Engineered adeno-associated viruses (aav) and uses thereof

Abstract

The present application belongs to the field of biological medicine, and relates to a modified adeno-associated virus (AAV) and use thereof. The present application is based on site-specific modification of unnatural amino acids, and site-specific chemical modification of AAV viral capsid proteins in combination with the design concept of protein prodrugs. The modified AAV virus is in an inert-inactive form in non-target tissues, and is converted into an active form only under special pathological microenvironments or exogenous stimulation, so as to realize targeted gene therapy on specific disease tissues.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to modified adeno-associated virus (AAV) and its uses. Background Technology

[0002] Gene therapy treats diseases that cannot be treated with traditional drugs or surgery by introducing normal genes into the patient's body to repair or replace defective genes. Thanks to its extremely high biocompatibility, efficient gene transduction and sustained expression time, and broad tissue targeting, alanine AV (AAV) is considered the most effective delivery vector for in vivo gene therapy. Currently, there are seven AAV gene therapies on the market globally, and 388 AAV-related trials have entered the clinical stage. As a leading gene therapy vector, AAV is widely used in the treatment of ophthalmic diseases, neurodegenerative diseases, muscle diseases, and cardiovascular diseases.

[0003] Achieving disease-targeted gene delivery using AAV viral vectors is a key focus in gene therapy. Currently available strategies for AAV targeted modification include: tissue screening based on directed evolution of AAV capsid protein libraries, AAV capsid modification guided by fusion nanobodies, chemical modification of AAV with small molecule ligands, capsid modification, and the discovery of novel serotypes of AAV in other species. Directed evolution strategies rely on screening large quantities of AAV capsid protein libraries in animal tissues. Due to tissue differences between species, it is impossible to directly transfer evolved AAVs into primates and humans. Fusion expression or chemical modification of ligands targeting specific functions can increase AAV enrichment at specific targets, but fusion expression affects the stability and infectivity of the AAV structure. Chemically modified small molecule ligands mainly couple to nucleophilic amino acid residues on the capsid protein. Random and uncontrollable chemical modifications can affect AAV infection, endosome escape, and nucleus entry, thus severely reducing gene transfection efficiency. Summary of the Invention

[0004] This invention is based on the site-specific modification of non-natural amino acids and combines the design concept of protein prodrugs to perform site-specific chemical modification of the AAV virus capsid protein. The modified AAV virus exists in an inert to inactive form in non-target tissues, and is only converted into an active form under specific pathological microenvironments (metalloproteinases, cathepsins, ROS) or exogenous stimulation (light, ultrasound, etc.) to achieve targeted gene therapy on specific diseased tissues. This provides the following aspects.

[0005] Modified AAV

[0006] In a first aspect, the present invention provides a modified adeno-associated virus (AAV) comprising a modified AAV capsid protein having at least one non-natural amino acid residue connected to a coupling portion, the coupling portion being covalently linked to the non-natural amino acid residue via a cleavable linker, the non-natural amino acid residue being derived from a non-natural amino acid containing a tetrazine group (i.e., a tetrazine non-natural amino acid).

[0007] In some embodiments, the non-natural amino acid containing a tetrazine group has the chemical structure shown in Formula I: (Formula I) R1 is selected from H, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C6-C 10 Aromatic group, 5 to 10-membered heteroaryl group, preferably, R1 is selected from methyl, ethyl, isopropyl, n-butyl, phenyl, 4-pyridyl; L1 is selected from direct bonds, CH2, NH2, O, and S, and m is an integer selected from 1 to 6; preferably, L1 is CH2 and m is 1; L2 is selected from C6-C 10 The aromatic subunit, n is an integer selected from 0 to 3; preferably, L2 is selected from 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, and n is 0 or 1; most preferably, L2 is 1,3-phenylene, and n is 0 or 1.

[0008] In some embodiments, the non-natural amino acid is selected from any of the following: ; In some implementations, the non-natural amino acid is mPet.

[0009] In some embodiments, the non-natural amino acid residues with the coupling moiety have the chemical structure shown in Formula II-1 or Formula II-2: (Formula II-1) (Formula II-2) Wherein, the non-natural amino acid residue is located at the Nth position of the capsid protein amino acid sequence, and the direction from label 1 to label 2 is the direction from the N end to the C end of the amino acid sequence. Label 1 represents the amino acid residues at positions 1 to N-1 of the capsid protein amino acid sequence, and label 2 represents the amino acid residues at positions N+1 to the C end of the capsid protein amino acid sequence. R1 is selected from H, halogen, hydroxyl, cyano, C1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C6-C 10 Aromatic group, 5 to 10-membered heteroaryl group, preferably, R1 is selected from methyl, ethyl, isopropyl, n-butyl, phenyl, 4-pyridyl; L1 is selected from direct bonds, CH2, NH2, O, and S, and m is an integer selected from 1 to 6; preferably, L1 is CH2 and m is 1; L2 is selected from C6-C 10 Aromatic subunit, where n is an integer selected from 0 to 3; preferably, L2 is selected from 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, where n is 0 or 1; most preferably, L2 is 1,3-phenylene, where n is 0 or 1; X is selected from direct bond, ether bond, etc. , , , , Preferably, X is selected from... , , One of them is connected to a carbon atom on cyclooctane, and L3 is connected at position 2; L3 is a pyrolytic linker; M represents the coupling part.

[0010] In some implementations, the coupling portion includes a mask moiety.

[0011] In some embodiments, the masking portion is selected from polymers, such as synthetic polymers (e.g., PEG) or peptide polymers (e.g., XTEN protein).

[0012] In some embodiments, the masking portion is selected from PEG, for example, with a molecular weight of 5k to 40k, such as 5k, 10k, 20k, or 40k. In some embodiments, the masking portion is selected from PEG with a molecular weight of 20k.

[0013] In some embodiments, the masking portion is selected from an XTEN protein. In some embodiments, the XTEN protein may comprise one or more sequence motifs of 5 to 14 (e.g., 9 to 14) amino acid residues, wherein the motif comprises 4 to 6 types of amino acids (e.g., 5 amino acids) selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P), and is substantially composed of said amino acids or composed of said amino acids, see, for example, US2010 / 0239554A1. In some embodiments, the XTEN protein has a length of 100 aa to 500 aa, for example 120 aa to 500 aa, 130 aa to 500 aa, 140 aa to 450 aa, for example 144 aa, 288 aa, 432 aa. In some embodiments, the XTEN protein comprises the sequence shown in any one of SEQ ID NOs: 11-13. In some embodiments, the cleavable linker contains at least one copy (e.g., 1 copy) of the restriction enzyme site sequence.

[0014] In some embodiments, the masking portion is selected from the extracellular domains of AAVR, such as PKD1 (e.g., SEQ ID NO: 14) and / or PKD2 (e.g., SEQ ID NO: 15). In some embodiments, the masking portion comprises both PKD1 and PKD2, as in SEQ ID NO: 16. In some embodiments, the masking portion comprises PKD2, as in SEQ ID NO: 15. In some embodiments, the cleavable linker comprises at least one copy (e.g., three copies in tandem) of a restriction enzyme cleavage site sequence.

[0015] In some embodiments, the masking portion comprises a fusion protein of an XTEN protein and an AAVR extracellular domain. In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus: (a) an AAVR extracellular domain (e.g., PKD1 and PKD2, or PKD2), an XTEN protein (e.g., SEQ ID NO: 11), the C-terminus of which is connected to an enzyme cleavage site sequence; or (b) an AAVR extracellular domain (e.g., PKD1 and PKD2, or PKD2), a first copy of the enzyme cleavage site sequence, an XTEN protein (e.g., SEQ ID NO: 11), the C-terminus of which is connected to a second copy of the enzyme cleavage site sequence; or (c) an AAVR extracellular domain (e.g., PKD1 and PKD2, or PKD2), a first copy of the enzyme cleavage site sequence, a first fragment of the XTEN protein (e.g., aa1-72 of SEQ ID NO: 11), a second copy of the enzyme cleavage site sequence, a second fragment of the XTEN protein (e.g., aa73-144 of SEQ ID NO: 11), and a third copy of the enzyme cleavage site sequence.

[0016] In some embodiments, the conjugation portion further includes a targeting portion (e.g., an antibody or its antigen-binding fragment, or an antibody mimic such as an affibody) connected to the masking portion. In some embodiments, the targeting portion is located at the N-terminus of the conjugation portion. In some embodiments, the conjugation portion includes an XTEN protein with the targeting portion (e.g., an antibody or its antigen-binding fragment, or an antibody mimic such as an affibody) attached to its N-terminus. In some embodiments, the conjugation portion includes an XTEN protein with an affibody attached to its N-terminus. In some exemplary embodiments, the conjugation portion includes the XTEN protein shown in SEQ ID NO: 11 and an affibody attached to its N-terminus. In some exemplary embodiments, the affibody includes the sequence shown in SEQ ID NO: 17.

[0017] In some embodiments, the cleavable linker is cleaved by factors in the pathological microenvironment (such as metalloproteinases, cathepsins, reactive oxygen species (ROS)) or by exogenous stimuli (such as light, ultrasound).

[0018] In some embodiments, the cleavable linker is cleaved by reactive oxygen species (ROS), for example, containing borate or borate ester groups.

[0019] In some embodiments, the cleavable linker is cleaved by photobleaching, for example, by a fluorescent group. In some embodiments, the cleavable linker is cleaved by near-infrared light-induced photobleaching, for example, by a group containing Cy7.

[0020] In some embodiments, the cleavable linker is cleaved by a protease, for example, by containing a restriction enzyme cleavage site sequence. In some embodiments, the cleavable linker contains at least one (e.g., one, two, or three) restriction enzyme cleavage site sequences.

[0021] In some embodiments, the protease is a type II transmembrane serine protease (Matriptase-2), Corin protease, matrix metalloproteinase (MMP), or urokinase-type plasminogen activator (UPA).

[0022] In some embodiments, the cleavage site sequence of Matriptase-2 comprises the sequence shown in any one of SEQ ID NOs: 20-27. In some embodiments, the cleavable linker comprises one, two, or three cleavage site sequences of Matriptase-2 (e.g., SEQ ID NO: 27). In some embodiments, the cleavable linker comprises three tandemly linked cleavage site sequences of Matriptase-2 shown in SEQ ID NO: 27.

[0023] In some implementations, the cleavage site sequence of Corin includes the sequence shown in any one of SEQ ID NOs: 28-34.

[0024] In some embodiments, the restriction site sequence of the MMP includes the sequence shown in any one of SEQ ID NOs: 35-36.

[0025] In some implementations, the UPA restriction site sequence includes the sequence shown in SEQ ID NO: 42.

[0026] In some embodiments, the cleavable linker is cleaved by a glycosidase (such as β-Glucuronidase) and contains, for example, a glucuronic acid group.

[0027] In some embodiments, the cleavable linker is cleaved by reactive oxygen species (ROS) and comprises the following structure: There is one connection to X and two connections to M.

[0028] In some embodiments, the cleavable linker is cleaved by near-infrared light-induced photobleaching and comprises the following structure: There is one connection to X and two connections to M.

[0029] In some embodiments, the cleavable linker is cleaved by a protease and comprises the following structure: , (Formula A) or Formula B, wherein X is linked at position 1 and M is linked at position 2; CS is the restriction enzyme site sequence. Preferably, when M is a synthetic polymer such as PEG, the cleavable linker comprises the structure shown in Formula A. Preferably, when M is a polypeptide polymer such as XTEN or a protein such as AAVR, the cleavable linker comprises the structure shown in Formula B.

[0030] In some implementations, M-L3-X is selected from: , , , .

[0031] In some embodiments, the non-natural amino acid residue is located at a site corresponding to the following positions: (a) S268, Q327, S448, T453, L462, T583, L585 of the amino acid sequence of the AAV-DJ capsid protein shown in SEQ ID NO: 8, for example Q327, T453, L585, for example L585; (b) The amino acid sequence of the AAV9 capsid protein shown in SEQ ID NO: 7, including G453, G455, S499, A591, and H584, ​​such as G455, S499, and H584, ​​such as H584; (c) H584 of the amino acid sequence of the AAV-PHP-eb capsid protein shown in SEQ ID NO: 9; (d) The amino acid sequence of the AAV8 capsid protein shown in SEQ ID NO: 10, specifically S266, Q328, S449, Q461, L463, D584, L586, Q588, T591, Q594; for example, D584, L586; or (e) The amino acid sequence of the AAV5 capsid protein shown in SEQ ID NO: 43, specifically D374, T444, A579, for example, A579.

[0032] In some embodiments, the non-natural amino acid residue is located at a site corresponding to the following positions: L585 of the AAV-DJ capsid protein amino acid sequence shown in SEQ ID NO: 8; H584 of the AAV9 capsid protein amino acid sequence shown in SEQ ID NO: 7; H584 of the AAV-PHP-eb capsid protein amino acid sequence shown in SEQ ID NO: 9; D584 of the AAV8 capsid protein amino acid sequence shown in SEQ ID NO: 10; or A579 of the AAV5 capsid protein amino acid sequence shown in SEQ ID NO: 43.

[0033] In some embodiments, the AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8.

[0034] In some implementations, the modified AAV further includes a nucleic acid payload of interest.

[0035] In some embodiments, the target nucleic acid payload is selected from therapeutic genes (e.g., antibodies, vaccines, cytokines such as angiogenic cytokines, hormones such as relaxin, coagulation factors, growth factors, enzymes, RNA interference agents such as shRNA, gene editing tools such as CRISPR / Cas systems, for example) or reporter genes (e.g., fluorescent proteins or luciferases).

