Squaramide modified adeno-associated virus vector

Abstract

The present invention relates to adeno-associated virus (AAV) vectors modified by covalently coupling at least one compound comprising a squaramide moiety to at least one amino group of an amino acid residue of a capsid of the AAV vector. The AAV vectors can be used for transduction of cells, in particular for gene therapy.

Description

Technical Field

[0001] This invention relates to adeno-associated virus (AAV) vectors modified by covalent coupling of a square ester with a ligand or functional moiety and the primary amino group of an amino acid residue in the capsid of the AAV vector. In some embodiments, the provided AAV vectors can be used for transducing cells, particularly for gene therapy. Background Technology

[0002] Gene therapy is based on modifying the genes of cells to deliver nucleic acids into the patient's cells to produce a therapeutic effect. In reality, sometimes all or part of a gene is defective or missing at birth, or genes may change or mutate during life. Any of these variations can disrupt protein synthesis, leading to health problems or diseases. Through gene therapy, defective genes or gene sequences causing medical problems can be replaced with healthy genes or gene sequences that do not cause medical problems; genes (or sequences) can be added to help the body fight or treat diseases; or problematic genes (or sequences) can be knocked down or eliminated. Therefore, gene therapy can be used to treat genetic diseases or acquired diseases.

[0003] Novel genes (or sequences) can be delivered into cells using various methods, such as the use of genetically engineered vectors to deliver target sequences (e.g., genes). Viral vectors, particularly adeno-associated virus (AAV) vectors, can be used for this purpose. AAV vectors have proven to be reliable, efficient, versatile, and safe tools for delivering targeted transgenes into a variety of tissues. AAV vectors offer advantages such as relatively broad tropism, high transduction efficiency, sustained free-type expression, and high safety, especially since wild-type AAV is not associated with any human diseases.

[0004] Clinical trials using AAV vectors for gene therapy for a variety of diseases have been initiated or are underway. However, some trials have shown limitations with these AAV vectors, including immunogenicity, non-selective distribution, and reduced therapeutic index.

[0005] In particular, it has recently been demonstrated that humoral immunity can be pre-existing in certain AAV serotypes, especially serotype 2AAV. Therefore, pre-existing anti-AAV neutralizing antibodies can prevent transduction in the target tissue, leading to a lack of efficacy, especially with systemic administration. Furthermore, when a second administration of the AAV vector is required to complete treatment, it can be excluded by the neutralizing antibodies present after the first administration. Therefore, there is a strong need for AAV vectors that can evade immune detection.

[0006] Another limitation of AAV vectors relates to their broad tropism. In fact, the widespread distribution of AAV vectors leads to transgene expression in tissues other than the target tissue, thus lacking specificity. This can result in a reduced therapeutic index. In reality, high doses of the vector may be required to achieve therapeutic efficacy in a given tissue. Such high doses not only pose challenges to vector production but also increase the risk of immune response. Therefore, there is a strong need for AAV vectors that can ensure cell-specific transduction.

[0007] To improve AAV vectors for evading the immune system and enhancing cell transduction and cell specificity, various strategies, particularly modifications to the capsid protein of the vector, have been explored. Such modifications to the capsid protein can be achieved by introducing mutations into the exposed amino acid residues on the surface of the AAV vector capsid. Alternatively, chemical modifications to the viral capsid have been proposed to add specific ligands or mask certain exposed amino acids. Such chemical modifications, for example, can be obtained by introducing non-natural amino acid residues containing reactive functional groups into the capsid protein, followed by orthogonal selective coupling of ligands with said reactive functional groups. Another strategy is to directly chemically modify the viral capsid without any primary mutations in the capsid protein. For example, WO2017 / 212019 provides surface-modified AAV vectors obtained by covalently coupling ligands with specific isothiocyanate groups to amino groups in the amino acid residues of the AAV capsid protein, thereby improving gene transfer to specific cells. Furthermore, WO2021 / 005210 reports the modification of tyrosine residues in the AAV capsid, providing a method for chemically modifying tyrosine residues in the AAV capsid to enhance the immunogenicity of the AAV vector. On the other hand, WO2022 / 096681 provides a surface-modified vector obtained by reacting a compound containing a lactam (e.g., β-lactam) with an amino group in the amino acid residues of the AAV capsid protein.

[0008] However, current strategies for modifying AAV supports still have several drawbacks. For example, in some cases, coupling chemistry involving certain isothiocyanate groups has been observed to be overly reactive, thus prone to significant self-coupling reactions. On the other hand, even if the use of β-lactams could address the issues associated with the aforementioned self-coupling reactions, their use may be subject to certain regulatory limitations that could affect the industrial scale-up design and overall cost of these products: FDA regulations require that β-lactams must be completely and thoroughly separated from non-β-lactam products during processing (see FDA Regulation April 2013, Case No.: FDA-2011-D-0104).

[0009] Therefore, new methods are still needed to modify the properties of AAV vectors to achieve efficient gene transduction, especially for gene therapy.

[0010] Invention Summary

[0011] This invention relates to adeno-associated virus (AAV) vector particles comprising a squaric acid amide linker portion formed by the reaction of a squaric acid ester with a primary amino group (e.g., an amino group of a lysine side chain) present in the AAV vector capsid.

[0012] In some aspects, the present invention relates to adeno-associated virus (AAV) vector particles comprising a portion of formula (II):

[0013]

[0014] in

[0015] N* is the nitrogen atom of the primary amino group of the amino acid residues exposed on the surface of the capsid polypeptide of the AAV carrier.

[0016] ---- indicates the connection point with the AAV carrier capsid; and

[0017] R L -NH- is a functional portion as defined and described in the categories and subclasses of this invention, which contains a nitrogen-containing group -NH-.

[0018] According to the present invention, AAV carrier particles include a square amide connector of formula (I):

[0019]

[0020] The functional part R L -NH- is covalently linked to the amino acid residues of the AAV carrier capsid of the AAV carrier particles disclosed in this invention to form a square amide linker of formula (I).

[0021] Furthermore, N is a nitrogen atom belonging to the functional part, and is therefore represented as R as defined in this invention. L -NH-, where N* is the nitrogen atom of the primary amino group of the amino acid residues exposed on the surface of the capsid polypeptide of the AAV carrier.

[0022] In some aspects, the present invention relates to techniques and / or methods for preparing the provided AAV carrier particles.

[0023] In some respects, the surface-exposed amino acid residue containing at least one primary amino group is lysine.

[0024] In some respects, N* is the nitrogen atom of the amino group of the lysine residue in the AAV carrier capsid.

[0025] Functional group R L -NH- includes the group -NH-, which forms part of a square amide linker of formula (I) as defined in this invention, and the functional group R. LIt comprises space shielding agents, labeling agents, cell type-specific ligands, drug components, and combinations thereof. Therefore, in some respects, R... L -NH- is a functional part containing or composed of a group selected from the group consisting of: space shielding agents, labeling agents, cell type-specific ligands, drug moieties, and combinations thereof.

[0026] In some respects, R L -NH- contains a labeling agent. In some aspects, the labeling agent comprises or is a fluorescent dye, such as fluoroalanine, fluorescein, rhodamine, or boron-dipyrrole methylene. Dyes and Alexa Or radioactive nuclides.

[0027] In some respects, R L -NH- contains cell type-specific ligands selected from the group consisting of: carbohydrates, hormones, peptides, glycosylated peptides, glycoproteins, proteins or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins and pharmaceutical fractions.

[0028] In some respects, R L -NH- contains space shielding agents selected from the group consisting of polyethylene glycol, pHPMA, and polysaccharides.

[0029] In some respects, the functional part R L -NH- contains a group Z and one or more spacer groups L, and the adeno-associated virus (AAV) vector particle contains a portion represented by formula (IIa):

[0030]

[0031] Wherein N*, ------, Z and L are as defined and described in the categories and subclasses of this invention.

[0032] In some respects, the functional part R L -NH- contains a group Z and one or more spacer groups L, wherein Z is H or contains a cell type-specific ligand selected from the group consisting of: carbohydrates, hormones, peptides, glycosylated peptides, proteins, glycoproteins or their functionally active fragments, membrane receptors or their functionally active fragments, antibodies or their functionally active fragments, spiegelmers, nucleic acids or peptide aptamers, vitamins and pharmaceutical portions.

[0033] In some respects, the functional part R L -NH- does not contain one or more spacer groups L, and the functional portion R L -NH- is composed of the group Z-NH-.

[0034] In some respects, the functional part RL -NH- contains one or more Z groups and one or more spacer groups L. In some aspects, the functional moiety R L -NH- contains 1-3 groups Z, each of which is attached to one or more spacer groups L.

[0035] In some respects, Z is a sugar. In other respects, Z is a peptide.

[0036] In some respects, Z is or contains sugars selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and their derivatives.

[0037] In some aspects, the sugar is selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, deoxyglucosamine, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose, and P1-fructose. In some preferred aspects, the sugar is selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, and P1-fructose, more preferably mannose.

[0038] In some aspects, Z is a linear or cyclic peptide or comprises a linear or cyclic peptide, wherein the peptide can be a peptide characterized by biological activity, particularly a blood-brain barrier-penetrating peptide (BBP). Specifically, the peptide is a blood-brain barrier (BBB) ​​shuttle peptide (also known as a BBB-penetrating peptide) that has enhanced transduction activity across the blood-brain barrier. BBB shuttle peptides have the ability to cross the BBB and are therefore capable of transporting a variety of substances into the brain parenchyma without disrupting the integrity of the BBB. Therefore, BBB shuttle peptides have the potential to increase AAV crossing the BBB and enhance brain transduction, particularly in neuronal cells. In some preferred aspects, the BBB shuttle peptide is selected from the group consisting of peptides THR or peptides having an RGD motif, including cyclic RGD peptides. Typically, peptide THR can bind to and internalize human transferrin receptor (hTfR), as described in Lee et al., 2004 (Eur J Biochem. 2001 Apr; 268(7): 2004-12. doi:10.1046 / j.1432-1327.2001.02073.x.PMID:11277922) and international patent application with publication number WO02 / 44329(A2). BBB shuttle peptides are also described in Sánchez-navarro et al., 2022 (Pharmaceutics. 2022 Sep 5; 14(9): 1874. Doi: 10.3390 / pharmaceutics14091874. PMID: 36145622; PMCID: PMC9505527) and, for example, international patent applications with publication numbers WO2008 / 025867(A1), WO2012 / 007625(A1), WO2013 / 127829(A1) and WO2015 / 001015(A1).

[0039] In some respects, the spacer group L comprises one or more groups selected from the group consisting of: arylene or heteroarylene Ar; saturated or unsaturated, straight or branched C1-C 40 Optionally substituted groups in the hydrocarbon chain; groups containing alkyleneamines; groups containing acyl groups; amino acids; alkyl ether groups, such as ethylene glycol or propylene glycol groups; polyethers, such as polyethylene glycol (PEG) or polypropylene glycol (PPG), or polyethers of branched polyols; polyamides, such as β-alanine polymers; vinyl polymers, such as pHPMA; polyesters, such as PLGA; polymers of alkylenediamines; and combinations thereof. In particular, the spacer group L comprises one or more groups selected from the group consisting of arylene or heteroarylene groups, comprising saturated or unsaturated, straight-chain or branched C1-C groups. 40Optionally substituted groups in the hydrocarbon chain, polyethylene glycol (PEG), polypropylene glycol (PPG), alkylene amines; acyl groups, amino acid moieties, polyethers of branched polyols, β-alanine polymers, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof.

[0040] In some respects, when Z is a peptide, Z is covalently linked to a C-terminal via its amide moiety or bioisosteric moiety. 1-6 The alkylene moiety is attached to the β-alanine moiety, or to the Ar group as defined herein, or to the acyl group, or to the squaramide moiety of formula (I), wherein the acyl group or nitrogen atom of the amide moiety corresponds to the acyl group of the C-terminal group of the peptide and the nitrogen atom of the N-terminal group of the peptide, respectively.

[0041] In some respects, when Z is a sugar, Z is bonded to PEG or PPG groups via ether bonds or to C. 1-6 The alkylene group is covalently linked, wherein the oxygen atom of the ether bond corresponds to an oxygen atom belonging to the sugar.

[0042] In some aspects, L comprises polyethylene glycol (PEG), which contains 1-40 ethylene glycol monomers. In some aspects, L comprises polyethylene glycol (PEG), which contains 1-10 ethylene glycol monomers. In some aspects, polyethylene glycol (PEG) is PEG1, PEG2, PEG3, PEG4, or PEG5.

[0043] In some aspects, L comprises a β-alanine polymer or a β-alanine moiety containing 1 to 40 β-alanine monomers or β-alanine units. In some aspects, the β-alanine polymer comprises 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 8, or 10 β-alanine monomers.

[0044] In some respects, L contains polyethylene glycol (PEG) and β-alanine polymers.

[0045] In some respects, L comprises polyethers containing branched polyols, such as branched C 3-12 Polyols, preferably branched C 3-6 Polyols, more preferably polyethers of branched C4 polyols.

[0046] In some aspects, L comprises an amino acid moiety. For the purposes of this disclosure, the term amino acid refers to a molecule comprising an amino and a carboxyl group, including but not limited to proteogenic and non-proteogenic amino acids. In some aspects, L comprises an arginine moiety and a β-alanine moiety. In particular, the amino acid moiety is linked to another portion of the spacer group L via its carboxylic ester group and / or amino group, or to a squaramide linker of formula (I) or to a functional moiety.

[0047] In some respects, L contains one or more arylene or heteroarylene Ar.

[0048] In some respects, L includes C 1-6 Alkylene, which can be a straight-chain C 1-6 Alkyl or branched C 3-6 Alkylene, more preferably L comprises a -CH2- group or a branched C4 alkylene group. In some aspects, L comprises C 1-6 An alkyleneamine group, preferably a -CH2-CH2-NH- group. In some aspects, L contains C. 1-6 An acyl group, preferably a -CH2-CH2-C(O)- group, is present between the PEG group and the β-alanine polymer, wherein n is 1-6, preferably a -CH2-CH2-C(O)- group, such that the PEG and the β-alanine polymer are linked by an amide moiety.

[0049] In some aspects, the arylene or heteroarylene Ar is a divalent aromatic group (or divalent aromatic moiety), i.e., it is connected to two different groups of the portion of formula (II) of the AAV carrier particle of the present invention (i.e., forming a bridge between the two portions of the portion of formula (II)), and it may additionally contain one or more optional substituents. Preferably, the arylene or heteroarylene Ar is a 6- to 10-membered arylene or a 5- or 12-membered heteroarylene containing one or more heteroatoms selected from the group consisting of N, O, S, and Se. In some specific aspects, the arylene or heteroarylene Ar is a phenylene or pyridylene group, which optionally contains one or more substituents. In some aspects, the arylene or heteroarylene Ar contains one or more substituents selected from the group consisting of halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkyl group.

[0050] In some respects, Z is a sugar or peptide, and L contains polyethylene glycol (PEG), which contains 1-40 glycol monomers.

[0051] In other respects, Z is a sugar or peptide, and L comprises polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers and arylene or heteroarylene Ar, preferably wherein the PEG and Ar groups are covalently linked through an amide moiety or its bioelectron isosteric moiety.

[0052] In other respects, Z is a sugar or peptide, L contains polyethylene glycol (PEG) with 1-40 ethylene glycol monomers, and one or more C 1-6 Alkylene and arylene or heteroarylene Ar, preferably wherein the PEG and Ar groups are covalently linked via an amide moiety or its bioelectron isosteric moiety, or wherein the PEG and C 1-6Alkyl groups are covalently linked via the amide moiety or its bioelectron isosteric moiety.

[0053] In other respects, the functional part R L -NH- contains 1-3 Z groups; where Z is a sugar or peptide, and L contains one or more polyethylene glycol (PEG) monomers comprising 1-40 ethylene glycol monomers, and one or more C groups. 1-6 Alkylene, branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyols, and arylene or heteroarylene Ar; preferably, wherein each Z is linked to a first C. 1-6 alkylene groups or PEG, the first C 1-6 The alkylene group is attached to the group Z via the amide moiety, and the PEG is covalently attached to the second C via the amide moiety or its bioelectron isosteric moiety. 1-6 alkylene groups; wherein the second C 1-6 The alkylene group is linked to the branched C via an ether bond. 3-12 Polyols, wherein the branched C 3-12 Polyols are linked to arylene or heteroarylene Ar via an amide moiety or its bioelectron isosteric moiety; or wherein the arylene or heteroarylene Ar is linked to a C group other than the first and second C groups. 1-6 One or more C of alkylene 1-6 Alkylene linkage, and wherein the branched C 3-12 Polyols interact with one or more C14 groups via their amide or bioelectron isosteric moiety. 1-6 A linker in an alkylene group. In one aspect, the functional moiety R... L -NH- contains 1-3 Z groups; where Z represents sugars. On the other hand, the functional moiety R... L -NH- contains 1-3 Z groups; each Z is independently a sugar or peptide.

[0054] In other respects, Z is a peptide and L contains polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and one or more C... 1-6 alkylene groups; or one or more C 1-6 alkyleneamine group, or one or more C 1-6 Acyl group.

[0055] In other respects, Z is a peptide and L contains a β-alanine polymer containing 1-40 β-alanine monomers.

[0056] In other respects, Z is a peptide and L comprises a β-alanine polymer containing 1-40, preferably 1-10, β-alanine monomers, and one or more C... 1-6 Alkyl groups, preferably C1-2 alkylene groups, or one or more C1-2 alkylene groups.1-6 alkylamine group, preferably -CH2-CH2-NH- group, or one or more C groups 1-6 Acyl group, preferably -CH2-CH2-C(O)- group.

[0057] In other respects, Z is a peptide and L comprises a β-alanine polymer containing 1-40, preferably 1-10, β-alanine monomers, and one or more C 1-6 Alkylene group, preferably C 1-2 alkylene group, or one or more C 1-6 alkyleneamine group, preferably -CH2-CH2-NH- group, or one or more C groups. 1-6 An acyl group, preferably a -CH2-CH2-C(O)- group, and an arylene or heteroarylene group Ar; wherein the PEG and Ar groups are covalently linked via an amide moiety or its bioisosteric moiety, or wherein the PEG and a C group are... 1-6 Alkyl groups are covalently linked via the amide moiety or its bioelectron isosteric moiety.

[0058] In other respects, Z is a peptide and L contains polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, one or more C 1-6 Alkylene group, preferably C 1-2 alkylene group, or one or more C 1-6 alkylamine group, preferably -CH2-CH2-NH- group, or one or more C groups 1-6 The acyl group, preferably -CH2-CH2-C(O)-, and the amino acid moiety, preferably the arginine moiety or the β-alanine moiety, more preferably the arginine moiety.

[0059] In other respects, Z is a peptide and L contains polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, a β-alanine polymer containing 1-40 β-alanine monomers, and one or more C 1-6 Alkylene group, preferably C 1-2 alkylene group, or one or more C 1-6 alkyleneamine group, preferably -CH2-CH2-NH- group, or one or more C groups. 1-6 The acyl group, preferably -CH2-CH2-C(O)-, and the amino acid moiety, preferably the arginine moiety or the β-alanine moiety, more preferably the arginine moiety.

[0060] In some respects, peptide Z and spacer L, especially spacer C 1-6 Alkyl groups are covalently linked via an amide moiety (e.g., amide-N(R1)C(O)-) or its bioelectron isosteric moiety; wherein R1 is selected from the group consisting of H, C 1-6 Alkyl, C1-6 Halogenated alkyl groups, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, even more preferably R1 is H.

[0061] In some aspects, L comprises polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and arylene or heteroarylene Ar as defined in this invention, wherein the PEG and Ar groups are covalently linked by an amide moiety (e.g., amide-N(R1)C(O)-) or its bioisosteric moiety; or L comprises polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and one or more C 1-6 Alkylene and arylene or heteroarylene Ar as defined in this invention, wherein the PEG and Ar groups are covalently linked by an amide moiety (e.g., amide-N(R1)C(O)-) or its bioelectron isosteric moiety, or wherein the PEG and C 1-6 Alkyl groups are covalently linked via an amide moiety (e.g., amide-N(R1)C(O)-_ or its bioelectron isosteric moiety); wherein R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, even more preferably R1 is H.

[0062] In some aspects, the one or more spacer bases L are selected from the group consisting of L1, L2, and L3, and the AAV carrier particles comprise portions selected from the group consisting of (IIa1), (IIa2), (IIa3), (IIa4), (IIa5), (IIa6), (IIa7), (IIa8), (IIa9), (IIa... 10 ), (IIa11 ), (IIa 12 ), (IIa 13 ), (IIa 14 ), (IIa 15 ), (IIa 16 ), (IIa 17 ), (IIa 18 ), (IIa 19 ) and (IIa 20 ):

[0063]

[0064]

[0065]

[0066] Wherein N*, ----, and Z are as defined and described in the categories and subclasses disclosed in this invention, and wherein L1 is one or more groups selected from the group consisting of: polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyols, and β-alanine polymers comprising 1-40 β-alanine monomers, or mixtures thereof; L2 comprises one or more arylene or heteroarylene Ar groups; and L3 is one or more groups selected from the group consisting of: amino acid moieties, C 1-6 alkyleneamine group, C 1-6 alkylene acyl groups and C 1-6 Alkylene, the C 1-6 Alkylene is a straight-chain C 1-6 Alkyl or branched C 3-6 Alkylene; and preferably Z is a peptide or sugar.

[0067] In some respects, L3 is covalently linked to L2 via a carbon atom of an arylene or via a carbon atom or a heteroatom of a heteroarylene; and wherein L1 and L2, or L1 and L3, are covalently linked via an amide moiety or its bioelectron isosteric moiety; or wherein L1 and L3 are covalently linked via an ether bond.

[0068] In some aspects, polyethylene glycol (PEG) is PEG1, PEG2, PEG3, PEG4, or PEG5. In some aspects, the β-alanine polymer contains 1-10 β-alanine monomers. In some aspects, L2 contains a phenylene or pyridylene group. In some aspects, L3 is C 1-3 The alkylene group, -CH2-CH2-NH- group, -CH2-CH2-C(O)- group, or amino acid moiety, preferably the arginine moiety or the β-alanine moiety.

[0069] In some respects, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers or a β-alanine polymer or mixture thereof containing 1-40 β-alanine monomers, and L2 is arylene or heteroarylene Ar; L1 and L2 are covalently linked through an amide moiety or its bioelectron isosteric moiety.

[0070] In some respects, L1 and L3 are covalently linked through the amide moiety or its bioelectron isosteric moiety.

[0071] In some respects, L3 is covalently linked to L2 via a carbon atom of the arylene group or via a carbon atom or a heteroatom of the heteroarylene group.

[0072] In some respects, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers or a β-alanine polymer containing 1-40 β-alanine monomers, or a mixture thereof; L2 is an arylene or heteroarylene Ar; and L3 is one or more groups selected from the group consisting of: amino acid moieties, C... 1-6 alkyleneamine, C 1-6 alkylene acyl and C 1-6 Alkylene; wherein L1 and L2 or L1 and L3 are covalently linked via an amide moiety or its bioelectron isosteric moiety.

[0073] In some respects, L1 is a branch C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyethers of polyols, where L3 is a C group. 1-6 Alkylene; L1 and L3 are covalently linked by ether bonds.

[0074] In some respects, L1 is a branch C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyethers of polyols, where L3 is a C group. 1-6 Alkylene; L1 and L3 are covalently linked via the amide moiety or its bioelectron isosteric moiety.

[0075] In some respects, L1 is a branch C 3-12 Polyethers of polyols, preferably branched C 3-6 The polyether of the polyol, wherein L2 contains an arylene or heteroarylene Ar; L1 and L2 are covalently linked through an amide moiety or its bioelectron isosteric moiety.

[0076] In some respects, when L2 contains an arylene or heteroarylene Ar, L1 and the square amide connector of formula (I) are covalently bonded to the arylene or heteroarylene Ar at the ortho, meta, or para positions, or when L2 contains an arylene or heteroarylene Ar and L3 is present, L1 and L3 are covalently bonded to the arylene or heteroarylene Ar at the ortho, meta, or para positions.

[0077] In some respects, when L2 contains an arylene or heteroarylene Ar and L3 is a group C 1-6 The alkylene group, L3, and the square amide linker of formula (I) are covalently bonded to the arylene or heteroarylene Ar at the ortho, meta, or para positions.

[0078] In some respects, when L2 contains an arylene or heteroarylene Ar and L3 is a group C 1-6 An alkylene group, one or more groups L3, are covalently bonded to the arylene or heteroarylene Ar at the ortho, meta, or para positions.

[0079] In some respects, L1 is polyethylene glycol (PEG), which contains 1-40 ethylene glycol monomers; L2 contains one or more arylene or heteroarylene groups; L3 is C 1-6 The alkylene group, L3, is covalently linked to L2 via a carbon atom of the arylene group or via a carbon atom or a heteroatom of the heteroarylene group; and L1 and L2 are covalently linked via an amide moiety or via their bioisosteric moiety.

[0080] In some respects, L1 is polyethylene glycol (PEG), which contains 1-40 ethylene glycol monomers; L2 contains one or more arylene or heteroarylene groups; L3 is C 1-6 The alkylene group, L3, is covalently linked to L2 via a carbon atom of the arylene group or via a carbon atom or a heteroatom of the heteroarylene group; and L1 and L3 are covalently linked via an amide moiety or via their bioisosteric moiety.

[0081] In some respects, L1 is polyethylene glycol (PEG), which contains 1-40 ethylene glycol monomers; L2 contains one or more arylene or heteroarylene groups; L3 is C 1-6 The alkylene group L3 is covalently connected to L2 via a carbon atom of the arylene group or via a carbon atom or a heteroatom of the heteroarylene group; and L1 and L2 are covalently connected via the amide moiety or its bioelectron isosteric moiety, or L1 and L3 are covalently connected via the amide moiety or its bioelectron isosteric moiety.

[0082] In some respects, Z is a sugar and L1 is or contains polyethylene glycol (PEG), which contains 1-40 glycol monomers, and Z and L1 are covalently linked by ether bonds.

[0083] In some respects, Z is a peptide and L3 is a C. 1-6 alkylene groups, Z and C 1-6 The alkylene groups are covalently linked via an amide moiety or its bioelectron isosteric moiety (e.g., amide moiety N(R1)C(O)- or its bioelectron isosteric moiety), as defined in the claims and embodiments of this specification.

[0084] In some respects, Z is a peptide, L1 is or contains polyethylene glycol (PEG), which contains 1-40 glycol monomers, and L3 is an arginine derivative. L1 and L3 are covalently linked by their amide moiety or bioelectron isosteric moiety.

[0085] In some aspects, the AAV carrier particles comprising the portion of formula (II) are AAV carrier particles comprising a portion selected from the group consisting of formulas (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), (IIh), (IIj), (IIk), (IIm), (IIIn), (IIp), (IIq), (IIr), (IIs), (IIt), (IIv), (IIw), and (IIx):

[0086]

[0087]

[0088]

[0089]

[0090] Where R a R b and R c Each is independently H or group R':

[0091]

[0092] R a R b and R cAt least one of them is a group R' and wherein n and n' are each independently selected from 1 to 40, m1 and m2 are each independently selected from 0, 1 or 2, m3 and m4, m5 and m6 are each independently selected from 1 to 6, preferably 1, 2 or 3, and N*, ----, Z, Ar and R1 are as defined and described in the categories and subcategories disclosed in this invention. In some specific aspects, n is 3, 4, or 5; Z is or contains a linear or cyclic peptide, wherein the peptide may be a biologically active peptide, preferably a blood-brain barrier (BBB) ​​shuttle peptide, more preferably a BBB shuttle peptide selected from the group consisting of: peptide THR or peptides having an RGD motif, including cyclic RGD peptides; or sugars selected from the group consisting of: monosaccharides, oligosaccharides, polysaccharides and their derivatives, preferably sugars selected from the group consisting of: mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, deoxyglycosamine, glucuronic acid, S6-galactose, S6-N-acetylglucosamine. Lactosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose, and P1-fructose, preferably selected from the group consisting of: mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, and P1-fructose, more preferably mannose; Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group containing one or more heteroatoms selected from the group consisting of: N, O, S, and Se, preferably phenylene or pyridinylene, optionally containing one or more substituents selected from the group consisting of: halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 alkoxy group; and R1 is selected from the following group: H, C 1-6 Alkyl, C 1-6 Haloalkyl, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n- and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, even more preferably R1 is H.

[0093] In some respects, the AAV carrier particles are selected from the portion shown in Table 1' below.

[0094] In some aspects, the AAV carrier is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, their pseudotypes, chimeras, and variants; preferably, the AAV carrier is selected from the group consisting of: AAV2, AAV5, AAV8, and AAV9.

[0095] In some respects, the AAV vector contains at least one transgene, and the transgene is optionally under the control of a promoter.

[0096] In some aspects, the AAV vector contains at least one transgene, which contains cDNA from the GBA gene, preferably the human GBA gene, and the transgene is optionally under the control of a promoter.

[0097] In some aspects, the present invention also provides pharmaceutical compositions comprising AAV carrier particles according to the invention and at least one pharmaceutically acceptable medium.

[0098] In some aspects, the present invention also relates to AAV carrier particles according to the invention or pharmaceutical compositions according to the invention, which are used as diagnostic agents and / or pharmaceuticals, preferably for gene therapy.

[0099] In other respects, the present invention relates to the use of AAV carrier particles according to the invention or pharmaceutical compositions according to the invention as diagnostic agents and / or pharmaceuticals, preferably for use in gene therapy.

[0100] In other respects, the present invention provides compounds of formula (III):

[0101]

[0102] Or a pharmaceutically acceptable salt thereof, wherein R2 is selected from the group consisting of: linear C 1-12 Alkyl, branched C 3-12 Alkyl, straight-chain C 1-12 Halogenated alkyl groups, branched C 3-12 Halogenated alkyl and benzyl; and R L -NH- is a functional part as defined and described in the categories and subclasses of this invention. In some aspects, R2 is methyl, ethyl, or benzyl, preferably ethyl.

[0103] In some respects, the functional part R L -NH- contains a Z group, one or more spacer groups L, and compounds of formula (III) or pharmaceutically acceptable salts thereof are represented by formula (IIIa):

[0104]

[0105] R2, Z, and L are as defined and described in the categories and subclasses of this invention.

[0106] In some aspects, compounds of formula (IIIa) or pharmaceutically acceptable salts thereof contain one or more spacer groups L, said spacer groups being selected from the group consisting of L1, L2, and L3, and specifically from the group consisting of (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), (IIIa8), (IIIa9), (IIIa... 10 (IIIa) 11 (IIIa) 12 (IIIa) 13 (IIIa) 14 (IIIa) 15 (IIIa) 16 (IIIa) 17 (IIIa) 18 (IIIa) 19 ) and (IIIa 20 ):

[0107]

[0108]

[0109]

[0110] R2, Z, L1, L2 and L3 are as defined and described in the categories and subclasses of this invention.

[0111] In other respects, compounds of formula (III) are selected from the group consisting of: (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIj), (IIIk), (IIIm), (IIIn), (IIIp), (IIIq), (IIIr), (IIIs), (IIIt), (IIIv), (IIIw) and (IIIx):

[0112]

[0113]

[0114]

[0115]

[0116] Or a pharmaceutically acceptable salt thereof, wherein R a R b and Rc The following are defined and described as in the categories and subclasses disclosed in this invention: n, n', m1, m2, m3, m4, Z, Ar, R1, and R2.

