTac, including an N-terminal H14n2-bdSUMO tag and a C-terminal ectopic cysteine for site-specific labeling. Plasmids were purified using mini prep and sequenced using Sanger sequencing.Step 2 : Protein expression and purification from E. coli cells

[0428] SHuffle® Express E. coli cells were transformed with the plasmid of interest and cultivated in TB media in shaker flasks overnight at 23°C. Media was centrifuged to harvest cells that were lysed using a high-salt buffer. The H14n2-bdSUMO tagged VHH was purified by affinity using IMAC resin. The affinity tag was cleaved during IMAC purification using bdSENPl protease taking the N-terminal SG tagged VHH of interest down. A Polishing step using size exclusion chromatography (SEC) with Superdex 75 in phosphate buffer pH 6.0 was carried out to obtain >95% purity.Step 3 : Azido labelling

[0429] The protein (3.65 mg / mL) was labelled on its C-terminal cysteine with Azido- PEG3-maleimide (1.5 eq.) in phosphate buffer pH 6.0 for Ih at 4°C. The excess of azido reactant was removed by size exclusion chromatography (Superdex™ 75 Increase 10 / 300 GL, Cytiva) and the VHH of interest eluted in PBS, concentrated at 1 mg / mL and lyophilized.EXAMPLE 3: SYNTHESIS AND COUPLING OF A Vs COMPRISING MOIETIES OF FORMULA (III) AND (I).

[0430] Conjugated AAVs were produced through a two-step modification process. In the first step, the squarate moieties, as described in our invention, were coupled to at least one primary amine exposed on the surface of the AAV. This resulted in the creation of afunctionalized AAV intermediary. In the subsequent step, our ligand moieties were introduced using click chemistry, as detailed below.3.1. Production and purification of AAVs.

[0431] AAVs were produced and purified according to well-known techniques in the art.3.2. Production and purification of chemically-conjugated AAVs.Materials

[0432] Compound (A), (B), (G), (K) and (L) are commercially available.

[0433] Compounds (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (E), (F), (H), (J), (M), (N), (P), (Q) and (R) were obtained as detailed above in examples 1 and 2.

[0434] The following AAVs, obtained as detailed in example 3.1, were used:- AAV2-CAG-eGFP: 1.0E13 vg / mL in DPBS + Ca2+, Mg2+, 0.001% Pluronic F68 at pH 7.4;- AAV2-4pmut-CAG-nanoLUC: 2.0E13 vg / mL in DPBS + Ca2+, Mg2+, 0.001% Pluronic F68 at pH 7.4;- AAV9-CAG-eGFP: 2.0E13 vg / mL in DPBS + Ca2+, Mg2+, 0.001% Pluronic F68 at pH 7.4.Table 5 - other material and reagents usedMethods

[0435] The coupling of the squarate compounds of formula (IV) on AAV2-CAG-eGFP, AAV2-4pmut-CAG-nanoLUC or AAV9-CAG-eGFP capsids (1 or 2 vg / mL) was carried out with a solution of TRIS buffer pH = 9.3 containing compounds of formula (IV) (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12) and (13) at a concentration of 3, 2 or 0.3 mM and incubated for 2h, 4h or 16h at 20°C to obtain, respectively, the AAV s comprisinga moiety of formula (III), AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13 as disclosed in table 6 below. At the end of the incubation period, unbound compounds of formula (IV) were removed using PD MidiTrap G-25 columns or PD SpinTrap G-25 columns. For PD MidiTrap G- 25 columns, columns were first equilibrated with 5x4 ml of formulation buffer (DPBS, Ca2+, Mg2+, 0.001 or 0.005% F68). The coupling reactions mixtures were then loaded onto the column, 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 dPCR titer was determined for each pooled fraction. For PD SpinTrap G-25 columns, columns were first equilibrated with 5x400 pl of formulation buffer (DPBS, Ca2+, Mg2+, 0.005% F68) and using centrifugation at 800 g for 1 min. The coupling reaction mixtures were then loaded onto the column and elution of rAAV vectors was performed by centrifugation at 800 g for 2 min. dPCR titers were determined for each sample. Pooled fractions were then sterile fdtrated using Acrodisc PP, PES, 0.2 pM 1.3 cm2.