[0036] In some implementations, the target nucleic acid payload is operatively linked to a promoter and is flanked by inverted terminal repeat (ITR) sequences.

[0037] In some exemplary embodiments, the non-natural amino acid residue at the N-position of the AAV capsid protein, to which the coupling portion is attached, has the structure shown in Formula IV-1 or Formula IV-2: (Formula IV-1) or (Formula IV-2); in: M is the coupling part, preferably a synthetic polymer, such as PEG (e.g., molecular weight of 5k~40k, such as 5k, 10k, 20k or 40k). The AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8; preferably, the AAV is: AAV-DJ, where the Nth position corresponds to L585 of the AAV-DJ capsid protein amino acid sequence shown in SEQ ID NO: 8; or AAV9, where the Nth position corresponds to H584 of the AAV9 capsid protein amino acid sequence shown in SEQ ID NO: 7; or AAV-PHP-eb, where the Nth position corresponds to H584 of the AAV-PHP-eb capsid protein amino acid sequence shown in SEQ ID NO: 9; or AAV8, where the Nth position corresponds to D584 of the AAV8 capsid protein amino acid sequence shown in SEQ ID NO: 10; or AAV5, where the Nth position corresponds to A579 of the AAV5 capsid protein amino acid sequence shown in SEQ ID NO: 43.

[0038] In some exemplary embodiments, the non-natural amino acid residue at the N-position of the AAV capsid protein, to which the coupling portion is attached, has the structure shown in Formula V-1 or Formula V-2: (Equation V-1) or (Equation V-2); in: M is the coupling part, preferably a synthetic polymer, such as PEG (e.g., molecular weight of 5k~40k, such as 5k, 10k, 20k or 40k). The AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8; preferably, the AAV is: AAV-DJ, where the Nth position corresponds to L585 of the AAV-DJ capsid protein amino acid sequence shown in SEQ ID NO: 8; or AAV9, where the Nth position corresponds to H584 of the AAV9 capsid protein amino acid sequence shown in SEQ ID NO: 7; or AAV-PHP-eb, where the Nth position corresponds to H584 of the AAV-PHP-eb capsid protein amino acid sequence shown in SEQ ID NO: 9; or AAV8, where the Nth position corresponds to D584 of the AAV8 capsid protein amino acid sequence shown in SEQ ID NO: 10; or AAV5, where the Nth position corresponds to A579 of the AAV5 capsid protein amino acid sequence shown in SEQ ID NO: 43.

[0039] In some exemplary embodiments, the non-natural amino acid residue at the N-position of the AAV capsid protein, to which the coupling portion is attached, has the structure shown in Formula VI-1 or Formula VI-2: (Formula VI-1) or (Formula VI-2); in: M is the coupling part, preferably a synthetic polymer, such as PEG (e.g., molecular weight of 5k~40k, such as 5k, 10k, 20k or 40k). The AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8; preferably, the AAV is: AAV-DJ, where the Nth position corresponds to L585 of the AAV-DJ capsid protein amino acid sequence shown in SEQ ID NO: 8; or AAV9, where the Nth position corresponds to H584 of the AAV9 capsid protein amino acid sequence shown in SEQ ID NO: 7; or AAV-PHP-eb, where the Nth position corresponds to H584 of the AAV-PHP-eb capsid protein amino acid sequence shown in SEQ ID NO: 9; or AAV8, where the Nth position corresponds to D584 of the AAV8 capsid protein amino acid sequence shown in SEQ ID NO: 10; or AAV5, where the Nth position corresponds to A579 of the AAV5 capsid protein amino acid sequence shown in SEQ ID NO: 43; CS is a restriction enzyme site sequence, such as the Matriptase-2 restriction enzyme site sequence shown in any one of SEQ ID NO: 20-27, the Corin restriction enzyme site sequence shown in any one of SEQ ID NO: 28-34, or the MMP restriction enzyme site sequence shown in SEQ ID NO: 35 or 36; preferably, CS is selected from the Matriptase-2 restriction enzyme site sequence shown in SEQ ID NO: 27, the Corin restriction enzyme site sequence shown in SEQ ID NO: 28, or the MMP restriction enzyme site sequence shown in SEQ ID NO: 35; more preferably, CS is the Matriptase-2 restriction enzyme site sequence shown in SEQ ID NO: 27; preferably, CS is a one-copy restriction enzyme site sequence.

[0040] In some exemplary embodiments, the non-natural amino acid residue at the N-position of the AAV capsid protein, to which the coupling portion is attached, has the structure shown in Formula VII-1 or Formula VII-2: (Equation VII-1) or (Equation VII-2); in: M is the coupling moiety, preferably a protein or polypeptide polymer, such as PEG (e.g., molecular weight of 5k~40k, such as 5k, 10k, 20k or 40k); preferably, M is selected from (i)-(iv): (i) XTEN, such as the sequence shown in any one of SEQ ID NOs: 11-13; preferably, wherein CS is a restriction site sequence of at least one copy (e.g., 1 copy); (ii) AAVR extracellular domain, such as PKD1 and / or PKD2; preferably wherein CS is a restriction site sequence of at least one copy (e.g., three copies in tandem); (iii) A fusion protein comprising an AAVR extracellular domain (e.g., PKD1 and / or PKD2) and an XTEN protein (e.g., the sequence shown in any one of SEQ ID NOs: 11-13), the fusion protein comprising, from the N-terminus to the C-terminus: (a) an AAVR extracellular domain (e.g., PKD1 and PKD2, or PKD2), an XTEN protein (e.g., SEQ ID NO: 11), wherein the C-terminus of the XTEN protein is connected to a restriction enzyme site sequence; or (b) an AAVR extracellular domain (e.g., PKD1 and PKD2, or PKD2), a first copy of the restriction enzyme site sequence, an XTEN protein (e.g., SEQ ID NO: 11), wherein the C-terminus of the XTEN protein is connected to a second copy of the restriction enzyme site sequence; or (c) an AAVR extracellular domain (e.g., PKD1 and PKD2, or PKD2), a first copy of the restriction enzyme site sequence, a first fragment of the XTEN protein (e.g., aa1-72 of SEQ ID NO: 11), a second copy of the restriction enzyme site sequence, and a second fragment of the XTEN protein (e.g., SEQ ID NO: 11). The sequence of the restriction enzyme site of the third copy (aa73-144) of 11; (iv) An XTEN (e.g., the sequence shown in any one of SEQ ID NOs: 11-13) with a targeting portion (e.g., an antibody or its antigen-binding fragment, or an antibody mimic such as an affibody) attached to its N-terminus; preferably, the targeting portion is an affibody, such as an affibody targeting HER2 (e.g., SEQ ID NO: 17); preferably, CS is a restriction site sequence of at least one copy (e.g., 1 copy); The AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8; preferably, the AAV is: AAV-DJ, where the Nth position corresponds to L585 of the AAV-DJ capsid protein amino acid sequence shown in SEQ ID NO: 8; or AAV9, where the Nth position corresponds to H584 of the AAV9 capsid protein amino acid sequence shown in SEQ ID NO: 7; or AAV-PHP-eb, where the Nth position corresponds to H584 of the AAV-PHP-eb capsid protein amino acid sequence shown in SEQ ID NO: 9; or AAV8, where the Nth position corresponds to D584 of the AAV8 capsid protein amino acid sequence shown in SEQ ID NO: 10; or AAV5, where the Nth position corresponds to A579 of the AAV5 capsid protein amino acid sequence shown in SEQ ID NO: 43; CS is an enzyme restriction site sequence, such as the Matriptase-2 restriction site sequence shown in any one of SEQ ID NO: 20-27, the Corin restriction site sequence shown in any one of SEQ ID NO: 28-34, or the MMP restriction site sequence shown in SEQ ID NO: 35 or 36; preferably, CS is selected from the Matriptase-2 restriction site sequence shown in SEQ ID NO: 27, the Corin restriction site sequence shown in SEQ ID NO: 28, or the MMP restriction site sequence shown in SEQ ID NO: 35.

[0041] Methods for preparing modified AAV

[0042] In a second aspect, the present invention provides a method for preparing a modified adeno-associated virus (AAV), comprising the following steps: - Provided: (1) an adeno-associated virus (AAV) incorporating non-natural amino acids, comprising a site-mutated capsid protein having at least one non-natural amino acid residue containing a tetrazine group; (2) a coupling portion connected to a trans-cyclooctene (TCO) group, wherein the coupling portion is connected to the TCO group via a cleavable linker. - Incubate (1) and (2) together to couple the non-natural amino acid to the coupling moiety via the reverse electron demand Diels-Alder (IEDDA) reaction.

[0043] In some embodiments, the IEDDA reaction is performed at room temperature, such as 20–30°C, or for example, 25°C. In some embodiments, incubation is carried out in phosphate-buffered saline (PBS) at 25°C.

[0044] In some implementations, the coupling portion is as defined in the first aspect.

[0045] In some implementations, the detachable connector is as defined in the first aspect.

[0046] In some embodiments, the positions of the non-natural amino acid residues are as defined in the first aspect.

[0047] In some embodiments, the AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8.

[0048] In some embodiments, the non-natural amino acid containing a tetrazine group has the chemical structure shown in Formula I: (Formula I) R1 is selected from H, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C6-C 10 Aromatic group, 5 to 10-membered heteroaryl group, preferably, R1 is selected from methyl, ethyl, isopropyl, n-butyl, phenyl, 4-pyridyl; L1 is selected from direct bonds, CH2, NH2, O, and S, and m is an integer selected from 1 to 6; preferably, L1 is CH2 and m is 1; L2 is selected from C6-C 10 The aromatic subunit, n is an integer selected from 0 to 3; preferably, L2 is selected from 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, and n is 0 or 1; most preferably, L2 is 1,3-phenylene, and n is 0 or 1.

[0049] In some embodiments, the non-natural amino acid is selected from any of the following: ; In some implementations, the non-natural amino acid is mPet.

[0050] In some embodiments, the coupling moiety with the trans-cyclooctene (TCO) group has the chemical structure shown in Formula III: (Formula III) Where X is selected from direct bond, ether bond, , , , , ; Preferably, X is selected from , , One of them is connected to the 4th carbon atom of trans-cyclooctene, and the 2nd position is connected to L3; L3 is a pyrolytic linker; Preferably, L3 is selected from , , , One link connects to X, and two links connect to M; CS is the restriction enzyme site sequence; M represents the coupling part.

[0051] In some embodiments, the cleavable linker comprises a borate group, and the coupling portion connected to the trans-cyclooctene (TCO) group is: M represents the coupling part, such as PEG.

[0052] In some embodiments, the cleavable linker comprises Cy7, and the coupling moiety linked to the trans-cyclooctene (TCO) group is: M represents the coupling part, such as PEG.

[0053] In some embodiments, the cleavable linker includes an enzyme cleavage site sequence, and the coupling moiety linked to the trans-cyclooctene (TCO) group is: , where CS is the restriction site sequence and M is the coupling part, such as PEG.

[0054] In some embodiments, the cleavable linker includes an enzyme cleavage site sequence, and the coupling moiety linked to the trans-cyclooctene (TCO) group is: , where CS is the restriction site sequence and M is the coupling part, such as XTEN or protein.

[0055] In some embodiments, the AAV incorporating non-natural amino acids is provided through the following steps: (a) Provide: (i) an AAV packaging system comprising a first nucleic acid molecule encoding a nucleotide sequence of the site-directed mutant capsid protein, wherein the nucleotide sequence contains a stop codon (e.g., TAG or TAA) at the position corresponding to the unnatural amino acid; (ii) a second nucleic acid molecule encoding an orthogonal tRNA synthetase; and (iii) a third nucleic acid molecule encoding an orthogonal tRNA; preferably, the orthogonal tRNA synthetase amidates the orthogonal tRNA with an unnatural amino acid containing a tetrazine group; (b) Introduce the AAV packaging system, the second nucleic acid molecule, and the third nucleic acid molecule simultaneously or in any order into a host cell (e.g., transform, transduce, or transfect). (c) The host cells obtained in step (b) are cultured in a culture medium containing non-natural amino acids to obtain the AAV incorporating non-natural amino acids.

[0056] In some embodiments, the AAV packaging system comprises: (i) a packaging plasmid containing a Rep gene and a Cap gene, wherein the Cap gene is a first nucleic acid molecule; (ii) an auxiliary plasmid containing an adenovirus auxiliary gene (E2A, E4, VA RNA); and (iii) an exogenous gene expression plasmid containing a nucleic acid payload of interest.

[0057] In some embodiments, the second and third nucleic acid molecules are present in the same vector, such as different expression cassettes, or in different vectors. In some embodiments, the second nucleic acid molecule is optionally operably linked to a regulatory element, such as a promoter, such as a constitutive promoter, such as the CMV, EF1α, CAG, or UBC promoter. In some embodiments, the third nucleic acid molecule is optionally operably linked to a regulatory element, such as a promoter, such as an RNA polymerase III promoter, such as the U6 or H1 promoter.

[0058] In some implementations, the target nucleic acid payload is operatively linked to a promoter and located between inverted terminal repeat (ITR) sequences at both ends.

[0059] In some embodiments, the orthocyanin-tRNA synthetase recognizes a tetrazine non-natural amino acid (e.g., mPtet) and amidates the orthocyanin-tRNA with the non-natural tetrazine amino acid (e.g., mPtet), wherein the anticodon of the orthocyanin-tRNA is the inverse complement of a stop codon (e.g., the inverse complement of TAG is CUA). Thus, the orthocyanin-tRNA synthetase loads the non-natural amino acid onto the orthocyanin-tRNA and subsequently incorporates the non-natural amino acid into the protein at the position corresponding to the stop codon.