[0117] Compounds of formula (III) can be used to obtain AAV carrier particles comprising the portion of formula (II) as defined and described in the categories and subclasses of this invention.

[0118] Therefore, in some aspects, the present invention also provides the use of compounds of formula (III) as defined and described in the categories and subclasses of the present invention for obtaining AAV carrier particles comprising a portion of formula (II) as defined and described in the categories and subclasses of the present invention.

[0119] In particular, the compound of formula (III) according to the invention comprises the squaric acid ester moiety of formula (IV):

[0120]

[0121] R2 is defined and described in the categories and subclasses disclosed in this invention, and wherein the squaric acid ester reacts with an amino group in the amino acid residues of the AAV carrier capsid under suitable conditions to form a squaric acid amide linker as defined in this disclosure (I).

[0122] In some aspects, the present invention also provides a method for synthesizing AAV carrier particles comprising a portion of formula (II) as defined and described in the categories and subcategories of the present invention, wherein the method comprises: incubating an AAV carrier with a compound of formula (III) as defined and described in the categories and subcategories of this specification under conditions suitable for reacting the squaric acid ester portion of a compound of formula (III) with at least one amino group of an amino acid residue of an AAV carrier capsid to form a squaric acid amide linker of formula (I). Detailed Implementation

[0123] Various coupling chemistry methods via amino groups (e.g., the amino group of the lysine side chain) have been described. However, not all such coupling chemistry methods are compatible with and / or effective under specific conditions and / or with specific substrates.

[0124] This disclosure relates to a coupling method for coupling amino acids of adeno-associated virus (AAV) using a squamamide linker of formula (I) as previously defined. This disclosure recognizes that not all coupling chemistry is effective for such coupling. For example, some coupling chemistry requires, or typically is carried out, under conditions that can disrupt one or more structural or functional properties of AAV (e.g., fail to maintain the integrity of AAV). Alternatively, various coupling chemistry requires, or typically is carried out, under conditions incompatible with certain biochemical ligands (such as sugars and / or (poly)peptides).

[0125] For example, some coupling strategies and conditions may favor self-coupling (intermolecular and / or intramolecular coupling) with another ligand molecule rather than coupling with AAV. In this regard, as previously mentioned, in certain cases, coupling chemistry involving certain isothiocyanate groups has been observed to be excessively reactive, thus readily facilitating significant self-coupling reactions.

[0126] Some coupling strategies involve a two-step coupling reaction. However, under certain conditions, AAV can become immunogenic when only the first coupling reaction occurs without the second, for example, due to non-native chemical structures modified on the capsid surface. In such cases, a one-step coupling chemistry method has been described, for example, in WO2017 / 212019, using isothiocyanate groups that have shown compatibility in certain circumstances. However, as disclosed in WO2022 / 096681, a potential problem with coupling AAV using such isothiocyanate groups is that, for example, this coupling reaction produces lipophilic linkers that can form immunogenic haptens.

[0127] WO2022 / 096681 discloses a surface-modified carrier obtained by a one-step reaction of a compound containing a lactam (e.g., β-lactam) with an amino group in the amino acid residues of the AAV capsid protein, which resolves issues involving self-coupling reactions and the formation of immunogenic haptens. However, due to the sensitizing properties of β-lactams, cross-contamination of such molecules into other products intended for human use must be prevented at the ppb level (1). Therefore, the area where β-lactams are processed must be completely and thoroughly separated from other areas where products intended for human use are processed. Thus, during development, β-lactams must be completely separated from other areas of development or production ((1) a) FDA, Guidance for Industry “Non-Penicillin Beta-Lactam Drugs: A CGMP Framework for Preventing Cross-Contamination, 2013; b) 21 CFR 211.176.

[0128] Therefore, this disclosure recognizes the particular need to provide suitable coupling chemistry that i) is compatible with a variety of AAV serotypes and maintains their integrity, ii) is a one-step reaction, iii) minimizes the self-coupling of ligands, iv) does not generate immunogenic linkers, v) provides synthetic methods with fewer limitations on industrial-scale production, and vi) is more flexible in terms of biochemical ligands and AAV serotypes.

[0129] The squaramide moiety is derived from the rigid cyclobutene ring of the squaric acid (diketoclyclobutenediol), which benefits from unique physical and chemical properties, making it unexpectedly suitable for conjugating a variety of adeno-associated virus vectors to different types of ligands. Furthermore, by selecting appropriate pH conditions, the primary and secondary substitutions of the squaric acid ester can be controlled, thus providing more selective substitution and resulting in a more flexible scaffold for conjugating a variety of adeno-associated virus vectors to different types of ligands compared to other solutions (linkers) known in the prior art.

[0130] In some aspects, the present invention relates to adeno-associated virus (AAV) vector particles comprising a portion of formula (II):

[0131]

[0132] Where N*, ---- and R L -NH- as defined and described in the categories and subclasses of this invention.

[0133] In particular, the present invention relates to adeno-associated virus (AAV) vector particles of formula (II) as defined and described herein, which are produced by the reaction of squaric acid esters of formula (III):

[0134]

[0135] Or a pharmaceutically acceptable salt thereof, wherein an amino group is present within the capsid of the AAV carrier (e.g., the amino group of the lysine side chain), for example, modified AAV carrier particles produced by said reaction;

[0136] Wherein the group R2 and the functional moiety R L -NH- as defined and described in the categories and subclasses of this invention.

[0137] For example, in some embodiments, the functional portion R L The -NH- group includes a Z group, one or more spacer groups L, and the adeno-associated virus (AAV) vector particle comprises a moiety represented by formula (IIa) as disclosed herein, and is produced by the reaction of a compound of formula (IIIa):

[0138]

[0139] Or a pharmaceutically acceptable salt thereof, wherein the amino group is present within the capsid of the AAV carrier;

[0140] R2, Z, and L are as defined and described in the categories and subclasses of this invention.

[0141] In some implementations, the functional portion R L-NH- contains a group Z and one or more spacer groups L, the portion represented by formula (IIa) is selected from formulas (IIa1), (IIa2) and (IIa3) as disclosed herein, and the AAV carrier particles contain portions selected from (IIa1), (IIa2) and (IIa3), which are respectively derived from reactions of compounds of formula (III) selected from the following group: (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), (IIIa8), (IIIa9), (IIIa 10 (IIIa) 11 (IIIa) 12 (IIIa) 13 (IIIa) 14 (IIIa) 15 (IIIa) 16 (IIIa) 17 (IIIa) 18 (IIIa) 19 ) and (IIIa 20 ):

[0142]

[0143]

[0144]

[0145] Or a pharmaceutically acceptable salt thereof, wherein the amino group is present within the capsid of the AAV carrier;

[0146] R2, Z, L1, L2 and L3 are as defined and described in the categories and subclasses of this invention.

[0147] In some specific embodiments, adeno-associated virus (AAV) vector particles comprising a portion of formula (II) as disclosed herein are reacted from compounds selected from the group consisting of formula (III): (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIe), (IIIg), (IIIh), (IIIj), (IIIk), (IIIm), (IIIn), (IIIp), (IIIq), (IIIr), (IIIs), (IIIt), (IIIv), (IIIw), and (IIIx).

[0148]

[0149]

[0150]

[0151] Or a pharmaceutically acceptable salt thereof, wherein the amino group is present within the capsid of the AAV carrier;

[0152] Where R a R b and R c The following are defined and described as in the categories and subclasses disclosed in this invention: n, n', m1, m2, m3, m4, Z, Ar, R1, and R2.

[0153] In some aspects, the present invention relates to methods for modifying AAV carriers, particularly by modifying at least one amino acid residue of the AAV capsid. In some embodiments, the invention provides a method for modifying the amino group of an amino acid residue of the capsid, preferably an amino group of a surface-exposed amino acid residue. As described herein, modification of AAV carriers has been successfully achieved by covalently coupling a linker comprising a squaric acid ester as a reactive group.

[0154] For proof of concept, several compounds of formula (III) containing a squaric acid ester moiety were prepared, wherein the functional moiety R L The -NH- group contains a labeling agent, sugar, or peptide used to modify the amino acid residues of the AAV carrier capsid. Results showed that capsid proteins of several AAV serotypes, including AAV2 and AAV5, could be effectively modified while maintaining the integrity of the AAV particles. Furthermore, these modified AAVs were demonstrated to remain infectious in cell lines such as the U87-MG glioblastoma.

[0155] AAV carrier

[0156] The AAV vector applicable to this invention may contain or be derived from any natural or recombinant AAV serotype.

[0157] “Serotype” is traditionally defined based on the lack of cross-reactivity between antibodies against one virus and antibodies against another. This difference in cross-reactivity is usually due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). As used herein, AAV includes a variety of naturally occurring and synthetic serotypes.

[0158] In some embodiments, the AAV vector according to the invention is selected from natural serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12; or their pseudotypes, chimeras and variants.

[0159] In some embodiments, the AAV vector according to the present invention belongs to AAV phylogenetic clades A, B, C, D, E, or F (see, for example, Gao et al., Clade of Adeno-Associated Viruses Are Widely Disseminated in Human Tissues. J. Virology. 2004). In some embodiments, the AAV vector according to the present invention belongs to AAV phylogenetic clade E. In some embodiments, the AAV vector according to the present invention belongs to AAV phylogenetic clade D. In some embodiments, the AAV vector according to the present invention belongs to AAV phylogenetic clade F. In some embodiments, the AAV vector according to the present invention belongs to AAV phylogenetic clade A. In some embodiments, the AAV vector according to the present invention belongs to AAV phylogenetic clade C. In some embodiments, the AAV vector according to the present invention belongs to AAV phylogenetic clade B. In some embodiments, the AAV vector according to the present invention is an AAV that does not belong to a classical phylogenetic clade.

[0160] As used herein, the term "pseudotype," when referring to an AAV vector or "pseudotyped AAV vector," refers to an AAV vector containing a portion of the AAV genome (particularly inverted terminal repeats (ITRs)) packaged by one AAV serotype within a capsid of another AAV serotype. These pseudotypes are indicated by a forward slash or hyphen; therefore, "AAV2 / 5" or "AAV2-5" indicates an AAV vector containing the genome of serotype 2, packaged within a capsid of serotype 5.

[0161] In some embodiments, the AAV vector is transcapitalized. In some embodiments, the transcapitalization method includes transfecting a combination of AAV serotype helper plasmids to produce a chimeric recombinant AAV capsid (see, for example, Rabinowitz et al. (2004), J.Virol. 78:4421-4432). In some embodiments, polyploid (when using two or more parental AAV helper cells) or haploid (when using only two) methods are utilized. In some embodiments, for example, the AAV capsid may be made from a serotype of VP1 / VP2 and a unique serotype of VP3 or a combination thereof. In some embodiments, haploid AAVs have the potential to uniquely bind the structural advantages of parental AAVs. In some embodiments, haploid AAVs have been shown to possess: 1) synergistic effects in transduction, 2) unexpected novelty, and 3) the ability to evade Nab (see, for example, Chai et al. (2019), Viruses 11:1138).

[0162] For example, in some implementations, pseudo AAV carriers 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.

[0163] As used herein, when referring to an AAV vector or a “chimeric AAV vector,” the term “chimera” means an AAV vector containing a capsid containing VP1, VP2, and VP3 proteins of at least two different AAV serotypes; or alternatively, it contains VP1, VP2, and VP3 proteins, wherein at least one of them contains at least a portion of another AAV serotype.

[0164] Examples of hybrid AAV carriers include, but are not limited to, AAV-DJ, AAV2G9, AAV2i8, AAV2i8G9, AAV8G9, and AAV9i1.

[0165] In some embodiments, the AAV vector according to the present invention is composed of: AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV106.1 / hu.37、AAV114.3 / hu.40、 AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.1 / hu.43、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV16 .12 / hu.11、AAV16.3、AAV16.8 / hu.10、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2i8、AAV2i8G9、AAV2-15 / rh.62、AAV22 3.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV2-3 / rh.61、AAV24.1、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV2.5T、AAV27.3、AAV29.3 / bb.1、 AAV29.5 / bb.2、AAV2G9、AAV3B、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-11 / rh.53、AAV3-3、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV3-9 / rh. 52、AAV3a、AAV3b、AAV4-19 / rh.55、AAV42.12、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4 、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-5、AAV4-4、AAV44.1、AAV44. 4.2、AAV44.5、AAV46.2 / hu.28、AAV46.6 / hu.29、AAV4-8 / rh.64、AAV4-9 / rh.54、AAV52.1 / hu.20、AAV52 / hu.19、AAV5-22 / rh.58、AAV5-3 / rh.57、AAV541 / hu.21、AAV54.2 / hu.22、AAV54.4R / hu.27、AAV54.5 / hu.23、AAV54.7 / hu.24、AAV58.2 / hu.25、AAV6.1、AAV6.1.2、AAV6.2、AAV7m8、AAV7.2、AAV7.3 / hu.7、AAV-8b、AAV8G9、AAV-8h、AAV9i1、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV9.84、AAV9.9、AAVcy .2、AAVcy.3、AAVcy.4、AAVcy.5、AAVcy.5R1、AAVcy.5R2、AAVcy.5R3、AAVcy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu. .6、AAVhu.7、AAVhu.8、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.12、AAVhu.13、AAVhu.14 / 9、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.19、AA Vhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AA Vhu.35、AAVhu.37、AAVhu.39、AAVhu.40、AAVhu.41、AAVhu.42、AAVhu.43、AAVhu.44、AAVhu.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、 AAVhu.47、AAVhu.48、AAVhu.48R1、AAVhu.48R2、AAVhu.48R3、AAVhu.49、AAVhu.51、AAVhu.52、AAVhu.53、AAVhu.54、AAVhu.55、AAVhu.56、AAVhu. 57、AAVhu.58、AAVhu.60、AAVhu.61、AAVhu.63、AAVhu.64、AAVhu.66、AAVhu.67、AAVpi.1、AAVpi.2、AAVpi.3、AAVrh.2、AAVrh.2R、AAVrh.8、AAVrh.8R、AAVrh8R R533A mutant、AAVrh8R A586R mutation、AAVrh.10、AAVrh.12、AAVrh.13、AAVrh.13R、AAVrh.14、AAVrh.17、AAVrh.18、AAVrh.19、AAVrh.20、AAVrh.21、AAVrh.22、AAVrh.23、AA Vrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37R2、AAVrh.38、AAVrh.39、AAVrh.40、AAVrh. rh.43、AAVrh.44、AAVrh.45、AAVrh.46、AAVrh.47、AAVrh.48、AAVrh.48.1、AAVrh.48.1.2、AAVrh.48.2、AAVrh.49、AAVrh.50、AAVrh.51、AAVrh.5 2、AAVrh.53、AAVrh.54、AAVrh.55、AAVrh.56、AAVrh.57、AAVrh.58、AAVrh.59、AAVrh.60、AAVrh.61、AAVrh.62、AAVrh.64、AAVrh.64、AAVrh.64、AAVrh.64 2、AAVrh.65、AAVrh.67、AAVrh.68、AAVrh.69、AAVrh.70、AAVrh.72、AAVrh.73、AAVrh.74、AAV-PHP.B、AAV-PHP.A、AAV-G2B-26、AAV-G2B-13、AAV-PHP.B TH1.1-32、AAV-TH1.1-35、AAV-PHP.B2、AAV-PHP.B3、AAV-PHP.N / PHP.B-DGT、AAV-PHP.B-EST、AAV-PHP.B-GGT、AAV-PHP.B-ATP、AAV-PHP.B-ATT T、AAV-PHP.B-DGT-T、AAV-PHP.B-GGT-T、AAV-PHP.B-SGS、AAV-PHP.B-AQP、AAV-PHP.B-QQP、AAV-PHP.B-SNP(3)、AAV-PHP.B-SNP、AAV-PHP.B-QGT 、AAV-PHP.B-NQT、AAV-PHP.B-EGS、AAV-PHP.B-SGN、AAV-PHP.B-EGT、AAV-PHP.B-DST、AAV-PHP.B-DST、AAV-PHP.B-STP、AAV-PHP.B-PQP、AAV-PHP.B-SQP、AAV-PHP.B-QLP、AAV-PHP.B-TMP、AAV-PHP.B-TTP、AAV-PHP.S / G2A12、AAV-G2A15 / G2A3、AAV-G2B4、AAV-G2B5、PHP.S、AAAV、AAV A3.3、AAV A3.4、AAV A3.5、AAV A3.7、AAV CBr-7.3、AAV CBr-7.1、AAVCBr-7.10、AAV CBr-7.2、AAV CBr-7.4、AAV CBr-7.5、AAV CBr-7.7、AAV CBr-7.8、AAV CBr-B7.3、AAVCBr-B7.4、AAV CBr-E1、AAVCBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-e5、AAVCBr-E6、AAV CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAVCHt-3、AAV CHt-6.1、AAVCHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAVCHt-6.7、AAV CHt-6.8,、AAV CHt-P1、AAV CHt-P2、AAV CHt-P5、AAVCHt-P6、AAV CHt-P8、AAV CHt-P9、AAV CKd-N4、AAV CKd-1、AAVCKd-10、AAV CKd-2、AAV CKd-3、AAV CKd-4、AAV CKd-6、AAVCKd-7、AAV CKd-8、AAV CKd-B1、AAVCKd-B2、AAV CKd-B3、AAVCKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAVCKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAVCKd-H5、AAV CKd-H6、AAV CKd-N3、AAVCKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAVCLg-F7、AAV CLg-F8、AAV CLv-M9、AAV CLv-R6、AAV CLv-1、AAV CLv1-1、AAV CLv1-10、AAVCLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV CLv1-7、AAV CLv1-8、AAVCLv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAVCLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、AAV CLv-D7、AAV CLv-D8、AAV CLv-E1、AAV CLv-K1、AAV CLv-K3、AAV CLv-K6、AAV CLv-L4、AAV CLv-L5、AAV CLv-L6、AAV CLv-M1、AAV CLv-M11、AAV CLv-M2、AAV CLv-M5、AAVCLv-M6、AAV CLv-M7、AAV CLv-M8、AAV CLv-R1、AAV CLv-R2、AAV CLv-R3、AAV CLv-R4、AAV CLv-R5、AAV CLv-R7、AAV CLv-R8、AAV CLv-R9、AAV CSp-8.10、AAV CSp-1、AAV CSp-10、AAV CSp-11、AAV CSp-2、AAV CSp-3、AAV CSp-4、AAV CSp-6、AAV CSp-7、AAV CSp-8、AAV CSp-8.2、AAV CSp-8.4、AAV CSp-8.5、AAV CSp-8.6、AAV CSp-8.7、AAV CSp-8.8、AAV CSp-8.9、AAV CSp-9、AAV-LK08、AAV-LK15、AAVShuffle 100-1、AAV Shuffle 100-2、AAV Shuffle 100-3、AAV Shuffle 100-7、AAVShuffle 10-2、AAV Shuffle 10-6、AAV Shuffle10-8、AAV SM 100-10、AAV SM 100-3、AAVSM 10-1、AAV SM 10-2、AAV SM 10-8、AAV.VR-355、AAV-b、AAVC1、AAVC2、AAVC5、AAVCh.5、AAVCh.5R1、AAV-DJ、AAV-DJ8、AAVF1 / HSC1、AAVF11 / HSC11、AAVF12 / HSC12、AAVF13 / HSC13、AAVF14 / HSC14、AAVF15 / HSC15、AAVF16 / HSC16、AAVF17 / HSC17、AAVF2 / HSC2、AAVF3、AAVF3 / HSC3、AAVF4 / HSC4、AAVF5、AAVF5 / HSC5、AAVF6 / HSC6、AAVF7 / HSC7、AAVF8 / HSC8、AAVF9 / HSC9、AAV-h、AAVH-1 / hu.1、AAVH2、AAVH-5 / hu.3、AAVH6、AAVhE1.1、AAVhEr1.14、AAVhEr1.16、AAVhEr1.18、AAVhER1.23、AAVhEr1.35、AAVhEr1.36、AAVhEr1.5、AAVhEr1.7、AAVhEr1.8、AAVhEr2.16、AAVhEr2.29、AAVhEr2.30、AAVhEr2.31、AAVhEr2.36、AAVhEr2.4、AAVhEr3.1、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVLG-9 / hu.39. AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK09, AAV-LK10, AAV-LK 11. AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAVN721-8 / rh.43, AAV-PAEC, AAV-PAEC 12. AAV-PAEC11, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, Anc80, Anc80L65, Anc81, Anc82, Anc83, Anc84, Anc94, Anc110, Anc113, Anc126, Anc127, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, goat AAV, Japanese AAV 10 serotype, UPENN AAV10, VOY101, and VOY201.

[0166] In some embodiments, “AAV vector variants” include genetically modified vectors, for example, by substitution, deletion, or addition of one or more amino acid residues in one or more capsid proteins VP1, VP2, and VP3. Examples of such variants include, but are not limited to, AAV vectors containing 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 more of their VP1, VP2, and / or VP3 capsid proteins.

[0167] Other examples of such variants include, but are not limited to, AAV1 with the Y731F mutation (or the corresponding site in other AAV serotypes); AAV2 with one or more of the following mutations: Y272F, Y444F, T491V, Y500F, S662V, and / or Y730F (or the corresponding site in other AAV serotypes), such as AAV2 with the Y444F mutation, AAV2 with the Y444F+Y500F+Y730F mutation, AAV2 with the Y272F+Y444F+Y500F+Y730F mutation, AAV2 with the Y444F+Y500F+Y730F+T491V mutation, and AAV2 with the Y272F+Y444F+Y500F+Y730F+T491V mutation; and AAV2 with the Y705F, Y731F, and / or T492V mutation (or other AAV serotypes). AAV3 having one or more of the corresponding sites in the AAV serotype; AAV5 having one or more of the Y263F and / or Y719F mutations (or the corresponding sites in other AAV serotypes); AAV6 having one or more of the Y445F, T492V, S663V, Y705F and / or Y731F mutations (or the corresponding sites in other AAV serotypes), such as AAV6 with the Y445F mutation, AAV6 with the Y705F+Y731F mutation, AAV6 with the T492V mutation, AAV6 with the Y705F+Y731F+T492V mutation, AAV6 with the S663V mutation and AAV6 with the S663V+T492V mutation; and AAV8 having one or more of the Y447F, T494V and / or Y733F mutations (or the corresponding sites in other AAV serotypes).

[0168] In some embodiments, the AAV carrier according to the present invention is selected from the group consisting of AAV2, AAV5, AAV8, and AAV9. In some embodiments, the AAV carrier according to the present invention is AAV2. In some embodiments, the AAV carrier according to the present invention is AAV5. In some embodiments, the AAV carrier according to the present invention is AAV8. In some embodiments, the AAV carrier according to the present invention is AAV9. In some embodiments, the AAV carrier according to the present invention is AAV2 / 2. In some embodiments, the AAV carrier according to the present invention is AAV2 / 5. In some embodiments, the AAV carrier according to the present invention is AAV2 / 8. In some embodiments, the AAV carrier according to the present invention is AAV2 / 9.

[0169] In some embodiments, the AAV vector according to the invention has a capsid selected from the group consisting of AAV serotypes 2, 5, 8, and 9. In some embodiments, the AAV vector according to the invention has a capsid of AAV serotype 2. In some embodiments, the AAV vector according to the invention has a capsid of AAV serotype 5. In some embodiments, the AAV vector according to the invention has a capsid of AAV serotype 8. In some embodiments, the AAV vector according to the invention has a capsid of AAV serotype 9.

[0170] In some embodiments, the AAV vector can target a variety of cells, tissues, and organs. Examples of cells targeted by the AAV vector in some embodiments include, but are not limited to, hepatocytes; retinal cells; i.e., photoreceptor cells, retinal pigment epithelium (RPE), Müller cells; muscle cells, i.e., myoblasts, satellite cells; cells of the central nervous system (CNS), i.e., neurons, glial cells; cardiac cells; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells; blood cells, such as lymphocytes, monocytes, basophils, eosinophils, neutrophils, mast cells; hematopoietic cells including hematopoietic stem cells; inner ear cells (e.g., inner hair cells and / or outer hair cells, Hensen cells, Deiters cells, column cells, phalangeal cells, limbic cells, etc.); induced pluripotent stem cells (iPS), etc. In some embodiments, examples of AAV-targetable tissues and organs include the eye, retina, liver, skeletal muscle, cardiac muscle, smooth muscle, ear, brain, spine, bone, connective tissue, heart, kidney, lung, lymph nodes, breast, myelin sheath, prostate, testis, thymus, thyroid gland, trachea, etc. In some embodiments, preferred cell types are hepatocytes, retinal cells, muscle cells, CNS cells, PNS cells, and hematopoietic cells. In some embodiments, preferred tissues and / or organs are the liver, muscle, heart, eye, and brain.

[0171] The tropism of AAVs can vary depending on their serotype. In some implementations, for example, AAV2 can be used to transduce the central nervous system (CNS), kidneys, and photoreceptor cells, while in other implementations, for example, AAV8 can effectively transduce the CNS, heart, liver, photoreceptor cells, retinal pigment epithelial cells (RPE), and skeletal muscle.

[0172] In some implementations, AAV can be generated by any method known in the art, such as transient transfection of a target cell line, for example, in HEK293 cells as described in the Examples section.

[0173] Recombinant AAV vector

[0174] In some embodiments, the AAV vector modified according to the present invention can be a recombinant AAV (rAAV) vector. Typically, wild-type (WT) AAV has a single-stranded linear DNA genome of approximately 5 kb, with two major open reading frames (ORFs) flanked by two inverted terminal repeats (ITRs). The 5' and 3' ORFs encode replication and capsid proteins, respectively. Typically, the ITR contains 145 nucleotides and serves as the origin of AAV genome replication and a packaging signal. In recombinant AAV, the viral ORF is replaced by a foreign gene expression cassette, while the replication and capsid proteins are provided in trans form. In some embodiments, the AAV vector modified according to the present invention can comprise a double-stranded, self-complementary DNA genome (scAAV) (see eg, Buie et al., Self-complementary AAV Virus (scAAV) Safe and Long-term Gene Transfer in the Trabecular Meshwork of Living Rats and Monkeys. Invest Opthalmol Vis Sci. 2010).

[0175] Therefore, as used herein, "recombinant AAV vector" or "rAAV" refers to an AAV in which a foreign nucleic acid sequence (e.g., a payload, such as a transgene) has been introduced into the viral genome. The foreign nucleic acid sequence can be of any type and is selected based on the intended use of the AAV vector. For example, the nucleic acid can contain and / or serve as a template for any RNA or DNA sequence. In some embodiments, the nucleic acid may preferably contain a DNA sequence. In some embodiments, the rAAV vector can be used as a gene vector for in vivo or in vitro applications.

[0176] For illustration, an exemplary rAAV vector modified according to the present invention may comprise a foreign gene expression cassette, which replaces the viral ORF and is positioned between two ITRs. In some embodiments, the foreign gene expression cassette may comprise a promoter sequence, a sequence encoding a target gene, and a terminator sequence. In some embodiments, the promoter and target gene are selected based on the target tissue and / or organ and known indications, such as for the treatment and / or prevention of disease states.

[0177] As other or alternative examples, in some embodiments, the rAAV vector used in the present invention may contain a DNA template for homologous recombination in cells. In some embodiments, rAAV may be used in combination with gene editing tools to promote homologous recombination in target cells in vivo, in vitro, and / or ex vivo. In some embodiments, the gene editing tools may be of any type and include, but are not limited to, CRISPR and its related systems (Cas proteins, guide RNAs), TALENs, zinc finger nucleases, a wide range of nucleases, and RNA and / or DNA encoding said proteins.

[0178] In some embodiments, the AAV vector modified according to the present invention contains at least one genetic material, selected according to the intended use of the AAV vector.

[0179] As used herein, the term "transgenic" refers to a polynucleotide introduced into a cell that is capable of being transcribed into RNA and optionally translated and / or expressed under suitable conditions. In some embodiments, the transgenic confers the properties required for its introduction into the cell, or otherwise achieves the desired therapeutic or preventative outcome. In some embodiments, the transgenic may be incorporated, wholly or partially, into the genome of the host cell, for example, through corrective gene editing using CRISPR-based methods, TALEN-based methods, ZFN-based methods, etc., in a suitable manner. In some embodiments, the transgenic may be transcribed into molecules that mediate RNA interference (i.e., gene silencing), such as miRNA, siRNA, shRNA, piRNA, etc.

[0180] In some embodiments, the at least one transgene comprises cDNA encoding a protein or a fragment thereof.

[0181] As used herein, the term "cDNA" refers to complementary DNA and corresponds to a DNA molecule typically synthesized from a single-stranded RNA (e.g., messenger RNA [mRNA] or microRNA [miRNA]) as a template in a reaction catalyzed by reverse transcriptase. Specifically, when cDNA is obtained from the reverse transcription of mRNA, it does not contain the complete gene encoding the protein, but only the coding sequence of said protein (i.e., exons without introns).

[0182] In some embodiments, the cDNA fragment may comprise a portion of the cDNA encoding the N-terminal or C-terminal portion of a protein. In some embodiments, such a fragment may be used, for example, when large cDNA cannot be easily carried by a single AAV vector, thus requiring the use of multiple vectors, such as a dual AAV vector.

[0183] In some embodiments, a fragment of cDNA may comprise a portion of the cDNA that encodes a functional and / or structural portion of a protein.

[0184] In some embodiments, the cDNA fragment may contain a sequence encoding a functional and / or structural portion of an RNA molecule. In some embodiments, such RNA molecules may be ribosomal RNA, transfer RNA, small nuclear RNA, small nucleolar RNA, microRNA, long noncoding RNA, short interfering RNA, guide RNA, and / or any type of functional RNA.

[0185] In some specific embodiments, the cDNA is derived from the GBA gene, preferably from the human GBA gene. Exemplary sequences of the GBA gene can be found in WO2022 / 096681.

[0186] In some embodiments, at least one transgene is controlled by at least one element that enhances transgene target specificity and / or expression. Examples of elements that enhance transgene target specificity and / or expression in some embodiments include, but are not limited to, promoters, post-transcriptional regulatory elements (PREs), polyadenylation (poly A) signal sequences, translational regulatory elements, targets controlled by endogenous RNA processing pathways, upstream enhancers (USEs), CMV enhancers, and introns.

[0187] In some implementations, at least one transgene is controlled by at least one promoter.

[0188] Those skilled in the art will recognize that the expression of transgenes in target cells may require specific promoters, including but not limited to species-specific, inducible, tissue-specific, time-specific, cell-specific, and / or cell cycle-specific promoters.

[0189] In some implementations, the promoter is a cell-specific promoter that is directed to the targeted cell.

[0190] In some implementations, promoter-driven transgene expression in target tissues lasts for a period of time. Promoter-driven expression can persist for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 15 days, 16 days, 17 days, 18 days, 19 days, etc. 20 days, 3 weeks, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years or more.

[0191] In some implementations, the promoter is a weak promoter for sustained transgene expression.

[0192] In some implementations, the promoter may be naturally occurring or non-naturally occurring. In some implementations, examples of promoters include, but are not limited to, viral promoters, plant promoters, and animal promoters (e.g., mammalian promoters). In some implementations, the promoter may be a human promoter.

[0193] In some implementations, the promoter may be truncated relative to a reference. In some implementations, the promoter may be mutated relative to a reference.

[0194] In some embodiments, the promoter may be a promoter that drives expression in multiple tissues. In some embodiments, such promoters that drive or promote expression in multiple tissues include, but are not limited to, human elongation factor 1a subunit (EF1a), cytomegalovirus (CMV) immediate early enhancer and / or promoter, chicken β-actin (CBA) and its derivative CAG, β-glucuronidase (GUSB), and ubiquitin C (UBC).