[0436] The click reaction on the functionalized AAV2-CAG-eGFP or AAV9-CAG-eGFP or AAV2-4pmut-CAG-nanoLUC capsids comprising a moiety of formula (III) (1E12 or 2E12 vg / mL) was carried out for 2h at 20°C in DPBS formulation buffer containing the compounds of formula (II), (A), (B), (E), (F), (G), (H), (J), (K), (L) or (M) at 135, 100 or 20 pM to obtain the AAVs particles comprising a moiety of formula (I) indicated in table 6 below. At the end of the incubation period, unbound compounds of formula (II) were removed using PD SpinTrap G-25 columns.. Columns were first equilibrated with 5x400 pl of formulation buffer (DPBS, Ca2+, Mg2+, 0.005% F68) and centrifugation at 800 g for 1 min. The coupling reactions mixtures were then loaded onto the column and elution of rAAV vectors was performed by centrifugation at 800 g for 2 min. dPCR titer was determined for each sample. The click reaction on the functionalized AAV2-4pmut- CAG-nanoLUC capsids comprising a moiety of formula (III) AAV-3 and AAV-7 was carried out for 2h at 20°C in DPBS formulation buffer containing the compounds of formula (II), (N), (P), (Q) or (R) at 25 pM to obtain the AAVs particles comprising a moiety of formula (I) AAV-3N, AAV-13P, AAV-7Q and AAV-7R indicated in table 6. At the end of the incubation period, unbound compounds of formula (II) were removed using Amicon Ultra Centrifugal Filter 100 KDa. Filters were first equilibrated with 1.5mL of formulation buffer (DPBS, Ca2+, Mg2+, 0.001% F68) and centrifuged at 2000 RPM for 2 min. Coupling reaction mixtures were then loaded onto the filter, the volume adjusted to 1.5 mL and centrifuged for 1 min at 2000 RPM. This step was repeated 8 times for each sample and the purified mixture recovered after the last centrifugation step. dPCR titer was determined for each sample.3.3. Characterization of chemically-conjugated AAVs

[0437] Characterization of chemically-conjugated AAVs3.3.a. Titration of Vector Genones (vg)

[0438] For all coupling reactions, titers were determined after the formulation step by digital PCR (dPCR) using a QIAcuity (Qiagen) or by droplet digital PCR (ddPCR) using a QX200 (Biorad) or by quantitative real time PCR (qPCR) titers using a LightCycler 480 (Roche).3.3.b. Analysis of coupling by SDS-PAGE, and mass photometry and detection of end group by Dot blot analysis.

[0439] The purity and integrity of the obtained conjugated AAV vectors was evaluated by silver staining of SDS-PAGE gels. Successful coupling should result in a shift of the VP proteins towards higher molecular masses.

[0440] The conjugation of the first molecule and the click chemistry step are validated using mass photometry with the SamuxMP (Refeyn). This analysis measures the mass of individual AAV capsids, including empty and full capsids. Successful conjugation should result in a shift in the mass of AAV capsids.

[0441] The functionalization of AAV-conjugated with biotin, RGD motif-containing peptides, Angiopep peptides or VHH was evaluated by DOT-blot analysis: a. Using Streptavidin-HRP (which binds selectively to biotin) b. Using biotinylated av|33 integrin (which binds selectively to the ligands of formula (II), (E) and (J)) followed by streptavidin-HRP detection. c. Using Fc conjugated LRP1 recombinant protein (which binds selectively to ligands of formula (II), (F), (H) and (M)) followed by anti-Fc-HRP detection.d. Using biotinylated TFR1 (which binds selectively to ligands of formula (II), (N), (P), (Q) and (R)) followed by streptavidin-HRP detection.

[0442] The results are summarized in Table 6 bellow, and illustrated in Figures 1, 2, 4 and 5.Table 6: Bioconjugations carried to obtain AAVs comprising moieties of formula (III) and formula (I); ND: not determined; NA: non applicable3.3.C. Evaluation of transduction efficiency

[0443] The transduction efficiency of conjugated vectors comprising a moiety of formula (I) AAV-3N and AAV-3P was evaluated on the HEK cell-line surexpressing the TFR1 receptor at a MOI of 300. Luciferase activity was measured after a 24h incubation with conjugated vectors using the Glomax Plate Reader (Promega). Results show an improvement in transduction efficiency of vectors conjugated with targeting moiety, compared to unconjugated vectors, of cells specifically expressing the receptor targeted.