[0060] In some embodiments, the orthogonal tRNA synthetase comprises the sequence shown in SEQ ID NO: 1. In some embodiments, the anticodon of the orthogonal tRNA is the inverse complement of the stop codon (e.g., the inverse complement of TAG is CUA). In some embodiments, the orthogonal tRNA comprises the sequence shown in SEQ ID NO: 2.

[0061] Applications of modified AAV

[0062] In a third aspect, the present invention provides a method for delivering a nucleic acid payload of interest to a subject, comprising administering the modified AAV described in the first aspect to the subject. The use of the modified AAV described in the first aspect for delivering a nucleic acid payload of interest to a subject, and its use in the preparation of reagents for delivering a nucleic acid payload of interest to a subject, are also provided.

[0063] In some implementations, the target nucleic acid payload is delivered to the subject's inflamed or infected tissue (e.g., liver tissue, such as liver fibrosis; or heart tissue, such as myocardial ischemia-reperfusion; or lung tissue, such as pulmonary fibrosis, pneumonia, or acute lung injury); preferably, the cleavable linker is cleaved by reactive oxygen species (ROS) and contains, for example, borate or borate ester groups; preferably, the AAV is AAV-DJ, AAV9, or AAV5.

[0064] In some embodiments, the target nucleic acid payload is delivered to the liver tissue of the subject, such as liver fibrosis; preferably, the cleavable linker is cleaved by reactive oxygen species (ROS) and contains, for example, borate or borate ester groups; preferably, the AAV is AAV-DJ.

[0065] In some embodiments, the target nucleic acid payload is delivered to the subject's cardiac tissue, such as myocardial ischemia-reperfusion; preferably, the cleavable linker is cleaved by reactive oxygen species (ROS) and contains, for example, borate or borate ester groups; preferably, the AAV is AAV9.

[0066] In some implementations, the target nucleic acid payload is delivered to the lung tissue of a subject, such as pulmonary fibrosis, pneumonia, or acute lung injury; preferably, the cleavable linker is cleaved by reactive oxygen species (ROS) and contains, for example, borate or borate ester groups; preferably, the AAV is AAV5.

[0067] In some embodiments, the target nucleic acid payload is delivered to the liver tissue of the subject; preferably, the cleavable linker is cleaved by a protease, for example, containing a Matriptase2 protease cleavage site sequence; preferably, the AAV is AAV8 or AAV-DJ.

[0068] In some embodiments, the target nucleic acid payload is delivered to the heart tissue of a subject; preferably, the cleavable linker is cleaved by a protease, for example, containing a Corin protease cleavage site sequence; preferably, the AAV is AAV9.

[0069] In some embodiments, the target nucleic acid payload is delivered to the subject's tumor tissue; preferably, the cleavable linker is cleaved by a protease, for example, containing a matrix metalloproteinase (MMP) cleavage site sequence; preferably, the coupling portion contains an XTEN protein; preferably, the coupling portion contains a targeting portion (such as an Affibody) that targets tumor cells; preferably, the coupling portion contains an XTEN protein and a targeting portion (such as an Affibody) linked to its N-terminus; preferably, the AAV is AAV-DJ.

[0070] In some embodiments, the target nucleic acid payload is delivered to the muscle tissue of the subject; preferably, the AAV is AAV9; preferably, the cleavable linker is photobleached and cleaved, for example, containing a fluorescent group, such as Cy7; preferably, the method further includes irradiating the muscle with near-infrared light.

[0071] In some embodiments, the target nucleic acid payload is delivered to the brain tissue of the subject; preferably, the AAV is PHP-eb; preferably, the cleavable linker is cleaved by photobleaching, for example, containing a fluorescent group, such as Cy7; preferably, the method further includes irradiating the head with near-infrared light.

[0072] In some embodiments, the modified AAV exhibits reduced humoral immunity levels in subjects. This reduced humoral immunity level refers to a decrease in total anti-AAV antibodies or neutralizing antibodies induced in the body compared to administration of the corresponding wild-type AAV or the corresponding type of AAV not modified as described herein.

[0073] In some implementations, the subject has pre-existing anti-AAV antibodies (e.g., neutralizing antibodies).

[0074] In some implementations, the subject has undergone AAV gene therapy.

[0075] In some implementations, the subject is a human being.

[0076] In some embodiments, the modified AAV is administered via injection, intratracheal administration, intranasal administration, or inhalation.

[0077] In some embodiments, the target nucleic acid payload is a therapeutic gene, such as an antibody, vaccine, cytokines like angiogenic cytokines, hormones like relaxin, coagulation factors, growth factors, enzymes, RNA interference agents like shRNA, or gene editing tools like CRISPR / Cas systems. In some embodiments, the target nucleic acid payload is a reporter gene.

[0078] In a fourth aspect, the present invention provides a method for treating a disease in a subject, the method comprising administering the modified AAV described in the first aspect to a subject in need of such treatment. The use of the modified AAV described in the first aspect for treating a disease in a subject, and its use in the preparation of a medicament for treating a disease in a subject, is also provided.

[0079] In some embodiments, the modified AAV contains a target nucleic acid payload selected from therapeutic genes (e.g., antibodies, vaccines, cytokines such as angiogenic cytokines, hormones such as relaxin, coagulation factors, growth factors, enzymes, RNA interference agents such as shRNA, gene editing tools such as CRISPR / Cas systems). In some embodiments, the therapeutic gene encodes a bioactive molecule that treats the disease.

[0080] In some implementations, the method includes delivering a target nucleic acid payload to a subject to treat a disease using the method described in the third aspect.

[0081] In some embodiments, the modified AAV exhibits reduced humoral immunity levels in subjects. This reduced humoral immunity level refers to a decrease in total anti-AAV antibodies or neutralizing antibodies induced in the body compared to administration of the corresponding wild-type AAV or the corresponding type of AAV not modified as described herein.

[0082] In some implementations, the subject has pre-existing anti-AAV antibodies (e.g., neutralizing antibodies).

[0083] In some implementations, the subject has undergone AAV gene therapy.

[0084] In some implementations, the subject is a human being.

[0085] Screening sites where non-natural amino acids can be incorporated

[0086] In a fifth aspect, the present invention provides a method for screening sites in adeno-associated virus (AAV) capsid proteins into which non-natural amino acids can be incorporated, the method comprising: (a) Provide: (i) a first modified AAV having a non-natural amino acid residue at a predetermined site in the capsid protein; (ii) a second modified AAV having a non-natural amino acid residue with a blocking group (such as PEG) attached to the predetermined site in the capsid protein; (iii) a third modified AAV having a non-natural amino acid residue with an amino group attached to the predetermined site in the capsid protein; wherein the non-natural amino acid in (i)-(iii) is a non-natural amino acid containing a tetrazine group; (b) The first modified AAV, the second modified AAV, and the third modified AAV were incubated with cells (e.g., HEK-293T cells) and their infectious activity was tested. When the infectious activity of the first modified AAV and the third modified AAV were comparable, and the second modified AAV showed weak or undetectable infectious activity, the preset site was selected as the site for incorporating non-natural amino acids.

[0087] In some embodiments, the ratio of the infectivity of the third modified AAV to the infectivity of the second modified AAV is not less than 10. 3 .

[0088] In some embodiments, the AAV includes a reporter gene, such as a fluorescent protein or luciferase, thereby generating a detectable signal proportional to the infectious activity. In some embodiments, the reporter gene is operatively linked to a promoter and flanked by inverted terminal repeats (ITRs).

[0089] In some embodiments, in the second modified AAV, the residue at the predetermined site is mutated to a non-natural amino acid containing a tetrazine group, the blocking group being linked to a TCO group (e.g., TCO-PEG20K), thereby being linked to the non-natural amino acid via an IEDDA reaction between the TCO group and the tetrazine group.

[0090] In some embodiments, in the third modified AAV, the residue at the predetermined site is mutated to a non-natural amino acid containing a tetrazine group, the amino group being linked to a TCO group (e.g., TCO-NH2), thereby being linked to the non-natural amino acid via an IEDDA reaction between the TCO group and the tetrazine group.

[0091] Mutant aaRS and Compositions

[0092] In a sixth aspect, the present invention provides a mutant aminoacyl-tRNA synthetase (aaRS) comprising the sequence shown in SEQ ID NO: 1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding said mutant aaRS is also provided. A vector comprising the isolated nucleic acid molecule as described above is also provided.

[0093] In a seventh aspect, the present invention provides a composition comprising: (a) the mutated aaRS as described in the sixth aspect or the first nucleic acid molecule encoding the mutated aaRS; and (b) tRNA or a second nucleic acid molecule encoding the tRNA, wherein the tRNA comprises the sequence shown in SEQ ID NO:2.

[0094] In some embodiments, the composition comprises one or more carriers, the one or more carriers comprising the first nucleic acid molecule and the second nucleic acid molecule.

[0095] In some implementations, the vector is a plasmid or a viral vector.

[0096] In some implementations, the first nucleic acid molecule is optionally operably linked to a first regulatory element, such as a promoter, like a constitutive promoter, such as the CMV, EF1α, CAG, or UBC promoter.

[0097] In some embodiments, the second nucleic acid molecule is optionally operably linked to a second regulatory element, such as a promoter, such as an RNA polymerase III promoter, like the U6 promoter or the H1 promoter.

[0098] In some embodiments, the composition further comprises a third nucleic acid molecule encoding the target protein, the third nucleic acid molecule containing a stop codon (e.g., TAG or TAA) recognized by the tRNA. In some embodiments, the target protein contains a non-natural amino acid, the stop codon corresponding to the position of the non-natural amino acid in the target protein. In some embodiments, the non-natural amino acid is a non-natural amino acid containing a tetrazine group, such as mPet.

[0099] Methods for generating target proteins

[0100] In an eighth aspect, the present invention provides a method for generating a target protein having at least one non-natural amino acid residue containing a tetrazine group, the method comprising: (a) Provide: (i) a first nucleic acid molecule comprising a nucleotide sequence encoding a target protein, wherein the codon at the position corresponding to the non-natural amino acid is a stop codon (e.g., TAG or TAA); (ii) a second nucleic acid molecule encoding the mutated aaRS described in the sixth aspect; and (iii) a third nucleic acid molecule encoding tRNA comprising the sequence shown in SEQ ID NO: 2. (b) Introduce the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule simultaneously or in any order into a host cell (e.g., transform, transduce, or transfect). (c) The host cells obtained in step (b) are cultured in a medium containing non-natural amino acids to allow the non-natural amino acids to be incorporated into the target protein being expressed in the cells; wherein the aaRS is capable of loading the non-natural amino acids onto tRNA and subsequently incorporating the non-natural amino acids into the protein at a position corresponding to the select codon.

[0101] In some implementations, the non-natural amino acid is mPet.

[0102] In some embodiments, the first, second, and third nucleic acid molecules are present on one or more vectors. In some embodiments, the first, second, and third nucleic acid molecules are present on different vectors. In some embodiments, the second and third nucleic acid molecules are present on the same vector, while the first nucleic acid molecule is present on a different vector.

[0103] In some embodiments, the one or more vectors are each independently selected from plasmids, cosmids, artificial chromosomes, bacteriophages, and viruses. In some embodiments, the one or more vectors are each independently selected from plasmids and viruses. In some embodiments, the one or more vectors are each independently selected from viral vectors. In some embodiments, the viral vector is adeno-associated virus, adenovirus, lentivirus, or baculovirus.

[0104] In some embodiments, the methods for introducing nucleic acids into cells are well known to those skilled in the art, and any of them can be used in this invention, including but not limited to electroporation, nuclear transfection, microinjection, gene gun, calcium phosphate-mediated transfection, cationic polymer transfection, liposome transfection, or viral infection.

[0105] In some embodiments, the target protein is selected from protein or polypeptide drugs (e.g., antibodies or antibody fragments thereof, vaccines, hormones, coagulation factors, growth factors or enzymes), detection or diagnostic reagents, or industrial proteins (e.g., industrial enzymes).

[0106] Terminology Definition

[0107] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0108] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0109] The term "corresponding to" regarding the position of a protein / peptide, such as the statement that a nucleotide or amino acid position "corresponds to" a nucleotide or amino acid position in a disclosed sequence (such as that shown in the sequence listing), refers to the nucleotide or amino acid position identified based on structural sequence alignment or by aligning the sequence to the disclosed sequence using a standard alignment algorithm (such as the GAP algorithm). For example, corresponding residues in similar sequences (e.g., fragments or species variants) can be determined by aligning a structural sequence to a reference sequence. By aligning the sequences, those skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guidance.

[0110] In this document, "the amino acid position corresponding to a given position" refers to the equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity. Therefore, the corresponding amino acid position can be identified by comparing the target AAV capsid protein sequence with the exemplary sequences (such as SEQ ID NO: 7-10) given herein (e.g., when obtaining the highest percentage of identity) through sequence alignment. For example, the statement "the position corresponding to L585 of the AAV-DJ capsid protein amino acid sequence shown in SEQ ID NO: 8" covers position 585 in the amino acid sequence shown in SEQ ID NO: 8 or the corresponding position in another AAV capsid protein amino acid sequence.

[0111] As used herein, the term "AAV" includes all types of AAV, including wild-type AAV and recombinant or variant AAV. AAV variants include, but are not limited to, AAVs with mutated or synthetic capsids, such as AAVs with heterozygous capsids, pseudotype AAVs, and self-complementary AAVs (scAAVs). Recombinant AAVs (rAAVs) are formed by replacing the Rep and Cap genes of wild-type AAVs entirely with exogenous target gene expression frames, retaining only the ITR sequences at both ends.