[0195] In some implementations, tissue or cell-specific expression elements can be used to restrict the expression of transgenes to certain cell types.

[0196] In some embodiments, tissue and / or cell-specific promoters may be neuron-specific promoters. Suitable examples of tissue or cell-specific expression elements suitable for neurons include, but are not limited to, neuron-specific enolase (NSE) promoters, platelet-derived growth factor (PDGF) promoters, platelet-derived growth factor B chain (PDGF-β) promoters, synaptic protein (Syn) promoters, myelin basic protein (MBP) promoters, methyl-CpG-binding protein 2 (MeCP2) promoters, and Ca2+ promoters. 2+ / Calmodulin-dependent protein kinase II (CaMKII) promoter, metabolotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin small gene ηβ2 promoter, proenkephalinogen (PPE) promoter, enkephalin (Enk) promoter, and excitatory amino acid transporter 2 (EAAT2) promoter.

[0197] In some embodiments, the promoter is a ubiquitous promoter. In some embodiments, the ubiquitous promoter may include, but is not limited to, CMV, CBA (including its derivatives CAG, CBh, etc.), EF-1a, PGK, UBC, GUSB (hGBp), and UCOE.

[0198] In some implementations, the promoter is not tissue- or cell-specific.

[0199] In some implementations, the promoter is an engineered promoter.

[0200] In some implementations, the promoter is the promoter of a naturally expressed protein.

[0201] In some preferred embodiments, the promoter is a CAG promoter (e.g., containing the CMV immediate early enhancer and the chicken β-actin promoter).

[0202] Coupled sites on AAV vectors

[0203] In some embodiments of the invention, the provided AAV vector is an AAV vector modified by covalently coupling at least one squaric acid ester to at least one amino acid of the AAV capsid (e.g., to at least one capsid protein of the AAV vector). A typical AAV capsid comprises three capsid proteins, named VP1, VP2, and VP3. In some embodiments, at least one squaric acid ester is covalently bound to at least one VP1 protein of the AAV vector. In some embodiments, at least one squaric acid ester is covalently bound to at least one VP2 protein of the AAV vector. In some embodiments, at least one squaric acid ester moiety is covalently bound to at least one VP3 protein of the AAV vector.

[0204] In some embodiments, the AAV vector is modified by covalently coupling at least one squaric acid ester to at least one surface-exposed amino acid residue of at least one capsid protein of the AAV vector.

[0205] As used herein, the term “surface exposure” refers to amino acid residues whose side chains are at least partially exposed on the outer surface of the AAV vector.

[0206] In some embodiments, at least one squaric acid ester is covalently bonded to at least one amino group of an amino acid residue exposed on the surface of the AAV carrier capsid. In particular, the at least one squaric acid ester is a compound of formula (III) or a specific embodiment thereof, selected from the group consisting of compounds of formulas (IIIa), (IIIa1), (IIIa2), (IIIb), and (IIIc) as defined and described in the categories and subclasses of the present invention.

[0207] "Amino" herein refers to a primary amine group (-NH2) or a secondary amine group (-NH-), or a salt thereof; preferably, the amino group is a primary amino group. In some preferred embodiments, the amino group is derived from a lysine residue, preferably from lysine residues exposed on the surface of the capsid of the AAV carrier.

[0208] As used herein, “at least one amino group of an amino acid residue of a 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 an amino acid residue.

[0209] In some embodiments, the AAV vector of the present invention contains multiple (e.g., several) modified amino acid residues in its capsid. In some embodiments, multiple (e.g., several) amino acid residues of the same capsid protein are modified. In some embodiments, multiple (e.g., several) amino acid residues of different capsid proteins are modified.

[0210] In some embodiments, the present invention relates to adeno-associated virus (AAV) vector particles comprising the portion of formula (II):

[0211]

[0212] in

[0213] N* is the nitrogen atom of the primary amino group of the amino acid residues exposed on the surface of the AAV carrier capsid polypeptide;

[0214] ---- indicates the connection point with the AAV carrier capsid; and

[0215] R L-NH- is a functional portion comprising or consisting of a cell type-specific ligand, said cell type-specific ligand being selected from the group consisting of: carbohydrates, hormones, peptides, glycosylated peptides, proteins, glycoproteins or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins and pharmaceutical portions.

[0216] In some implementations, the functional portion R L -NH- contains a Z group and one or more spacer groups L, wherein

[0217] Z is H or contains or is composed of cell-type-specific ligands selected from the group consisting of: carbohydrates, hormones, peptides, glycosylated peptides, proteins, glycoproteins or their functionally active fragments, membrane receptors or their functionally active fragments, antibodies or their functionally active fragments, spiegelmers, nucleic acids or peptide aptamers, vitamins and pharmaceutical portions; preferably, Z is or contains a peptide or a carbohydrate. In some embodiments, the peptide is or contains a linear or cyclic peptide, wherein the peptide may be a biologically active peptide. In particular, the peptide is a blood-brain barrier (BBB) ​​shuttle peptide with enhanced transduction activity across the blood-brain barrier. In some preferred aspects, the BBB shuttle peptide is selected from the group consisting of: peptide THR or peptides having an RGD motif, including cyclic RGD peptides. In some embodiments, the sugar is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and their derivatives, more preferably from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, deoxyglycosamine, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose and P1-fructose, more preferably from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid and P1-fructose, more preferably mannose; and

[0218] L contains or consists of one or more groups selected from the group consisting of: alkyl ether groups, such as polyethylene glycol (PEG) or polypropylene glycol (PPG), preferably polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers; branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyol; arylene or heteroarylene Ar; preferably, Ar is phenylene or pyridylene, optionally containing one or more substituents selected from the group consisting of: halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkoxy; C 1-6 Alkylene; C 1-6 alkyleneamines; C1-6 Acyl groups; amino acid moieties, preferably arginine or β-alanine moieties; polyamides, such as β-alanine polymers, preferably containing 1-40 β-alanine monomers; vinyl polymers, such as pHPMA; polyesters, such as PLGA; and polymers of alkylene diamines; and combinations thereof.

[0219] In some specific embodiments, L comprises one or more polyethylene glycol (PEG) monomers containing 1-40 ethylene glycol monomers; C 1-6 Alkylene; C 1-6 alkyleneamines; C 1-6 Acyl group; β-alanine polymers containing 1-40 β-alanine monomers; branched C 3-12 Polyethers of polyols; amino acid moiety, and arylene or heteroarylene Ar.

[0220] In some respects, the PEG and Ar groups are covalently linked via the amide moiety -N(R1)C(O)- or its bioisosteric moiety, wherein R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, even more preferably R1 is H.

[0221] In some respects, L contains polyethylene glycol (PEG), which comprises 1-40 ethylene glycol monomers and one or more C atoms. 1-6 Alkyl groups, wherein the PEG and C 1-6 Alkyl groups are covalently linked via the amide moiety or its bioelectron isosteric moiety.

[0222] In some aspects, L comprises polyethylene glycol (PEG), which contains 1-40 ethylene glycol monomers and arylene or heteroarylene groups Ar, preferably wherein the PEG and Ar groups are covalently linked through an amide moiety or its bioelectron isosteric moiety.

[0223] In some respects, L comprises polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, β-alanine polymers containing 1-40 β-alanine monomers, and C 1-6Acyl group. In other respects, L comprises a β-alanine polymer containing 1-40 β-alanine monomers, and C... 1-6 Alkylamine group. In other respects, L comprises a β-alanine polymer containing 1-40 β-alanine monomers and an arylene or heteroarylene Ar.

[0224] In other respects, L comprises polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and one or more C... 1-6 Alkyl groups, wherein the PEG and C 1-6 Alkyl groups are covalently linked via ether bonds.

[0225] In other respects, L contains polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and one or more C 1-6 Alkylene, arylene or heteroarylene Ar, and branched C 3-12 Polyethers of polyols, in which PEG and C 1-6 Alkyl groups are covalently linked through their amide moiety or bioelectronic isosteric moiety; wherein the branched C 3-12 Polyol polyethers and C 1-6 Alkyl groups are covalently linked via ether bonds; and the branched C-chains are... 3-12 The polyether and Ar groups of polyols are covalently linked through the amide moiety or its bioelectron isosteric moiety.

[0226] In some embodiments, the one or more spacer groups L are selected from the group consisting of L1, L2, and L3, wherein L1 is one or more groups selected from the group consisting of polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, or branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyols, and β-alanine polymers comprising 1-40 β-alanine monomers, or mixtures thereof; L2 comprises one or more arylene or heteroarylene Ar, preferably L2 comprises phenylene or pyridinium; and L3 is one or more groups selected from the group consisting of: an amino acid moiety, preferably an arginine moiety, β-alanine; C 1-6 alkyleneamine group; C 1-6 alkylene acyl groups and C 1-6 Alkylene; the C 1-6 Alkylene is a straight-chain C 1-6 Alkyl or branched C 3-6 Alkylene.

[0227] In some embodiments, portions of formula (II) are selected from the group consisting of formulas (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), (IIh), (IIj), (IIk), (IIm), (IIIn), (IIp), (IIq), (IIr), (IIs), (IIt), (IIv), (IIw), and (IIx):

[0228]

[0229]

[0230]

[0231]

[0232] Where R a R b and R c Each is independently H or group R':

[0233]

[0234] R a R b and R c At least one of them is a group R';

[0235] n and n' are each independently selected from 1 to 40, and preferably n and n' are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0236] m1 and m2 are each independently selected from 0, 1 or 2;

[0237] m3, m4, m5 and m6 are each independently selected from 1 to 12, preferably 1 to 6, and more preferably 1 or 2;

[0238] N* is the nitrogen atom of the amino group of the amino acid residue in the AAV carrier capsid;

[0239] Indicates the connection point with the AAV carrier capsid;

[0240] Z is or contains linear or cyclic peptides or sugars, wherein the peptides may be bioactive peptides, such as blood-brain barrier (BBB) ​​shuttle peptides, preferably selected from the group consisting of peptides THR or peptides having RGD motifs, including cyclic RGD peptides, and the sugars are preferably selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides and their derivatives, preferably sugars selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, deoxyglycosamine, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose and P1-fructose, preferably selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid and P1-fructose, more preferably mannose;

[0241] Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group comprising one or more heteroatoms selected from the group consisting of N, O, S, and Se, preferably phenylene or pyridinium, optionally comprising one or more substituents selected from the group consisting of halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 alkoxy group; and R1 is selected from the following group: H, C 1-6 Alkyl, C 1-6 Haloalkyl, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably, R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 The alkyl haloide and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H.

[0242] General description and definition

[0243] The term "alkyl" refers to a monovalent or divalent, straight-chain or branched saturated hydrocarbon chain (also known as (C1-C8)alkyl) containing 1-8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, tert-butylmethyl, n-pentyl, n-hexyl, n-heptyl, or n-octyl. The term "alkylene" corresponds to a divalent group obtained by removing a hydrogen atom from an alkyl group as defined above herein, resulting in a moiety with two connection points.

[0244] The term "acyl" refers to a -C(O)R group, where R is an alkyl or phenyl group as previously defined. Acyl groups include, for example, acetyl, ethyl carbonyl, or benzoyl.

[0245] The term "alkoxy" or "alkyloxy" refers to -O-alkyl, where Alk is an alkyl group as defined above. Aloxy groups include, for example, methoxy, ethoxy, n-propoxy, or tert-butoxy.

[0246] "Aryl" herein refers to an aromatic monocyclic (i.e., phenyl) or bicyclic (i.e., phenyl) system containing 4-12 carbon atoms, preferably 6-10 carbon atoms. It should be understood that in the case of a bicyclic system, one ring is aromatic and the other ring is aromatic or unsaturated. Aryl groups include, for example, phenyl, naphthyl, indenyl, or benzocyclobutenyl, optionally substituted with one or more groups, said groups optionally containing one or more substituents selected from the group consisting of halogens, C... 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkoxy. The preferred aryl group used herein is phenyl. The term "arylene" corresponds to a divalent group obtained by removing a hydrogen atom from an aryl group as defined above herein, yielding a portion having two connection points. The preferred arylene group used herein is a phenylene group optionally substituted with one or more substituents selected from the group consisting of halogens, C... 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkyl group.

[0247] "Heteroaryl" herein refers to an aromatic ring or ring system having 5-12 carbon atoms, the aromatic ring or ring system comprising 1-2 fused or covalently linked rings, typically each ring comprising 5-6 atoms; at least one of which is an aromatic ring, and one or more carbon atoms in one or more of the rings are substituted with oxygen, nitrogen, sulfur, or selenium atoms, wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatomium may optionally be quaternized. Such rings may be fused with aryl rings. Non-limiting examples of such heteroaryl groups include: triazolyl, pyrroleyl, furanyl, thiopheneyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiazolyl, tetrazolyl, oxtriazolyl, thiazolyl, pyridinyl, pyrazinyl, pyrazinyl, pyrazinyl, dioxinyl, thiazolyl, triazinyl, imidazo[2,1-b][1,3]thiazolyl. Thiophene[3,2-b]furanyl, thieneno[3,2-b]thienyl, thieneno[2,3-d][1,3]thiazolyl, thieneno[2,3-d]imidazolyl, tetrazol[1,5-a]pyridyl, indoleyl, indoleazinyl, isoindoleyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, indazoleyl, benzimidazolyl, 1,3-benzene benzoxazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1,3-benzothiazolyl, 1,2-benzisoxiazolyl, 2,1-benzisoxiazolyl, benzotriazolyl, 1,2,3-benzoxadiazolyl, 2,1,3-benzoxadiazolyl, 1,2,3-benzothiadiazolyl, 2,1,3-benzothiadiazolyl, thienylpyridinyl, purine, imidazo[ 1,2-a]pyridinyl, 6-oxopyridazin-1(6H)-yl, 2-oxopyridin-1(2H)-yl, 6-oxopyridazin-1(6H)-yl, 2-oxopyridin-1(2H)-yl, 1,3-benzodioxanepentenyl, quinolinyl, isoquinolinyl, cenolinyl, quinazolinyl, quinoxalinyl, optionally substituted by one or more groups selected from the group consisting of: halogen, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkoxy. The preferred heteroaryl group used herein is pyridyl. The term "heteroaryl" corresponds to a divalent group obtained by removing a hydrogen atom from a heteroaryl group as defined above herein to obtain a portion having two connection sites. The preferred heteroaryl group used herein is pyridylene, optionally substituted with one or more groups selected from the group consisting of: halogens, C... 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkyl group.

[0248] The term "heteroatom" refers to one or more of oxygen, sulfur, nitrogen, phosphorus, selenium, or silicon (including any oxidized form of nitrogen, sulfur, phosphorus, selenium, or silicon; any quaternized form of basic nitrogen; or a substituted nitrogen of a heterocycle, such as N (e.g., in 3,4-dihydro-2H-pyrrole), NH (e.g., in pyrrolealkyl), or NR). + (e.g., in N-substituted pyrroleyl groups).

[0249] As used in this article, the term "unsaturated" refers to a portion having one or more unsaturated units.

[0250] The term "halogen" refers to F, Cl, Br, or I.

[0251] The term "arylalkyl" refers to the -Alk-Ar group, where Alk represents the alkyl group as defined above, and Ar represents the aryl group as defined above.

[0252] The term "heteroalkyl" refers to a straight-chain or branched saturated hydrocarbon chain containing 1-5 carbon atoms and at least 1 or 2 heteroatoms, such as sulfur, nitrogen, or oxygen atoms, particularly alkoxy, alkylamine, dialkylamine, thioether, and other similar groups. Heteroalkyl groups include, for example, -O(CH2)nOCH3, -(CH2)nOCH3, -N(CH2)nN(CH2CH3)2, -N(CH2CH3)2, or -(CH2)nS-(CH2)n-CH3, where n is selected from 1-4, etc.

[0253] The term "cycloalkyl" refers to a saturated monocyclic or polycyclic system, such as a fused or bridged bicyclic system, containing 3-12 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, naphthyl, or norbornyl.

[0254] The term "haloalkyl" refers to a straight or branched saturated hydrocarbon chain containing 1-6 carbon atoms and substituted by one or more, particularly 1-6, halogen atoms, such as trifluoromethyl or 2,2,2-trifluoroethyl.

[0255] The term "OR" a "" refers to the fact that the R group can be an alkyl, aryl, haloalkyl, or arylalkyl group as defined above, connected to the rest of the molecule by an oxygen atom. O-cycloalkyl includes, for example, O-cyclopentyl or O-cyclohexyl.

[0256] As described herein, compounds may contain an "optionally substituted" moiety. Generally, the term "substituted," regardless of whether it is preceded by the term "optionally," means that one or more hydrogen atoms in the specified moiety of the compound are replaced by suitable substituents. "Substituent" applies to one or more hydrogen atoms explicitly or implicitly present in the structure (e.g., It means at least as well as It means at least Unless otherwise specified, the "optionally substituted" group may have suitable substituents at each substituted position, and the substituents at each position may be the same or different when more than one position in any given structure may be substituted by more than one substituent selected from a particular group. The combinations of substituents contemplated in this disclosure are preferably combinations capable of forming stable or chemically viable compounds. As used herein, the term "stable" means that a compound does not undergo substantial change when subjected to conditions that allow it to be generated, detected, and, in some embodiments, recovered, purified, and used for one or more purposes disclosed herein.

[0257] The suitable substituent on the substituted carbon atom of the "optionally substituted" group is independently a halogen; alkyl, acyl, aryl, heteroaryl, arylalkyl, heteroalkyl, cycloalkyl, alkoxy, haloalkyl, haloalkoxy, or OR group. a , where R a And each substituent is as defined above in this paper.

[0258] When the terms “part of formula (II)” and “compound of formula (III)” are used, the terms also include pharmaceutically acceptable salts that the part and the compound may form. As used herein, the term “pharmaceutically acceptable salt” includes conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases, as well as quaternary ammonium salts. More specific examples of suitable acid salts include hydrochlorides, hydrobroms, sulfates, phosphates, nitrates, perchlorates, fumarates, acetates, propionates, succinates, glycolates, formates, lactates, maleates, tartrates, citrates, palmitates, malonates, hydroxymaleates, phenylacetates, glutamates, benzoates, salicylates, fumarates, toluenesulfonates, methanesulfonates, naphthalene-2-sulfonates, benzenesulfonates, hydroxynaphthoates, hydroiodates, malates, stearates, tannates, etc. More specific examples of suitable basic salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucosamine, and procaine. For example, preferred salt forms include sodium salts of compounds of formula (III) disclosed within the scope of this specification.

[0259] Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in detail by SMBerge et al. in J. Pharmaceutical Sciences, 1977, 66, 1–19, which are incorporated herein by reference. For the purposes of this specification, pharmaceutically acceptable salts also include zwitterionic pharmaceutical forms.

[0260] Many organic compounds can form complexes with solvents, react therein, or precipitate or crystallize therefrom. These complexes are called "solvents." For example, complexes with water are called "hydrates." Solvents of compounds of formula (III) are within the scope of this invention.

[0261] The term "isomer" refers to the compounds of the present invention having the same molecular formula as described herein, but differing in properties, atomic bonding sequence, or spatial arrangement of atoms. Isomers with different atomic spatial arrangements are called "stereoisomers." Stereoisomers that are not mirror images of each other are called "diastereomers," and stereoisomers that are non-superimposed mirror images of each other are called "enantiomers" or "optical isomers." "Stereoisomer" refers to racemic, enantiomer, and diastereomer. A pair of diastereomers is called an epimer. Unless otherwise stated, the structures described herein also include all isomeric forms of that structure (e.g., enantiomers, diastereomers, and geometric (or conformations)); for example, R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention, as well as mixtures of enantiomers, diastereomers, and geometric (or conformations), are within the scope of this disclosure. Unless otherwise stated, all tautomerizations are within the scope of this disclosure.

[0262] The term "anomer" refers to a cyclic monosaccharide that is an epimer. If the monosaccharide is an aldose, its C-1 carbon atom configuration is different; if the monosaccharide is a ketose, its C-2 carbon atom configuration is different. The C-1 or C-2 carbon atom is referred to as the "anomer carbon".

[0263] The term "bioisosterone" when referring to a specific group or part, particularly an amide group in the embodiments and aspects defined in this invention, refers to other possible groups or parts that are equivalent in electronic and spatial arrangement to the specific group, meaning that bioisosterone groups share some common biological characteristics in addition to their physicochemical similarity.

[0264] Additionally, unless otherwise stated, this disclosure also includes compounds distinguished only by the presence of one or more isotopically enriched atoms. For example, compounds having the structure of this invention include those with hydrogen replaced by deuterium or tritium, or carbon replaced by... 13 C- or 14 C-enriched carbon substitutions are all within the scope of this disclosure. Such compounds can be used, for example, as analytical tools, probes in bioassays, or therapeutic agents according to this disclosure. In some embodiments, the compounds of this disclosure contain one or more deuterium atoms.

[0265] The combinations of substituents and variables contemplated in this disclosure are limited to those capable of forming stable compounds. As used herein, the term "stable" means that a compound has sufficient stability to be manufactured and to maintain its integrity for a sufficiently long period of time for use in the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject).

[0266] Functional part R L -NH-

[0267] Functional part R L -NH- can be of any type and is usually selected based on the biological effects sought when chemically modifying the AAV carrier capsid.

[0268] In some implementations, R L -NH- contains cell type-specific ligands, labelers, space shielding agents, pharmaceutical moieties, or combinations thereof. In some embodiments, the functional portion R L -NH- may also contain (nano)particles, including magnetic (nano)particles and quantum dots. For example, in some embodiments, R L -NH- can contain iron, dyes, silicon, gold, or carbon (nano) particles.

[0269] In some implementations, R L -NH- is a functional moiety containing, or composed of, the following: labeling agents, such as fluorescent dyes such as fluorescein, fluoroalanine, rhodamine, boron-dipyrrolemethylene. Dyes and Alexa Or radioactive nuclides.

[0270] In some implementations, R L -NH- is a functional part containing or composed of a steric shielding agent, such as an agent capable of shielding certain epitopes of the capsid, thereby preventing neutralizing antibody binding. For example, in some embodiments, R L -NH- may contain polyethylene glycol (PEG), pHPMA, peptides, or polysaccharides. In some embodiments, R L -NH- contains polyethylene glycol (PEG), which contains 1-40 ethylene glycol monomers, for example 1-10 ethylene glycol monomers, such as -(OCH2CH2)- (referred to herein as "PEG1"), -(OCH2CH2)2- (referred to herein as "PEG2"), -(OCH2CH2)3- (referred to herein as "PEG3"), -(OCH2CH2)4- (referred to herein as "PEG4"), or -(OCH2CH2)5- (referred to herein as "PEG5"). In some particularly preferred embodiments, R L-NH- represents a functional part comprising a sugar or peptide or composed thereof, as defined in the claims and embodiments of this specification.

[0271] In some embodiments, the functional portion R L -NH- contains a Z group and optionally one or more spacer groups L. In other embodiments, the functional portion R L -NH- consists of the group Z-NH- and does not contain one or more spacer groups L.

[0272] Group Z

[0273] In some embodiments, Z is a functional portion that contains or is composed of cell type-specific ligands, i.e., ligands capable of targeting a specific type of cell. In some embodiments, such ligands can modify the tropism of the AAV vector, i.e., its ability to selectively infect and / or transduce specific cell lines, tissues, and / or organs. For example, in some embodiments, Z may contain or be composed of ligands that specifically bind to membrane bioentities (e.g., membrane receptors) of target cells. In some embodiments, such ligands may be, for example, sugars, hormones (including steroid hormones), peptides (e.g., peptides with RGD motifs), vascular peptide-2 or muscle-targeting peptides, proteins or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, CB1 and CB2 ligands, antibodies (including heavy chain antibodies) or functionally active fragments thereof (e.g., Fab, Fab', and VHH), scFv, biantibodies, spiegelmers, aptamers (including nucleic acid aptamers and peptide aptamers), small chemical molecules known to bind target bioentities (such as vitamins and drugs), and / or any suitable combination thereof.

[0274] As used herein, the term "cell type-specific ligand" refers to a compound (chemical or biological) that mediates the specific binding and transduction of a target cell type, and is therefore used to selectively deliver transgenes to specific cell types in vivo, thereby increasing the number of AAV carrier particles reaching target cells and tissues and reducing adverse reactions in non-target cells and tissues. Cell type-specific ligands are drugs that exhibit high affinity for target cells but little affinity for non-target cells. For example, target cells are specific tissue types, and cell type-specific ligands specifically bind to marker proteins, surface antigens, and receptor proteins expressed by target tissue cells.

[0275] "Functionally active fragments" refer to fragments of proteins, membrane receptors, or antibodies that retain the functional activity of their full-length counterparts.

[0276] In some implementations, Z comprises or is composed of cell-type-specific ligands derived from sugars. Details regarding sugars are provided below.

[0277] In some embodiments, Z comprises or is composed of cell type-specific ligands derived from proteins such as transferrin, epidermal growth factor (EGF), and basic fibroblast growth factor (FGF).

[0278] In some embodiments, Z comprises or consists of cell-type-specific ligands derived from vitamins, such as folic acid.

[0279] In some embodiments, Z is or comprises a linear or cyclic peptide, wherein the peptide may be a biologically active peptide. For example, the peptide may be a peptide that targets a transmembrane receptor, which may or may not be associated with cellular transcytosis mechanisms, thereby enabling it to cross natural barriers such as the blood-brain barrier (BBB). In particular, the peptide is a blood-brain barrier (BBB) ​​shuttle peptide (or BBB-penetrating peptide) with enhanced transduction activity across the blood-brain barrier. In some preferred aspects, the BBB shuttle peptide is selected from the group consisting of peptide THR or peptides having an RGD motif, including cyclic RGD peptides. In particular, peptide THR targets the transferrin receptor TfR1, and RGD-based peptides target integrin subclasses. The expression of TfR1 and integrins in various tissues is key to targeting the CNS (via the BBB) and muscle tissue. In a preferred embodiment, Z comprises peptide THR (SEQ ID No.: 1).

[0280] In some embodiments, Z comprises or is composed of a cell type-specific ligand derived from or consisting of a muscle-targeting peptide (MTP). In some embodiments, Z is a cancer cell-targeting peptide and comprises peptides, such as peptides having an RGD motif, including cyclic RGD peptides.

[0281] In some embodiments, Z comprises or is composed of cell type-specific ligands derived from small molecules or hormones, such as naproxen, ibuprofen, cholesterol, progesterone, or estradiol.

[0282] In some embodiments, Z comprises an antibody or its antigen-binding portion. In some such embodiments, the antibody may be or comprise, for example, a single-chain antibody or a variable domain, such as a camel antibody, a heavy-chain antibody, a nanobody, a shark antibody, etc. In some embodiments, the antibody or its antigen-binding portion may be or comprise Fab, Fab', VHH, scFv, a biantibody, etc. In some specific embodiments, the antibody or its antigen-binding portion is characterized by having specific affinity for specific cell-specific proteins, membrane proteins, and / or membrane protein receptors.

[0283] In some embodiments, Z comprises or consists of a cell type-specific ligand selected from the group consisting of carbohydrates, hormones, peptides, glycosylated peptides, proteins, glycoproteins or fragments thereof, membrane receptors or fragments thereof, antibodies or fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins and pharmaceutical portions.

[0284] In one specific embodiment, Z comprises or consists of sugars selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides; preferably, the sugar is a monosaccharide, wherein the monosaccharide is preferably selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, deoxyglycosamine, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose, and P1-fructose, more preferably selected from the group consisting of mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid, and P1-fructose, and even more preferably mannose.

[0285] In some specific embodiments, suitable examples of sugar include, but are not limited to, monosaccharides, oligosaccharides, polysaccharides and their derivatives; or peptide-substituted sugars.

[0286] As used herein, the term "derivative" when referring to a monosaccharide, oligosaccharide, or polysaccharide means a sugar that includes one or more non-hydroxyl groups. Examples of such non-hydroxyl groups include, but are not limited to, hydrogen, alkyl, amino (such as NH2, alkylamino, dialkylamino), N-acetamido, and / or thiol groups.

[0287] In some embodiments, the non-hydroxyl group is a negatively charged group, such as phosphate, phosphonate, sulfate, sulfonate and carboxyl groups.

[0288] Monosaccharides, also known as simple sugars, are the simplest form of sugar and the most basic unit of carbohydrates. Monosaccharides can be classified according to the number of carbon atoms they contain, such as 3 (triose), 4 (tetraose), 5 (pentose), 6 (hexose), 7 (heptaose), etc.

[0289] Examples of monosaccharides include, but are not limited to, glycolaldehyde, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythulose, arabinose, lysose, ribose, xylose, ribulose, xylulose, allose, azoose, galactose, glucose, gulose, idulose, mannose, tarose, fructose, allulose, sorbose, tagatose, mannoheptulose, and sedoheptulose.

[0290] Deoxymonosaccharides are common derivatives of the monosaccharides included in this invention, namely monosaccharides in which the hydroxyl group is replaced by a hydrogen atom.

[0291] Examples of deoxymonosaccharides include, but are not limited to, deoxyribose, fucose, fucose, rhamnose, quinoose, and pneumose.

[0292] 2-Amino-2-deoxymonosaccharides are also common derivatives of the monosaccharides included in this invention, namely monosaccharides in which the hydroxyl group is replaced by an amino group.

[0293] Examples of 2-amino-2-deoxymonosaccharides include, but are not limited to, glucosamine, galactosamine and glucosamine, and their acetylated forms, including, but not limited to, N-acetylglucosamine and N-acetylglucosamine.

[0294] In some embodiments, the monosaccharide contains negatively charged groups, such as phosphate ester groups, sulfate ester groups, or carboxylic ester groups.

[0295] Examples of monosaccharides containing phosphate groups include, but are not limited to, glucose-6-phosphate, mannose-6-phosphate, and fructose-1-phosphate.

[0296] Examples of monosaccharides containing sulfate groups include, but are not limited to, galactose-6-sulfate (S6-galactose) and N-acetylgalactosamine-6-sulfate (S6-N-acetylgalactosamine).

[0297] Examples of monosaccharides containing a carboxyl group include, but are not limited to, glucuronic acid and sialic acid.

[0298] It should be understood that the monosaccharides and their derivatives mentioned in this article include both acyclic (open-chain) and cyclic forms.

[0299] It should also be understood that the monosaccharides and their derivatives mentioned herein also include D-stereoisomers and L-stereoisomers, as well as mixtures of D- and L-stereoisomers (e.g., racemic mixtures).

[0300] It should also be understood that the monosaccharides and their derivatives mentioned in this article also include α-terminal isomers and β-terminal isomers, as well as racemic mixtures of α- and β-terminal isomers.

[0301] Oligosaccharides are carbohydrate polymers that contain a small number (usually 2-10) of monosaccharides.

[0302] In some embodiments, the oligosaccharides according to the invention comprise at least two, three, four, five, six, seven, eight, nine, or ten monosaccharides, such as those selected from the monosaccharides disclosed above, including their derivatives.

[0303] In some embodiments, such oligosaccharides can be homooligosaccharides (i.e., composed of units of the same monosaccharide, including its derivatives) or heterooligosaccharides (i.e., composed of units of at least two different monosaccharides, including their derivatives).

[0304] In some implementations, examples of oligosaccharides include, but are not limited to, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, heptasaccharides, octasaccharides, nonasaccharides, and decasaccharides.