[0444] Mobility shifts and dot blot staining confirmed the effective coupling of squarate linkers of formula (IV) (compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13) on AAVs, obtaining the resulting AAVs comprising a moiety of formula (III) (AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV- 12 and AAV-13), followed by ligand of formula (II) addition (compounds A, B, E, F, G, H, J, K, L, M, N, P, Q and R) using click chemistry to obtain the resulting AAVs comprising a moiety of formula (I) of table 6 and Figures 1, 2, 4 and 5. Coupling efficiency can be modulated by the design of the first coupling molecule of formula (IV), offering a versatile approach to achieve varying ligand loading on AAVs. This approach facilitates creating intermediates AAVs comprising a moiety of formula (III) with the right amount of reactive moieties for easy payload attachment of ligands, using the compounds of formula (II), in a “plug and play” manner, without affecting upstream and downstream AAV production processes. The mild coupling conditions preserved ligand and AAV integrity and functionality, ensuring effective interactions with their target. This preservation is crucial for maintaining biological activity and binding specificity. Consequently, the functionalized AAVs of formula (I) can engage effectively in their intended biological interactions improving their transduction efficiency and selectivity. This demonstrates the robustness and efficacy of the two step-coupling process while validating that the mild coupling conditions are optimal for producing functionalized AAVs with preserved ligands functionality, essential for targeted delivery and therapeutic interventions.

Claims

CLAIMS1. A viral capsid comprising a viral capsid protein covalently linked to a functional group, wherein the functional group comprises a crosslinked group resulting from a click-chemistry reaction between a first group and a second group forming a click-chemistry reactive pair; the viral capsid protein is covalently attached to a squaramide moiety of formula (SQ):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; and N is a nitrogen atom of the functional group.

2. The viral capsid according to claim 1, wherein the viral capsid is selected from the group consisting of non-enveloped viruses and enveloped viruses, preferably the non-enveloped virus is selected from the group consisting of adenoviruses and parvoviruses, more preferably a non-enveloped virus selected from the group consisting of bocavirus and AAVs, preferably the enveloped virus is selected from the group consisting of herpes simplex virus, lenti viruses and retroviruses; more preferably the viral capsid is a viral capsid of a parvovirus, even more preferably an adeno-associated virus (AAV) capsid.

3. The viral capsid according to any of claims 1 or 2, wherein the click-chemistry reaction is selected from the group consisting of a strained-promoted alkyne-azideclick-chemistry (SPAAC) reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC); and an inverse electron-demand Diels- Alder (IEDDA) reaction.

4. The viral capsid according to any of claims 1 to 3, wherein R3 and R4 are, respectively, either the first group or the second group forming the click-chemistry reactive pair, the click-chemistry reactive pair being selected from the group consisting of i. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine; viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyne;5. The viral capsid according to any of claims 1 to 4, wherein said viral capsid comprises a moiety of formula (I):whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of a viral capsid protein;— represents the point of attachment to the viral capsid protein;Z is H or is a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof; each Si and S2 comprise independently a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethyleneglycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branched polyol, a P-alanine polymer, pHPMA, PLGA, a sarcosine polymer and combinations thereof; andR is a crosslinked group resulting from a click-chemistry reaction between R3 and R4.

6. The viral capsid according to claim 5, wherein Z comprises a cyclic or a linear peptide or a polypeptide, preferably the polypeptide is an antibody or , an antigenbinding fragment, preferably a single-chain variable fragment (scFv), a VHH or nanobody.

7. The viral capsid according to any of claims 5 or 6, wherein each Si and S2 comprise independently a group selected from the group consisting of an arylene group, a heteroarylene group, a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, a P-alanine group comprising 1 to 40 P-alanine units and a sarcosine polymer comprising 1 to 40 sarcosine monomers.

8. A viral vector particle comprising a nucleic acid and the viral capsid according to any of claims 2 to 7, preferably wherein the viral vector particle is an AAV vector particle, more preferably the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11,AAV12, AAV13, AAVrhlO and AAVrh74; or pseudotypes, chimeras, and variants thereof.

9. A pharmaceutical composition comprising a viral vector particle according to any of claims 1 to 8, and at least one pharmaceutically acceptable vehicle.