[0112] As used in this text, the term "cleavable linker" refers to a linker that is sensitive to cleavage, such as enzyme cleavage, acid-induced cleavage, light-induced cleavage, and disulfide bond cleavage; it can also be called a cleavable linker. Enzyme cleavage includes, but is not limited to, protease-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, glycosidase-induced cleavage, phosphatase-induced cleavage, and sulfatase-induced cleavage.

[0113] As used in this text, the term "masking portion" refers to a coupling portion of the AAV capsid protein linked to a non-natural amino acid residue. When the AAV capsid protein binds to the masking portion, it prevents AAV from binding to its receptor on the surface of target cells. When the masking portion is linked to the AAV capsid protein via a cleavable linker, the binding of AAV to the receptor can be restored by cleaving the linker.

[0114] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen. When referring to the term "antibody," unless the context explicitly indicates otherwise, it includes not only the complete antibody but also the antigen-binding fragment capable of specifically binding to a target antigen. A "complete antibody" typically consists of two pairs of polypeptide chains, each pair containing one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a variable region (VL) and a constant region (CL). The constant region consists of a single CL domain. While not directly involved in antibody-antigen binding, the constant domain exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites. The distribution of amino acids in different regions or domains can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0115] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to an antigen; it is also referred to as an "antigen-binding moiety." Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, disulfide-linked Fv, scFv, di-scFv, diabody, single-domain antibody, and polypeptides that contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0116] As used herein, "antibody mimic" refers to a peptide that has antigen-binding activity similar to that of an antibody, but is not derived from an antibody; rather, it is derived from a non-immunoglobulin backbone protein. Antibody mimics consist of single chains, unlike conventional full-length antibodies where two heavy chains and two light chains form a heterotetrameric quaternary structure to exert their function. Specifically, such antibody mimics include affiliates (Nygren, PA, FEBS) derived from the Z domain of protein A. J.275(11):2668-2676,2008), ubiquitin-derived γ-B crystal protein or Affilin (Ebersbach et al., J.Mol.Biol.372(1):172-185,2007), cysteine ​​protease inhibitor-derived Affimer (Johnson et al., Anal.Chem.84(15):6553-6560,2012), Sac7d-derived Affitin (Krehenbrink et al., J.Mol.Biol.383(5):1058-1068,2008), triple-helical coiled helical protein-derived Alphabody (Desmet et al., Nat.Commun.23(12):1556-1561,2005), ankyrin repeat motif-derived DARPin (Stumpp et al., Drug. Discov. Today. 13(15-16):695-701, 2008), Fynomer derived from the SH3 domain of Fyn protein (Grabulovski et al., J. Biol. Chem. 282(5):3196-3204, 2007), Kunitz domain peptide derived from the Kunitz domain of various protein inhibitors (Nixon and Wood, Curr. Opin. Drug Discov. Dev. 9(2):261-268, 2006), Monobody derived from the 10th type 3 domain of fibronectin (Koide and Koide, Methods Mol. Biol. 352:95-109, 2007), nanoCLAMP derived from carbohydrate-binding module 32-2 (Suderman et al., Protein (Exp. Purif. 134:114-124, 2017), variable lymphocyte receptor (VLR) derived from hagfish (Boehm et al., Ann. Rev. Immunol. 30:203-220, 2012), and VLR-based engineered repebody to enhance antigen affinity (Lee et al., Proc. Natl. Acad. Sci. USA, 109:3299-3304, 2012).

[0117] As used herein, in the context of tRNA and aminoacyl-tRNA synthetase, the term "orthogonal" refers to, for a cell or translation system, the reduced (e.g., less than 20%, less than 10%, less than 5%, or less than 1%) or even zero efficiency of orthogonal tRNA (O-tRNA) aminoacylation by any endogenous aminoacyl-tRNA synthetase compared to the aminoacylation of endogenous tRNA by endogenous aminoacyl-tRNA synthetase, or the reduced (e.g., less than 20%, less than 10%, less than 5%, or less than 1%) or even zero efficiency of orthogonal aminoacyl-tRNA synthetase (O-RS) aminoacylation of any endogenous tRNA compared to the aminoacylation of endogenous tRNA by endogenous aminoacyl-tRNA synthetase. An "orthogonal pair" consists of orthogonal tRNA (O-tRNA) and orthogonal aminoacyl-tRNA synthetase (O-RS). O-tRNA is not acylated by endogenous synthases and can mediate the incorporation of non-natural amino acids into proteins encoded by polynucleotides containing a select codon recognized by O-tRNA in the cell; O-RS recognizes O-tRNA in the cell and preferentially aminoacylates O-tRNA with non-natural amino acids. The term "select codon" refers to a codon recognized by O-tRNA during translation but not by endogenous tRNA. The O-tRNA anticodon loop recognizes the select codon on mRNA and incorporates its amino acid, such as a non-natural amino acid, at this site in the polypeptide. In some exemplary embodiments, the select codon is the stop codon TAG.

[0118] In some embodiments, the orthocyanin-tRNA synthetase described herein recognizes a tetrazine non-natural amino acid (e.g., mPtet) and amidates the orthocyanin-tRNA with the non-natural tetrazine amino acid (e.g., mPtet), wherein the anticodon of the orthocyanin-tRNA is the inverse complement of the stop codon TAG (i.e., CUA). Thus, the orthocyanin-tRNA synthetase loads the non-natural amino acid onto the orthocyanin-tRNA and subsequently incorporates the non-natural amino acid into the protein at the position corresponding to the stop codon.

[0119] As used herein, the term "operably ligated" refers to a relationship in which the described components are positioned to allow them to function in their intended manner. A regulatory sequence operably ligated to a target nucleic acid sequence is ligated in such a way that expression of the target nucleic acid sequence is achieved under conditions compatible with the control sequence.

[0120] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0121] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0122] Beneficial effects of the invention

[0123] This invention first provides a method for site-specific and uniform chemical modification of AAV capsid protein. Based on this method and combined with the design concept of protein prodrugs, the AAV viral capsid protein is chemically modified at specific sites. The modified AAV virus blocks AAV activity through a masking module and deblocks AAV activity in specific tissues, thereby achieving precise tissue-targeted gene delivery of AAV. At the same time, it avoids the recognition of pre-existing anti-AAV neutralizing antibodies in vivo and effectively reduces AAV-induced immune responses, enabling secondary AAV injection. This method not only has broad application prospects but also has the potential for clinical translation.

[0124] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0125] Figure 1 The mPtet insertion efficiency determination is shown. (a) Schematic diagram of plasmid structure; (b) Determination results of the insertion efficiency of Mb, Mm, Mf and G1 into mPtet.

[0126] Figure 2 A schematic diagram of the AAV virus packaging system is shown.

[0127] Figure 3 The schematic diagrams show the preparation processes of tetrazine-modified non-natural amino acid-modified AAV and NAEK-modified AAV.

[0128] Figure 4 This shows a comparison between AAV containing tetrazine non-natural amino acids and AAV containing NAEK. (A) AAV-DJ mPTet and AAV-DJ NAEK (A) Coupling efficiency with PEG of different molecular weights (5K, 10K, 20K); (B) AAV-DJ modified with TCO-PEG mPTet With DBCO-PEG modified AAV-DJ NAEK Transduction activity in HEK293T cells. (C) AAV-DJ modified with TCO-PEG. mPTet Transduction activity in mice.

[0129] Figure 5 The working principle and synthesis of the ROS-stimulated-responsive linker are shown. (A) Schematic diagram of the structure of the ROS-stimulated-responsive linker; (B) Schematic diagram of the working principle of the ROS-stimulated-responsive linker; (C) Flowchart of the synthesis of TCO-BR-PEG.

[0130] Figure 6 The synthesis flow chart of TCO-NIR-PEG is shown.

[0131] Figure 7 The screening of linkers for protease responses is shown. (a) Schematic diagram of the report molecular structure of the screening experiment; (bc) Linker screening results for Matriptase2 protease; (d) Schematic diagram of the linker screening principle and screening results for Corin protease.

[0132] Figure 8 The flowchart of the synthesis of TCO-linker (enzyme-cleaved linker)-PEG is shown.

[0133] Figure 9 The synthesis flowchart of TCO-linker (enzyme-cleaved linker)-XTEN is shown.

[0134] Figure 10The results of screening for non-natural amino acid insertion sites are shown. (a) Candidate insertion sites and cell infection experiment results based on structure prediction for AAV-DJ; (b) Candidate insertion sites and cell infection experiment results based on structure prediction for AAV9; (c) Candidate insertion sites and cell infection experiment results based on structure prediction for AAV8; (d) Candidate insertion sites and cell infection experiment results based on structure prediction for AAV5.

[0135] Figure 11 AAV-PEG modified with PEG and different responsive groups (BR / NIR / MT2) are shown. BR / NIR / MT2 The results of in vitro bond breaking verification. (a) AAV-PEG BR (b) AAV-PEG NIR (c) AAV-PEG MMP .

[0136] Figure 12 The images show AAV-PEG modified with PEG and different responsive groups (BR / NIR / MT2). BR / NIR / MT2 Cell infection validation results. (a) AAV-PEG BR (b) AAV-PEG NIR (c) AAV-PEG MT2 .

[0137] Figure 13 AAV-DJ-PEG was displayed. BR Targeted delivery results in a liver fibrosis model.

[0138] Figure 14 AAV-9-PEG was displayed. BR Targeted delivery results in a cardiac ischemia-reperfusion model.

[0139] Figure 15 AAV-9-PEG was displayed. BR Therapeutic outcomes of gene delivery tools in a cardiac ischemia-reperfusion model. Left: Flowchart; Middle: Echocardiogram; Right: Ejection fraction (EF) and fractional shortening (FS).

[0140] Figure 16 AAV9-PEG was displayed. NIR Results of targeted delivery to muscle tissue.

[0141] Figure 17 PHP-eb-PEG was displayed. NIRResults of targeted delivery to brain tissue. (A) Flowchart; (B) Bioluminescence imaging of mice; (C) In vitro bioluminescence imaging of major organs of mice.

[0142] Figure 18 The in vivo delivery results of Mask-AAV (PEG) based on liver cathepsin response are shown.

[0143] Figure 19 The in vivo delivery results of Mask-AAV (XTEN) based on liver cathepsin response are shown.

[0144] Figure 20 The in vivo delivery results of AAV-DJ with hPKD1,2 as the mask module are shown.

[0145] Figure 21 The in vivo delivery results of AAV-DJ with PKD1,2 and XTEN as mask modules are shown.

[0146] Figure 22 The results of in vivo delivery based on the AAV8 vector are shown.

[0147] Figure 23 The in vivo delivery results of Mask-AAV based on the response of high MMP expression in tumor tissue are shown.

[0148] Figure 24 The results of humoral immune level detection for ROS-responsive Mask-AAV are shown. (a) Neutralizing antibody (NAbs) detection; (b) Total IgG level detection.

[0149] Figure 25 The results show the humoral immune level detection, total IgG level detection, and neutralizing antibody (NAbs) detection of the enzyme-responsive Mask-AAV.

[0150] Figure 26 The in vivo delivery effect of Mask-AAV for secondary injection is shown. (a) Bioluminescence imaging; (b) Statistical graph.

[0151] Figure 27 The delivery performance of Mask AAV with pre-stored neutralizing antibodies is shown.

[0152] Explanation of sequence information

[0153] Information about the sequence involved in this invention is provided in Table 1.

[0154] Table 1: Sequence Information

[0155] Example

[0156] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0157] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0158] Example 1: Mask-AAV technology

[0159] The Mask-AAV technology provided in this application is based on site-specific modification of non-natural amino acids. It combines the design concept of protein prodrugs to perform site-specific chemical modification of the AAV virus capsid protein. The modified AAV virus is in an inert-inactive form in non-target tissues. It is only converted into an active form under special pathological microenvironments (metalloproteinases, cathepsins, ROS) or exogenous stimulation (light, ultrasound, etc.) to achieve targeted gene therapy on specific disease tissues.

[0160] 1. Site-specific modification technology of non-natural amino acids

[0161] Site-directed modification of non-natural amino acids (NAA) involves altering the cellular translation machinery. Utilizing aminoacyl-tRNA synthetase / tRNA pairs orthogonal to the host cell's biological structure, and through orthogonal recognition of nonsense codons, NAAs with chemical properties not found in nature are inserted at any selected site on the target protein. In the following examples, a NAA with a tetrazine side chain was introduced onto the AAV capsid protein for the first time using NAA. The click reaction (IEDDA reaction) between tetrazine and trans-cyclooctene enables efficient site-directed coupling of functional molecules onto AAV. Through uniform and site-directed chemical modification of the AAV capsid protein, different functional molecules can be used to modify the capsid protein, conferring different functions to AAV. For example, modification with targeted antibody molecules increases AAV's tropism towards specific tissues; modification with polymers such as polyethylene glycol and XTEN, as well as the AAVR domain, prolongs the in vivo half-life of AAV and enhances its anti-neutralization ability. Because of the selectivity of the sites, site screening can be performed to select sites that interfere with AAV activity and sites that do not affect AAV activity after coupling, according to different needs.

[0162] 2. Prodrug Modification Strategy

[0163] Prodrugs are defined as small molecules, biological macromolecules, etc., that can be transformed into inactive forms through chemical modification or gene fusion. This allows these molecules to remain inert in the cycle, and only become active when stimulated by specific disease or tissue microenvironments or external signals. This ensures that these drug molecules exert their effects only in the tissues or diseases to which we want them to work, avoiding off-target toxicity caused by non-specific distribution.