[0305] In some embodiments, specific examples of disaccharides include, but are not limited to, cellobiose, chitobiose, gentiobiose, 6-O-β-D-glucopyranosyl-D-fructose, isomaltose, kosherbose, lactose, lactulose, laminarinose, maltose, maltoluose, mannose, melibiose, melibiose, aspergillus niger, paraginose, rutinose, rutinose, sophorose, sucrose, trehalose, pinebiose, and xylobiose.

[0306] In some implementations, specific examples of trisaccharides include, but are not limited to, fructotriose, maltotriose, maltotriulose, pinotriose, aspergillus trisaccharide, and raffinose.

[0307] In some implementations, specific examples of tetrasaccharides include, but are not limited to, succinate, maltodextrose, aspergillus niger tetrasaccharide, fructose tetrasaccharide, sesame sugar, and stachyose.

[0308] In some implementations, specific examples of oligosaccharides include, but are not limited to, acarbose, fructooligosaccharides, galactooligosaccharides, isomaltooligosaccharides, and maltodextrin.

[0309] In some embodiments, oligosaccharides can be multi-tentacle structures, wherein some or all of the monosaccharides in the oligosaccharide are not linked to each other by O-glycosidic bonds, but by branched linker structures. An example of a multi-tentacle sugar is triantennary N-acetylgalactosamine, which is a ligand for the desialyl glycoprotein receptor ASGPR (see, for example, Zhou et al., Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins; ACS Cent. Sci. 2021).

[0310] "Polysaccharides" are sugar polymers containing a large number (usually more than 10) of monosaccharides. They range in structure from linear to highly branched.

[0311] In some embodiments, the polysaccharide comprises more than 10 monosaccharides (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more), such as those selected from the monosaccharides disclosed above, including their derivatives. In a similar manner to the oligosaccharides described above, the polysaccharide can be a homopolysaccharide or a heteropolysaccharide.

[0312] In some embodiments, examples of polysaccharides include, but are not limited to, β-glucan, lentinan, cizoran, yeast polysaccharide, cellulose, hemicellulose, chitin, chitosan, dextrin, glucan, fructan, inulin, galactan, glucan, glycogen, fructan β2→6, lignin, mannan, pectin, starch, amylopectin, amylose, and xanthan gum.

[0313] In some embodiments, the sugar or its derivative according to the invention is a monosaccharide, preferably a hexose. In some embodiments, the preferred sugar or its derivative according to the invention is mannose, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, S6-galactose, S6-N-acetylgalactosamine, glucuronic acid, P6-galactose, or P1-galactose. In some embodiments, the preferred sugar or its derivative according to the invention is mannose, galactose, N-acetylglucosamine, or N-acetylgalactosamine.

[0314] In some embodiments, the sugar or a derivative thereof is mannose. In some embodiments, the sugar or a derivative thereof is galactose. In some embodiments, the sugar or a derivative thereof is N-acetylglucosamine. In some embodiments, the sugar or a derivative thereof is N-acetylglucosamine.

[0315] In some embodiments, the sugar or its derivative according to the invention is a deoxymonosaccharide. In some preferred embodiments, the deoxymonosaccharide is preferably fucose.

[0316] In some embodiments, the sugar or its derivative is a non-hydroxy sugar containing a dialkylamino group. In some preferred embodiments, the non-hydroxy sugar containing a dialkylamino group is a deoxyglycosamine.

[0317] In some embodiments, the sugar or its derivative is a non-hydroxy sugar containing a sulfate group. In some preferred embodiments, the non-hydroxy sugar containing a sulfate group is S6-galactose or S6-N-acetylgalactosamine.

[0318] In some embodiments, the sugar or its derivative is a non-hydroxy sugar containing a phosphate ester group. In some preferred embodiments, the non-hydroxy sugar containing a phosphate ester group is P6-glucose, P6-mannose, or P1-fructose.

[0319] In some embodiments, the sugar or its derivative is a non-hydroxy sugar containing a carboxyl group. In some preferred embodiments, the non-hydroxy sugar containing a carboxyl group is glucuronic acid or sialic acid.

[0320] In some embodiments, the sugar is selected from or composed of the group consisting of: mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, deoxyglucosamine, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose, and P1-fructose. In some preferred embodiments, the sugar is selected from the group consisting of: mannose, fructose, glucose, xylose, trehalose, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid, and P1-fructose, more preferably mannose.

[0321] spacer base L

[0322] In some implementations, the functional portion R L -NH- contains a group Z and at least one spacer group L. In particular, in some embodiments, one or more spacer groups L are present for attaching the group Z to the square amide connector of formula (I).

[0323] In some respects, the functional part R L -NH- contains one or more Z groups and one or more spacer groups L. In some respects, the functional moiety R L -NH- contains 1-3 groups Z, each of which is attached to one or more spacer groups L, which are used to attach each of the groups Z to the square amide connector of formula (I).

[0324] In some implementations, L can be any chemical chain that may contain heteroatoms and cyclic moieties, such as aryl and / or heteroaryl.

[0325] In some implementations, L may contain up to 1000 carbon atoms, or even more. The length and chemical properties of L can be optimized based on the groups Z to be coupled to the AAV support as needed and the desired biological effects.

[0326] In some embodiments, L is a chemical chain group containing 2-1000 carbon atoms, preferably 2-500 carbon atoms, 2-300 carbon atoms, such as 2-100 carbon atoms, 2-40 carbon atoms, 4-30 carbon atoms, or 4-20 carbon atoms.

[0327] In some embodiments, L connects the group Z to a square amide linker of formula (I) as defined in this disclosure, and preferably contains up to 1,000 carbon atoms, and preferably in the form of a chemical chain, which optionally contains heteroatoms (e.g., O, NH, S, Se or P) and / or cyclic moieties, such as aryl and / or heteroaryl groups.

[0328] In some embodiments, L may comprise one or more groups or portions selected from: alkyl (e.g., C 1-20 C 1-12 C 1-6 Alkyl, aryl, heteroaryl, alkyl ether, polyether, polyester, acyl, alkylamide, polyamide, guanidine, or combinations thereof. As used herein, “combination” means that L may comprise several hydrocarbon chains, oligomer chains, polymer chains (e.g., 2, 3, 4, 5, or 6), optionally comprising one or more heteroatoms, aryl or heteroaryl groups, and linked by any suitable group, such as -O-, -S-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-, -NH-CO-NH-, -O-CO-, -NH-(CS)-NH-, -NH-CS-phosphodiester or thiophosphate groups. Various alkyl groups are considered, including but not limited to -(CH2)n-, where “n” is from about 2 to about 20 or more. In some embodiments, L comprises C 2-20 Straight-chain or branched alkyl chain.

[0329] In some embodiments, L is or contains a polyether (e.g., polyethylene glycol or polypropylene glycol). Various ethers and polyethers are considered, including but not limited to -(OCH2CH2). n - where "n" is an integer from approximately 1 to approximately 40 or greater. In some implementations, L is or includes the formula -(OCH2CH2). n - of polyethylene glycol (“PEG”), where “n” is an integer from 1 to 10, an integer from 1 to 6, an integer from 3 to 6, an integer from 3 to 5, or an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, L is or contains polypropylene glycol, for example, of the formula -(OCH(CH3)CH2) n - where "n" is an integer from 1 to 10, an integer from 1 to 6, an integer from 3 to 6, an integer from 3 to 5, or an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0330] In some embodiments, L is or comprises an alkylamide. Various alkylamides are considered, including but not limited to -(CH2). y -C(O)NH-(CH2) p - and -(OCH2CH2) y -C(O)NH-(OCH2CH2) p - where "y" and "p" may be the same or different, and "y" and "p" are approximately 1 to approximately 20 or greater. In some embodiments, L is or includes the formula -(CH2). y -C(O)NH-(CH2) p -or formula-(OCH2CH2)y -C(O)NH-(OCH2CH2) p - Alkylamides, wherein "y" and "p" are each independently selected from integers 1-10, 1-6, 3-6, and 3-5, or independently selected from integers 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Various amides with alkyl or ether linkages are considered, including but not limited to -R4-C(O)NH-R5-, wherein "R4" and "R5" are each independently selected from alkyl groups (e.g., C...). 1-20 C 1-12 C 1-6 Alkyl), ether, or polyether (e.g., PEG with a molecular weight of about 200-2,000 g / mol). In a preferred embodiment, L is a polymer containing β-alanine, preferably containing 1-40 β-alanine monomers, more preferably 1-10 β-alanine monomers.

[0331] In some embodiments, L may also comprise an alkylene diamine, such as -NH(CH2). r NH-, where "r" is an integer from 2 to 20, such as 2 to 10, or an integer selected from 2, 3, 4, or 5. In some embodiments, L is a polymer of an alkylene diamine (also known as a polyamine), such as -NH-[(CH2)] r -NH] t - compounds, where "r" is as defined above and herein, and "t" is an integer of at least 2, such as at least 3, 4, 5, 10, or greater. The polymer of the target alkyl diamine is, for example, spermidine and spermine.

[0332] In some embodiments, L may also comprise a polyamide obtained from a vinyl monomer, such as poly(N-(2-hydroxypropyl)methacrylamide)(pHPMA) (e.g., pHPMA with a molecular weight of about 200 to about 5000 g / mol).

[0333] In some embodiments, L may also comprise polyester, such as polycaprolactone (e.g., polycaprolactone with a molecular weight of about 200 to about 5000 g / mol) or poly(D,L-lactic-co-glycolic acid) (PLGA) (e.g., PLGA with a molecular weight of about 200 to about 5000 g / mol).

[0334] In some embodiments, L may also contain an acyl group, such as -(CH2). r C(O)-, where “r” is an integer from 2 to 20, such as 2 to 10, or an integer selected from 2, 3, 4 or 5.

[0335] In some embodiments, L may comprise one or more optionally substituted groups, said groups comprising or consisting of the following groups: arylene or heteroarylene, saturated or unsaturated, straight-chain or branched C2-C 40 Hydrocarbon chains, groups containing alkylamines, groups containing acyl groups, amino acid moieties, polyethylene glycol, polypropylene glycol, polyethers of branched polyols, β-alanine polymers, pHPMA, PLGA, polymers of alkyldiamines, and combinations thereof.

[0336] In some embodiments, L is or comprises polyethylene glycol (PEG), which contains 1-40 glycol monomers, such as 2-10, such as -(OCH2CH2)2- (referred to herein as "PEG2"), -(OCH2CH2)3- (referred to herein as "PEG3"), -(OCH2CH2)3- (referred to herein as "PEG3"), -(OCH2CH2)4- (referred to herein as "PEG4") or -(OCH2CH2)5- (referred to herein as "PEG5").

[0337] In some embodiments, L may comprise one or more arylene or heteroarylene Ar groups. In certain aspects, the arylene or heteroarylene Ar group is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group, said group comprising one or more heteroatoms selected from the group consisting of N, O, S, and Se. In some embodiments, the Ar group is substituted with an acyl or amide moiety or its bioelectron isosteric derivative. In certain aspects, the arylene or heteroarylene Ar group is selected from the group consisting of phenylene and pyridylene. For example, in some embodiments, L comprises an optionally substituted phenylene moiety. For example, in some embodiments, L comprises an optionally substituted pyridylene moiety. In other embodiments, said phenylene or pyridylene is partially substituted with one or more moieties selected from the group consisting of halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkyl group.

[0338] In some embodiments, L may comprise an alkylene group, an ether, a polyether, an alkylene amide, an aryl group, a heteroaryl group, an acyl group, or a combination thereof. In one specific embodiment, L comprises a polyether, an aryl group, a heteroaryl group, an acyl group, or a combination thereof. In one specific embodiment, L comprises an aryl or heteroaryl Ar. Preferably, the aryl or heteroaryl Ar is a 6- to 10-membered aromatic carbocyclic group or a 5- or 12-membered heterocyclic group, the group comprising one or more heteroatoms selected from N, O, S, and Se. In some specific aspects, the aryl or heteroaryl Ar is selected from the group consisting of phenylene and pyridylene, optionally partially substituted by one or more of the group consisting of halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C1-6 Acyl and C 1-6 Alkyl group.

[0339] In one embodiment, L comprises PEG. In another embodiment, L comprises PEG and one or more aromatic groups, such as arylene and / or heteroarylene Ar. In yet another embodiment, L comprises PEG and one or more C groups. 1-6 Alkyl groups and one or more aromatic groups, such as arylene and / or heteroarylene Ar. In one specific embodiment, L comprises PEG and aryl or heteroarylene Ar selected from the group consisting of phenylene and pyridyl groups, which are optionally partially substituted by one or more of the group consisting of halogens, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkyl group. In another embodiment, L comprises PEG and an amino acid, preferably an arginine moiety. In another embodiment, L comprises PEG, a β-alanine polymer, and one or more aromatic groups, such as arylene and / or heteroarylene Ar groups. In another embodiment, L comprises PEG, a β-alanine polymer, and one or more C groups. 1-6 Alkyl, C 1-6 Alkylamine or C 1-6 Acyl group; and amino acid, preferably arginine moiety.

[0340] In a specific embodiment, L comprises one or more polyethylene glycol (PEG) monomers, which contain 1-40 PEG monomers and one or more C... 1-6 Alkylene, branched C 3-12 The polyether of a polyol, and one or more aromatic groups, such as arylene and / or heteroarylene Ar.

[0341] In some embodiments, L consists of one or more groups selected from the group consisting of arylene or heteroarylene, comprising saturated or unsaturated, straight-chain or branched C2-C groups. 40 The hydrocarbon chain may have optional substituted groups, preferably one or more C groups. 1-6 Alkyl, C 1-6 Alkylamine or C 1-6 Acyl group; polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, polypropylene glycol (PPG) containing 1-40 propylene glycol monomers, branched C 3-12Polyethers of polyols, arginine derivatives, β-alanine polymers containing 1-40 β-alanine monomers, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof; wherein L contains one or more groups selected from at least the group consisting of: arylene or heteroarylene, polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and β-alanine polymers containing 1-40 β-alanine monomers.

[0342] R1 and R2

[0343] In some implementations, R1 is selected from the group consisting of: H, C 1-6 Alkyl, C 1-6 Haloalkyl, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed herein. In some embodiments, R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Haloalkyl and Z-(OCH2-CH2)n-, wherein n is selected from 1-40. In a preferred embodiment, R1 is selected from the group consisting of H, C. 1-6 Alkyl and C 1-6 Halogenated alkyl group. Even more preferably, R1 is H.

[0344] In some implementations, R2 is selected from the group consisting of linear C 1-12 Alkyl, branched C 3-12 Alkyl, straight-chain C 1-12 Halogenated alkyl groups, branched C 3-12 Halogenated alkyl, aryl, heteroaryl, and benzyl. In some preferred embodiments, R2 is methyl, ethyl, trifluoromethyl, trifluoroethyl, phenyl, pyridyl, or benzyl, more preferably ethyl.

[0345] Arene- or heteroarylene Ar

[0346] In some embodiments, the Ar group is an arylene, wherein the arylene is defined and described in the categories and subcategories disclosed herein. In other embodiments, the Ar group is a heteroarylene, wherein the heteroarylene is defined and described in the categories and subcategories disclosed herein.

[0347] In some embodiments, the arylene or heteroarylene Ar is a 6- to 10-membered arylene or a 5- or 12-membered heteroarylene comprising one or more heteroatoms selected from the group consisting of N, O, S, and Se. In some specific embodiments, the arylene or heteroarylene Ar is a group selected from the group consisting of phenylene and pyridylene, optionally partially substituted by one or more of the group consisting of halogens, C, etc.1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Acyl and C 1-6 Alkyl group.

[0348] AAV carrier molecular formula

[0349] In some embodiments, the present invention relates to AAV carrier particles comprising the portion of formula (II):

[0350]

[0351] in

[0352] The AAV carrier particles include a square amide connector of formula (I):

[0353]

[0354] Group R L The amino acid residues of the AAV carrier capsid are covalently linked to the squaramide linker of formula (I); N is the functional moiety R as defined in this invention. L -NH- is the nitrogen atom of the amino group, and N* is the nitrogen atom of the primary amino group of the amino group of the surface-exposed amino acid residues of the AAV carrier capsid polypeptide.

[0355] In some embodiments, the present invention relates to AAV carrier particles comprising a portion of formula (II) as defined in this disclosure, wherein the functional portion R L -NH- contains a Z group, one or more spacer groups L, and the adeno-associated virus (AAV) vector particle is represented by formula (IIa):

[0356]

[0357] N*, Z and L are as defined and described in the categories and subclasses of this invention.

[0358] In some embodiments, L comprises one or more optionally substituted groups selected from the group consisting of arylene or heteroarylene Ar, saturated or unsaturated, straight or branched C2-C. 40 Hydrocarbon chain, preferably with one or more C groups 1-6 Alkylene, C 1-6 Alkylamine or C 1-6 Acyl group, polyethylene glycol, polypropylene glycol, amino acid moiety, β-alanine polymer, branched C 3-12 Polyethers of polyols, pHPMA, PLGA, polymers of alkyl diamines, and combinations thereof; preferably, L is or contains one or more groups selected from the group consisting of polyethylene glycol, aryl or heteroaryl Ar, C 1-6 Alkylene, C 1-6 Alkylamine, C1-6 Acyl group, amino acid moiety, preferably arginine moiety, branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyols, and β-alanine polymers containing 1-40 β-alanine monomers. In some embodiments, L is or contains polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers. In some preferred embodiments, the polyethylene glycol (PEG) is PEG3, PEG4, or PEG5.

[0359] In some specific embodiments, the present invention relates to AAV carrier particles comprising a portion of formula (II) as defined in this disclosure, comprising one or more spacer bases L selected from the group consisting of L1, L2, and L3, wherein the portion of formula (II) is selected from the group consisting of (IIa1), (IIa2), (IIa3), (IIa4), (IIa5), (IIa6), (IIa7), (IIa8), (IIa9), (IIa... 10 ), (IIa 11 ), (IIa 12 ), (IIa 13 ), (IIa 14 ), (IIa 15 ), (IIa 16 ), (IIa 17 ), (IIa 18 ), (IIa 19 ) and (IIa 20 ):

[0360]

[0361]

[0362]

[0363]

[0364] N*, Z, L1, L2 and L3 are defined and described as in the categories and subclasses disclosed in this invention.

[0365] In some embodiments, L1 is an optional substituted group selected from the group consisting of: polyethylene glycol, polypropylene glycol, pHPMA, PLGA, β-alanine polymers, branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyols, polymers of alkyl diamines, and combinations thereof; preferably, L1 is selected from the group consisting of: polyethylene glycol, β-alanine polymers, and branched C-type polymers. 3-12 Polyethers of polyols, preferably branched C 3-6Polyols. In some embodiments, polyethylene glycol (PEG) comprises 1-40 ethylene glycol monomers. In some preferred embodiments, polyethylene glycol (PEG) is PEG3, PEG4, or PEG5. In some embodiments, the β-alanine polymer comprises 1-40 β-alanine monomers, preferably 1-10 β-alanine monomers.

[0366] In some embodiments, L2 comprises one or more arylene or heteroarylene Ar groups as defined herein. In some preferred embodiments, L2 comprises phenylene or pyridylene.

[0367] In some aspects, L1 comprises one or more of the following: polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, a β-alanine polymer containing 1-40 β-alanine monomers, and branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyols; L2 contains one or more arylene or heteroarylene groups; and L3 contains C 1-6 Alkylene, C 1-6 Alkylamine, C 1-6 The acyl or amino acid moiety is preferred, with the arginine moiety being the most desirable.

[0368] In some embodiments, L3 is covalently connected to L2 via a carbon atom of an arylene or via a carbon atom or a heteroatom of a heteroarylene; and L1 and L2 or L1 and L3 are covalently connected via an amide moiety or its bioelectron isosteric moiety, preferably an amide moiety -N(R1)C(O)- or its bioelectron isosteric moiety, wherein R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1 to 3, n is selected from 0 to 40, and Z is as defined and described in the categories and subcategories disclosed in this invention; more preferably, R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, even more preferably R1 is H.

[0369] In some respects, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers; L2 contains one or more arylene or heteroarylene groups; L3 is C 1-6Alkylene; L3 is covalently linked to L2 via a carbon atom of the arylene group or via a carbon atom or a heteroatom of the heteroarylene group; and L1 and L3 are covalently linked via an amide moiety -N(R1)C(O)- or its bioisosteric moiety, wherein R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 The alkyl halogroup and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, and even more preferably, R1 is H.

[0370] In some respects, L1 and L2 are covalently linked via an amide moiety -N(R1)C(O)- or its bioelectron isosteric moiety, wherein R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Halogenated alkyl groups, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 The alkyl halogroup and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, more preferably R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, and even more preferably, R1 is H.

[0371] In some respects, L1 and L3 are covalently linked via an amide moiety -N(R1)C(O)- or its bioelectron isosteric moiety, wherein R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6 Haloalkyl, Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n-, and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-, wherein q is selected from 1-3, n is selected from 0-40, and Z is as defined and described in the categories and subcategories disclosed in this invention; preferably, R1 is selected from the group consisting of H, C 1-6 Alkyl, C 1-6The alkyl group is a haloalkyl group and Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40. More preferably, R1 is H or Z-(OCH2-CH2)n-, wherein n is selected from 1 to 40, and even more preferably, R1 is H.

[0372] In some embodiments, L1 comprises one or more polyethylene glycol (PEG) monomers comprising 1-40 ethylene glycol monomers, and branched C 3-12 Polyethers of polyols, preferably branched C 3-6 Polyols; L2 contains one or more arylene or heteroarylene Ars; and L3 contains one or more Cs. 1-6 Alkylene; PEG and a C 1-6 Alkyl groups are covalently linked via an amide moiety or its bioelectronic isosteric group, with branched C-terminals. 3-12 Polyol polyethers and C 1-6 Alkyl groups are covalently linked via ether bonds, with branched C-chains. 3-12 The polyether of the polyol and the arylene or heteroarylene Ar are covalently linked through the amide moiety or its bioelectron isostere, preferably the amide moiety -N(R1)C(O)- or its bioelectron isostere.

[0373] In some embodiments, L1 comprises one or more of the following: polyethylene glycol (PEG) comprising 1-40 ethylene glycol monomers and a β-alanine polymer comprising 1-40 β-alanine monomers; L2 comprises one or more arylene or heteroarylene Ar; and L3 comprises one or more C 1-6 Hydroxyl group, C 1-6 Alkylamines and C 1-6 Acyl group; and L1 and L2 or L1 and L3 are covalently linked via the amide moiety -N(R1)C(O)- or its bioelectron isosteric moiety.

[0374] In some embodiments, L1 comprises one or more of the following: polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers and a β-alanine polymer containing 1-40 β-alanine monomers, and L3 comprises one or more C 1-6 alkylene groups, C 1-6 alkylamine group, C 1-6 The acyl group and amino acid moiety are preferred, with the arginine moiety being the preferred component.

[0375] In some preferred embodiments, n is 1, 2, 3, 4 or 5; n' is selected from 2-8; m1 and m2 are each independently 0, 1 or 2; m3, m4, m5 and m6 are each independently 1-3, preferably 2; Ar is phenylene or pyridylene; and R1 is H.

[0376] Examples of bioisosteric moieties of the amide moiety include N-substituted and unsubstituted moieties, such as amide-N(R1)C(O)- and its bioisosteres, which can be selected from -C(O)N(R1)-, -N(R3)C(O)N(R1)-, -N(R1)C(O)N(R3)-, -N(R1)C(S)-, -C(S)N(R1)-, -N(R1)C(S)N(R3)-, -N(R3)C(S)N(R1)-, -S(O)2-N(R1)-, -N(R1)-S(O)2- and triazolyl groups, wherein R3 and R1 are each independently selected from the group consisting of H, C 1-6 Alkyl, C 1-6 The terms R3 and R1 are: alkyl haloalkyl, aryl, alkylaryl, Z-(OCH2–CH2)n–, ZC(O)NH-(CH2)q–(OCH2-CH2)n–, and Z-NHC(O)–(CH2)q–(OCH2-CH2)n–, wherein q is selected from 1 to 3, n is selected from 1 to 40, and R3 may be the same as or different from R1.

[0377] In some embodiments, when L2 contains an arylene or heteroarylene Ar, L1 and the square amide connector of formula (I), or L1 and L3 (if present) are covalently bonded to the arylene or heteroarylene Ar at the ortho, meta, or para positions.

[0378] In some embodiments, when L2 contains an arylene or heteroarylene Ar and L3 is a group C 1-6 When alkylene, L3 and the square amide linker of formula (I) are covalently bonded to the arylene or heteroarylene Ar at the ortho, meta, or para positions.

[0379] In some embodiments, when L2 contains an arylene or heteroarylene Ar and L3 is a group C 1-6 When alkylene, one or more groups L3 are covalently bonded to the arylene or heteroarylene Ar at the ortho, meta, or para positions.

[0380] In some embodiments, L1 or L3 may be selected from alkyl groups (e.g., C4). 1-20 C 1-12 C 1-6 Alkyl groups, ethers, polyethers, polyesters, polyamides, alkylamides, or combinations thereof. As used herein, "combination" means that L1 or L3 may contain several hydrocarbon chains, oligomer chains, or polymer chains (e.g., 2, 3, 4, 5, or 6) linked by any suitable group, such as -O-, -S-, -NHC(O)-, -OC(O)-, -C(O)-OC(O)-, -NH-, -NH-CO-NH-, -O-CO-, -NH-(CS)-NH-, -NH-CS- phosphate diester or thiophosphate groups. Various alkyl groups are considered, including but not limited to -(CH2).m - where "m" is about 2 to about 20 or more. In some implementations, L3 includes C 2-20 Straight-chain or branched alkyl chain.

[0381] In some embodiments, L1 or L3 may also comprise polyesters, such as polycaprolactone (e.g., polycaprolactone with a molecular weight of about 200 to about 5000 g / mol) or poly(D,L-lactic-co-glycolic acid) (PLGA) (e.g., PLGA with a molecular weight of about 200 to about 5000 g / mol).

[0382] In some embodiments, L1 or L3 may be selected from optionally substituted groups, said groups comprising or consisting of the following groups: saturated or unsaturated, straight-chain or branched C2-C. 40 Hydrocarbon chain, polyethylene glycol, polypropylene glycol, branched C 3-12 Polyethers of polyols, β-alanine polymers, pHPMA, PLGA, polymers of alkylene diamines, amino acid moieties such as arginine or β-alanine moieties, and combinations thereof. Various alkylamides are considered, including but not limited to -(CH2) m3 -C(O)NH-(CH2) m4 - and -(OCH2CH2) m3 -C(O)NH-(OCH2CH2) m4 - where "m3" and "m4" may be the same or different, and "m3" and "m4" are approximately 1 to approximately 20 or greater. In some embodiments, L1 or L3 is of formula -(CH2). m3 -C(O)NH-(CH2) m4 -or-(OCH2CH2) m3 -C(O)NH-(OCH2CH2) m4 - Alkylamides, wherein "m3" and "m4" are each independently selected from integers 1-10, 1-6, 3-6, and 3-5, or independently selected from integers 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Various amides using linking units having alkyl or ether bonds are considered, including but not limited to -R5-C(O)NH-R6-, wherein "R5" and "R6" are each independently selected from alkyl (e.g., C... 1-20 C 1-12 C 1-6 Alkyl), ether or polyether.

[0383] In some implementations, L1 may also include a branch C. 3-12 Polyethers of polyols (preferably branched C) 3-6Polyols), acyl-C(O)-(CH2)r or alkyleneamines, such as -NH(CH2)r or alkylene diamines (such as -NH(CH2)rNH-), where "r" is an integer from 2 to 20, for example 2 to 10, or an integer selected from 2, 3, 4, or 5. In some embodiments, L1 is a polymer of an alkylene diamine (also known as a polyamine), such as -NH-[(CH2)rNH-]. r -NH] t - compounds, wherein "r" is as defined above and herein, and "t" is an integer of at least 2, such as at least 3, 4, 5, 10, or greater. The polymer of the target alkyl diamine is, for example, spermidine and spermine. In some embodiments, L1 may also comprise a polyamide, such as a β-alanine polymer having 1-40 β-alanine monomers, or a polyamide obtained from an amide vinyl monomer, such as poly(N-(2-hydroxypropyl)methacrylamide)(pHPMA) (e.g., pHPMA with a molecular weight of about 200-about 5000 g / mol).

[0384] In some embodiments, L3 is a group C 1-6 Alkylene group, preferably -CH2- or -CH2-CH2- group; or group C 1-6 Alkylamine, preferably -CH2-CH2-NH-; group C 1-6 Acyl group, preferably -CH2-CH2-C(O)- group; or amino acid moiety, preferably arginine or β-alanine moiety.

[0385] In some specific embodiments, the AAV carrier particles comprise portions of formula (II) selected from the group consisting of: formulas (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), (IIh), (IIj), (IIk), (IIm), (IIIn), (IIp), (IIq), (IIr), (IIs), (IIt), (IIv), (IIw), and (IIx):

[0386]

[0387]

[0388]

[0389]

[0390] Where R a R b and R c Each is independently H or group R':

[0391]

[0392] Furthermore, n, n', m1, m2, m3, m4, m5, m6, N*, Z, Ar, and R1 are defined and described as in the categories and subclasses disclosed in this invention.

[0393] In another preferred embodiment, the AAV carrier particle has a portion of formula (II) as defined herein, R L It contains a group Z and a spacer group L, wherein Z is a sugar and L is a polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and the part of formula (II) is represented by formula (IIb) as defined herein.

[0394] In a preferred embodiment, the AAV carrier particle has a portion of formula (IIa1) comprising a group Z and a spacer group L1, wherein Z is a sugar and L1 is polyethylene glycol (PEG) comprising 1-40 ethylene glycol monomers, and the portion of formula (II) is represented by formula (IIb) as defined herein.

[0395] In a preferred embodiment, the AAV carrier particles have a portion of formula (IIa2) or (IIa3) comprising a group Z and spacer groups L1, L2, and L3, where Z is a sugar, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, L2 is an arylene or heteroarylene Ar, and L3 is C 1-6 The alkylene groups, L1 and L2, are covalently linked by the amide moiety -N(R1)C(O)-, where R1 is as defined herein, and the part of formula (II) is represented by formula (IIc).

[0396] In another preferred embodiment, the AAV carrier particles have a portion of formula (IIa4) or (IIa5) comprising a group Z and spacer groups L1, L2, and L3, where Z is a sugar, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, L2 is an arylene or heteroarylene Ar, and L3 is a group C. 1-6 The alkylene groups, L1 and L3, are covalently linked via an amide moiety -N(R1)C(O)-, where R1 is as defined herein, and the part of formula (II) is represented by formula (IId).

[0397] In another preferred embodiment, the AAV carrier particle has a portion of formula (IIa6) comprising a group Z and spacer groups L1 and L3, where Z is a peptide, L1 is polyethylene glycol (PEG) comprising 1-40 ethylene glycol monomers, and L3 is a group C. 1-6Alkyl groups L1 and L3 are covalently linked by an ether bond, and when the N-terminus of peptide Z and L3 are covalently linked by an amide or its bioelectroisosteryl portion, the portion of formula (II) is represented by formula (IIe), wherein the N-terminus of peptide Z is the nitrogen atom of the amide; and when the C-terminus of peptide Z and L3 are covalently linked by an amide or its bioelectroisosteryl portion, the portion of formula (II) is represented by formula (IIf), wherein the C-terminus of peptide Z is the carbonyl group of the amide, and wherein the amide may be N-substituted or unsubstituted, for example, amide-N(R1)C(O)-, wherein R1 is as defined herein.