10. A viral vector particle according to any of claims 1 to 8, or a pharmaceutical composition according to claim 9, for use as a medicament.

11. A viral vector particle according to any of claims 1 to 8, or a pharmaceutical composition according to claim 9, for use in gene therapy.

12. A viral vector particle according to any of claims 1 to 8, or a pharmaceutical composition according to claim 9, for use in a method of delivering a nucleic acid to a cell for the treatment of a disease.

13. A non-therapeutic method of delivering a nucleic acid to a cell, the method comprising contacting a cell with a viral vector particle according to any of claims 1 to 8, comprising a nucleic acid to be expressed in the contacted cell.

14. Use of a pair of compounds for manufacturing a viral vector particle comprising a viral capsid according to any of claims 5 to 8, wherein said pair of compounds consists of a compound of formula (II):and a compound of formula (IV), or a pharmaceutically acceptable salt thereof:whereinRi is selected from the group consisting of a linear Ci-12 alkyl, a branched C3-12 alkyl, a linear Ci-12 haloalkyl, a branched C3-12 haloalkyl, benzyl, phenyl and pyridyl; Z is H or is a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof; each Si and S2 comprise independently a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branchedpolyol, a P-alanine polymer, pHPMA, PLGA, a sarcosine polymer, and combinations thereof; andR3 and R4 are, respectively, either the first group or the second group of a clickchemistry reactive pair, said click-chemistry reactive pair selected from the group consisting of i. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine; viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyne.

15. A viral capsid comprising a viral capsid protein and a moiety of formula (III):wherein R4 is, either the first group or the second group forming a click-chemistry reactive pair; preferably R4 is selected from the group consisting of an azide, a strained alkyne, a tetrazine, a cyclic olefine, an alkyne, a 1,2,3-triazine, an amidine, and a 1,2,4-triazine; and whereinN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein; and Si comprises a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines,an acyl group, an amino acid moiety, a polyether of a branched polyol, a P-alanine polymer, pHPMA, PLGA, a sarcosine polymer, and combinations thereof.

16. A method for manufacturing a viral vector particle comprising: reacting a viral vector with a compound of formula (IV):in conditions suitable for reacting a squarate moiety of the compound of formula(IV) with at least one amino group from a surface-exposed natural amino acid residue of a viral capsid protein from the viral vector so as to form a viral vector particle comprising a moiety of formula (III):reacting the viral vector particle comprising moiety of formula (III) with a compound of formula (II):in conditions suitable for the groups R3 and R4 forming a click-chemistry reactive pair to react and obtain a viral vector particle comprising a moiety of formula (I):whereinRi is selected from the group consisting of a linear Ci-12 alkyl, a branched C3-12 alkyl, a linear Ci-12 haloalkyl, a branched C3-12 haloalkyl, benzyl, phenyl and pyridyl;R is a group resulting from a click-chemistry reaction between a first and a second group of a click-chemistry reactive pair, wherein R3 and R4 are, respectively, either the first group or the second group of said click-chemistryreactive pair, preferably R3 and R4 are, respectively, either the first group or the second group of said click-chemistry reactive pair selected from the group consisting of i. an azide and a strained alkyne; ii. a tetrazine and a cyclic olefine; iii. a tetrazine and an alkyne; iv. a tetrazine and a strained alkyne; v. a 1,2,3-triazine and an amidine; vi. a 1,2,3-triazine and a guanidine; vii. a 1,2,4-triazine and a cyclic olefine; viii. a 1,2,4-triazine and an alkyne; and ix. 1,2,4-triazine and a strained alkyneN* is a nitrogen atom of a primary amino group from a surface-exposed natural amino acid residue of the viral capsid protein;— represents the point of attachment to the viral capsid protein;Z is H or is a group selected from the group consisting of a cell-type targeting ligand, a receptor targeting ligand, a labelling agent, a steric shielding agent, a drug moiety and any combination thereof; and each Si and S2 comprise independently a group selected from the group consisting of an arylene or a heteroarylene group, a C1-C40 hydrocarbon chain, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; a polymer of alkylene diamines, an acyl group, an amino acid moiety, a polyether of a branched polyol, a P-alanine polymer, pHPMA, PLGA, a sarcosine polymer and combinations thereof.

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