[0164] By using the aforementioned non-natural amino acid site-directed insertion technology, polymers or proteins and other macromolecules can be modified through IEDDA-mediated chemical reactions to block the binding of AAV to the receptor, thus transforming AAV into a non-transductional inactive form. Furthermore, by introducing a breakable linker between the polymer and AAV, the linker breaks under specific pathological microenvironments or after exogenous stimulation, thereby relieving the polymer's blocking effect on AAV and restoring AAV function.

[0165] 3. Stimulus-responsive breakable connectors

[0166] In the following embodiments, three different stimulus-response bond-breaking systems were introduced: (1) Based on the excessive ROS level in diseased tissues, ROS-responsive bond-breaking linkers were developed to enable AAV to unblock in specific disease microenvironments and infect specific diseased tissues; (2) Based on the overexpressed protease in tissues, a series of linkers that can respond to the highly expressed protease in specific tissues and efficiently break bonds were screened to enable AAV to infect and express in specific tissues; (3) Based on the exogenous stimulus signal of near-infrared light response, AAV in specific light-irradiated tissues was unblocked and infected.

[0167] Example 2: Synthesis of non-natural amino acids from tetrazine

[0168] Reference J. Am. Chem. Soc. 2020, 142, 16, 7245-7249: Synthesis of the tetrazine non-natural amino acid mPtet (i.e., Tet-v3.0Me). The synthetic steps are shown below.

[0169]

[0170] Other reported non-natural amino acids with tetrazine groups include the following structures, the synthetic steps of which can be found in Angew Chem Int Ed Engl. 2025, 64(35):e202508922.

[0171]

[0172] Example 3: Mutant aminoacyl-tRNA synthetase

[0173] To introduce tetrazine non-natural amino acids (such as mPtet) into the AAV viral vector at a specific site, an additional set of orthogonal aminoacyl-tRNA synthetase (aaRS) and tRNA needs to be introduced. aaRS is responsible for recognizing mPtet, and the anticodon corresponding to tRNA recognizes the TAG stop codon at a predetermined site on the AAV capsid protein. By introducing the stop codon at the predetermined site on the capsid protein and simultaneously introducing aaRS / tRNA, mPtet can be introduced at the predetermined site on the capsid protein.

[0174] We identified and transferred the homologous aminoacyl-tRNA synthetase aaRS / tRNA to the mPtet site, and then used the existing aaRS / tRNA (SEQ ID NO: 3) capable of insertion. MbAn enzyme (SEQ ID NO: 6, tRNA) (J. Am. Chem. Soc. 2020, 142, 16, 7245-7249) was introduced into a G1-derived aminoacyl-tRNA synthetase. By combining the crystal structure of the aaRS and tetrazine interactions, and through rational site mutation, a more efficient aaRS enzyme with an inserted mPtet was obtained, the sequence of which is shown in SEQ ID NO: 1. The corresponding tRNA sequence is shown in SEQ ID NO: 2.

[0175] The efficiency of the above-mentioned mutant enzyme insertion into mPtet was determined. Specifically, the insertion efficiency was verified as follows: 293T cells were seeded into 48-well plates and cultured to a cell density of 90%. The cells were then incubated with PEI containing plasmids, including two plasmids (e.g., ...). Figure 1 (As shown on the left) A reporter gene plasmid carrying the Mcherry-TAG-EGFP fluorescent reporter gene and orthogonal pair plasmids carrying PylRS / tRNAs from different species (Mb, Mm, Mf, and G1) were transfected. Six hours after transfection, 500 μM DMEM cell culture medium containing mPtet was added. After 48 hours, the expression of the reporter gene was measured by flow cytometry. The reading efficiency was verified by comparing the fluorescence intensity of Mcherry with that of EGFP.

[0176] The results are as follows Figure 1 As shown, compared with other aaRS including Mb, Mm, and Mf, the efficiency of mutant aaRS enzyme insertion provided by the present invention is significantly improved.

[0177] Example 4: AAV virus packaging

[0178] A schematic diagram of the AAV virus packaging system is shown below. Figure 2As shown, the 4-plasmid packaging system includes: ① pHelper plasmid for AAV assembly: containing the E2a, E4, and VA genes; ② packaging plasmid pRC-TAG: containing the capsid protein (Cap-TAG) with the stop codon TAG and the replication gene Rep; ③ plasmid pAAV-POI plasmid for delivery carrying the target transduction gene (POI): containing a 5' ITR, CMV promoter, target gene (such as green fluorescent protein EGFP, luciferase Fluc, and angiogenic cytokine VEGFA, etc., any expression gene), SV40 pA sequence, and 3' ITR; and ④ plasmid pCMV-aaRS-tRNA responsible for non-natural amino acid recognition: containing orthogonal aaRS and tRNA, the anticodon of which recognizes the TAG stop codon on the AAV capsid protein. Orthogonal aaRS recognizes the non-natural amino acid and amidates the orthogonal tRNA with the non-natural amino acid, thereby loading the non-natural amino acid onto the orthogonal tRNA, and subsequently incorporating the non-natural amino acid into the protein at the position corresponding to the stop codon.

[0179] The specific experimental procedure is as follows: (1) 293FT cells (obtained from ATCC) were placed in a 10cm... 2 Cells were cultured in culture dishes for 24 hours until the cell density approached 90%. Then, 293FT cells were transfected using PEI as the transfection vector, with the four plasmids in a 1:1:1:1 ratio. One plasmid was 10 cm². 2 20 μg of plasmid needs to be transfected. The ratio of PEI to plasmid is 3:1. Add Opti-MEM buffer according to this ratio for transfection.

[0180] (2) After 6 hours, the culture medium needs to be replaced. 400 μM mPtet is added to the DMEM medium. After adding the culture medium, the cultured cells are collected after 72 hours. The supernatant is removed by centrifugation and the cells are collected for AAV purification.

[0181] (3) AAV was purified by PEG8000 precipitation, and the titer of the packaged AAV was determined by qPCR. Thus, AAV virus particles carrying a stop codon were obtained.

[0182] (4) For the detection of AAV virus, Western blot analysis was used. Hybridization with anti-AAV antibody (PROGEN, 65158) was performed to confirm whether AAV particles were fully expressed. The results showed that AAV virus particles could not be packaged without the addition of mPtet, and complete AAV particles could only be produced in the system with the addition of non-natural amino acid mPtet.

[0183] Example 5: Comparison of reaction efficiency between AAV containing tetrazine non-natural amino acids and AAV containing NAEK.

[0184] Similar to the packaging and purification of tetrazine-containing non-natural amino acids (AAV), NAEK-containing AAVs were packaged and purified (see US10087217B2), and the modification efficiency of the two AAVs was evaluated at the cellular and animal levels. AAV-DJ was used as the base vector to package AAV-DJ... mPTet With AAV-DJ NAEK In this process, the codon corresponding to position 585 of the AAV-DJ capsid protein was replaced with the TAG stop codon, thereby introducing a non-natural amino acid at that site. Different lengths of TCO-PEG (5K, 10K, 20K) and DBCO-PEG (5K, 10K, 20K) (obtained from CONFLUORE, catalog numbers BCTG-1-25mg, BCTG-11-250mg, BGNH-45-250mg, BCDG-9-250mg, BCDG-10-100mg, respectively) were reacted at different concentrations (0μM, 1μM, 10μM, 10μM) as shown in the flowchart below. Figure 3 As shown in the figure. Subsequently, the modification level and blocking effect of the two reactions on AAV were verified at the Western blot, cellular, and animal levels, respectively.

[0185] Specifically, AAV-DJ NAEK Or AAV-DJ mPTet Viral particles were incubated with DBCO-PEG or TCO-PEG reagents of different molecular weights in phosphate-buffered saline (PBS) at 25 °C. The final concentration of the PEG reagent was adjusted according to experimental needs. The reaction time was 2 hours to achieve covalent coupling between the viral capsid and the PEG molecules. After the reaction was complete, free NAEK or mPTet molecules, at a molar ratio of 10 times that of the PEG reagent, were added to the system, and incubation was continued at 25 °C for 30 minutes to fully block unreacted active groups, thereby terminating the coupling reaction.

[0186] To analyze coupling efficiency and modification status, the reacted samples were mixed with loading buffer, subjected to heat denaturation, and then MBP-sfGFP protein was added as an internal control fluorescence standard. Subsequently, the samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking the membrane with 5% (w / v) skim milk powder, the GFP fluorescence signal in each lane was scanned and quantified using Typhoon Imager (Cytiva). AAV capsid proteins were detected using a mouse monoclonal antibody against AAV VP1 / VP2 / VP3 (clone B1). The gray intensity of the VP3 band was quantified using ImageJ software and normalized to the MBP-sfGFP fluorescence signal to evaluate the PEG modification efficiency.

[0187] In the in vitro transduction experiment, the obtained AAV-DJ-PEG conjugate was added to HEK293T cells for infection culture with a multiplicity of infection (MOI) of 1000, and the expression level of the transgene was detected 48 hours after infection.

[0188] In in vivo experiments, the AAV-PEG conjugate was administered via intramuscular injection (5 × 10⁶ mice per mouse). 9 Viral genome (vg) or intravenous injection (1×10⁻⁶ per mouse) 10 (vg) was administered to experimental animals. On day 7 after administration, the expression of the transgene was detected and analyzed using bioluminescence imaging technology.

[0189] The results are as follows Figure 4 As shown, the results indicate that PEGs of different molecular weights (5K, 10K, 20K) can be successfully coupled to the AAV-DJ variants containing non-natural amino acids, among which AAV-DJ... mPTet The coupling efficiency with TCO-PEG is significantly higher than that with AAV-DJ. NAEK Coupling efficiency with DBCO-PEG (4A).

[0190] In in vitro cell experiments, AAV-DJ modified with TCO-PEG... mPTet Almost no detectable EGFP expression was produced in HEK293T cells, indicating that PEG conjugation effectively blocked the cellular transduction of AAV-DJ; in contrast, AAV-DJ modified with DBCO-PEG showed significantly reduced EGFP expression. NAEK It still retains some transduction activity (4B).

[0191] In in vivo experiments, the AAV-DJ-mediated transgene expression level gradually decreased with increasing PEG molecular weight. Specifically, AAV-DJ modified with PEG20K showed the highest expression level. mPTet In mice, almost no detectable luciferase signal was produced, and its expression level was close to the background value, indicating that high molecular weight PEG modification can effectively inhibit the transduction activity of AAV-DJ in vivo (4C).

[0192] Example 6: Design and Construction of Stimulus-Response Connectors

[0193] Taking PEG as a mask module as an example, PEG, as an FDA-approved pharmaceutical excipient, exhibits excellent stability and biocompatibility in vivo. Modifying the surface of AAV with PEG as a module, as described above, allows PEG to mask the infectious activity of AAV. Simultaneously, introducing TCO groups onto PEG that can undergo bioorthogonal reactions with mPTet on AAV, and introducing a stimulus-responsive linker between the TCO and PEG, enables temporary masking of AAV activity and restoration of activity under specific pathological microenvironments or exogenous light stimulation. We have designed three types of linker modules to meet the needs of different diseases or tissues.

[0194] (a) ROS stimulus-response linkers

[0195] In various acute and chronic inflammations, ROS play a crucial role in the activation of inflammatory cells, the release of inflammatory mediators, and the inflammatory response. Overexpressed ROS are also widely used as important biomarkers in pathological inflammation for detection and targeted drug delivery. Figure 5 As shown, phenylboronic acid was used as the ROS initiator, and a ROS-responsive bond-breaking linker was developed by combining a 1,6 elimination reaction. Trans-cyclooctene (TCO) was used as a molecular handle for rapid and efficient coupling of the ROS-responsive module. PEG units were used as the module terminator to shield the receptor-binding domain of the protein, thereby obtaining a linker module based on endogenous stimulation signals of inflammatory response.

[0196] Specifically, targeting ROS overexpressed in inflammatory tissues, a ROS-responsive mask module, TCO-BR-PEG, was designed. Boric acid, acting as the ROS-responsive unit, undergoes a 1,6-elimination reaction upon ROS oxidation, causing the PEG portion to detach from AAV, thus releasing the blocking effect of PEG on AAV infectious activity. This allows AAV to infect only at the site of inflammatory disease.

[0197] Synthesis of compound molecules TCO-BR-PEG as follows Figure 5 As shown in C, the specific steps are as follows.

[0198] The synthesis of 1a was carried out according to the previously reported method (J. Am. Chem. Soc. 2022, 144, 51, 23614-23621). 1a (740 mg, 1 mmol) and N,N-diisopropylethylamine (155 mg, 1.2 mmol, from BIDE) were dissolved in tetrahydrofuran (5 mL) and stirred at 0 °C, followed by the addition of trans-cyclooctene-amino (92 mg, 0.4 mmol, from BIDE). The reaction mixture was gradually heated to room temperature and stirred for 2 hours. After the reaction was complete, the mixture was diluted with ethyl acetate (30 mL), washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:1) to give a white solid 1b (400 mg, 50% yield). HRMS (ESI) + ):C 42 H 52 BN3O 12 [M+H] + The theoretical value is 801.3753; the measured value is [M+H2O]. + 819.3968, [M+Na] + 824.3514.

[0199] In the subsequent reaction, 1b (50 mg, 0.062 mmol) was dissolved in tert-butanol (1 mL, from BIDE), and N,N-diisopropylethylamine (16 mg, 0.124 mmol, from BIDE) was added, followed by amino-PEG20k (1240 mg, 0.062 mmol, from jenkem). The reaction mixture was stirred overnight, poured into 50 mL of anhydrous diethyl ether, and filtered. The resulting crude product was further purified by dialysis (molecular weight cutoff 10 kDa) using deionized water (ddH2O) as the dialysis medium for 16 hours. Finally, the dialyzed solution was freeze-dried to obtain a white powder, TCO-BR-PEG.