[0398] In another preferred embodiment, the AAV carrier particles have formula (IIa7) or (IIa8) or (IIa9) or (IIa... 10 The portion comprises a group Z and spacers L1, L2 and 1-3 spacers L3, as defined herein; wherein Z is a peptide, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, L2 is an arylene or heteroarylene Ar, and L3 is a group C. 1-6 Alkylene; wherein L1 and the first spacer group L3 are covalently linked by an ether bond; L1 and L2 or L1 and the second spacer group L3 are covalently linked by an amide moiety -N(R1)C(O)-; and when the N-terminus of peptide Z and L3 are covalently linked by an amide or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIh), wherein the N-terminus of peptide Z is the nitrogen atom of the amide; and when the C-terminus of peptide Z and L3 are covalently linked by an amide or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIg), wherein the C-terminus of peptide Z is the carbonyl group of the amide; and wherein the amide may be N-substituted or unsubstituted, for example, amide -N(R1)C(O)-, wherein R1 is as defined herein.

[0399] In a preferred embodiment, the AAV carrier particles have the formula (IIa) as defined herein. 11 Part R L -NH is composed of the group Z-NH-, where Z is a peptide and the nitrogen atom of the group Z-NH corresponds to the N-terminal group of the peptide, and part of formula (II) is represented by formula (IIb) as defined herein.

[0400] In another preferred embodiment, the AAV carrier particles comprise a group Z and spacer groups L1, L2 and 1-3 spacer groups L3, as defined herein; wherein Z is a peptide, L1 is a β-alanine polymer comprising 1-40 β-alanine monomers, L2 is an arylene or heteroarylene Ar, and the L3 spacer group is a group C. 1-6 alkyleneamines or C groups 1-6 Acyl group or group C 1-6Alkylene; wherein L1 and L2 or L1 and a spacer group L3 are covalently linked by an amide moiety -N(R1)C(O)-; and when the N-terminus of peptide Z and L1 are covalently linked by an amide or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIj), wherein the N-terminus of peptide Z is the nitrogen atom of the amide; and when the C-terminus of peptide Z and L3 are covalently linked by an amide or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIk), wherein the C-terminus of peptide Z is the carbonyl group of the amide; and wherein the amide may be N-substituted or unsubstituted, for example, amide -N(R1)C(O)-, wherein R1 is as defined herein.

[0401] In another preferred embodiment, the AAV carrier particles comprise a group Z and spacer groups L1 and L3, where Z is a peptide, L1 is a β-alanine polymer comprising 1-40 β-alanine monomers, and L3 is a group C. 1-6 alkyleneamines or C groups 1-6 Alkylenes, L1 and L3 are covalently linked via an amide moiety, and when the C-terminus of peptide Z and L1 are covalently linked via an amide moiety or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIm), wherein the N-terminus of peptide Z is the nitrogen atom of the amide; and when the N-terminus of peptide Z and L1 are covalently linked via an amide moiety or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIn); and wherein the amide may be N-substituted or unsubstituted, for example, amide-N(R1)C(O)-, wherein R1 is as defined herein.

[0402] In another preferred embodiment, the AAV carrier particle comprises a group Z, a spacer group L1, and a spacer group L3, where Z is a peptide, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and the first group L3 is a group C. 1-6 alkyleneamines or C groups 1-6 Acyl group, and the second spacer group L3 is an arginine moiety; wherein the group C 1-6 alkyleneamines or C groups 1-6 The acyl group and the spacer group L1 are covalently linked via an ether bond. Both L1 and the square amide linker are covalently linked to the arginine moiety. Furthermore, when the N-terminus of peptide Z and the C-terminus group... 1-6 When the acyl group is covalently linked through the amide moiety or its bioisosteric moiety, the portion of formula (II) is represented by formula (IIp), wherein the N-terminus of peptide Z is the nitrogen atom of the amide; and when the C-terminus of peptide Z and the C-group are covalently linked... 1-6 When an alkylene amine is covalently linked via an amide moiety or its bioelectron isosteric moiety, the portion of formula (II) is represented by formula (Iq), wherein the C-terminus of the peptide Z is a carbonyl group of the amide; and wherein the amide may be N-substituted or unsubstituted, for example, amide-N(R1)C(O)-, wherein R1 is as defined herein.

[0403] In another preferred embodiment, the AAV carrier particle comprises a group Z, two spacer groups L1 and two spacer groups L3, where Z is a peptide, the first L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, the second L1 is a β-alanine polymer containing 1-40 β-alanine monomers, and one spacer group L3 is a group C. 1-6 alkyleneamines or C groups 1-6 Acyl group, and the second spacer group L3 is an arginine moiety; wherein the group C 1-6 alkyleneamines or C groups 1-6 The acyl group and the spacer group L1 are covalently linked via an ether bond. Both the PEG and the square amide linker are covalently linked to the arginine moiety, and the β-alanine polymer is linked to the C group. 1-6 alkyleneamines or C groups 1-6 The acyl group forms an amide moiety, and when the N-terminus of peptide Z and the β-alanine polymer are covalently linked through the amide moiety or its bioelectron isosteric moiety, the part of formula (II) is represented by formula (IIr), wherein the N-terminus of peptide Z is the nitrogen atom of the amide; and when the C-terminus of peptide Z and the β-alanine polymer are covalently linked through the amide moiety or its bioelectron isosteric moiety, the part of formula (II) is represented by formula (IIs), wherein the C-terminus of peptide Z is the carbonyl group of the amide; and wherein the amide may be N-substituted or unsubstituted, for example, amide-N(R1)C(O)-, wherein R1 is as defined herein.

[0404] In another preferred embodiment, the AAV carrier particle comprises a group Z, two spacer groups L1, one L2, and one to three spacer groups L3, as defined herein; wherein Z is a peptide, the first spacer group L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, the second spacer group L1 is a β-alanine polymer containing 1-40 β-alanine monomers; L2 is an arylene or heteroarylene Ar; and each L3 is a group C. 1-6 alkylene or group C 1-6 alkyleneamines or C groups 1-6 Acyl group; wherein both spacer groups L1 are connected to a first spacer group L3, and the first spacer group L3 is a group C. 1-6 alkyleneamines or C groups 1-6 Acyl group; PEG and L2 or PEG and the second spacer group L3 are C groups 1-6The alkylene group is covalently linked via an amide moiety -N(R1)C(O)-; and when the N-terminus of peptide Z and the β-alanine polymer are covalently linked via an amide moiety or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIt), wherein the N-terminus of peptide Z is the nitrogen atom of the amide; and when the C-terminus of peptide Z and the β-alanine polymer are covalently linked via an amide moiety or its bioelectroisosteryl moiety, the portion of formula (II) is represented by formula (IIv), wherein the C-terminus of peptide Z is the carbonyl group of the amide; and wherein the amide may be N-substituted or unsubstituted, for example, amide-N(R1)C(O)-, wherein R1 is as defined herein.

[0405] In another preferred embodiment, the AAV carrier particle comprises a group Z as defined herein, a spacer group L2, and 0, 1, or 2 spacer groups L3; wherein Z is a peptide, L2 is an aryl or heteroaryl group, and L3 is a group C. 1-6 alkylene or group C 1-6 alkyleneamines or C groups 1-6 Acyl group; and when the N-terminus of peptide Z and L2 or Z and L3 are covalently linked by an amide moiety or a bioelectron isosteric moiety, the part of formula (II) is represented by formula (IIw), wherein the N-terminus of peptide Z is a nitrogen atom of an amide; and wherein the amide may be N-substituted or unsubstituted, for example, amide-N(R1)C(O)-, wherein R1 is as defined herein.

[0406] In another preferred embodiment, the AAV carrier particles have formula (IIa) 12 ) or (IIa 13 ) or (IIa 14 ) or (IIa 15 The component comprises three groups Z, which are sugars or peptides, and four spacer groups L1, one spacer group L2, and three to five spacer groups L3; wherein the three spacer groups L1 are polyethylene glycol (PEG) groups, each independently containing 1-40 ethylene glycol monomers; one spacer group L1 is a branched C 3-12 Polyethers of polyols; L2 is arylene or heteroarylene Ar; and each L3 is a C group. 1-6 Alkylene; wherein the portion of formula (II) is represented by formula (IIx) as defined herein.

[0407] It is worth noting that when L (or L1) contains a PEG group directly linked to sugar Z, the terminal oxygen atom of the PEG group (when located on the Z side) can be part of Z. This is the case, for example, when Z is a sugar and its anomeric carbon has a PEG linker. Similarly, when Z is a peptide, the nitrogen atom of amide Z-NR3C(O)- is located at the N-terminus of the peptide, and the carbonyl group of amide ZC(O)NR3- is located at the C-terminus of the peptide.

[0408] In one illustrative embodiment, the AAV carrier particles of the present invention are selected from:

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416] For the purposes of this disclosure, when the portion of formula (II) contains a sugar, the stereochemistry of the anomeric carbon of the sugar (if present) is not shown in the figures and diagrams of the AAV carrier particles of the present invention, or in the figures and diagrams of the portion of formula (II) disclosed in the present invention, or in the figures and diagrams of the compound of formula (III).

[0417] In one illustrative embodiment, the AAV carrier of the present invention comprises a portion selected from the following formula (II):

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424] In some embodiments, the AAV carrier particles of the present invention are selected from the group consisting of (1)-AAV, (2)-AAV, and (3)-AAV. The same nomenclature is used for other AAV serotypes and modification groups.

[0425] In some embodiments, the AAV vector of the present invention is (3)-AAV2 containing at least one transgene, said transgene containing cDNA from the GBA gene, preferably the human GBA gene, optionally said transgene under the control of at least one regulatory element, preferably a promoter as defined above, more preferably a CAG promoter.

[0426] Compounds of formula (III) containing squaric acid ester groups

[0427] In some embodiments, the present invention also relates to compounds of formula (III) comprising squaric acid esters for obtaining the AAV carrier of the present invention.

[0428] In some embodiments, the present invention provides compounds of formula (III):

[0429]

[0430] Or a pharmaceutically acceptable salt thereof, wherein R2 and R L -NH- as defined and described in the categories and subclasses of this invention.

[0431] For example, in some implementations, the functional portion R L -NH- contains a Z group, one or more spacer groups L, and compounds of formula (III) are represented by formula (IIIa):

[0432]

[0433] R2, Z, and L are as defined and described in the categories and subclasses of this invention.

[0434] In some implementations, the functional portion R L -NH- contains a group Z and one or more spacer groups L selected from L1, L2 and L3, and compounds of formula (IIIa) are selected from the group consisting of: (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), (IIIa8), (IIIa9), (IIIa 10 (IIIa) 11 (IIIa) 12 (IIIa) 13 (IIIa) 14 (IIIa) 15 (IIIa) 16 (IIIa) 17 (IIIa) 18 (IIIa) 19 ) and (IIIa 20 ):

[0435]

[0436]

[0437]

[0438] R2, Z, L1, L2, and L3 are as defined and described in the categories and subclasses of this invention. In some aspects, L1 is polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers; L2 contains one or more arylene or heteroarylene groups; L3 is C 1-6 The alkylene group, L3, is covalently linked to L2 via a carbon atom of the arylene group or via a carbon atom or a heteroatom of the heteroarylene group; and L1 and L2 or L1 and L3 are covalently linked via an amide moiety or its bioelectron isosteric moiety.

[0439] In some specific embodiments, compounds of formula (III) disclosed herein are selected from the group consisting of: formulas (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIj), (IIIk), (IIIm), (IIIn), (IIIp), (IIIq), (IIIr), (IIIs), (IIIt), (IIIv), (IIIw), and (IIIx):

[0440]

[0441]

[0442]

[0443]

[0444]

[0445] Or a pharmaceutically acceptable salt thereof, wherein n, n', m1, m2, m3, m4, Z, Ar, R a R b R c R1 and R2 are as defined and described in the categories and subclasses disclosed in this invention.

[0446] In some embodiments, the compound of formula (III) comprises a labeler (fluorescein) or a sugar (mannose, glucose, mannose phosphate) or a peptide (THR, SEQ ID No.:1) selected from the compounds in Table 2 or their salts.

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453] Method for obtaining AAV carrier particles modified with squaramide containing the part of formula (II)

[0454] In some embodiments, the present invention also relates to a method for preparing an AAV carrier according to the invention. In some embodiments, the method of the present invention comprises incubating an AAV carrier with a compound of formula (III) as defined and described in the categories and subclasses of this specification under conditions suitable for reacting the squaric acid ester portion of the compound of formula (III) as defined and described in the categories and subclasses of this invention with at least one amino group of amino acid residues of the AAV carrier capsid to form a squaric acid amide linker of formula (I) as defined in the present invention.

[0455] In some embodiments, suitable conditions for obtaining at least one portion of formula (II) include suitable conditions that promote the formation of covalent bonds between the amino groups of the amino acid residues of the AAV carrier capsid and the squaric acid ester portion, without compromising the structural integrity of the AAV.

[0456] In some embodiments, the AAV carrier is incubated with compounds as described herein and in some examples: formula (III), and formulas (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), (IIIa8), (IIIa9), (IIIa... 10 (IIIa) 11 (IIIa) 12 (IIIa) 13 (IIIa) 14 (IIIa) 15 (IIIa) 16 (IIIa) 17 (IIIa) 18 (IIIa) 19 (IIIa) 20 ), (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIj), (IIIk), (IIIm), (IIIn), (IIIp), (IIIq), (IIIr), (IIIs), (IIIt), (IIIv), (IIIw) or (IIIx).

[0457] In some embodiments, incubation can be performed in an aqueous buffer solution with a pH range of 5.5-10, preferably 7-10, such as 9-10, or 9.3. In some preferred embodiments, the pH is 9.3.

[0458] In some embodiments, the incubation buffer may be selected from TRIS buffer, borate buffer, Hepes buffer, acetate buffer, phosphate buffer, such as PBS or Dulbecco phosphate-buffered saline (dPBS). In some preferred embodiments, the buffer is a TRIS buffer.

[0459] In some embodiments, incubation can last from several minutes to several hours, for example, 5 minutes to 6 hours, or 3 to 5 hours. In some preferred embodiments, the incubation lasts about 4 hours. In some embodiments, incubation can last from several hours to several days, for example, 6 to 72 hours, or 12 to 48 hours or 16 to 24 hours. In some embodiments, incubation ends when a sufficient coupling yield is achieved.

[0460] In some embodiments, the incubation temperature is typically 4°C–50°C. In some preferred embodiments, the incubation is carried out at room temperature, i.e., at a temperature of 18°C–30°C, for example, about 20°C. In some embodiments, the incubation solution may be stirred.

[0461] In some embodiments, the molar ratio of the compound of formula (III) to the AAV support may be 1.10. 5 -1.10 7 For example, 1.10 6 -5.10 6 In some preferred embodiments, the molar excess of compound (III) is 3.10. 6 equivalent.

[0462] In some embodiments, the method of the present invention may include one or more other steps before or after the incubation step as described above.

[0463] For example, in some embodiments, the method of the present invention may include a preparatory step of providing or generating an AAV carrier to be modified.

[0464] In some embodiments, the method of the present invention may further include one or more other steps following the incubation step, such as:

[0465] - A step at the end of the incubation step to remove unreacted compounds containing squaric acid ester groups (e.g., compounds of formula (III)), for example by dialysis or tangential flow filtration, and / or

[0466] - The steps for collecting chemically modified AAV particles, and / or

[0467] - Steps to purify the AAV vector, and / or

[0468] - Steps for recovering AAV carriers, and / or

[0469] - Steps for preparing and / or packaging AAV vectors.

[0470] In some embodiments, the method of the present invention may further include a preparatory step of providing or preparing a compound of formula (III) prior to incubating the AAV carrier with a compound of formula (III) as defined and described in the categories and subclasses of this specification, forming a formula (I) squaric acid amide linker under conditions suitable for the squaric acid ester moiety of the compound of formula (III) to react with at least one amino group of an amino acid residue of the AAV carrier capsid.

[0471] Specifically, compounds of formula (III), including all compounds of formula (III) disclosed in this invention, are selected from (IIIa), (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), (IIIa8), (IIIa9 ... 10 (IIIa) 11 (IIIa) 12 (IIIa) 13 (IIIa) 14 (IIIa) 15 (IIIa) 16 (IIIa) 17 (IIIa) 18 (IIIa) 19 (IIIa) 20 (IIIb), (IIIc), (IIId), (IIIe), (IIIf), (IIIg), (IIIh), (IIIj), (IIIk), (IIIm), (IIIn), (IIIp), (IIIq), (IIIr), (IIIs), (IIIt), (IIIv), (IIIw), and (IIIx) can be prepared by a variety of methods. The starting products are commercial products or products prepared from commercial compounds according to known synthesis methods, or products known to those skilled in the art.

[0472] In some instances, the synthesis of compounds of formula (III) involves adding amino group R... L The functional moiety of -NH2 reacts with the squaric acid diester of formula (V), wherein R L As defined and described in the categories and subclasses of this specification:

[0473]

[0474] The reaction is carried out under suitable conditions to obtain compound (III); wherein R2 is defined according to the category and subclass of the present invention.

[0475] In some instances, the synthesis of compounds of formula (III) includes:

[0476] -Provide precursor compounds of formula (VI):

[0477] Z-L1-NH2 (VI),

[0478] Wherein Z and L1 are defined by the categories and subcategories disclosed in this invention;

[0479] - Under suitable conditions, the compound of formula (VI) is reacted with a squaric acid diester of formula (V) as defined herein to obtain a compound of formula (IIIa1):

[0480]

[0481] R2 is defined according to the categories and subcategories disclosed in this invention.

[0482] For example, compounds of formula (VI) can be prepared from the corresponding azide precursor disclosed in document WO2022096681 by reducing the azide functional groups using methods and conditions known in the art for reducing azide.

[0483] In other examples, the synthesis of compounds of formula (III) includes:

[0484] - Provide the precursor of formula (VIIa) or formula (VIIIa):

[0485]

[0486] Z, L2, L3, and R2 are defined according to the categories and subcategories disclosed herein;

[0487] - Reacting the compound of formula (VIIa) or the compound of formula (VIIIa) with the compound of formula (IX),

[0488] Z-L1-NHR1 (IX),

[0489] Under suitable conditions, compounds of formula (IIIa4) or formula (IIIa5) are obtained, wherein L1 and L3 are covalently linked by an amide moiety -N(R1)C(O)-, and wherein R1 is defined according to the class and subclass definition disclosed herein.

[0490] The appropriate conditions for reacting compounds of formula (VIIa) or (VIIIa) with compounds of formula (IX) as defined above are well known in the art for peptide coupling.

[0491] The intermediate compounds of formulas (VIIa) and (VIIIa) can be prepared by reacting the corresponding amino acid precursors of formulas (VIIa') and (VIIIa') with the compound of formula (V) in the presence of a base and an alcohol solvent, respectively.

[0492]

[0493] The compounds of formula (V) are defined according to the categories and subclasses disclosed herein.

[0494] In other examples, the synthesis of compounds of formula (III) includes:

[0495] - Provide a precursor to formula (VIIb) or formula (VIIIb):

[0496]

[0497] Z, L2, L3, and R2 are defined according to the categories and subcategories disclosed herein;

[0498] - Reacting the compound of formula (VIIb) or the compound of formula (VIIIb) with the compound of formula (IX),

[0499] Z-L1-NHR1 (IX),

[0500] Compounds of formula (IIIa2) or (IIIa3) are obtained under suitable conditions, wherein L1 and L2 are covalently linked by an amide moiety -N(R1)C(O)-, and wherein R1 is defined according to the class and subclass definitions disclosed in this invention.

[0501] The appropriate conditions for reacting compounds of formula (VIIb) or formula (VIIIb) with compounds of formula (IX) as defined above herein are well known in the art for peptide coupling.

[0502] The intermediate compounds of formulas (VIIb) and (VIIIb) can be prepared by reacting the corresponding amino acid precursors of formulas (VIIb') and (VIIIb') with the compound of formula (V) in the presence of a base and an alcohol solvent, respectively.

[0503]

[0504]

[0505] Among them, L2 and L3, as well as compounds of formula (V, are defined according to the categories and subclasses disclosed herein.

[0506] In some embodiments, the group Z of the compound of formula (III) is a peptide, which can be prepared by any method known in the art (such as peptide synthesis on a solid support according to a standard procedure) and coupled to an amino or carboxyl group of a spacer group L as described above under suitable conditions (e.g., on a solid support according to a standard procedure) to obtain an amide moiety. Subsequently, after releasing the group ZL-NH2 or peptide Z-NH2 (where the amino group is the N-terminus of peptide Z) from the solid support, the method for preparing the compound of formula (III) includes reacting the compound of formula (V) with the primary amine group of Z-NH2 or ZL-NH2 under suitable conditions, such as in an aqueous environment, to obtain the compound of formula (III).

[0507] Uses of the provided AAV carrier

[0508] In some embodiments, the AAV vector of the present invention can be used as a research tool. In some embodiments, the AAV vector of the present invention can be used as a drug, such as a carrier for delivering therapeutic nucleic acids (such as DNA or RNA) in gene therapy. In some embodiments, the AAV vector of the present invention can be used in diagnostic methods, such as as an imaging agent. In some embodiments, the AAV vector of the present invention can be used as a combination of therapeutic and diagnostic tools, such as for diagnostic purposes.

[0509] Modification of the biological functionality and / or properties of AAV vectors

[0510] In some embodiments, the chemical modification of the AAV vector capsid can modify one or more of its biological functions and / or properties. In some embodiments, the biological functions and / or properties may depend on the functional portion R introduced to modify the AAV vector in this invention. L The properties of the modified AAV vector. In some embodiments, one or more biological characteristics of the modified AAV vector may be altered compared to the unmodified AAV vector, for example:

[0511] - AAV vector modification selectivity for specific organs, tissues, and / or cell types (e.g., increased selectivity or transfer selectivity from one tissue / organ / cell to another); and / or

[0512] - Modifications to the immunoreactivity of the AAV vector, such as reduced immunogenicity and / or decreased affinity for neutralizing antibodies, and / or altered humoral responses upon in vivo administration of the AAV vector, such as failure to produce AAV-directed neutralizing antibodies; and / or

[0513] - Improve the infection efficiency of AAV vectors; and / or

[0514] - Improve the transduction efficiency of AAV vectors to specific cells, tissues, and / or organs; and / or

[0515] - Reduce cytotoxicity during cell transduction; and / or

[0516] - Inducing targeted cell death in cancer cells; and / or

[0517] - To enable visualization / monitoring of AAV vectors after in vivo administration or in vitro cell modification; and / or

[0518] - To enable diagnostic applications; for example, by combining therapeutic and diagnostic agents.

[0519] In some embodiments, when the AAV vector is used as a drug, such as a gene vector for gene therapy, this modified property can lead to an increase in the therapeutic index of the AAV vector. In some embodiments, the increase in the therapeutic index of the AAV vector can be achieved by reducing the relative dose of AAV administered to the subject to achieve the desired therapeutic effect, and this dose reduction can reduce the relative toxicity of the AAV treatment regimen.

[0520] In some embodiments, the AAV vector of the present invention exhibits preference for organs or cells selected from the liver, heart, brain, joints, retina, and / or skeletal muscle. In some embodiments, the AAV vector of the present invention exhibits preference for cultured cells selected from, but not limited to, hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigment cells, photoreceptor cells, chondrocytes, hematopoietic stem cells (HSCs), and / or induced pluripotent stem cells (iPSCs).

[0521] Uses and methods for transducing cells

[0522] In some embodiments, the present invention relates to an AAV vector according to the invention, which is used for transducing cells of a subject.

[0523] "Transduced cells" herein refers to the delivery of nucleic acids into cells. The target nucleic acid for transduction can be of any type and is selected based on the desired effect. In some embodiments, when the AAV vector according to the invention is used for transduced cells, it contains transgenes.

[0524] In some embodiments, for example, the AAV vector may comprise a foreign gene expression cassette. In some embodiments, the cassette may comprise a promoter, a target gene, and a terminator. In some embodiments, as other or alternative examples, the AAV vector of the present invention may comprise a DNA template for homologous recombination in cells. In some embodiments, such AAV vectors may be used in combination with gene editing tools to promote homologous recombination in targeted cells. In some embodiments, the gene editing tools may be of any type and include, but are not limited to, CRISPR and related systems (including, but not limited to, Cas proteins such as Cas9 protein or its fusion protein, crRNA and tracrRNA, the latter two being separate or linked together in a single gRNA), TALEN, zinc finger nucleases, large-scale nucleases, and RNA and DNA encoding said gene editing proteins and related systems.

[0525] In some embodiments, the present invention also relates to the use of the AAV vector according to the invention for transducing subject cells.

[0526] In some embodiments, the invention also relates to a method for transducing subject cells, comprising administering the AAV vector according to the invention to the subject.

[0527] In some embodiments, the present invention also relates to a method of delivering transgenes to cells, the method comprising contacting cells with an AAV vector particle of formula (II) as defined and described in the categories and subclasses disclosed herein, and nucleic acids to be expressed in the contacted cells, particularly transgenes to be expressed in the contacted cells.

[0528] In some embodiments, the invention also relates to a method of delivering a transgene into the cells of a subject, comprising administering to the subject an AAV vector according to the invention containing the transgene.

[0529] In some embodiments, the invention also relates to in vitro or ex vivo methods for transducing cells, including contacting the cells with an AAV vector according to the invention. In some embodiments, the cells may be derived from a subject (e.g., a patient). In some embodiments, after transduction, the cells may be transplanted to a subject in need (e.g., a patient and / or another subject).

[0530] In some embodiments, the AAV vector can be applied to cells in vivo, in vitro, or ex vivo. In some embodiments, the cells may be derived from mammals (e.g., humans, non-human primates, cattle, mice, sheep, goats, pigs, rats, etc.). In some embodiments, the cells may be derived from humans. In some embodiments, the cells may be, but are not limited to, hepatocytes, cardiomyocytes, myocytes, neurons, motor neurons, retinal pigment cells, photoreceptor cells, chondrocytes, hematopoietic stem cells (HSCs), or induced pluripotent stem cells (iPSCs).

[0531] In some embodiments, the AAV vector according to the invention specifically transduces any one or more of the following cells: neurons (e.g., pyramidal neurons, Purkinje neurons, spindle neurons, intermediate spindle neurons and / or interneurons [e.g., Golgi cells, Lugaro cells, basket cells, astrocytes, candlestick cells, monopolar brush cells, granule cells, Lemsa cells, 1a inhibitory neurons, 1b inhibitory neurons, interneurons expressing parvalbumin, interneurons expressing CCK, interneurons expressing VIP, interneurons expressing SOM, cholinergic interneurons, interneurons expressing tyrosine hydroxylase, interneurons expressing calciretinoin or interneurons expressing nitric oxide synthase]), oligodendrocytes, astrocytes, microglia, ependymal cells, radial glial cells and / or posterior pituitary cells).

[0532] In some embodiments, the AAV vector according to the invention nonspecifically transduces one or more (or all) of the following cells: oligodendrocytes, astrocytes, microglia, ependymal cells, radial glial cells, and / or posterior pituitary cells.

[0533] In some embodiments, the AAV vector according to the present invention can target a variety of cells, tissues, and / or organs for therapeutic and / or preventative interventions. For example, in some embodiments, the AAV vector targets include, but are not limited to, hepatocytes; retinal cells; i.e., photoreceptor cells, retinal pigment epithelium (RPE), Müller cells; inner ear cells (e.g., inner hair cells and / or outer hair cells, Hensen cells, Deiters cells, column cells, phalangeal cells, limbic cells, etc.); muscle cells, i.e., myoblasts, satellite cells; cells of the central nervous system (CNS), i.e., neurons, glial cells; cardiac cells; cells of the peripheral nervous system (PNS); osteoblasts; tumor cells; blood cells, such as lymphocytes, monocytes, basophils, eosinophils, neutrophils, mast cells; hematopoietic cells, including hematopoietic stem cells; induced pluripotent stem cells (iPS), etc. Examples of tissues and organs that can be targeted by AAV include the eye, retina, ear, liver, skeletal muscle, cardiac muscle, smooth muscle, brain, spine, bone, connective tissue, heart, kidney, lung, lymph nodes, breast, myelin sheath, prostate, testis, thymus, thyroid gland, trachea, etc. In some embodiments, preferred cell types are hepatocytes, retinal cells, muscle cells, CNS cells, PNS cells, and / or hematopoietic cells. In some embodiments, preferred tissues and / or organs are the liver, muscle, heart, eye, and / or brain.

[0534] In some embodiments, if the AAV target described herein includes one or more cell types such as retinal cells, it is considered to target CNS cells; in some embodiments, targeting retinal cells is not considered to represent targeting the CNS.

[0535] Applications in gene therapy

[0536] In some implementations, the AAV vectors described herein can be used specifically for gene therapy, such as delivering targeted therapeutic nucleic acids to a subject.

[0537] Therefore, in some embodiments, the present invention also relates to an AAV vector according to the invention for gene therapy.

[0538] In some embodiments, the invention also relates to a method of gene therapy in subjects in need, comprising administering the AAV vector according to the invention to the subjects.

[0539] In some embodiments, the AAV of the present invention can be delivered to the subject via any suitable route. In some embodiments, suitable routes of administration include, but are not limited to, inhalation, local administration, intratissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal), intraconjunctival (e.g., intraretinal, subretinal), mucosal (e.g., oral cavity, nasal cavity), intraarticular, intravitreal, intracranial, intravascular (e.g., intravenous), intraventricular, intracisional, intraperitoneal, and intralymphatic routes. In some embodiments, the choice of route of administration depends on the target tissue and / or organ, i.e., on the tissue and / or organ for which transduction is sought.

[0540] In some embodiments, the AAV carrier according to the invention is administered via intraspinal and / or intracerebral administration.

[0541] In some embodiments, the AAV carrier according to the invention is administered intraspinally. In some embodiments, intraspinal administration includes or consists of intrathecal and epidural administration.

[0542] In some embodiments, the AAV carrier according to the invention is administered intracerebrally.

[0543] In some embodiments, the AAV carrier according to the invention is administered intracerebrally, wherein the intracerebral administration is performed at a site selected from or composed of the following groups: striatum (e.g., putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, globus pallidus and / or globus pallidus), thalamus, hypothalamus, epithalamus, thalamic base, parenchyma, cerebrum, medulla oblongata, deep cerebellar nuclei (e.g., substantia nigra, dentate nucleus, emboliated nucleus, globular nucleus and / or parietal nucleus), cerebrospinal fluid (CSF), meninges, dura mater, arachnoid mater, pia mater, subarachnoid cistern (e.g., cerebellomedullary cistern, pontine cistern, interpeduncular cistern, optic chiasm cistern, lateral ventricle cistern, superior cistern and / or lamina terminalis cistern), subarachnoid space, cortex, septum, pons and / or cerebellum.

[0544] In some embodiments, the AAV carrier according to the invention is applied intrastriatally (i.e., in the striatum, for example, in the putamen, caudate nucleus, nucleus accumbens, olfactory tubercle, globus pallidus and / or globus pallidus), intrathecally (i.e., in the thalamus), and intracisionally (i.e., in the subarachnoid cistern, for example, in the cerebellomedullary cistern, pontine cistern, interpeduncular cistern, optic chiasm cistern, lateral ventricle cistern, superior cistern and / or endplate cistern); preferably in the cerebellomedullary cistern).