[0200] (ii) Photosensitive stimulus-response connectives

[0201] The exogenous stimulus signal is near-infrared light, and the photoresponsive unit is heptamethrin cyanine dye (Cy7). Due to the photobleaching phenomenon of Cy7 under near-infrared light irradiation, the mechanism involves photoinduced activation leading to singlet-state-induced double bond scission. Through rational structural design, after hydrolysis, TCO units and PEG are released, thereby achieving the unblocking of the near-infrared photoresponsive Mask-AAV. This yields a connecting submodule based on the exogenous stimulus signal.

[0202] This process can be achieved using photosensitive chemically responsive groups. Red light can be used to photobleach the fluorescent molecule Cy7. The principle is that the Cy7 luminescent group absorbs near-infrared light. During the radiation recovery to the ground state, the generated singlet oxygen chemically breaks the bonds of the luminescent group Cy7. Based on rational chemical design, a red light-responsive mask module TCO-NIR-PEG was designed. Under near-infrared irradiation, the Cy7 module undergoes chemical bond breakage. Through a 1,4 elimination reaction, the modified PEG is detached from AAV, releasing the blocking effect of PEG on the infection activity of AAV, thereby enabling AAV to infect only at the light-irradiated tissue sites.

[0203] Synthesis of compound TCO-NIR-PEG as follows Figure 6 As shown, the specific steps are as follows.

[0204] A dialdehyde compound (bisaldehyde, 562 mg, 2 mmol, from BIDE) and anhydrous sodium acetate (410 mg, 5 mmol, from BIDE) were dissolved in acetic anhydride (15 mL, from BIDE) and stirred at room temperature, followed by the addition of a quaternary ammonium salt (1.5 g, 5 mmol). The reaction mixture was heated at 100 °C for 1 hour. After cooling, the mixture was poured into 200 mL of anhydrous diethyl ether and filtered. The resulting solid was dissolved in methanol, dried over anhydrous MgSO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (methanol / dichloromethane = 1:6) to give a dark green solid compound 2a (1.1 g, 70% yield). Chemical formula: C 41 H 48 ClN2O7S2; HRMS ([M+H2O) + Theoretical value: 797.26, Measured value: [M+H2O] + 797.53.

[0205] Compound 2a (780 mg, 1 mmol) was dissolved in acetonitrile (5 mL) and stirred at 0 °C. N,N'-diethylethylenediamine (580 mg, 5 mmol, from Bioderm) was then added. The reaction mixture was gradually heated to room temperature and stirred for 2 hours. The reaction solution was then poured into 50 mL of anhydrous diethyl ether and filtered to give a blue solid crude product 2b (64 mg, approximately 80%), which was used directly in the next step of the reaction without further purification.

[0206] Next, the dialdehyde compound (172 mg, 0.2 mmol, from BIDE) and DIPEA (52 mg, 0.4 mmol, from BIDE) were dissolved in dichloromethane (5 mL) and stirred at 0 °C. Then, TCO-PNB ester (58 mg, 0.2 mmol, from BIDE) was added. The reaction mixture was gradually heated to room temperature and stirred overnight. After the reaction was complete, the mixture was diluted with dichloromethane (30 mL), washed successively with saturated Na₂CO₃ aqueous solution and saturated brine, and the organic phase was dried over anhydrous Na₂SO₄, filtered, and concentrated. The residue was purified by silica gel column chromatography (methanol / dichloromethane = 1:6) to give the target compound 2c as a blue solid (50 mg, yield 25%). Chemical formula: C 56 H 75 N4O9S2; ESI-MS (theoretical value): 1011.50, measured value: 1011.4971.

[0207] Compound 2c (5 mg, 0.0049 mmol) was dissolved in tert-butanol (1.5 mL), and deionized water (ddH2O, 1 mL) was added. Under nitrogen protection, CuSO4·5H2O (0.611 mg, 0.0024 mmol, from BIDE), sodium L-ascorbate (0.97 mg, 0.0049 mmol, from BIDE), and azide-PEG (98 mg, 0.0049 mmol, from BIDE) dissolved in 0.5 mL ddH2O were added sequentially. The reaction mixture was stirred overnight, poured into 50 mL of anhydrous diethyl ether, and filtered. The resulting crude product was further purified by dialysis (molecular weight cutoff 10 kDa) using ddH2O as the dialysis medium overnight. Finally, the dialyzed solution was freeze-dried to obtain a blue powder, TCO-NIR-PEG.

[0208] (iii) Linkers for protease response

[0209] Using tissue-specific proteases as response signals, different tissues in normal physiological tissues express relatively specific proteases, such as the type II transmembrane serine protease Matriptase-2, which is highly expressed in liver tissue; the protease Corin, which is highly expressed in heart tissue; and the matrix metalloproteinases (MMPs), which are highly expressed in common tumor tissues. Using these as stimulation signals, linkers capable of efficient bond breaking in specific tissues were screened, and a Mask-AAV that specifically infects liver, heart, and tumor tissues was constructed. After circulating to specific tissues, it responds to the tissue-specific proteases, releasing the AAV activity barrier and achieving specific infection of liver, heart, and tumor tissues. This yields a linker module based on endogenous stimulation signals responsive to tissue-specific proteases.

[0210] Specifically, for liver, heart, or tumor tissues that overexpress proteases, a substrate containing the corresponding enzyme-cleaved peptide is introduced between PEG and TCO to design a protease-responsive TCO-linker-PEG. In liver, heart, or tumor tissues, a specific enzymatic cleavage reaction occurs, allowing PEG to release the blocking effect on AAV infection activity, thereby enabling AAV to infect specific tissues or tumor sites.

[0211] For the enzyme-cleaved peptide substrates, linkers capable of efficiently breaking bonds on the cell surface of specific tissues were screened by in vitro incubation with ground tissue. Linkers for high expression of Matriptase2 protease in liver tissue, high expression of Corin protease in heart tissue, and high expression of MMP in tumor tissue were screened. Specifically, 6-8 week old Babl / c mice were sacrificed, and liver and heart tissues were separated. 50 mg of tissue was collected, homogenized, and filtered through a single-cell membrane to obtain cell suspensions of liver and heart tissue, respectively. High expression of MMP in tumor tissue was achieved in SKOV3 breast cancer cells. A fusion protein of Flag-MBP-substrate sequence-affibody was constructed as a reporter molecule and recombinantly expressed in *E. coli*. The expressed fusion protein was incubated with the cell suspensions. After incubation, Western blotting was performed using anti-Flag and anti-MBP antibodies. If the substrate sequence was cleaved, the molecular weight of the intact fusion protein band decreased, and smaller bands appeared. The resulting highly efficient linker was then used for the synthesis of an enzyme-responsive series of TCO-linker-PEGs.

[0212] Linker screening results for liver tissue with high expression of Matriptase2 protease are as follows: Figure 7 As shown in Figure ac, the final screened linker sequences MT2-1 to MT2-8 are shown in SEQ ID NO: 20-27. MT2-8 is used in the following examples, and is abbreviated as MT2. The linker screening results for high expression of Corin protease in cardiac tissue are shown in Figure ac. Figure 7 As shown in d, after corin cleavage, the cleaved FLAG-MBP bands can be observed, with CL1~CL7 sequences as shown in SEQ ID NO: 28-34. The sequences of linkers highly expressing MMP in tumor tissue were obtained through screening using the same method, as shown in SEQ ID NO: 35-36.

[0213] Enzyme-responsive series TCO-linker-PEG synthesis, such as Figure 8 As shown, CS represents the linker for the protease response.

[0214] TCO-Mal (trans-cyclooctene-maleimide, CONFLUORE), Cys-Linker-N3 (CONFLUORE), and DBCO-PEG (CONFLUORE) were reacted in dichloromethane at room temperature for 2 hours. After the reaction was complete, the mixture was recrystallized with diethyl ether, the precipitate was collected, and TCO-linker-PEG was obtained by freeze-drying.

[0215] Example 7: Coupling of different Mask modules / active targeting modules with TCO

[0216] In addition to the PEG module, we tried using other modules to block AAV activity, including XTEN, AAVR, and active targeting modules.

[0217] (1) XTEN module

[0218] XTEN is a non-structured polypeptide that can be recombinantly expressed in *E. coli*, exhibiting an extended conformation similar to that of a polypeptide. Due to its ease of recombinant expression, we fused a protease-responsive linker with the XTEN protein, thereby introducing a responsive linker (CS) to the C-terminus of the XTEN protein. We then introduced a cys residue at the C-terminus of this fusion protein to provide a thiol group (SH), using this as the reactive group to prepare TCO-linker-XTEN. The preparation process is as follows: Figure 9 As shown.

[0219] TCO-Mal (trans-cyclooctene-maleimide, CONFLUORE) was dissolved in DMSO to prepare a 10 mM stock solution. The fused expression of XTEN-cys was reacted with TCO-Mal in water at a molar ratio of 1:5 at room temperature for 2 h. After the reaction was complete, unreacted small molecules were removed using a 10 kDa ultrafiltration tube to obtain TCO-linker-XTEN, which was verified by SDS-PAGE.

[0220] (2) AAVR module

[0221] AAVR is an auxiliary receptor for AAV and can bind to multiple AAVs. Considering that AAVR is the natural receptor for AAV, recombinant expression of AAVR onto AAV can directly mask AAV; therefore, AAVR was also chosen as the mask module for Mask-AAV. We fused a protease-responsive linker with a portion of the extracellular domain of AAVR to a PKD protein, introduced a responsive linker (CS) into the C-terminus of the protein, and introduced a cys residue into the C-terminus of the fusion protein to provide a thiol group (SH). Using this as a reactive group, we prepared the TCO-linker-PKD protein under similar conditions to the XTEN module.

[0222] (3) Active targeting module

[0223] For tissues where AAV struggles to actively target, such as tumor tissue, we introduced an active targeting module. This module (anti-HER2 affibody, Z342, sequence SEQ ID NO: 17) was fused with the XTEN protein and a linker (sequence SEQ ID NO: 35 or 36) was introduced to respond to the high expression of MMPs in tumor tissue. The resulting fusion protein, from N-terminus to C-terminus, contains: anti-HER2 affibody, XTEN protein, and linker. A cys residue was introduced at the C-terminus of this fusion protein to provide a thiol (SH) group, and the preparation conditions were similar to those of the XTEN module. The active targeting module acts as a mask module, blocking AAV infection of other tissues, while simultaneously increasing the tropism of Mask-AAV towards tumor tissues through its active targeting function. It also unblocks AAV activity in response to MMPs in tumor tissue, enabling efficient AAV infection of tumor tissues.

[0224] Example 8: Selection of insertion sites for non-natural amino acids

[0225] To ensure that the modified modules can temporarily block the infectivity of AAV while the residual chemical groups released do not affect AAV infection (this can be understood as PEG or other modules having a large space, and after bond breaking, a very small chemical group; theoretically, through our molecular design, the residue after bond breaking is the product of the reaction between tetrazine and TCO), our screening method and procedure are as follows: Based on the crystal structure of AAV, we mutated it into a tetrazine non-natural amino acid at the site where AAV might interact with the receptor. The specific procedure is similar to that described above. We obtained AAV with tetrazine non-natural amino acids introduced at different sites through purification, and reacted it with TCO-PEG20K (CONFLUORE) (the theoretically blocked module) or TCO-NH2 (CONFLUORE) (the unblocked remaining module). The reacted virus was added to 293T cells for infection. After 48 hours, after cell digestion, the average fluorescence intensity of the cells was analyzed by flow cytometry to verify the expression level of the EGFP gene, thereby exploring the infectivity of each AAV. Due to the differences in the interaction between different serotypes and receptors, this method may be necessary for preliminary screening of a particular serotype to obtain a site with the best blocking effect and the best recovery of infectious activity after unblocking.

[0226] The results are as follows Figure 10As shown in Figure a, candidate sites for AAV-DJ interaction near the AAV-DJ-receptor interface were rationally selected, including S268, Q327, S448, T453, L462, T583, and L585. Cell infection experiments showed that PEGylation modification at different sites could effectively inhibit the infectivity of AAV. At the same time, the L585 site showed the highest difference in infection (blocking and unblocking). Therefore, L585 was selected as the best coupling site for subsequent modification.

[0227] Candidate sites for AAV9 include: G453, G455, S499, A591, and H584. Figure 10 b). Cell infection experiments showed that PEGylation modification at different sites could effectively inhibit the infectivity of AAV, while the H584 site showed the highest difference in infection (blocking and unblocking). Therefore, H584 was selected as the best coupling site for subsequent studies.

[0228] Candidate sites for AAV8 include: S266, Q328, S449, Q461, L463, D584, L586, Q588, T591, and Q594. After cleavage by MT2, the protease-responsive linker may retain short peptide sequences CRKRR or GSRGC. Therefore, in AAV8 cell infection experiments, tetrazine-containing non-natural amino acids of AAV were introduced at different sites and reacted with TCO-CRKRR or TCO-GSRGC to obtain AAV conjugated with CRKRR or GSRGC, mimicking the residue remaining after MT2 cleavage of the liver. The reacted virus was added to 293T cells for infection. After 48 hours, the cells were digested, and the mean fluorescence intensity was analyzed by flow cytometry to verify the expression level of the EGFP gene, thereby exploring the infectivity of each AAV. The results are as follows: Figure 10 As shown in Figure c, the simulated enzyme-cleaved AAV-CRKRR or AAV-GSRGC exhibits infectivity no less than that of the wild-type virus, indicating that the fragmentation does not affect infection. Specifically, because the wild-type aav8 itself has weak cellular infectivity, the conjugation with CRKRR, due to the presence of positively charged amino acids such as R and K, leads to an increase in the infectivity of AAV-CRKRR.