[0545] In some embodiments, the condition treated by applying the AAV vector of the present invention can be of any type. For example, in some embodiments, the condition to be treated can be selected from infectious diseases and hereditary and acquired genetic diseases. In some embodiments, the target genetic disease includes, but is not limited to, hereditary muscle diseases such as Duchenne muscular dystrophy, leukodystrophy, spinal muscular atrophy (SMA), hemophilia, sickle cell disease, and hereditary retinal dystrophy. In some embodiments, the AAV vector of the present invention can also be used to treat diseases such as, but not limited to, cancer, arthritis, arthropathy, congenital and acquired heart disease, Parkinson's disease, Alzheimer's disease, and infectious diseases (e.g., hepatitis C).

[0546] In some preferred embodiments, the AAV carrier described herein can be used specifically for the prevention and / or treatment of ophthalmic diseases. Therefore, in some embodiments, the invention also relates to an AAV carrier according to the invention for the prevention and / or treatment of ophthalmic diseases. In some embodiments, the invention also relates to the use of the AAV carrier according to the invention in the preparation of medicaments for the prevention and / or treatment of ophthalmic diseases. In some embodiments, the invention also relates to a method of preventing and / or treating ophthalmic diseases in subjects in need, comprising administering the AAV carrier according to the invention to the subjects.

[0547] In some preferred embodiments, the AAV carriers described herein can also be used specifically for the prevention and / or treatment of central nervous system (CNS) diseases. "Central nervous system" or "CNS" refers to the brain and spinal cord, and is contrasted with "peripheral nervous system" or "PNS," which does not include the brain and spinal cord. In some embodiments, the eye, particularly the retina, is not considered part of the CNS. In some embodiments, the eye, and particularly the retina, may be considered part of the peripheral nervous system (PNS). Therefore, in some embodiments, the invention also relates to a modified AAV carrier according to the invention for the prevention or treatment of CNS diseases. In some embodiments, the invention also relates to the use of the modified AAV carrier according to the invention in the preparation of medicaments for the prevention or treatment of CNS diseases. In some embodiments, the invention also relates to a method of preventing and / or treating CNS diseases in a subject in need, comprising administering the modified AAV carrier according to the invention to the subject.

[0548] In some embodiments, the brain tissue may be or includes the striatum, thalamus, substantia nigra, parietal cortex, hippocampus, and / or globus pallidus. In some embodiments, the CNS sites are located in the striatum. In some embodiments, the CNS sites are located in the thalamus. In some embodiments, the CNS sites are located in the cerebellomedullary cistern.

[0549] As used herein, the term "prevent / preventing / prevention" refers to preventive and avoidance measures aimed at reducing the likelihood that a subject will develop a specific disease within a given time period. This reduction may be reflected, for example, in delaying the onset of at least one symptom of the subject's disease.

[0550] As used herein, the terms "treating" or "remission" refer to therapeutic treatment, excluding preventative or avoidance measures; the aim is to slow (relieve) the progression of a specific disease. The population requiring treatment includes those already suffering from the disease and those suspected of having it. A subject's specific disease is successfully "treated" if, after receiving a therapeutic dose of the AAV carrier according to the invention, one or more of the following are observed and / or measurably reduced or eliminated: one or more symptoms associated with the disease; reduced morbidity and mortality; and / or improved quality of life. The parameters described above used to assess successful treatment and improvement of the target disease can be easily measured using routine procedures familiar to physicians.

[0551] As used herein, the term "subject" refers to a mammal, preferably a human. In some embodiments, the subject may be a "patient," i.e., a warm-blooded animal, more preferably a human, who is awaiting or currently receiving medical care or has been / is about to undergo medical procedures, or is being monitored for disease progression. "Mammal" herein refers to any mammal, including humans, non-human primates, livestock and farm animals, as well as 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.

[0552] Composition

[0553] In some embodiments, the present invention also relates to compositions comprising an AAV vector according to the present invention. In some embodiments, the AAV vector in the compositions according to the present invention comprises at least one genetic material.

[0554] In some embodiments, the composition is a pharmaceutical composition comprising an AAV carrier according to the invention and at least one pharmaceutically acceptable carrier.

[0555] The term "pharmaceuticalally acceptable," when referring to mediators, excipients, carriers, and / or preservatives, means a molecular entity or composition that, when administered to a subject, preferably a human, does not produce allergic reactions or similar adverse effects. For human use, the pharmaceutical composition should meet the sterility, pyrogenicity, and general safety and purity standards required by regulatory agencies (e.g., the FDA Office or EMA).

[0556] In some embodiments, pharmaceutically acceptable mediators, excipients, carriers, and preservatives that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, proteins (e.g., serum albumin, gelatin, immunoglobulins, etc.), buffering substances (e.g., phosphates, citrates, or other organic acids, etc.), amino acids (e.g., glycine, glutamine, asparagine, arginine, lysine, etc.), antioxidants (e.g., ascorbic acid, etc.), chelating agents (e.g., EDTA), sorbic acid, potassium sorbate, mixtures of metaglycerides of saturated vegetable fatty acids, water, salts or electrolytes (e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, etc.), hydrophilic polymers (e.g., polyvinylpyrrolidone, polyethylene-polyoxypropylene block polymers, etc.), cellulose-based substances (e.g., sodium carboxymethyl cellulose), polyacrylates, waxes, nonionic surfactants (e.g., Tween, Pluronics, polyethylene glycol, etc.), lanolin, and suitable combinations thereof.

[0557] In some embodiments, the pharmaceutical compositions according to the invention comprise pharmaceutically acceptable mediators for preparing formulations to be injected into subjects. In some embodiments, these may be, in particular, isotonic, sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of these salts), or dry, in particular lyophilized compositions, which, depending on the circumstances, can be formulated into injectable solutions by adding sterile water or physiological saline.

[0558] In some embodiments, the pharmaceutical composition according to the invention comprises one or more agents that promote the entry of the AAV carrier described herein into mammalian cells, such as natural and / or synthetic polymers, such as poloxamer, chitosan, cyclodextrin, dendrimers, poly(lactic acid-co-glycolic acid) polymers, etc.

[0559] In some embodiments, an AAV vector comprising at least one genetically modified organism according to the invention is included as part of a medicament. Therefore, in some embodiments, the present invention relates to a medicament comprising an AAV vector containing at least one genetically modified organism according to the invention.

[0560] Genetically modified organisms / disease combination

[0561] As described above, the AAV vector according to the present invention may contain at least one transgene, selected according to the intended use of the AAV vector. Examples of transgenes that can be used to treat ophthalmic diseases or CNS diseases are provided below. In some embodiments, the eye is not considered a "CNS" site. In some embodiments, the eye may be considered a "PNS" site.

[0562] In some embodiments, the ophthalmic diseases include hereditary retinal diseases. In some embodiments, hereditary retinal diseases include, but are not limited to, Leber congenital amaurosis, retinitis pigmentosa, punctate retinal degeneration, choroidal agenesis, Stargardt's disease, retinal dystrophy, choroidal dystrophy, cone dystrophy, cone-rod dystrophy, rod-cone dystrophy, macular dystrophy, and macular degeneration. In some embodiments, the ophthalmic diseases include infectious diseases, such as infectious diseases (e.g., viral, bacterial, fungal, etc.). In some embodiments, the ophthalmic diseases include trauma. In some embodiments, the ophthalmic diseases include autoimmune diseases. In some embodiments, the ophthalmic diseases include cancer.

[0563] In some implementations, CNS disease is an infectious disease of the CNS, a degenerative disease of the CNS, an autoimmune disease of the CNS, a neoplastic disease of the CNS, a cerebrovascular disease, a damage to the CNS, or a structural defect of the CNS.

[0564] In some implementations, CNS diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Friedreich ataxia, Canavan disease, muscular dystrophy, spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), Gaucher disease, adrenoleukodystrophy, Angelman syndrome, or epilepsy.

[0565] In some specific embodiments, the CNS disease is Parkinson's disease or Gaucher disease.

[0566] In some specific implementations, the gene involved in CNS diseases is the GBA gene, preferably the human GBA gene.

[0567] In some embodiments, the AAV carrier of the present invention can effectively transduce certain regions of the brain, including the striatum, thalamus, substantia nigra, parietal cortex, hippocampus, and / or globus pallidus. Therefore, in some embodiments, the AAV carrier of the present invention is of great significance for targeting the striatum, thalamus, substantia nigra, parietal cortex, hippocampus, and globus pallidus, and / or for treating diseases affecting these regions.

[0568] In some embodiments, the AAV carrier of the present invention is particularly suitable for treating diseases of the striatum, substantia nigra, thalamus, substantia nigra, globus pallidus, parietal cortex and / or hippocampus, including but not limited to Huntington's disease, Parkinson's disease, multiple sclerosis atrophy, Lewy body dementia (LBD), progressive supranuclear palsy and Angelman syndrome.

[0569] In some embodiments, the AAV vector of the present invention can effectively transduce neurons. Therefore, in some embodiments, CNS diseases are neurological diseases or diseases affecting neurons.

[0570] In some embodiments, the AAV vector of the present invention can effectively transduce neurons involved in motor function control. Therefore, in some embodiments, CNS diseases are diseases that affect motor function. Non-limiting examples of diseases affecting motor function include, but are not limited to, Parkinson's disease and Huntington's disease.

[0571] In some specific embodiments, the CNS disease is Parkinson's disease, and the AAV vector of the present invention has at least one transgene containing cDNA of the GBA gene, preferably the human GBA gene.

[0572] In some embodiments, the CNS disease is muscular dystrophy, and the AAV vector of the present invention has at least one transgene, wherein the transgene comprises or consists of cDNA selected from the group consisting of the DMD gene.

[0573] In some embodiments, the CNS disease is Gaucher disease, and the AAV vector of the present invention has at least one transgene, wherein the transgene comprises cDNA of the GBA gene.

[0574] Those skilled in the art will recognize that genes can have multiple transcriptional and / or translational isotypes, and that transgenes containing cDNA of the genes described herein cover the potential uses of transcriptional variants and / or splicing variants of the target gene.

[0575] Application plan

[0576] In some embodiments, the modified AAV carrier according to the invention is administered to a subject in need at a therapeutically effective amount.

[0577] In some embodiments, the modified AAV carrier according to the invention is administered in the following dosage range: about 10 8 Viral genome (vg) - approximately 10 15 vg, such as about 10 8 vg - approximately 10 14 vg, approximately 10 8 vg - approximately 10 13 vg, approximately 10 8 vg - approximately 10 12 vg, approximately 10 8 vg - approximately 10 11 vg, approximately 10 8 vg - approximately 10 10 vg, approximately 10 8 vg - approximately 10 9 vg, approximately 10 9vg - approximately 10 15 vg, approximately 10 9 vg - approximately 10 14 vg, approximately 10 9 vg - approximately 10 13 vg, approximately 10 9 vg - approximately 10 12 vg, approximately 10 9 vg - approximately 10 11 vg, approximately 10 9 vg - approximately 10 10 vg, approximately 10 10 vg - approximately 10 15 vg, approximately 10 10 vg - approximately 10 14 vg, approximately 10 10 vg - approximately 10 13 vg, from about 10 10 vg - approximately 10 12 vg, approximately 10 10 vg - approximately 10 11 vg, approximately 10 11 vg - approximately 10 15 vg, approximately 10 11 vg - approximately 10 14 vg, approximately 10 11 vg - approximately 10 13 vg, approximately 10 11 vg - approximately 10 12 vg, approximately 10 12 vg - approximately 10 15 vg, approximately 10 12 vg - approximately 10 14 vg, approximately 10 12 vg - approximately 10 13 vg, or approximately 10 13 vg - approximately 10 15 vg.

[0578] The term "vector genome," abbreviated as "vg," refers to one or more polynucleotide sequences comprising a vector (e.g., a viral vector). The vector genome may be encapsulated within a viral particle. Depending on the specific viral vector, the vector genome may contain single-stranded DNA, double-stranded DNA, or single-stranded or double-stranded RNA. The vector genome may include endogenous sequences associated with the specific viral vector and / or any heterologous sequences inserted into the specific viral vector via recombination techniques (e.g., transgenics). In some embodiments, the nucleic acid titer of the viral vector may be measured in vg / mL. Suitable methods for determining this titer are known in the art and include, for example, quantitative PCR.

[0579] As used herein, the term “about” when placed before a numerical value means that the value is approximate and that small variations will not significantly affect the practice of the disclosed embodiments. Such small variations are, for example, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10% or more.

[0580] In some embodiments, the modified AAV carrier according to the invention is administered at a dose of approximately 1 × 10⁻⁶. 8 vg±0.5×10 8 Approximately 2×10 8 vg±0.5×10 8 Approximately 2.75 × 10 8 vg±0.5×10 8 Approximately 3×10 8 vg±0.5×10 8 Approximately 4×10 8 vg±0.5×10 8 Approximately 5×10 8 vg±0.5×10 8 Approximately 6×10 8 vg±0.5×10 8 Approximately 7×10 8 vg±0.5×10 8 Approximately 8×10 8 vg±0.5×10 8 Approximately 9×10 8 vg±0.5×10 8 Approximately 1×10 9 vg±0.5×10 9 Approximately 2×10 9 vg±0.5×10 9 Approximately 3×10 9 vg±0.5×10 9 Approximately 4×10 9 vg±0.5×10 9 Approximately 5×10 9 vg±0.5×10 9 Approximately 6×10 9 vg±0.5×10 9 Approximately 7×10 9 vg±0.5×10 9 Approximately 8×10 9 vg±0.5×10 9 Approximately 9×10 9 vg±0.5×10 9 Approximately 1×10 10 vg±0.5×10 10 Approximately 2×10 10vg±0.5×10 10 Approximately 3×10 10 vg±0.5×10 10 Approximately 4×10 10 vg±0.5×10 10 Approximately 5×10 10 vg±0.5×10 10 Approximately 6×10 10 vg±0.5×10 10 Approximately 7×10 10 vg±0.5×10 10 Approximately 8×10 10 vg±0.5×10 10 Approximately 9×10 10 vg±0.5×10 10 Approximately 1×10 11 vg±0.5×10 11 Approximately 2×10 11 vg±0.5×10 11 Approximately 3×10 11 vg±0.5×10 11 Approximately 4×10 11 vg±0.5×10 11 Approximately 5×10 11 vg±0.5×10 11 Approximately 6×10 11 vg±0.5×10 11 Approximately 7×10 11 vg±0.5×10 11 Approximately 8×10 11 vg±0.5×10 11 Approximately 9×10 11 vg±0.5×10 11 Approximately 1×10 12 vg±0.5×10 12 Approximately 2×10 12 vg±0.5×10 12 Approximately 3×10 12 vg±0.5×10 12 Approximately 4×10 12 vg±0.5×10 12 Approximately 5×10 12 vg±0.5×10 12 Approximately 6×10 12 vg±0.5×10 12 Approximately 7×10 12 vg±0.5×10 12 Approximately 8×10 12 vg±0.5×1012 Approximately 9×10 12 vg±0.5×10 12 Approximately 1×10 13 vg±0.5×10 13 Approximately 2×10 13 vg±0.5×10 13 Approximately 3×10 13 vg±0.5×10 13 Approximately 4×10 13 vg±0.5×10 13 Approximately 5×10 13 vg±0.5×10 13 Approximately 6×10 13 vg±0.5×10 13 Approximately 7×10 13 vg±0.5×10 13 Approximately 8×10 13 vg±0.5×10 13 Approximately 9×10 13 vg±0.5×10 13 Approximately 1×10 14 vg±0.5×10 14 Approximately 2×10 14 vg±0.5×10 14 Approximately 3×10 14 vg±0.5×10 14 Approximately 4×10 14 vg±0.5×10 14 Approximately 5×10 14 vg±0.5×10 14 Approximately 6×10 14 vg±0.5×10 14 Approximately 7×10 14 vg±0.5×10 14 Approximately 8×10 14 vg±0.5×10 14 Approximately 9×10 14 vg±0.5×10 14 Approximately 1×10 15 vg±0.5×10 15 Approximately 2×10 15 vg±0.5×10 15 Approximately 3×10 15 vg±0.5×10 15 Approximately 4×10 15 vg±0.5×10 15 Approximately 5×10 15 vg±0.5×10 15 Approximately 6×1015 vg±0.5×10 15 Approximately 7×10 15 vg±0.5×10 15 Approximately 8×10 15 vg±0.5×10 15 or approximately 9×10 15 vg±0.5×10 15 .

[0581] In some embodiments, the modified AAV carrier according to the invention is administered at a dose of approximately 1 × 10⁻⁶. 6 vg / kg±0.5×10 6 Approximately 2×10 6 vg / kg±0.5×10 6 Approximately 3×10 6 vg / kg±0.5×10 6 Approximately 4×10 6 vg / kg±0.5×10 6 Approximately 5×10 6 vg / kg±0.5×10 6 Approximately 6×10 6 vg / kg±0.5×10 6 Approximately 7×10 6 vg / kg±0.5×10 6 Approximately 8×10 6 vg / kg±0.5×10 6 Approximately 9×10 6 vg / kg±0.5×10 6 Approximately 1×10 7 vg / kg±0.5×10 7 Approximately 2×10 7 vg / kg±0.5×10 7 Approximately 3×10 7 vg / kg±0.5×10 7 Approximately 4×10 7 vg / kg±0.5×10 7 Approximately 5×10 7 vg / kg±0.5×10 7 Approximately 6×10 7 vg / kg±0.5×10 7 Approximately 7×10 7 vg / kg±0.5×10 7 Approximately 8×10 7 vg / kg±0.5×10 7 Approximately 9×10 7 vg / kg±0.5×107 Approximately 1×10 8 vg / kg±0.5×10 8 Approximately 2×10 8 vg / kg±0.5×10 8 Approximately 3×10 8 vg / kg±0.5×10 8 Approximately 4×10 8 vg / kg±0.5×10 8 Approximately 5×10 8 vg / kg±0.5×10 8 Approximately 6×10 8 vg / kg±0.5×10 8 Approximately 7×10 8 vg / kg±0.5×10 8 Approximately 8×10 8 vg / kg±0.5×10 8 Approximately 9×10 8 vg / kg±0.5×10 8 Approximately 1×10 9 vg / kg±0.5×10 9 Approximately 2×10 9 vg / kg±0.5×10 9 Approximately 3×10 9 vg / kg±0.5×10 9 Approximately 4×10 9 vg / kg±0.5×10 9 Approximately 5×10 9 vg / kg±0.5×10 9 Approximately 6×10 9 vg / kg±0.5×10 9 Approximately 7×10 9 vg / kg±0.5×10 10 Approximately 8×10 9 vg / kg±0.5×10 9 Approximately 9×10 9 vg / kg±0.5×10 9 Approximately 1×10 10 vg / kg±0.5×10 10 Approximately 2×10 10 vg / kg±0.5×10 10 Approximately 3×10 10 vg / kg±0.5×10 10 Approximately 4×10 10 vg / kg±0.5×10 10 Approximately 5×10 10vg / kg±0.5×10 10 Approximately 6×10 10 vg / kg±0.5×10 10 Approximately 7×10 10 vg / kg±0.5×10 10 Approximately 8×10 10 vg / kg±0.5×10 10 Approximately 9×10 10 vg±0.5×10 10 Approximately 1×10 11 vg / kg±0.5×10 11 Approximately 2×10 11 vg / kg±0.5×10 11 Approximately 3×10 11 vg / kg±0.5×10 11 Approximately 4×10 11 vg / kg±0.5×10 11 Approximately 5×10 11 vg / kg±0.5×10 11 Approximately 6×10 11 vg / kg±0.5×10 11 Approximately 7×10 11 vg / kg±0.5×10 11 Approximately 8×10 11 vg / kg±0.5×10 11 Approximately 9×10 11 vg / kg±0.5×10 11 Approximately 1×10 12 vg / kg±0.5×10 12 Approximately 2×10 12 vg / kg±0.5×10 12 Approximately 3×10 12 vg / kg±0.5×10 12 Approximately 4×10 12 vg / kg±0.5×10 12 Approximately 5×10 12 vg / kg±0.5×10 12 Approximately 6×10 12 vg / kg±0.5×10 12 Approximately 7×10 12 vg / kg±0.5×10 12 Approximately 8×10 12 vg / kg±0.5×10 12 Approximately 9×10 12 vg / kg±0.5×10 12 Approximately 1×1013 vg / kg±0.5×10 13 Approximately 2×10 13 vg / kg±0.5×10 13 Approximately 3×10 13 vg / kg±0.5×10 13 Approximately 4×10 13 vg / kg±0.5×10 13 Approximately 5×10 13 vg / kg±0.5×10 13 Approximately 6×10 13 vg / kg±0.5×10 13 Approximately 7×10 13 vg / kg±0.5×10 13 Approximately 8×10 13 vg / kg±0.5×10 13 Approximately 9×10 13 vg / kg±0.5×10 13 or about 1×10 14 vg / kg±0.5×10 14 .

[0582] In some embodiments, the dosage of the modified AAV vector required to achieve the desired or therapeutic effect will vary depending on several factors, including but not limited to the specific route of administration, the gene, RNA, or protein expression level required to achieve the therapeutic effect, the specific disease being treated, and the stability of the gene, RNA, and / or protein product. Those skilled in the art can adjust the dosage and / or determine dosage ranges based on the foregoing factors and other factors well known in the art to treat specific subjects and / or specific diseases.

[0583] In some implementations, the volume of the modified AAV carrier administered to the subject will also depend on the size of the subject, the dose of AAV carrier required to achieve a therapeutic effect, the concentration of the AAV carrier, and the recommended route of administration, etc.

[0584] In some embodiments, the administration rate of the AAV carrier delivered to the subject will also depend on the size of the subject, the dose of AAV carrier required to achieve a therapeutic effect, the concentration of the AAV carrier, the volume of the AAV carrier solution, and the recommended route of administration. For example, in some embodiments, for intracerebral administration, an administration rate of about 0.1 μL / min to about 1 μL / min, or about 1 μL / min to about 5 μL / min, or about 5 μL / min to about 10 μL / min can be used.

[0585] In some embodiments, the administration rate of the AAV carrier to the subject is approximately 0.1 μL / min ± 0.05 μL / min, approximately 0.2 μL / min ± 0.05 μL / min, approximately 0.3 μL / min ± 0.05 μL / min, approximately 0.4 μL / min ± 0.05 μL / min, approximately 0.5 μL / min ± 0.05 μL / min, approximately 0.6 μL / min ± 0.05 μL / min, approximately 0.7 μL / min ± 0.05 μL / min, approximately 0.8 μL / min ± 0.05 μL / min, and approximately 0.9 μL / min ± 0.05 μL / min. μL / min±0.05μL / min, about 1μL / min±0.5μL / min, about 2μL / min±0.5μL / min, about 3μL / min±0.5μL / min, about 4μL / min±0.5μL / min, about 5μL / min±0 .5μL / min, about 6μL / min±0.5μL / min, about 7μL / min±0.5μL / min, about 8μL / min±0.5μL / min, about 9μLmin±0.5μL / min or about 10μL / min±0.5μL / min.

[0586] In some embodiments, the total dose or total volume of the AAV carrier may be administered continuously (i.e., wherein the total dose or total volume of the modified AAV carrier is administered in a single injection or infusion); or discontinuously (i.e., wherein a portion of the total dose or total volume of the AAV carrier is administered intermittently between each injection, preferably in short intermittent intervals such as 15 seconds, 30 seconds, 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes).

[0587] Reagent test kit

[0588] This invention also relates to reagent kits and kits for use in:

[0589] - Transduced cells from the subject; and / or

[0590] - Delivery of transgenic material to subjects; and / or

[0591] -Prevention and / or treatment of diseases in the subject.

[0592] In some embodiments, the kit or kit package contains one or more AAV vectors and / or compositions according to the present invention.

[0593] In some embodiments, the kit or kit package further includes means for delivering one or more AAV carriers and / or compositions according to the invention.

[0594] In some embodiments, the kit further includes instructions for delivering one or more AAV vectors and / or compositions according to the invention. In some embodiments, the kit includes instructions for using the compositions and / or methods described herein for the prevention and / or treatment of a target disease.

[0595] In some embodiments, the kits described herein may further include other materials required from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and / or packaging inserts with instructions for carrying out any of the methods described herein. Attached Figure Description

[0596] Figure 1 After a single bilateral injection of AAV2-GFP or (3)-AAV2-GFP into the striatum, mouse brain sections were stained with GFP at the striatum level.

[0597] Example

[0598] The starting products used are commercial products or products prepared from commercial compounds according to known synthetic methods or products known to those skilled in the art.

[0599] The structures of the compounds described in the examples were determined using commonly used spectrophotometric techniques (nuclear magnetic resonance (NMR), liquid chromatography-mass spectrometry (LC / MS), and purity determination by high performance liquid chromatography (HPLC)).

[0600] The synthetic intermediates and compounds of this invention are named according to the IUPAC (The International Union of Pure and Applied Chemistry) nomenclature and described in a neutral form.

[0601] Use the following abbreviations:

[0602] ACN: Acetonitrile

[0603] BnBr: Benzyl bromide

[0604] Boc: tert-Butoxycarbonyl

[0605] CH2Cl2 or DCM: Dichloromethane

[0606] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0607] DIC: N,N'-Diisopropylcarbodiimide

[0608] DIEA: N,N'-Diisopropylethylamine

[0609] DIPEA: N,N-Diisopropylethylamine

[0610] DMF: Dimethylformamide

[0611] DMSO: Dimethyl sulfoxide

[0612] DTT: Dithiothreitol

[0613] EtOAc: Ethyl acetate

[0614] EtOH: Ethanol

[0615] FA: Formic acid

[0616] Fmoc: fluorenylmethoxycarbonyl

[0617] HATU: Hexafluorophosphate azirzotriazole tetramethylurea

[0618] HCl: hydrochloric acid

[0619] H2O: water

[0620] m-CPBA: m-chloroperoxybenzoic acid

[0621] MeOH: Methanol

[0622] MgSO4: Magnesium sulfate

[0623] Na2SO4: Sodium sulfate

[0624] NaH: Sodium hydride

[0625] NaHCO3: Sodium bicarbonate

[0626] NaN3: Sodium azide

[0627] NEt3 or TEA: Triethylamine

[0628] NH3: ammonia

[0629] PEG: Polyethylene Glycol

[0630] Pd / C: Palladium on carbon

[0631] RT: Room temperature

[0632] tBu: tert-butyl

[0633] TFA: Trifluoroacetic acid

[0634] TBAF: Tetra-n-Butylammonium Fluoride

[0635] Bu4NBr: Tetrabutylammonium bromide

[0636] TBDMSCl: tert-butyldimethylchlorosilane

[0637] TMSOTf: Trimethylsilyl trifluoromethanesulfonate

[0638] TIPS: Triisopropylsilane

[0639] LC / MS: Liquid Chromatography-Mass Spectrometry

[0640] HPLC: High Performance Liquid Chromatography

[0641] UPLC: Ultra-high performance liquid chromatography

[0642] NMR: Nuclear Magnetic Resonance

[0643] HPLC / MS Method for Determination of Z-Terminal Purity of Sugars

[0644] Equipment: Shimadzu LCMS-2020 single quadrupole liquid chromatography-mass spectrometry, HPLC-Shimadzu Nexera-iLC-2040C 3D with DAD detector

[0645] Column: Gemini-NX 3μm C18, 4.6*50mm, or equivalent

[0646] Column temperature: 30℃

[0647] Eluent: A = H₂O, B = ACN

[0648] Flow rate: 0.5 mL / min

[0649] Gradient condition:

[0650]

[0651] Analysis time: 12 min

[0652] UV / UPLC method for determining the purity of peptide Z-terminal groups:

[0653] Tools: UPLC Acquity HClass

[0654] Column: BEHC18 (WATERS), 150*2.1mm, or equivalent.

[0655] Column temperature: 60℃

[0656] Eluent: A = H₂O (0.1% TFA), B = ACN (0.1% TFA)

[0657] Flow rate: 0.5 mL / min

[0658] Gradient condition: 0-100% B within 10 minutes

[0659] Detection: 214nm

[0660] Preparation of 1:4-(((2-ethoxy-3,4-dioxobut-1-en-1-yl)amino)methyl)benzoic acid

[0661]

[0662] Add 3,4-diethoxycyclobut-3-ene-1,2-dione (3.1 g) to a mixture of 4-(aminomethyl)benzoic acid (2.5 g) and DIPEA (4.3 mL) in ethanol. Stir the reaction mixture at room temperature for 3 hours, then dilute with H₂O and treat with 1 M HCl until pH = 4. Filter off the resulting precipitate, wash with H₂O, and dry. Purify the crude substance by preparative reversed-phase HPLC using ACN / H₂O as eluent. Combine the fractions containing the pure product, concentrate to dryness under reduced pressure to give the desired intermediate (0.42 g, yield 43.7%) as a yellow solid. LC / MS (6 min): RT = 2.05 min, Measured [M+H] + =275.75. 1 H NMR(300MHz,DMSO-d6)δ(ppm):12.95(s,1H),9.40–8.99(m,1H),8.01–7.84(m,2 H),7.42(d,J=8.0Hz,2H),4.65(ddd,J=34.1,26.5,6.4Hz,4H),1.49–1.18(m,3H)

[0663] Preparation of 2:4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoic acid

[0664]

[0665] 3,4-diethoxycyclobut-3-ene-1,2-dione (6.8 g) was added to a mixture of 4-aminobenzoic acid (5.0 g) and DIPEA (9.5 mL) in ethanol. The reaction mixture was stirred at room temperature for 3 hours, then diluted with H2O and treated with 1 M HCl until pH = 4. The resulting precipitate was filtered, washed with H2O, and dried. The crude sample was purified by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated under reduced pressure to give the desired intermediate (0.249 g, yield 36.8%) as a yellow solid. LC / MS (6 min): RT = 2.1 min, Measured [M+H] + =261.75. 1H NMR (300MHz, DMSO-d6) δ (ppm): 12.80 (s, 1H), 11.00 (s, 1H), 7.92 (d, J = 8.8Hz, 2H), 7.49 (d, J = 8.8Hz, 2H), 4.80 (q, J = 7.1Hz, 2H), 1.44 (t, J = 7.1Hz, 3H).