[0229] Candidate sites for AAV5 include: D374, G444, and A579. Figure 10 d) Cell infection experiments showed that PEGylation modification at different sites could effectively inhibit the infection activity of AAV. Meanwhile, the A579 site showed the highest infection difference (blocking and unblocking). Therefore, A579 was selected as the best coupling site for subsequent modification.

[0230] Example 9: In vitro cell infection and controlled gene transduction of Mask-AAV

[0231] (1) Coupling of AAV and Mask modules

[0232] AAV serotypes (AAV-DJ, AAV-9, AAV8, PHP-eb, etc.) with tetrazine-intercalated non-natural amino acids were placed in PBS buffer. Stock solutions of different response units, such as TCO-BR / NIR / MMP-PEG / XTEN / AAVR, were prepared at varying concentrations. PEG compounds, due to their good solubility, were prepared at 5 mM, while protein molecules were prepared at 300 μM - 1 mM. The reaction of PEG with AAV was carried out at a final concentration of 100 μM, while the reaction of protein molecules was carried out at a concentration of 30 μM. The reaction was incubated overnight at 25°C. The resulting AAV viral particles were ultrafiltered and centrifuged using a 100K ultrafiltration tube to remove uncoupled compounds. The obtained viral particles were used for subsequent cell infection and animal experiments.

[0233] (2) In vitro bond breakage verification

[0234] AAV-PEG was obtained by reacting AAV-DJ, which has a mutation of mPTet at position 585 of the capsid protein, with TCO-BR-PEG20K, TCO-NIR-PEG20K, and TCO-MT2-PEG20K, respectively. BR AAV-PEG NIR AAV-PEG MT2 The PEG bond cleavage was tested under different signal stimuli.

[0235] AAV-PEG BR The samples were placed in 0, 100, 250, and 500 μM hydrogen peroxide solutions and incubated at 37°C for 4 hours. DTT was then added for denaturation, followed by incubation at 100°C for 5 minutes. These were then used for subsequent Western blotting (WB) detection. The primary antibody used was an anti-AAV antibody (PROGEN, clone B1). WB procedures were performed according to standard operating procedures. Figure 11 As shown in Figure a, after the reaction with PEG20K, the bands of VP1, VP2, and VP3 shifted upwards, indicating that site-directed coupling between AAV and PEG was achieved. After incubation with different concentrations of hydrogen peroxide, the upper bands disappeared, suggesting AAV-PEG... BR This allows for complete bond breaking at a hydrogen peroxide concentration of 100 μM. This demonstrates that PEG can achieve responsive bond breaking in a hydrogen peroxide environment.

[0236] AAV-PEG NIRIrradiated with 690 nm near-infrared light at 0, 5, 10, 25, and 50 J, followed by incubation at 37°C for 30 min, then denaturation with DTT, and incubation at 100°C for 5 min, for subsequent Western blotting (WB) detection. Figure 11 As shown in b, PEG can achieve responsive bond breaking in near-infrared illumination environments.

[0237] AAV-PEG MT2 Placed in the buffer of matriptase-2 enzyme (SEQ ID NO: 19), incubated at 37°C for 4 hours, then denatured with DTT at 100°C for 5 minutes for subsequent Western blotting. Figure 11 As shown in c, PEG can achieve responsive bond breaking in an environment containing the matriptase-2 enzyme.

[0238] (3) Cell infection verification

[0239] 293T plates were seeded, with 2E4 cells per well in a 48-well plate. Cells were cultured overnight, and the above-mentioned AAV-PEG was added to each well. BR The corresponding mutant AAV (AAV-DJ with a mutation at position 585 to mPTet) does not contain a mask module, and AAV-PEG treated with different concentrations of ROS. BR AAV-packaged Flux-T2A-EGFP (SEQ ID NO: 39) or EGFP reporter genes were added to cells at an MOI of 1000. After 48 hours, EGFP expression was observed using a confocal microscope. For the EGFP reporter gene, flow cytometry was performed directly after 48 hours. For the Flux-T2A-EGFP reporter gene, 100 μL of cell lysis buffer was added after removing the culture medium, along with luciferase substrate, to quantitatively detect the expression of the target gene after AAV infection and assess the AAV's infectious activity.

[0240] Similarly, AAV-PEG is added to the cell culture medium. NIR and AAV-PEG after NIR light treatment NIR Verify the recovery of AAV infection controlled by light.

[0241] Similarly, AAV-PEG is added to the cell culture medium. MT2 and AAV-PEG treated with different concentrations of enzyme MT2 To verify the enzyme response to AAV infection recovery.

[0242] The results are as follows Figure 12 As shown, ac represents the results for the responsive groups BR, NIR, and MT2, respectively. The results indicate that AAV-PEG modified with PEG has different responsive groups (BR / NIR / MT2).BR / NIR / MT2 The vector's infectivity was significantly inhibited, essentially losing its ability to infect target cells; in the case of the corresponding AAV-PEG... BR / NIR / MT2 After treatment with different stimuli (ROS / red light / matriptase-2), the cleavable linker broke, and the PEG masking group was removed from the AAV surface, allowing the unmasked AAV to regain its infectivity against cells. These results demonstrate that the system of this invention can achieve controllable removal of the PEG mask through stimuli, thereby achieving controllable recovery of AAV infectivity.

[0243] In addition, the effects of inserting tetrazine non-natural amino acids on other AAV serotypes (including AAV-9, AAV-8, PHP-eb, etc.) were verified. The results showed that they could also achieve controllable removal of PEG masking through stimulation signals, thereby achieving controllable recovery of AAV infection activity, indicating the universality of this modification strategy for different serotypes.

[0244] Example 10: Disease-targeted gene delivery for liver fibrosis and myocardial ischemia-reperfusion based on ROS response

[0245] To explore the activity recovery and controllable gene transduction of Mask AAV under disease stimulation at the in vivo level, we plan to demonstrate the targeted delivery capability of Mask-AAV through various animal models of chronic or acute inflammation.

[0246] 1. Liver fibrosis model

[0247] Using mouse liver fibrosis as a model, BABL / C mice (obtained from the Department of Animal Science and Technology, Peking University School of Medicine) were intraperitoneally injected with carbon tetrachloride for 4 weeks to establish a mouse liver fibrosis model. Using luciferase (Fluc-T2A-EGFP) as the reporter gene, the selected AAV-DJ-PEG was intravenously injected. BR The mutant (DJ has a mutation site of L585 and a PEG molecular weight of 20K) was used as a control, and the expression of luciferase in the liver tissue of healthy mice and model mice was verified by small animal in vivo imaging.

[0248] The results are as follows Figure 13 As shown, the experimental results indicate that, in healthy mice, site-specific PEG-modified AAV-DJ-PEG... BR Virtually no gene transduction occurred, indicating that this PEG masking effectively blocked the infectivity of AAV; however, in mice with liver fibrosis, AAV-DJ-PEG... BRThe transduction activity was significantly restored, indicating that highly expressed reactive oxygen species (ROS) in inflammation-related tissues can trigger the breakage of the cleavable linker, thereby achieving selective restoration of AAV infection activity.

[0249] 2. Myocardial ischemia-reperfusion model

[0250] To achieve heart tissue-specific gene delivery, a mouse model of myocardial ischemia-reperfusion was established. The left descending artery of BABL / C mice (obtained from the Department of Animal Science and Technology, Peking University School of Medicine) was surgically ligated to induce myocardial ischemia. After 30 minutes, the ligation was removed, and reperfusion was initiated to obtain the myocardial ischemia-reperfusion model. Using luciferase (Fluc-T2A-EGFP) as the reporter gene, the aforementioned AAV-9-PEG was selected via intravenous injection. BR The mutant (the mutation site of AAV9 is H584, ​​and the molecular weight of PEG is 20K) and the corresponding mutant AAV (AAV9) that does not contain the mask module. mPTet Using a sham-operated group as a control, the study verified its ROS response to ischemia-reperfusion injury, and using intravenous injection of wild-type AAV9 as a control, the study verified its targeting to cardiac tissue. The results are as follows: Figure 14 As shown, bioluminescence imaging results indicate that after receiving AAV9-PEG... BR In the treated myocardial ischemia-reperfusion (I / R) model mice, significant luciferase expression signals were detected in the cardiac region; in contrast, only near-background signals were detected in the sham-operated control group. Figure 14 Comparative analysis was conducted after systemic administration of the virus. Results showed that unmasked AAV9... mPTet Luciferase expression signals were detected in multiple tissues, while AAV9-PEG... BR Gene expression is primarily restricted in cardiac tissue ( Figure 14 The above results indicate that AAV9-PEG BR It can respond to highly expressed reactive oxygen species (ROS) under pathological conditions during myocardial ischemia-reperfusion injury, thereby achieving local restoration of gene expression in damaged myocardial tissue.

[0251] Example 11: Non-invasive drug delivery of VEGF-A via AAV 165 Achieving gene therapy for myocardial ischemia-reperfusion in mice

[0252] Replace the target gene in the AAV packaging gene with VEGF-A. 165 (Sequence SEQ ID NO: 40), wild-type and mutant AAV9 are produced via a three- or four-plasmid packaging system, wherein VEGF-A 165Containing a secretory signal peptide, it was used to infect 293T cells. Western blotting was used to detect VEGFA expression in the supernatant (the antibody used was anti-VEGFA monoclonal antibody, Proteintech, 66828-1-Ig). In gene therapy for myocardial ischemia-reperfusion, VEGF-A was delivered intravenously after perfusion. 165 AAV9-WT and AAV-9-PEG genes BR Using FLUC as a control gene, relevant cardiac function parameters (ejection fraction FS and left ventricular ejection fraction LVEF) were measured by echocardiography on day 2. These parameters were re-measured on day 28. The improvement in these parameters validated the effectiveness of VEGF-A. 165 Gene therapy efficacy, to demonstrate the effectiveness of AAV-9-PEG BR As an effective gene delivery tool for the treatment of myocardial ischemia-reperfusion.

[0253] The results are as follows Figure 15 As shown, with AAV9-PEG BR Compared to the (luciferase) control group, those receiving AAV9-PEG BR Mice treated with VEGF-A165 exhibited higher ejection fraction (EF) and fractional shortening (FS). These results demonstrate that masked AAV delivery of VEGF-A165 can improve myocardial function without invasive procedures and provides an effective delivery strategy for precision gene therapy of myocardial ischemia-reperfusion injury.

[0254] Example 12: NIR-responsive Mask AAV for targeted gene delivery to muscle and brain tissue

[0255] 1. Targeted delivery to muscle tissue

[0256] To verify that AAV-responsive photocontrolled gene transduction can be achieved based on near-infrared responsive units, AAV-PEG was obtained by reacting with TCO-NIR-PEG. NIR Using Flux-T2A-EGFP as the reporter gene, 6-8 week old female BALB / c mice (obtained from the Department of Animal Science and Technology, Peking University School of Medicine) were selected and intravenously injected with AAV9 virus (AAV9) without a mask module one week after feeding. mPTet ), and AAV9-PEG NIR (The mutation site of AAV9 is H584, ​​and the molecular weight of PEG is 20K), among which AAV9-PEG is injected.NIR Take 4 mice, such as Figure 16 As shown, near-infrared light was applied to the leg muscles at an intensity of 200 mw / cm². 2 The irradiation time was 10 minutes. Seven days after intravenous injection, the substrate for luciferase was injected intraperitoneally at a dose of 15 mg / kg. Luciferase expression was detected by in vivo chemiluminescence imaging 10 minutes later. Results are as follows: Figure 16 As shown, bioluminescence imaging results performed on day 28 post-drug administration revealed significant and localized luciferase expression signals in the near-infrared irradiated region, with signal intensity increased compared to the unirradiated control group, while unmodified AAV9... mPTet It exhibits extensive transduction and transgenic expression in various tissues throughout the body; in contrast, AAV9-PEG... NIR The expression of the transgene is mainly limited to muscle tissue exposed to near-infrared irradiation. Figure 16 The above results demonstrate that masked AAVs activated by near-infrared triggering can achieve precise temporal and tertiary control of gene delivery to specific irradiated areas under systemic drug delivery conditions.

[0257] 2. Targeted delivery to brain tissue

[0258] Brain-targeted gene delivery was studied using the PHP-eb serotype as the research vector. While a brain-targeted delivery vector for PHP-eb was obtained through directed evolution, off-target effects in the liver still exist. This study explored whether the Mask AAV strategy could reduce off-target transduction of PHP-eb in the liver, achieving brain-targeted gene delivery under near-infrared light control. The non-natural amino acid mPtet was introduced at the H584 site of the PHP-eb capsid protein, and after purification, a mutant virus, PHP-eb, was obtained. This mutant virus was then reacted with TCO-NIR-PEG to obtain PHP-eb-PEG. NIR (PEG molecular weight is 20K). Female c57bl / 6j mice (obtained from the Department of Animal Science and Technology, Peking University School of Medicine) were intravenously injected with wild-type PHP-eb and PHP-eb-PEG, respectively. NIR The virus, in which 4 mice showed symptoms 30 minutes after intravenous injection, such as... Figure 17 As shown in Figure a, near-infrared light (690nm, 400mw / cm²) was applied to the head of the mouse. 2 After 10 minutes, and 7 days later, the substrate for luciferase was injected intraperitoneally for chemiluminescence imaging. The detection of ex vivo tissues was performed as before.