[0666] Preparation of 3:4-(2-((2-ethoxy-3,4-dioxane-1-en-1-yl)amino)ethyl)benzoic acid

[0667]

[0668] Add 560 mg of 3,4-diethoxycyclobut-3-ene-1,2-dione to a mixture of 500 mg of 4-(2-aminoethyl)benzoic acid and 0.79 mL of DIPEA in ethanol. Stir the reaction mixture overnight at room temperature, then dilute with H₂O and treat with 1 M HCl until pH = 4. Filter the resulting precipitate, wash with H₂O, and dry. Purify the crude product by rapid column chromatography using DCM / MeOH as eluent. Combine the fractions containing the purified product, concentrate to dryness under reduced pressure to give the desired intermediate (310 mg, 35% yield) as a yellow solid. LC / MS (6 min): RT = 2.1 min, Measured [M+H] + 289.75, [MH] - 287.70. 1 H NMR(300MHz,DMSO-d6)δ(ppm):12.72(s,1H),8.92–8.61(m,1H),7.94–7.80(m,2H),7.39–7.25(m,2H),4 .68–4.52(m,2H),3.74(d,J=6.7Hz,1H),3.54(q,J=6.6Hz,1H),2.96–2.85(m,2H),1.33(q,J=7.3Hz,3H)

[0669] Preparation of 4:3-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoic acid

[0670]

[0671] 3,4-Diethoxycyclobut-3-ene-1,2-dione (1.4 g) was added to a mixture of 3-aminobenzoic acid (1.0 g) and DIPEA (1.9 mL) in ethanol. The reaction mixture was stirred overnight at room temperature, then diluted with H2O and treated with 1 M HCl until pH = 4. The resulting precipitate was filtered, washed with H2O, and dried. The crude product was purified by rapid column chromatography using DCM / MeOH as eluent. Fractions containing the purified product were combined and concentrated to dryness under reduced pressure to give the desired intermediate (1.17 g, 61% yield) as a yellow solid. LC / MS (6 min): RT = 2.07 min, Measured [M+H] + 261.70, [MH] - 259.65. 1 H NMR (400MHz, DMSO-d6) δ (ppm): 13.05 (s, 1H), 10.89 (s, 1H), 7.98 (t, J = 1.9 Hz, 1H), 7.67 (dt, J = 7.6, 1 .3Hz,1H),7.60(d,J=8.1Hz,1H),7.47(t,J=7.9Hz,1H),4.78(q,J=7.0Hz,2H),1.42(t,J=7.1Hz,3H)

[0672] Preparation of 5:2-(4-((2-ethoxy-3,4-dioxane-1-en-1-yl)amino)phenyl)acetic acid

[0673] Add 1.2 g of 3,4-diethoxycyclobut-3-ene-1,2-dione to a mixture of 1.0 g of 4-aminophenylacetic acid and 1.7 mL of DIPEA in ethanol. Stir the reaction mixture overnight at room temperature, then dilute with H₂O and treat with 1 M HCl until pH = 4. Filter the resulting precipitate, wash with H₂O, and dry. Purify the crude product by rapid column chromatography using DCM / MeOH as eluent. Combine the fractions containing the purified product, concentrate to dryness under reduced pressure to give the desired intermediate (1.3 g, 71% yield) as a yellow solid. LC / MS (6 min): RT = 2.3 min, Measured [M+H] + 276.0, [MH] - 274.15. 1H NMR (400MHz, DMSO-d6) δ (ppm): 12.31 (s, 1H), 10.74 (s, 1H), 7.29 (d, J = 8.1Hz, 2H) ,7.22(d,J=8.6Hz,2H),4.76(q,J=7.1Hz,2H),3.53(s,2H),1.41(t,J=7.1Hz,3H)

[0674] Preparation of 6:5-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)pyridinecarboxylic acid

[0675] Step 1: Synthesis of 5-aminopyridinecarboxylic acid

[0676]

[0677] Pd / C was added to a solution of 500 mg 5-nitropyridinecarboxylic acid in methanol under an argon atmosphere. The inert gas was evacuated and backfilled with hydrogen (three times in total). The reaction mixture was stirred overnight under a hydrogen atmosphere (balloon). The resulting mixture was stirred overnight at room temperature. It was then filtered through a diatomaceous earth mat and concentrated under reduced pressure to give 5-aminopyridinecarboxylic acid (425 mg, yield quantified) as a solid. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 7.96 (d, J = 2.6Hz, 1H), 7.74 (d, J = 8.5Hz, 1H), 6.93 (dd, J = 8.5, 2.7Hz, 1H), 6.17–5.99 (m, 2H).

[0678] Step 2: 5-((2-ethoxy-3,4-dioxobut-1-en-1-yl)amino)pyridinecarboxylic acid

[0679]

[0680] 3,4-diethoxycyclobut-3-ene-1,2-dione (572 mg) was added to a mixture of 5-aminopyridinecarboxylic acid (422 mg) and TEA (0.56 mL) in ethanol. The reaction mixture was stirred at room temperature for 3 hours, then diluted with H2O and treated with 1 M HCl until pH = 4. The resulting precipitate was filtered off, washed with H2O, and dried to give the desired intermediate (609 mg, yield 76%) as a yellow solid. LCMS (6 min): RT = 2.0 min, measured [M+H] + 263.00; [MH] - 261.15. 1HNMR(300MHz,DMSO-d6)δ(ppm):11.16(s,1H),10.48(s,1H),8.76–8.50(m,2H),8.13– 8.00(m,3H),7.92(dd,J=8.6,2.7Hz,1H),4.81(q,J=7.1Hz,2H),1.44(t,J=7.1Hz,3H)

[0681] Example 1: Preparation of compound (III).

[0682] Example 1.1 - Synthesis of compound (1):

[0683] 3-Ethoxy-4-((2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)amino)cyclobut-3-ene-1,2-dione

[0684] Step 1: Synthesis of (3S,4S,5S,6R)-2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0685]

[0686] Under an argon atmosphere, Pd / C (0.16 g) was added to a methanol solution of (3S,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (1.0 g) as described in application WO2022096681. The reaction mixture was stirred overnight under a hydrogen atmosphere, filtered through a diatomaceous earth pad, and the filtrate was concentrated to dryness under reduced pressure. The crude product was used in the next step without further purification (0.74 g, 80% yield). LC / MS (6 min): RT = 0.5 min, measured [M+H] + =312.10

[0687] Step 2: Synthesis of 3-ethoxy-4-((2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)amino)cyclobut-3-ene-1,2-dione

[0688]

[0689] Under an argon atmosphere, 0.09 mL of TEA was added to a solution of the aforementioned compound (150 mg) in EtOH, followed by 90 mg of 3,4-diethoxycyclobut-3-ene-1,2-dione. The reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure to dryness. The residue was purified by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated to dryness under reduced pressure. The resulting oil was dissolved in a small amount of H2O, the solution was frozen, and then freeze-dried to give the final substance (0.111 g, yield 53%) as a colorless, hard oil. LC / MS (12 min): RT = 6.28 min, measured [M+H] + =436.1; [MH] - =434.0. HPLC purity: 95.6% (200nm), 96.6% (270nm) 1 H NMR(300MHz,DMSO-d6)δ(ppm):8.92–8.50(m,1H),4.80–4.59(m,5H),4.54(d, J=5.8Hz,1H),4.42(t,J=6.0Hz,1H),3.77–3.35(m,18H),1.37(t,J=7.0Hz,3H)

[0690] Example 1.2 - Synthesis of compound (2):

[0691] 4-(((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)methyl)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)benzamide

[0692]

[0693] Under an argon atmosphere, the compound (150 mg) described in step 1 of Example 1 was added to a mixture of intermediate 4-(((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)methyl)benzoic acid (159 mg), HATU (220 mg), and DIPEA (0.25 mL) in DMF. The reaction mixture was stirred at room temperature for 2 hours, and then the solvent was removed under reduced pressure. The residue was purified by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated to dryness under reduced pressure. The resulting oily substance was dissolved in a small amount of H2O, the solution was frozen, and then freeze-dried to give the final substance (36 mg, yield 13.2%) as a white powder. LC / MS (12 min): RT = 7.28 min, [M+H+ =569.1; [MH] - =567.1 HPLC purity: 95.5% (200nm), 98.2% (270nm). 1 H NMR(300MHz, DMSO-d6)δ(ppm): δ9.39–8.96(m,1H),8.50(t,J=5.5Hz,1H),7.84(d,J=8.1Hz,2H),7.38(d,J=8.1Hz,2 H),4.85–4.60(m,5H),4.55(d,J=5.9Hz,2H),4.43(t,J=5.9Hz,1H),3.76–3.34(m,16H),1.35(dt,J=14.2,7.0Hz,3H)

[0694] Example 1.3 - Synthesis of compound (3):

[0695] 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)benzamide

[0696]

[0697] Under an argon atmosphere, the compound obtained in step 1 of Example 1 was added to a mixture of the intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoic acid (151 mg), HATU (220 mg), and DIPEA (0.25 mL) in DMF. The reaction mixture was stirred at room temperature for 2 hours. The solvent was then evaporated under reduced pressure. The residue was purified by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated under reduced pressure. The resulting oil was dissolved in a small amount of H2O, the solution was frozen, and then freeze-dried to give the final substance (66 mg, yield 40.3%) as a white powder. LC / MS (12 min): RT = 4.79 min, [M+H] + =555.1; [MH] - =553.1. HPLC purity: 99.9% (200nm), 99.8% (315nm). 1H NMR (300MHz, DMSO-d6) δ (ppm): 10.86 (s, 1H), 8.44 (t, J = 5.6Hz, 1H), 7.84 (d, 2H), 7.43 (d, J = 8.5Hz, 2H), 4.79 (q, J = 7.1Hz, 2H), 4.70 ( dd,J=5.8,4.6Hz,2H),4.63(d,J=1.6Hz,1H),4.54(d,J=5.8Hz,1H),4.43(t,J=6.0Hz,1H),3.76–3.35(m,18H),1.44(t,J=7.1Hz,3H)

[0698] Example 1.4 - Synthesis of compound (4):

[0699] 3-Ethoxy-4-((14-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)amino)cyclobut-3-ene-1,2-dione

[0700] Step 1: Preparation of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate

[0701]

[0702] Mannosylpentaacetate (5.0 g) was dissolved in anhydrous DCM under an argon atmosphere, morpholine (4.4 mL) was added, and the solution was stirred overnight at room temperature. The reaction mixture was then washed twice with 2 M HCl (2 x 40 mL) and water (50 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The resulting yellow oil was dissolved in anhydrous DCM (100 mL) under an argon atmosphere, cooled to 0 °C, and treated with trichloroacetonitrile. After stirring at 0 °C for 1 hour, DBU (0.38 mL) was added, and the reaction mixture was stirred at 0 °C for 1 hour, then at room temperature for 1 hour. The solvent was removed under reduced pressure, and the brown oily residue was purified by silica gel column chromatography using hexane / EtOAc (1:1) as eluent to give the product (2.65 g, 42% yield) as a pale yellow oil. No UV absorbance was detected by LC / MS (no chromophore) and no ionization was detected (no ion carrier). 1 H NMR(300MHz,DMSO-d6)δ(ppm):10.15(s,1H),6.22(s,1H),5.36–5.19(m,3H),4.26–4.04(m,3H),2.16(s,3H),2.07–1.96(m,10H)

[0703] Step 2: Preparation of a mixture of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-((2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azanonane-19-yl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate and (2R,3R,4S,5S)-2-(acetoxymethyl)-6-((14-amino-3,6,9,12-tetraoxatetradecyl)oxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate:

[0704]

[0705] At room temperature, under an argon atmosphere, the compound (0.5 g) obtained in the previous step and the molecular sieve were... (To keep the reaction mixture anhydrous) Commercially available N-Boc-PEG5-ol (0.514 g) was added to the suspension in anhydrous DCM. The mixture was cooled to -25 °C and TMSOTf (0.203 mL) was added. The suspension was stirred at -25 °C for 1 hour, then stirred overnight at room temperature. The reaction was then quenched with saturated NaHCO3 solution, DCM (10 mL) was added, the phases were separated, and the organic phase was washed with water (15 mL), brine (15 mL), and dried over MgSO4. The resulting solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was filtered through a silica gel pad to give 600 mg of crude material (yellow oil) containing both Boc-deprotected and Boc-protected products. This crude material was used directly in the next step without further purification. Ionization showed Boc-deprotection (LC / MS (6 min): RT = 2.19 min, [M+H)). + =568.35) and Boc-protected (LC / MS (6min): RT=3.16min[M+H]) + =668.40) mixture. 1 H NMR(300MHz, DMSO-d6)δ(ppm):7.76(t,J=5.6Hz,1H),5.29–5.05(m,3H),4.57(t,J=5.4Hz,1H),4.21–3.98(m,4H), 3.56–3.50(m,16H),3.49–3.41(m,4H),3.20–3.13(m,1H),2.16–2.10(m,4H),2.06–2.01(m,10H),1.98–1.93(m,4H)

[0706] Step 3: (3S,4S,5S,6R)-2-((14-amino-3,6,9,12-tetraoxatetradecyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0707]

[0708] The solution of the compound (0.6 g crude) in DCM was cooled to 0°C and trifluoroacetic acid (3.44 mL) was added. The reaction mixture was stirred at 0°C for 30 min, then stirred overnight at room temperature. The solvent was removed under reduced pressure to give 580 mg of crude product. The residue was purified by reversed-phase rapid column chromatography to give 112 mg of pure compound (31% yield). The resulting product was then dissolved in a solution of 7N NH3 in methanol and stirred overnight at room temperature. The solvent was removed under reduced pressure to dryness. The crude product was used in the next step without further purification (85 mg, yield quantification). LC / MS (6 min): RT = 1.30 min, [M+H] + =399.60

[0709] Step 4: Preparation of compound (4), 3-ethoxy-4-((14-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-3,6,9,12-tetraoxatetradecyl)amino)cyclobut-3-ene-1,2-dione

[0710]

[0711] TEA (44 μL) was added to the solution of the previous compound (80 mg) in EtOH. After stirring for 5 minutes, 3,4-diethoxycyclobut-3-ene-1,2-dione (33 mg) was added, and the reaction mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure, and the residue was purified by preparative HPLC (ACN / water) to give 38 mg of the desired compound (yield 34%). HPLC purity: 99.4% (261 nm). 1 H NMR(300MHz, DMSO-d6)δ(ppm):8.71(d,J=50.5Hz,1H),4.71–4.69(m,1H),4.67(d,J=3.0Hz,1H),4.66–4.61(m,2H),4.54 (d,J=5.8Hz,1H),4.42(t,J=6.0Hz,1H),3.72–3.47(m,20H),3.47–3.42(m,2H),3.41–3.32(m,3H),1.37(t,J=7.1Hz,3H).

[0712] Example 1.5: Synthesis of compound (5):

[0713] 3-((3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthon]-5-yl)amino)-4-ethoxycyclobut-3-ene-1,2-dione

[0714]

[0715] A mixture of commercially available 5-aminofluorescein (50 mg) and 3,4-diethoxycyclobut-3-ene-1,2-dione (25 mg) in anhydrous EtOH was cooled to 0 °C under an argon atmosphere. Then, DIPEA (28 μL) was added to the solution, and the reaction mixture was stirred overnight at room temperature. Next, 1 M HCl was added dropwise to adjust the pH to 3. The resulting mixture was extracted with DCM (3 × 15 mL). The combined organic layers were dried over Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure to give 46 mg of crude product.

[0716] The reaction was repeated under similar conditions, except that TEA (22 μL) was used instead of DIPEA. The crude product (52 mg) was combined with previous batches, and the substances were purified by preparative HPLC to obtain a total of 57 mg of the desired compound as an orange solid. LC / MS (6 min): RT = 2.82 min, [M+H] + =471.55. HPLC purity: 99.92% (222nm), 99.96% (303nm). 1 HNMR(300MHz,DMSO-d6)δ10.17(s,1H),7.96(d,J=2.1Hz,1H),7.73(dd,J=8.4,2.2Hz,1 H),7.31–7.21(m,1H),6.70–6.48(m,6H),4.81(q,J=7.1Hz,2H),1.45(t,J=7.1Hz,3H).

[0717] Example 1.6 - Synthesis of compound (6): 4-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)benzamide

[0718]

[0719] Under an argon atmosphere, the compound obtained in step 1 of Example 1 was added to a mixture of intermediate 4-(2-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)ethyl)benzoic acid (167 mg), HATU (219 mg), and DIPEA (0.25 mL) in DMF. The reaction mixture was stirred overnight at room temperature. The solvent was then evaporated under reduced pressure. The residue was purified twice by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated under reduced pressure. After lyophilization, the final compound (20 mg, 7% yield) was obtained as a white powder. LC / MS (12 min): RT = 4.8 min, [MH] - =581.2. HPLC purity: 99.6% (200nm), 99.7% (260nm). 1 HNMR (400MHz, DMSO-d6) δ (ppm): 8.87 (s, 0.5H), 8.68 (s, 0.5H), 8.45 (d, J = 6.0Hz, 1 H),7.79(d,J=8.0Hz,2H),7.34–7.22(m,2H),4.71(dd,J=7.4,4.8Hz,2H),4.63(d,J =1.6Hz,2H),4.59(d,J=7.0Hz,1H),4.55(d,J=5.9Hz,1H),4.43(t,J=6.0Hz,1H),3. 66(dt,J=11.1,5.8Hz,2H),3.61–3.36(m,18H),2.89(s,2H),1.35(q,J=7.4Hz,3H).

[0720] Example 1.7 - Synthesis of compound (7): 3-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)benzamide

[0721]

[0722] Under an argon atmosphere, the compound obtained in step 1 of Example 1 (250 mg) was added to a mixture of intermediate 3-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoic acid (251 mg), HATU (366 mg), and DIPEA (0.42 mL) in DMF. The reaction mixture was stirred overnight at room temperature. The solvent was then evaporated under reduced pressure. The residue was purified by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated under reduced pressure. After lyophilization, the final compound (71 mg, 33% yield) was obtained as a white powder. LC / MS (12 min): RT = 4.9 min, [M+H] Measured values. + =555.1; [MH] - =553.1. HPLC purity: 99.9% (200nm), 99.9% (315nm). 1 H NMR (400MHz, DMSO-d6) δ (ppm): 10.87 (s, 1H), 8.47 (t, J = 5.6Hz, 1H), 7.80 (t, J = 1.9Hz, 1H),7.57(dt,J=7.6,1.4Hz,1H),7.50(d,J=8.1Hz,1H),7.43(t,J=7.8Hz,1H),4.77(q ,J=7.1Hz,2H),4.70(dd,J=7.9,4.8Hz,2H),4.62(d,J=1.6Hz,1H),4.54(d,J=5.9Hz,1 H),4.42(t,J=5.8Hz,1H),3.70–3.61(m,2H),3.58–3.32(m,16H),1.41(t,J=7.1Hz,3H)

[0723] Example 1.8 - Synthesis of compound (8): 2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)phenyl)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)acetamide

[0724]

[0725] Under an argon atmosphere, the compound obtained in step 1 of Example 1 was added to a mixture of intermediate 2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)phenyl)acetic acid (159 mg), HATU (219 mg), and DIPEA (0.25 mL) in DMF. The reaction mixture was stirred overnight at room temperature. The solvent was then evaporated under reduced pressure. The residue was purified twice by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated under reduced pressure. After lyophilization, the final compound (21 mg, 10% yield) was obtained as a white powder. LC / MS (12 min): RT = 4.8 min, [MH] - =567.1. HPLC purity: 99.6% (200nm), 99.7% (300nm). 1 H NMR (400MHz, DMSO-d6) δ (ppm): 10.62 (s, 1H), 8.10 (t, J = 5.7Hz, 1H), 7.28 (d ,J=8.3Hz,2H),7.22(d,J=8.6Hz,2H),4.82–4.72(m,3H),4.74–4.69(m,2H), 4.64(d,J=1.7Hz,1H),4.55(d,J=5.9Hz,1H),4.44(t,J=5.9Hz,1H),3.73–3 .62(m,2H),3.62–3.35(m,16H),3.21(q,J=5.8Hz,2H),1.42(t,J=7.1Hz,3H)

[0726] Example 1.9 - Synthesis of compound (9): 5-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)pyridineamide

[0727]

[0728] Under an argon atmosphere, the compound (310 mg) obtained in step 1 of Example 1 was added to a mixture of intermediate 5-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)pyridinecarboxylic acid (328 mg), HATU (454 mg), and DIPEA (0.52 mL) in DMF. The reaction mixture was stirred at room temperature for 3 hours. The solvent was then evaporated under reduced pressure. The residue was purified by preparative reversed-phase HPLC using ACN-FA / H2O-FA as eluent. Fractions containing the purified product were combined, and ACN was partially evaporated under reduced pressure. The aqueous solution was frozen and then lyophilized to give the final compound (36 mg, 6% yield) as a yellow solid foam. LC / MS (12 min): RT = 4.59 min, Measured [M+H] + =556.0; [MH] - =554.0. HPLC purity: 95.7% (200nm), 95.6% (323nm). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 11.11 (s, 1H), 8.66 (dd, J=2.6, 0.8Hz, 1H), 8.61 (t, J= 5.8Hz,1H),8.02(dd,J=8.5,0.7Hz,1H),7.92(dd,J=8.5,2.6Hz,1H),4.80(q,J=7.1H z,2H),4.68(s,2H),4.63(d,J=1.6Hz,1H),4.47(d,J=32.8Hz,2H),3.67(td,J=9.7,8 .0,4.9Hz,2H),3.61–3.52(m,8H),3.48(dd,J=11.7,5.8Hz,8H),1.44(t,J=7.1Hz,3H)

[0729] Example 1.10 - Synthesis of compound (10): 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)benzamide

[0730] Step 1: Synthesis of (2R,3R,4S,5R)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate

[0731]

[0732] Morpholine (8.8 mL) was added to a solution of α-D(+)-glucose pentaacetate (10 g), and the mixture was stirred overnight at room temperature. The reaction mixture was then washed with 2 M HCl (2 × 200 mL) and water (200 mL), dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting yellow oil was dissolved in anhydrous DCM under an argon atmosphere, cooled to 0 °C, and treated with trichloroacetonitrile (25.7 mL). After stirring at 0 °C for 1 hour, DBU (0.76 mL) was added, and the reaction mixture was stirred at 0 °C for 1 hour, then stirred overnight at room temperature. The solvent was removed under reduced pressure, and the residue was purified by column chromatography, eluting with a mixture of hexane and EtOAc to give the desired product (10.3 g, 82% yield) as a yellow oil. 1 H NMR (300MHz, DMSO-d6) δ (ppm): 9.99 (s, 1H), 6.43 (s, 1H), 5.41 (t, J = 9.9Hz, 1H), 5.19–5.05 (m, 2H), 4.25–4.07 (m, 3H), 2.01–1.94 (m, 12H)

[0733] Synthesis of step 2: (2R,3R,4S,5R)-2-(acetoxymethyl)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate

[0734]

[0735] Under an argon atmosphere, 6.9 g of 2-(2-(2-azidoethoxy)ethoxy)ethanol-1-ol, synthesized as described in application WO2022096681, was added to a suspension of the compound (10.3 g) obtained in the previous step and the molecular sieve in anhydrous DCM. The mixture was cooled to -25 °C and 4.5 mL of TMSOTf was added. The reaction mixture was stirred at -25 °C for 1 hour, and then stirred overnight at room temperature. The reaction was quenched with saturated NaHCO3 solution, DCM was added, the phases were separated, and the organic phase was washed with water and brine and dried over Na2SO4. The resulting suspension was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography by elution with a mixture of hexane and EtOAc to give the desired product (3.8 g, 29% yield) as a yellow oil. 1HNMR(300MHz, DMSO-d6)δ(ppm):5.25(t,J=9.4Hz,1H),4.90(t,J=9.7Hz,1H),4.85–4.72(m,2H),4.21–4.12(m,1H),4.05–3.99( m,1H),3.99–3.92(m,1H),3.84–3.75(m,1H),3.61–3.52(m,9H),3.40–3.37(m,2H),2.02(s,3H),2.00–1.96(m,6H),1.94(s,3H)

[0736] Synthesis of step 3: (3R,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0737]

[0738] The compound obtained in the previous step (3.8 g) was dissolved in a solution of 7N NH3 in methanol and stirred overnight at room temperature. The solvent was then evaporated under reduced pressure, and the crude desired product was used in the next step without further purification (3.0 g, yield quantification). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 4.99–4.84 (m, 2H), 4.57–4.40 (m, 1H), 4.15 (d, J = 7.8Hz, 1H), 3.92–3.81 (m, 1H), 3.70–3.22 (m, 18H)

[0739] Synthesis of step 4: (3R,4S,5S,6R)-2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0740]

[0741] Pd / C was added to a methanol solution (500 mg) of the compound obtained in the previous step under an argon atmosphere. The inert gas was evacuated and backfilled with hydrogen (three times in total). The reaction mixture was stirred overnight under a hydrogen atmosphere (balloon), filtered through a diatomaceous earth pad, and the filtrate was concentrated to dryness under reduced pressure. The crude product was used in the next step without further purification (460 mg, yield quantification). LC / MS (6 min): RT = 0.5 min, measured [M+H] + =312.05.

[0742] Step 5: 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(2-(2-(((3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)benzamide

[0743]

[0744] Under an argon atmosphere, the compound obtained in the previous step (254 mg) was added to a mixture of intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoic acid (256 mg), HATU (372 mg), and DIPEA (0.43 mL) in DMF. The reaction mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure. The residue was purified by preparative reversed-phase HPLC using ACN / H2O as eluent. Fractions containing the purified product were combined and concentrated under reduced pressure. The resulting oily substance was dissolved in a small amount of H2O, the solution was frozen, and then freeze-dried to give the final product (74 mg, yield 16%) as a white solid foam. LC / MS (12 min): RT = 4.79 min, measured [M+H] + =555.1; [MH] - =553.1. HPLC purity: 99.8% (200nm), 99.8% (315nm). 1 H NMR (300MHz, DMSO-d6) δ (ppm): 10.91 (s, 1H), 8.44 (t, J = 5.5Hz, 1H), 7.90–7.7 9(m,2H),7.44(d,J=8.5Hz,2H),4.96(d,J=4.9Hz,1H),4.89(m,2H),4.79(m,2H ),4.48(t,J=5.9Hz,1H),4.15(d,J=7.7Hz,1H),3.92–3.78(m,1H),3.71–3.61 (m,1H),3.61–3.49(m,9H),3.43(m,3H),3.08(m,3H),2.95(m,1H),1.42(t,3H)

[0745] Example 1.11 - Synthesis of compound (11): 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)benzamide

[0746] Step 1: Synthesis of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2-bromoethoxy)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate

[0747]

[0748] (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2,2,2-trichloro-1-iminoethoxy)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate (1.0 g) obtained in step 1 of Example 1.4 was added to a stirred solution of anhydrous DCM with 0.115 mL of 2-bromoethanol, followed by 0.01 mL of boron trifluoride diethyl ether. The reaction mixture was stirred overnight at room temperature. Triethylamine (0.2 mL) was added, and the resulting mixture was concentrated under reduced pressure. The crude substance was purified by rapid chromatography with hexane / EtOAc as eluent to give the desired product (710 mg, 76% yield) as a white solid. 1 ¹H NMR (300MHz, chloroform-d) δ (ppm): 5.38–5.24 (m, 3H), 4.87 (d, J = 1.7 Hz, 1H), 4.27 (dd, J = 12.6, 5.9 Hz, 1H), 4.17–4.09 (m, 2H), 4.03–3.83 (m, 2H), 3.52 (dd, J = 6.3, 5.7 Hz, 2H), 2.16 (s, 3H), 2.10 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H).

[0749] Step 2: Synthesis of (2R,3R,4S,5S)-2-(acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-2H-pyran-3,4,5-trimethyltriacetate

[0750]

[0751] The compound (700 mg) obtained in the previous step was added to a stirred solution of anhydrous DMF with NaN3 (800 mg), and the mixture was stirred overnight at 60 °C. The solvent was removed under reduced pressure, the crude material was treated with EtOAc, and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by rapid chromatography using hexane / EtOAc as eluent to give the desired product (600 mg, 93% yield) as a colorless oil. 1H NMR (400MHz, chloroform-d) δ (ppm): 5.36 (dd, J=10.1, 3.3Hz, 1H), 5.31 (d, J=9.8Hz, 1H), 5.28 (dd ,J=3.4,1.8Hz,1H),4.87(d,J=1.8Hz,1H),4.29(dd,J=12.3,5.3Hz,1H),4.16–4.10(m,2H ), 4.05 (ddd, J=9.8, 5.3, 2.4Hz, 1H), 3.87 (ddd, J=10.6, 6.8, 3.8Hz, 1H), 3.67 (ddd, J=10. 6,6.0,3.6Hz,1H),3.54–3.39(m,2H),2.16(s,3H),2.11(s,3H),2.05(s,3H),2.00(s,3H)

[0752] Step 3: Synthesis of (3S,4S,5S,6R)-2-(2-azidoethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0753]

[0754] The compound obtained in the previous step (600 mg) was dissolved in a solution of 7N NH3 in methanol and stirred overnight at room temperature. The solvent was removed under reduced pressure until dry, and the crude product was used in the next step without further purification (358 mg, quantitative yield). 1 H NMR (400MHz, DMSO-d6) δ (ppm): 4.75 (t, J = 5.0Hz, 2H), 4.67 (d, J = 1.6Hz, 1H), 4.59 (d, J = 5.9Hz, 1H), 4.46 (t, J = 6.0Hz, 1H), 3.78 (ddd, J=11.0,6.4,3.5Hz,1H),3.70–3.64(m,1H),3.62(ddd,J=4.7,3.4,1.6Hz,1H),3.55(ddd,J=11.0,6.5,3.3Hz,1H),3.51–3.35(m,6H).

[0755] Step 4: Synthesis of (3S,4S,5S,6R)-2-(2-aminoethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

[0756]

[0757] Under a gentle argon atmosphere, 10% Pd / C was added to a methanol solution of the compound obtained in the previous step (358 mg). The inert gas was evacuated and backfilled with hydrogen (three times in total). The reaction mixture was stirred overnight under a hydrogen atmosphere (balloon). The resulting mixture was filtered through a diatomaceous earth pad, washed with MeOH, and concentrated under reduced pressure to give the product as a colorless oil. The crude product was used in the next step without further purification (320 mg, yield quantification). 1 ¹H NMR (300MHz, deuterium oxide) δ (ppm): 4.89 (d, J = 1.8Hz, 1H), 3.98 (dd, J = 3.4, 1.8Hz, 1H), 3.91 (dd, J = 12.0, 1.5Hz, 1H), 3.87–3.72 (m, 3H), 3.70–3.62 (m, 2H), 3.55 (ddd, J = 10.4, 6.0, 4.4Hz, 1H), 2.93–2.78 (m, 2H)

[0758] Step 5: Synthesis of 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-N-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)benzamide

[0759]

[0760] DIPEA (0.35 mL) was added to a stirred solution of DMF containing 211 mg of the intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoic acid described in Preparation 2. The mixture was stirred for 5 minutes, then HATU (307 mg) was added, followed by a DMF solution of the compound obtained in the previous step (150 mg). The mixture was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and the crude substance was purified by reversed-phase rapid column chromatography to give the desired product (97 mg, 31% yield) as a white powder. LC / MS (12 min): RT = 7.65 min, Measured [MH] - =465.0. HPLC purity: 98.5% (200nm), 98.9% (315nm). 1¹H NMR (400MHz, deuterium oxide) δ (ppm): 7.79–7.74 (m, 2H), 7.41 (d, J = 8.5 Hz, 2H), 4.91 (d, J = 1.7 Hz, 1H), 4.84–4.76 (m, 2H), 3.96 (dd, J = 3.5, 1.7 Hz, 1H), 3.90 (ddd, J = 10.8, 7.1, 4.1 Hz, 1H), 3.83–3.77 (m, 2H), 3.77–3.55 (m, 6H), 1.48 (t, J = 7.1 Hz, 3H)

[0761] Example 1.12 - Synthesis of compound (12): ((2R,3S,4S,5S)-6-(2-(2-(2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzamido)ethoxy)ethoxy)ethoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methylphosphodiester

[0762] Step 1: Synthesis of (3S,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,4,5-triol

[0763]

[0764] Under Ar conditions, a solution of (3S,4S,5S,6R)-2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (1.0 g) synthesized as described in application WO2022096681, containing TBDMSCl (490 mg) and imidazole (400 mg), was added to DMF. The reaction mixture was stirred overnight at room temperature. The reaction mixture was then diluted with EtOAc (20 mL) and washed with water and brine. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting oily crude product (1.1 g, 83% yield) was used for the next step without further purification. LC / MS (6 min): RT = 3.25 min, Measured [M+H] + 452.25. 1 ¹H NMR (300MHz, chloroform-d) δ (ppm): 4.84–4.69 (m, 1H), 3.87 (dd, J = 3.3, 1.6Hz, 1H), 3.81–3.65 (m, 5H), 3.61–3.47 (m, 10H), 3.30 (t, J = 5.0Hz, 2H), 0.80 (s, 9H), 0.00 (d, J = 2.7Hz, 6H)

[0765] Step 2: Synthesis of (((2R,3R,4S,5S)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methoxy)(tert-butyl)dimethylsilane

[0766]

[0767] Under an argon atmosphere, the compound obtained in the previous step (1.0 g) was dissolved in anhydrous DMF, and the solution was cooled to 0 °C. NaH (60%) (440 mg) was slowly added, and the mixture was stirred at 0 °C for 10 min. BnBr (1.31 mL) was added, and the reaction mixture was stirred at 0 °C for 10 min, then stirred overnight at room temperature. The reaction was quenched with methanol, and the mixture was concentrated to dryness under vacuum. The crude material was diluted with EtOAc and washed with water and brine. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The product was purified by column chromatography using hexane / EtOAc as eluent to give the desired product (1.2 g, 76% yield) in an oily form. 1 ¹H NMR (300MHz, chloroform-d) δ (ppm): 7.57–6.85 (m, 16H), 4.93–4.80 (m, 2H), 4.75–4.51 (m, 5H), 3.95–3.83 (m, 2H), 3.78 (dd, J = 4.8, 2.8Hz, 3H), 3.74–3.68 (m, 1H), 3.63–3.47 (m, 10H), 3.27 (t, J = 5.1Hz, 2H), 0.83 (s, 9H), 0.01 (d, J = 3.4Hz, 6H)

[0768] Step 3: Synthesis of ((2R,3R,4S,5S)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methanol

[0769]

[0770] Under an argon atmosphere, the compound obtained in the previous step (1.1 g) was dissolved in THF, and the mixture was cooled to 0 °C. A solution of 1 M TBAF in THF (2.3 mL) was slowly added, and the reaction mixture was stirred at 0 °C for 10 min, then at room temperature for 3 h. The reaction was quenched with water, and the resulting mixture was extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude substance was purified by column chromatography using DCM / MeOH as eluent to give the desired product (750 mg, 81% yield) in an oily form. LC / MS (6 min): RT = 4.33 min, Measured [M+Na] + =630.5; [M+H2O] + =625.5. 1 ¹H NMR (300MHz, chloroform-d) δ (ppm): 7.63–7.06 (m, 15H), 4.92–4.76 (m, 2H), 4.75–4.53 (m, 5H), 3.88 (dd, J = 5.3, 2.5Hz, 2H), 3.81–3.46 (m, 14H), 3.26 (t, J = 5.0Hz, 2H)

[0771] Step 4: Synthesis of ((2R,3R,4S,5S)-6-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methyldibenzyl phosphate

[0772]

[0773] Under an argon atmosphere, the compound (550 mg) obtained in the previous step and A molecular sieve was added to a suspension of anhydrous DCM with a solution of 0.45 M tetrazolium in a can (6.2 mL). The mixture was stirred at room temperature for 2 hours, and then dibenzyl diisopropylphosphamide (0.64 mL) was added under an argon atmosphere. The reaction mixture was stirred at room temperature overnight, then cooled to 0 °C, and m-CPBA (470 mg) was added. The resulting mixture was stirred at room temperature for 1 hour, and the reaction was quenched with water. The suspension was filtered through a diatomaceous earth mat, the filtrate was washed with water, dried with Na2SO4, filtered, and concentrated under reduced pressure. The product was purified by column chromatography using hexane / EtOAc and DCM / MeOH as eluents to give the desired product (320 mg, yield 41%) as an oil. LC / MS (6 min): RT = 4.92 min, measured value [M+H] + =868.75. 1H NMR(300MHz, DMSO-d6)δ(ppm):7.66–7.14(m,25H),5.00(ddd,J=9.6,6.1,2.1Hz,4H),4.78(d,J=10.9Hz,1H),4.64(d,J=8.7Hz,3H),4. 53(d,J=11.5Hz,2H), 4.19(dd,J=6.7,3.2Hz,2H), 3.90(d,J=1.9Hz,1H), 3.83–3.61(m,4H), 3.58–3.47(m,10H), 3.28(t,J=4.9Hz,2H). 31 P NMR(300MHz,DMSO-d6)δ(ppm):-1.0(s)

[0774] Step 5: Synthesis of ((2R,3R,4S,5S)-6-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)-3,4,5-tris(benzyloxy)tetrahydro-2H-pyran-2-yl)methyldibenzyl phosphate

[0775]

[0776] PPh3 (91 mg) was added to a suspension of the compound (200 mg) obtained in the previous step in a THF / H2O mixture. The reaction mixture was stirred overnight at room temperature and then extracted with EtOAc. The organic layers were combined, washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude substance was purified by column chromatography to give the desired product (117 mg, 60% yield) in an oily form. 1 HNMR(300MHz, DMSO-d6)δ(ppm):7.66–7.14(m,25H),5.00(ddd,J=9.6,6.1,2.1Hz,4H),4.78(d,J=10.9Hz,1H),4.64(d,J=8.7Hz,3H),4.53(d, J=11.5Hz,2H),4.19(dd,J=6.7,3.2Hz,2H),3.90(d,J=1.9Hz,1H),3.83–3.61(m,4H),3.58–3.47(m,10H),3.28(t,J=4.9Hz,2H),2.70(m,2H). 31 P NMR(300MHz,DMSO-d6)δ(ppm):-1.0(s)

[0777] Step 6: Synthesis of dibenzyl(((2R,3R,4S,5S)-3,4,5-tris(benzyloxy)-6-(2-(2-(2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzamido)ethoxy)ethoxy)ethoxy)tetrahydro-2H-pyran-2-yl)methyl)phosphate)

[0778]

[0779] Under an argon atmosphere, the previously obtained compound (80 mg) was added to a mixture of the intermediate 4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoic acid (30 mg), HATU (43 mg), and DIPEA (0.05 mL) in DMF. The reaction mixture was stirred at room temperature for 2 hours. The solvent was then evaporated under reduced pressure. The residue was purified by preparative reversed-phase HPLC using ACN / H₂O as eluent. Fractions containing the purified product were combined and concentrated under reduced pressure. The resulting oil was dissolved in a small amount of H₂O, the solution was frozen, and then freeze-dried to give the final substance (50 mg, 49% yield) as a white powder. LC / MS (6 min): RT = 4.25 min [M+H] + =1085.6.

[0780] Step 7: Synthesis of ((2R,3S,4S,5S)-6-(2-(2-(2-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzamido)ethoxy)ethoxy)ethoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methylphosphophosphate dihydrogen ester

[0781]

[0782] Under an argon atmosphere, 10% Pd / C was added to a solution of the compound (37 mg) obtained in the previous step in THF. The reaction mixture was stirred under a hydrogen atmosphere (balloon) until the starting material was exhausted, and then filtered through a diatomaceous earth pad. The filtrate was evaporated to dryness under reduced pressure. The residue was purified by preparative reversed-phase HPLC using ACN / H2O as the eluent. Fractions containing the purified product were combined and concentrated under reduced pressure. The resulting oily substance was dissolved in a small amount of H2O, the solution was frozen, and then freeze-dried to give the final substance (1.5 mg, yield 7%) as a white powder. LC / MS (6 min): RT = 1.61 min, Measured [MH] - =633.10. HPLC purity: 92.1% (200nm), 93.1% (315nm).

[0783] Example 1.13 - Synthesis of a compound of formula (III) containing a peptide group Z.

[0784]

[0785] (&) Bold text indicates R L -NH- NH group / (#)R L -NH- is not shown (bold T indicates the amino group of threonine). The peptide group THRPPMWSPVWP is the peptide THR and corresponds to SEQ ID No:1.

[0786] 1.13.1. General Procedure for Peptide Synthesis:

[0787] peptide synthesis :

[0788] All peptides were synthesized on a solid support [Rink amide resin for compounds (14), (17), (15), (18), (19), (20), and (22); 2-chlorotriphenylmethyl resin for compounds (13), (16), (21), and (23)] on a GYROS PROTEIN Symphony X peptide synthesizer at a scale of 0.25 mmol. Synthesis was performed according to the standard protocol in the Fmoc / tBu strategy, with the Fmoc protecting group deprotected using DIC / oxyma Pure coupling activator and a piperidine solution in DMF.

[0789] Deprotection :

[0790] The resin was treated for 90 minutes with a mixture of TFA / H2O / TIPS / DTT (90:5:2.5:2.5) to cleave the resin and deprotect the side chains. 15 minutes before deprotection was complete, Bu4NBr (20 equivalents) was added to the mixture to prevent methionine oxidation. After filtration, the resin was washed with TFA, the filtrate was concentrated, the peptide was precipitated in diethyl ether, separated by centrifugation, and freeze-dried to remove residual TFA to the greatest extent possible.

[0791] 1.13.2. Grafting:

[0792] General method for grafting 3,4-diethoxycyclobut-3-ene-1,2-dione Add DIEA (until pH reaches 7-7.5) and a solution of 3,4-diethoxycyclobut-3-ene-1,2-dione (1.2 equivalents, 0.12 mmol) in ethanol (0.5 mL) to a solution of the peptide (0.1 mmol) in distilled water (0.5 mL). Stir the reaction at room temperature for 2 hours.

[0793] 1.13.3. Purification and Salt Exchange:

[0794] Using a reverse-phase column (Vydac Denali prep C-18, 10μm), The crude peptide was directly purified from the reaction mixture using a reversed-phase preparative HPLC system (Waters Delta Prep4000) with 50-300 nm as eluent and a suitable gradient of CAN+TFA 0.1% / H2O+TFA 0.1%. Fractions containing the purified target peptide were identified by UV measurement at 214 nm (UV / Visible Waters 2489 detector), and the selected fractions were then combined and lyophilized.

[0795] During the purification process, trifluoroacetate is exchanged for acetate using a special buffer solution.

[0796] All peptides were synthesized using the general method described above on a solid-phase carrier, and then purified / salt-exchanged by a suitable grafting method in solution.

[0797] 1.13.4. Synthesis of compound (13):

[0798] ESI-MS (m / z): [M+H] + Calculated value of C92H125N22O21S: 1907.20, measured value: 1906.37. [M+2H] 2+ Measured value: 954.10, Actual value: 953.75, [M+3H] 3+ Measured value: 636.40, actual value: 636.48. UPLC purity: 91.6% (214nm), RT = 5.20min

[0799] 1.13.5. Synthesis of compound (14):

[0800] ESI-MS (m / z): [M+H] + Calculated value of C84H110N21O18S: 1733.99, measured value: 1733.47. [M+2H] 2+ Measured value: 867.49, Actual value: 866.68, [M+3H] 3+ Measured value: 578.66, Actual value: 578.45. UPLC purity: 95.6% (214nm), RT = 4.52min

[0801] 1.13.6. Synthesis of compound (15):

[0802] ESI-MS (m / z): [M+H] + Calculated value of C91H123N22O21S: 1893.17, measured value: 1892.56. [M+2H] 2+Measured value: 947.08, actual value: 946.41, [M+3H] 3+ Measured value: 631.72, Actual value: 631.58. UPLC purity: 95.6% (214nm), RT = 4.55

[0803] 1.13.7. Synthesis of compound (16):

[0804] ESI-MS (m / z): [M+H] + Calculated value of C85H120N21O20S: 1788.07, measured value: 1787.37. [M+2H] 2+ Measured value: 894.53, Actual value: 893.69, [M+3H] 3+ Measured value: 596.34. UPLC purity: 95.6% (214nm), RT = 4.61min.

[0805] 1.13.8. Synthesis of compound (17):

[0806] ESI-MS (m / z): [M+H] + Calculated value of C77H105N2O17S: 1614.86, measured value: 1614.44. [M+2H] 2+ Measured value: 807.93, actual value: 807.09, [M+3H] 3+ Measured value: 538.95, Actual value: 538.86. UPLC purity: 95.0% (214nm), RT = 4.38min

[0807] 1.13.9. Synthesis of compound (18):

[0808] ESI-MS (m / z): [M+H] + Calculated value of C84H118N21O20S: 1774.05, measured value: 1773.12, [M+2H] 2+ Measured value: 887.02, Actual value: 886.95, [M+3H] 3+ Measured value: 591.68, actual value: 591.86. UPLC purity: 95.5% (214nm), RT = 4.06min

[0809] 1.13.10. Synthesis of compound (19):

[0810] ESI-MS (m / z): [M+H] + Calculated value of C105H151N22O28S: 2201.54, Measured value: Non-spectral, [M+2H] 2+ Measured value: 1101.27, Actual value: 1101.23, [M+3H]3+ Measured value: 734.51. UPLC purity: 96.6% (214nm), RT = 5.25min.

[0811] 1.13.11. Synthesis of compound (20):

[0812] ESI-MS (m / z): [M+H] + Calculated value of C93H125N24O21S: 1946.22, measured value: 1946.37, [M+2H] 2+ : 974.11, Measured value: 973.96, [M+3H] 3+ Measured value: 649.74, Actual value: 649.54. UPLC purity: 97.8% (214nm), RT = 4.94min.

[0813] 1.13.12. Synthesis of compound (21):

[0814] ESI-MS (m / z): [M+H] + Calculated value of C87H122N23O20S: 1842.13, measured value: 1841.35, [M+2H] 2+ Measured value: 921.56, Actual value: 920.83, [M+3H] 3+ Precipitation value: 614.71, Measured value: 614.51. UPLC purity: 95.1% (214nm), RT = 4.77min.

[0815] 1.13.13: Synthesis of compound (22)

[0816] ESI-MS (m / z): [M+H] + Calculated value of C108H150N29O26S: 2302.62, Measured value: Non-spectral, [M+2H] 2+ Measured value: 1151.81, Actual value: 1151.56, [M+3H] 3+ 768.20, measured value: 767.91. UPLC purity: 95.0% (214nm), RT = 4.63min

[0817] 1.13.14: Synthesis of compound (23)

[0818] ESI-MS (m / z): [M+H] + Calculated value of C102H147N28O25S: 2197.52; Measured value: non-spectral, [M+2H] 2+ Measured value: 1099.26, actual value: 1099.12; [M+3H] 3+Measured value: 733.17, actual value: 732.88; [M+4H] 4+ 550.13, measured value: 549.95. UPLC purity: 95.0% (214nm), RT = 4.42min

[0819] Example 2: Synthesis and coupling of AAV.

[0820] As described below, coupled AAV is generated by coupling the squaric acid ester portion of the present invention with at least one primary amino group exposed on the surface of AAV.

[0821] II.1. Production and purification of AAV.

[0822] AAV is produced and purified using techniques well-known in the art.

[0823] II.2. Production and purification of chemically coupled AAV.

[0824] Material

[0825] Compounds (1)-(11), (13)-(17) and (19)-(23) were obtained as described in Example 1 above.

[0826] Use the following AAV obtained as described in Example II.1:

[0827] -AAV2-eGFP: 1.0E13 vg / mL in DPBS + Ca 2+ Mg 2+ In 0.001% Pluronic F68, pH 7.4;

[0828] -AAV5-eGFP: 1.0E13 vg / mL in DPBS + Ca 2+ Mg 2+ In 0.001% Pluronic F68, pH 7.4;

[0829] -AAV9-eGFP: 1.0E13 vg / mL in DPBS + Ca 2+ Mg 2+ In 0.001% Pluronic F68, pH 7.4.

[0830] Table 3 - Other Materials and Reagents

[0831] describe Specification TBS, pH 9.3 50mM Tris or 150mM Tris, 150mM NaCl, pH: 9.3 Formulation buffer <![CDATA[DPBS+Ca 2+ ,Mg 2+ ,0.001%Pluronic F68]]> Concanavalin A-HRP SIGMA(L6397)

[0832] method

[0833] Conjugation of squaric acid ester adapters to AAV2-eGFP, AAV5-eGFP, or AAV9-eGFP capsids (2E11 or 1E12 vg) was performed using a solution of TRIS buffer (pH = 9.3) containing compounds (1)-(11), (13)-(17), and (19)-(23) in molar ratios of 1E6 or 3E6 equivalents, and incubated at 20°C for 4 h, 16 h, 48 h, or 72 h. At the end of the incubation period, unbound adapters were removed using a PDMidiTrap G-25 column. The column was first loaded with 5 × 4 ml formulation buffer (DPBS, Ca2+). 2+ Mg 2+ Equilibrate with 0.001% F68. Then, load the coupling reaction mixture onto each column and allow the sample to enter the bed. Elute the rAAV vector with 1.5 ml of formulation buffer. Add 0.3 ml of each of the five fractions to 1.5 ml PP tubes. Combine fractions 2–5 and determine the qPCR or ddPCR titer for each combined fraction. Then, sterilely filter the combined fractions using Acrodisc PP, PES, 0.2 μM 1.3 cm².

[0834] II.3. Characterization of chemically coupled AAV

[0835] II.3.a. Titration of vector gene (vg)

[0836] For all coupling reactions, quantitative real-time PCR (qPCR) titers were determined using a LightCycler 480 (Roche) or droplet digital PCR (ddPCR) titers were determined using a QX200 (Biorad) for samples collected after preparation / filtration steps.

[0837] II.3.b. Coupling analysis by SDS-PAGE and Lectin WB

[0838] The purity and integrity of the obtained AAV-conjugated vectors were assessed by SDS-PAGE gel silver staining. The conjugation efficacy of the mannose moiety on AAVs was further investigated by Western blot analysis using concanavalin A-HRP lectin (ConA) staining, which selectively binds mannose. Successful conjugation resulted in the migration of VP protein to higher molecular weights, and mannose-conjugated AAVs were specifically stained with ConA.

[0839] result

[0840] The results are summarized in Table 4 below.

[0841] Table 4

[0842]

[0843]

[0844] ND: Undetermined; NA: Not applicable.

[0845] Conclusion: Observation of migration, mass variation, and / or ConA staining of all modified AAVs demonstrates the effective coupling of squaric acid ester linkers to AAVs. Furthermore, the coupling rate can be tuned based on linker design, providing a general method for achieving different ligand loadings on AAVs.

[0846] Parallel evaluations of ligand stability in any buffer solution showed that over 90% of the ligands retained their intact structure after 24 hours of incubation.

[0847] II.3.c. Infectivity assay (U87-MG glioblastoma cells)

[0848] The infectivity of the conjugated AAV was tested. To this end, U87-MG cells were transduced with MOI 1E5 using (1)-AAV2, (2)-AAV2 or (3)-AAV2 vectors, and the transduced (eGFP positive) and untransduced cell populations were analyzed by fluorescence microscopy 72 hours after transduction.

[0849] Conclusion: Mannose-conjugated AAV2-eGFP particles are infectious in the U87-MG cell line.

[0850] II.3.d. Infectivity assay in HEK-TfR1(+) cells

[0851] The infectivity of the conjugated AAV was tested. For this purpose, HEK-293 cells (HEK-TfR1(+)) overexpressing human transferrin receptor were transduced with (13)-AAV9, (16)-AAV9, or (23)-AAV9 vectors at an MOI of 1E4. Twenty-four hours post-infection, transduction was measured by quantitative PCR to determine the vector genome copy number per cell.

[0852] Results: For the (13)-AAV9 and (16)-AAV9 vectors, 10 copies were measured per cell. For the (23)-AAV9 vector, 28 copies were measured per cell.

[0853] Conclusion: The AAV9 vector coupled with a peptide-based ligand is infectious in HEK-TfR1(+) cells.

[0854] Example 3: Evaluation of the transduction properties of two AAV vectors (including conjugated vectors) in the mouse brain

[0855] The purpose of this study was to investigate the transduction characteristics of two GFP-expressing recombinant AAV2 vectors (AAV2 and (3)-AAV2) in the mouse brain after a single bilateral striatal injection.

[0856] Material

[0857] animal

[0858] Six (6) adult male C57BL / 6 mice (Mus musculus) were purchased from Charles River Laboratories.

[0859] Test Project

[0860] “AAV2” is a recombinant AAV2 vector containing an unmodified capsid and carrying a CAG-eGFP expression cassette.

[0861] "(3)-AAV2" is a recombinant AAV2 vector containing a modified capsid with a surface-bound mannose linker and carrying a CAG-eGFP expression cassette.

[0862] method

[0863] Test Project

[0864] Compound (III) was covalently linked to the primary amino group of an amino acid exposed on the capsid surface after incubating with the AAV2 carrier for 4 hours at 20°C in Tris buffer, pH 9.3. The mixture was then tangentially filtered with sterile buffered saline (BSSS) + 0.001% poloxamer to prepare and remove unbound free molecules. The coupled AAV solution was filtered through a 0.22 μm filter.

[0865] Research Design

[0866] According to Table 5, stereotactic surgery was performed on six (6) mice, and test items were randomly injected into the striatum.

[0867] Table 5: Processing Schedule.

[0868] Group n Test Project target dose G1 3 AVV2 left and right striatum 5.5E+8vg / Hemisphere G2 3 (3)-AVV2 left and right striatum 5.5E+8vg / Hemisphere

[0869] Surgical Procedure

[0870] Anesthesia was maintained during the operation using 1.5-2% isoflurane. Buprenorphine 0.04 mg / kg was administered subcutaneously to control pain. Each animal was placed in a stereotactic apparatus. The midline of the scalp was incised. A hole was drilled above the target area using Bregma as a reference point. The test sample was injected using a 33G Hamilton syringe. The injection rate of the test sample solution was 0.25 μL / min. After injection, the wound was sutured with surgical sutures.

[0871] In vitro analysis

[0872] Euthanasia and Organ Treatment

[0873] Six weeks after stereotactic injection, all animals were euthanized via intraperitoneal (ip) injection of 600 mg / kg pentobarbital. The whole brain was removed and fixed at room temperature for 3 hours after cardiac perfusion with 0.9% saline and 4% paraformaldehyde (PFA) phosphate buffer. It was then transferred to 15% sucrose / PBS and stored for several days. Next, it was transferred to cryomolds, embedded in OCT medium, frozen in isopentane cooled on dry ice, and coronal sections were prepared on a cryostat to a thickness of 10 μm. The sections were mounted on glass slides and stored at -20°C.

[0874] GFP immunofluorescence

[0875] Unless otherwise specified, all steps were performed at room temperature in Dulbecco phosphate-buffered saline (DPBS), pH 7.2–7.8. Frozen sections were air-dried and washed. Non-specific binding sites were blocked, and sections were washed again. Sections were incubated overnight at 4°C with primary antibody in 1% normal donkey serum / DPBS: goat anti-GFP polyclonal antibody (Abcam, ab5450; 1:5000). Sections were washed and incubated with a secondary antibody: donkey anti-goat IgG (H+L), Alexa Fluor 488 (Abcam, ab150129), 1:500. Sections were washed and incubated with DAPI (Applichem, Cat. No. 1001). Sections were washed, covered, and sealed with a coverslip.

[0876] Full slide scanning of all stained sections was performed on a Zeiss AxioScan Z1 automated microscope with high-aperture lenses, equipped with a Zeiss Axiocam 506 mono and Hitachi 3CCD HV-F202SCL camera and ZeissZEN 3.3 software.

[0877] result

[0878] Representative images of groups G1 and G2 at the striatal level are shown below. Figure 1 As shown in the figure. These results demonstrate that (3)-AAV2 is fully functional and transduced to the target region, the striatum, after injection into the striatum. Furthermore, GFP expression was increased after injection of (3)-AAV2 compared to AAV2.

Claims

1. Adeno-associated virus (AAV) vector particle, comprising part of formula (II): in N* is the nitrogen atom of the primary amino group of the amino acid residues exposed on the surface of the capsid polypeptide of the AAV carrier. ---- indicates the connection point with the AAV carrier capsid; and R L -NH- includes the nitrogen-containing group -NH- and the group R. L The functional portion, and wherein the functional portion R L -NH- includes space shielding agents, markers, cell type-specific ligands, drug moieties, or combinations thereof.

2. The AAV carrier particle according to claim 1, wherein the functional portion R L -NH- contains a group Z and one or more spacer groups L, and the adeno-associated virus (AAV) vector particle is represented by formula (IIa): in Z is H or a cell type-specific ligand selected from the group consisting of: carbohydrates, hormones, peptides, glycosylated peptides, proteins, glycoproteins or their functionally active fragments, membrane receptors or their functionally active fragments, antibodies or their functionally active fragments, spiegelmers, nucleic acids or peptide aptamers, vitamins and drug fractions. L contains one or more groups selected from the group consisting of arylene or heteroarylene, comprising saturated or unsaturated, straight-chain or branched C1-C groups. 40 The hydrocarbon chain may have optional substituted groups, preferably one or more C groups. 1-6 Alkyl, C 1-6 Alkylamine or C 1-6 Acyl groups, polyethylene glycol (PEG), polypropylene glycol (PPG), alkylene amines; acyl groups, amino acid moieties, polyethers of branched polyols, β-alanine polymers, pHPMA, PLGA, polymers of alkylene diamines and combinations thereof.

3. The AAV carrier particle according to claim 2, wherein L comprises one or more spacer groups selected from the group consisting of L1, L2, and L3, and the portion of formula (IIa) is selected from the group consisting of (IIa1), (IIa2), (IIa3), (IIa4), (IIa5), (IIa6), (IIa7), (IIa8), (IIa9), (IIa... 10 ), (IIa 11 ), (IIa 12 ), (IIa 13 ), (IIa 14 ), (IIa 15 ), (IIa 16 ), (IIa 17 ), (IIa 18 ), (IIa 19 ) and (IIa 20 ): as well as Wherein L1 is one or more groups selected from the group consisting of: polyethylene glycol (PEG) containing 1-40, preferably 1-20, ethylene glycol monomers, branched C 3-12 Polyols, preferably branched C 3-6 Polyethers of polyols, and β-alanine polymers comprising 1-40, preferably 1-10, β-alanine monomers, or mixtures thereof; L2 comprises one or more arylene or heteroarylene groups; L3 is one or more groups selected from the following: an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, C 1-6 Alkylene, the C 1-6 Alkylene is a straight-chain C 1-6 Alkyl or branched C 3-6 Alkylene; C 1-6 alkyleneamine and C 1-6 Acyl group; wherein L3 is covalently connected to L2 via a carbon atom of the arylene group or via a carbon atom or a heteroatom of the heteroarylene group; and wherein, L1 and L2, or L1 and L3, are covalently linked through an amide moiety or its bioelectron isosteric moiety; or L1 and L3 are covalently linked through an ether bond.

4. The AAV carrier particle or a pharmaceutically acceptable salt thereof according to any one of claims 2 or 3, wherein Z comprises a linear or cyclic peptide or sugar, preferably wherein the peptide is biologically active, and the sugar is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.

5. The AAV carrier particle according to any one of claims 2-4, wherein the peptide is a blood-brain barrier (BBB) ​​shuttle peptide, preferably selected from the group consisting of: peptide THR or peptide having an RGD motif, including cyclic RGD peptides; and wherein the sugar is selected from the group consisting of: mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose, trehalose, deoxyglycosamine, glucuronic acid, S6-galactose, S6-N-acetylglucosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose, and P1-fructose.

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

7. The AAV carrier particle according to any one of claims 1-6, wherein a portion of formula (II) is selected from the group consisting of formulas (IIb), (IIc), (IId), (IIe), (IIIf), (IIg), (IIh), (IIj), (IIk), (IIm), (IIIn), (IIp), (IIq), (IIr), (IIs), (IIt), (IIv), (IIw), and (IIx): Where R a R b and R c Each is independently H or group R': Group R' is R a R b and R c At least one of the following, wherein Z is a peptide or sugar, Ar is an arylene or a heteroarylene containing one or more heteroatoms selected from the group consisting of: N, O, S, and Se; and R1 is selected from the group consisting of: H, C 1-6 Alkyl, C 1-6 The alkyl halogroups are Z-(OCH2-CH2)n-, ZC(O)NH-(CH2)q-(OCH2-CH2)n- and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-; preferably, R1 is H; m1 and m2 are each independently 0, 1 or 2, m3, m4, m5 and m6 are each independently selected from 1 to 6, preferably 1 or 2, and n and n' are each independently selected from 1 to 40, preferably 1 to 20, and q is selected from 1 to 3.

8. A pharmaceutical composition comprising AAV carrier particles according to any one of claims 1-7 and at least one pharmaceutically acceptable carrier.

9. The AAV carrier particles according to any one of claims 1-7 or the pharmaceutical composition according to claim 8, used as a drug.

10. The AAV carrier particles according to any one of claims 1-7 or the pharmaceutical composition according to claim 8, for use in gene therapy.

11. A method for delivering nucleic acids to cells, the method comprising contacting cells with AAV carrier particles according to any one of claims 1-7 and nucleic acids to be expressed in the contacted cells.

12. Compounds of formula (IIIa): in Z is H or a cell type-specific ligand selected from the group consisting of: carbohydrates, hormones, peptides, glycosylated peptides, proteins, glycoproteins or their functionally active fragments, membrane receptors or their functionally active fragments, antibodies or their functionally active fragments, spiegelmers, nucleic acids or peptide aptamers, vitamins and drug fractions. L is composed of one or more groups selected from the group consisting of arylene or heteroarylene, comprising saturated or unsaturated, straight-chain or branched C2-C groups. 40 Optionally substituted groups in the hydrocarbon chain, polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, polypropylene glycol (PPG) containing 1-40 propylene glycol monomers, branched C 3-12 Polyethers of polyols, arginine derivatives, β-alanine polymers containing 1-40 β-alanine monomers, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof; wherein L contains one or more groups selected from at least the group consisting of: arylene or heteroarylene, polyethylene glycol (PEG) containing 1-40 ethylene glycol monomers, and β-alanine polymers containing 1-40 β-alanine monomers.

13. The compound of claim 12, wherein L comprises one or more spacer groups selected from the group consisting of L1, L2, and L3, and the compound of formula (III) is selected from the group consisting of (IIIa1), (IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), (IIIa8), (IIIa9), (IIIa... 10 (IIIa) 11 (IIIa) 12 (IIIa) 13 (IIIa) 14 (IIIa) 15 (IIIa) 16 (IIIa) 17 (IIIa) 18 (IIIa) 19 ) and (IIIa 20 ): Wherein L1 is one or more groups selected from the following: polyethylene glycol (PEG) containing 1-40, preferably 1-20, ethylene glycol monomers, branched C 3-12 Polyols, preferably branched C 3-6 Polyethers of polyols, and β-alanine polymers comprising 1-40, preferably 1-10, β-alanine monomers, or mixtures thereof; L2 comprises one or more arylene or heteroarylene groups; L3 is one or more groups selected from the group consisting of: an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, C 1-6 Alkylene, the C 1-6 Alkylene is a straight-chain C 1-6 Alkyl or branched C 3-6 Alkylene; C 1-6 alkyleneamine and C 1-6 Acyl group, the C 1-6 Alkylene is a straight-chain C 1-6 Alkyl or branched C 3-6 Alkylene; wherein L3 is covalently connected to L2 via a carbon atom of an arylene or via a carbon atom or a heteroatom of a heteroarylene; and wherein L1 and L2 or L1 and L3 are covalently connected via an amide moiety or its bioelectron isosteric moiety, or wherein L1 and L3 are covalently connected via an ether bond.

14. A method for preparing AAV carrier particles according to any one of claims 1-7, wherein the method comprises reacting a compound of formula (III) according to any one of claims 12 or 13 with an amino group present in the AAV carrier capsid.

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