[0259] The results are as follows Figure 17As shown in figures b and c, bioluminescence imaging results revealed that, compared to the control group that did not receive near-infrared irradiation, the luciferase expression signal in the brains of irradiated mice was increased by approximately 134.4 times. In vitro bioluminescence imaging results showed that transgene expression was detectable in both brain and liver tissues of mice injected with AAV-PHP.eB, with a signal intensity ratio of approximately 3:1 between the brain and liver. In contrast, AAV-PHP.eB-PEG... NIR Under near-infrared light irradiation, transduction was primarily achieved in brain tissue, with the signal intensity ratio between the brain and liver significantly increased to 100.6. These results indicate that site-specific PEG modification combined with near-infrared light triggering can achieve specific AAV transduction in brain tissue without reducing gene delivery efficiency in the central nervous system, while significantly reducing gene delivery levels in non-target tissues such as the liver.

[0260] Example 13: Liver tissue-specific gene delivery based on Mask-AAV (PEG) response to liver cathepsin

[0261] Mask elements of different PEG lengths, including TCO-MT2-PEG10k / PEG20k / PEG40k, were synthesized using the previously mentioned synthesis method. Flux-T2A-EGFP was used as the reporter gene, and AAV-DJ was used as the vector. The L585 site was mutated to mPTet for coupling. AAV-DJ-PEG was then prepared by coupling under the previously mentioned reaction conditions. MT2 Complex. Male Balb / C mice aged 6-8 weeks (obtained from the Department of Animal Science and Technology, Peking University School of Medicine) were intravenously injected with AAV-DJ. mPTet and AAV-DJ-PEG MT2 The complex was observed in vivo at 7 and 14 days after intravenous injection to monitor luciferase expression in mice. Tissues from major mouse organs (heart, liver, spleen, lung, kidney, and muscle) were further quantitatively assessed for liver-targeting specificity. Animal experiments also showed that longer PEG lengths exhibited higher liver expression specificity.

[0262] The results are as follows Figure 18 As shown, bioluminescence imaging results revealed that the infectivity of AAV blocked with PEG without the introduction of the hepatic cleavable linker was significantly inhibited, while TCO-MT2-PEG20k with the introduction of the hepatic cleavable linker MT2 showed a recovery of infectivity. Furthermore, the tissue distribution results showed that, compared to unmodified AAV, TCO-MT2-PEG20k only exhibited highly efficient gene transduction in liver tissue, while unmodified AAV showed off-target gene transduction in organs such as the heart. Figure 18 ).

[0263] Example 14: Liver tissue-specific gene delivery based on Mask-AAV (XTEN) response to liver cathepsin

[0264] Using XTEN as the mask module, we expressed a series of XTEN proteins of different lengths (144aa, 288aa, 432aa) in *E. coli* (sequences SEQ ID NO: 11-13, respectively). These proteins were fused with an MT2 linker (SEQ ID NO: 27) that can be cleaved after a protease response, and a non-cleavable NC (SEQ ID NO: 18) linker. For the 144aa protein, purification was performed using NI-NTA, followed by anion exchange purification after labeling with trans-cyclooctene (TCO)-maleimide (MAL) (TCO-mal) (obtained from Xi'an Kangfuno Biotechnology Co., Ltd.). Longer XTEN proteins, due to their stronger binding ability to anion exchange columns, were directly purified using anion exchange, followed by secondary anion exchange purification after labeling with TCO-mal. This yielded TCO-MT2 / NC-XTEN proteins of different linker lengths for viral conjugation labeling. Similarly, using Fluc-T2A-EGFP as the reporter gene and AAV-DJ as the vector, the L585 site was mutated to mPTet for conjugation, and AAV-DJ-XTEN was prepared by conjugation according to the previously mentioned reaction conditions. MT2 / NC Complex. Male Balb / C mice aged 6-8 weeks were intravenously injected with AAV-DJ. mPTet and AAV-DJ-XTEN MT2 / NC Complex. In vivo imaging was performed on mice 7 and 14 days after intravenous injection to observe luciferase expression. Tissues from major mouse organs (heart, liver, spleen, lung, kidney, and muscle) were further quantitatively assessed for liver-targeting specificity. Similarly, for XTEN, longer XTEN proteins showed higher specificity in liver expression; however, considering the ease of protein purification and TCO labeling, a 144aa XTEN protein (hereinafter referred to as XT144) was ultimately selected for subsequent experiments.

[0265] The results are as follows Figure 19As shown, bioluminescence imaging results revealed that AAV infection activity was significantly inhibited in XTEN-blocked AAV without the introduction of the hepatic cleavable linker MT2, while TCO-MT2-XTEN144a / 288a / 432a with the introduction of the hepatic cleavable linker MT2 all showed restored infection activity. Furthermore, tissue distribution results showed that, compared to unmodified AAV, TCO-MT2-XTEN144a / 288a / 432a only exhibited efficient gene transduction in liver tissue, while unmodified AAV showed off-target gene transduction in organs such as the heart. Figure 19 ).

[0266] Example 15: Liver tissue-specific gene delivery based on mask-AAV (AAVR) response to liver cathepsin

[0267] Using AAVR as the mask module, Flu-T2A-EGFP as the reporter gene, and AAV-DJ as the vector, the L585 site was mutated to mPTet for coupling. We first attempted to directly express the PKD1 and PKD2 domains of the extracellular domain of AAVR that directly interact with AAV, expressing human PKD1,2 proteins (hPKD1,2, sequence SEQ ID NO:16) and PKD2 protein (sequence SEQ ID NO:15), respectively. The C-terminus of hPKD1,2 was linked to an unbreakable linker NC, 1MT2 (1 copy of the hepatically cleavable linker MT2), 2MT2 (two copies of MT2 in tandem), or 3MT2 (three copies of MT2 in tandem). The Mask module modified by TCO was covalently modified with AAV-DJ (mutated to mPTet at position 585), and the infectious activity of AAV was verified by intravenous injection into BABL / C (from the College of Animal Science and Technology, Peking University School of Medicine) and in vivo fluorescence imaging to detect the expression of the reporter gene FLUC.

[0268] The results are as follows Figure 20 As shown, bioluminescence imaging results indicate that PKD1,2-modified AAV-DJ- can effectively restore transduction efficiency in the liver; non-cleavable AAV-DJ-hPKD1,2 NC And the AAV-DJ-hPKD1,2 with insufficient connecting arm length. MT2 All showed only negligible levels of transgene expression.

[0269] To further enhance the recovery of infection activity, the XTEN protein was introduced, and different numbers of MT2 linkers were introduced into the AAV-XTEN linker region to construct different XTEN-based mask modules (NC, v1~v4). The specific construct structures are as follows: Figure 21As shown in Figure a, NC from N to C consists of a His tag, hPKD1,2 (SEQ ID NO: 16), and an NC connector (SEQ ID NO: 18); v1 from N to C consists of a His tag, hPKD1,2 (SEQ ID NO: 16), and an MT2 connector (SEQ ID NO: 27); v2 from N to C consists of a His tag, hPKD1,2 (SEQ ID NO: 16), XTEN(144aa) (SEQ ID NO: 11), and an MT2 connector (SEQ ID NO: 27); v3 from N to C consists of a His tag, hPKD1,2 (SEQ ID NO: 16), an MT2 connector (SEQ ID NO: 27), XTEN(144aa) (SEQ ID NO: 11), and an MT2 connector (SEQ ID NO: 27); v4 from N to C consists of a His tag, hPKD1,2 (SEQ ID NO: 18), and an NC connector (SEQ ID NO: 18). 16) MT2 connector (SEQ ID NO: 27), XTEN(72aa) (aa1-72 of SEQ ID NO: 11), MT2 connector (SEQ ID NO: 27), XTEN(72aa) (aa73-144 of SEQ ID NO: 11) and MT2 connector (SEQ ID NO: 27).

[0270] The results are as follows Figure 21 As shown in b, bioluminescence imaging results show that the introduction of XTEN protein into the Mask module significantly improved transduction efficiency in the liver.

[0271] Further optimize the PKD1,2 structural domain, such as Figure 21 As shown in c, based on the above v4 structure, hPKD1,2 are replaced with human hPKD2 (SEQ ID NO: 15) or mouse mPKD2 (SEQ ID NO: 41) to obtain hPKD2-v4 and mPKD2-v4 as Mask modules, respectively.

[0272] The results are as follows Figure 21 As shown in Figure d, bioluminescence imaging results indicate that PKD2 alone and the mouse-derived PKD domain exhibit more efficient and longer-lasting expression in the liver, with AAV-DJ-mPKD2 showing the best results. MT2 The highest and longest-lasting luciferase expression was achieved in the liver.

[0273] The above results indicate that masked AAV activated by protease triggering can achieve robust liver-specific transduction in vivo and significantly reduce gene expression in non-target tissues.

[0274] Example 16: Study on liver tissue-specific gene delivery based on AAV8 vector

[0275] AAV8, a commonly used liver expression vector, is highly efficient in transfecting the liver, but it also exhibits high expression in tissues such as the heart and muscle, leading to significant off-target effects. We also transferred a liver-responsive enzyme bond-breaking system to AAV8 to evaluate its effectiveness. Based on the results from AAV-DJ, and considering both liver expression specificity and low immunogenicity, we selected TCO-MT2-PEG20K and TCO-mPKD2-3MT2 for our experiments. Using Flux-T2A-EGFP as the reporter gene and AAV8 as the vector, we mutated the D584 position to mPTet for coupling, and coupled a mask module according to the previously mentioned reaction conditions. We also used in vivo imaging and ex vivo tissue grinding to evaluate the liver-specific expression of the constructed vectors.

[0276] The results are as follows Figure 22 As shown, bioluminescence imaging results revealed that TCO-MT2-PEG20k and TCO-mPKD2-3MT2-modified AAV8 effectively restored transduction efficiency in the liver. In vitro organ lysate analysis further confirmed that the modified AAV8 primarily achieved transgene expression in liver tissue, with no significant infection observed in other tissues such as the heart and kidney, indicating that its infectivity was blocked in non-target tissues. In contrast, unmodified AAV8 showed significant transduction in both heart and skeletal muscle tissues. Figure 22 ).

[0277] Example 17: Cardiac tissue-specific gene delivery via Mask-AAV based on cardiac tissue protease response

[0278] Other tissue-specific proteases can also be used to construct tissue-specific AAV vectors, such as the heart tissue-specific Corin protease, which is also a member of the serine protease family. Based on the previously screened linker (Cr-linker) that can efficiently respond to Corin protease cleavage, using XT144 as the mask module and AAV9 as the base vector, the 584th position was mutated to mPTet for conjugation, thus constructing the AAV9-Cr-XT144 vector. Female Balb / C animals aged 6-8 weeks were intravenously injected, and the expression and distribution of the AAV vector were observed by in vivo imaging at 7 and 14 days. Tissue samples were also taken for grinding to quantify the expression of the vector in different tissues. The results showed that bioluminescence imaging indicated that AAV9 modified with AAV9-Cr-XT1442 could effectively restore transduction efficiency in heart tissue. Detection of ex vivo organ lysate further confirmed that transgene expression was mainly achieved in heart tissue.

[0279] Example 18: Lung tissue-specific gene delivery based on mask-aav, which overexpresses ROS response in lung inflammatory diseases.

[0280] In common diseases such as pulmonary fibrosis, pneumonia, or acute lung injury, overexpressed ROS can act as a response element to activate masked AAV constructed from the AAV5 serotype, such as... Figure 10 As shown in d, based on library screening, the TCO-BR-PEG module was coupled to A591 to construct AAV5-PEG. BR Vector. An acute lung injury model was established using 6-8 week old female c57bl / 6j via intratracheal infusion of LPS. Subsequently, AAV5-PEG was used as the vector. BR The vector was intravenously injected, and the expression and distribution of the AAV5 vector were observed by in vivo imaging at 7 and 14 days. Tissue samples were also collected and ground to quantify the expression of the vector in different tissues. Results showed that bioluminescence imaging revealed AAV5-PEG... BR It can effectively restore transduction efficiency in lung tissue, and the results of in vitro organ lysate analysis further confirm that transgene expression is mainly achieved in lung tissue.

[0281] Example 19: Achieving tumor tissue-specific gene delivery based on high expression of MMPs in tumor tissue

[0282] Matrix metalloproteinases (MMPs) are widely expressed in tumor tissues and have been extensively used in cancer treatment. Numerous prodrug molecules based on high MMP expression in tumor tissues have been studied, such as IFN-γ, IL2, and IL12 molecules that respond to MMP release activity. In this application, the enzyme-responsive element is highly replaceable; it can be replaced with a linker (sequence SEQ ID NO: 35) that responds to MMP bond cleavage. Using XTEN (XT144) as the mask unit, complete blocking of AAV activity can be achieved. When the vector is circulated to the tumor, the mask unit is released in response to MMP bond cleavage in the tumor tissue, enabling tumor-specific gene delivery. In this embodiment, Flux-T2A-EGFP is used as the reporter gene, and XTEN (XT144) is used as the mask unit. Alternatively, an Affibody (Z342, sequence SEQ ID NO: 17) that targets HER2 can be fused to the N-terminus of the XTEN protein for active mask-AAV targeting (AAV-DJ-Z342). 3T Using AAV-DJ as a vector, the L585 site was mutated to mPTet for coupling, and the mask module was coupled according to the previously mentioned reaction conditions. Here, we take the common N87 tumor model (N87 tumor cells obtained from ATCC) as an example. On day 0, 50 μL of N87 tumor cells (5 × 10⁻⁶ cells) were collected. 6A mixture of 100 cells and 50 μL of matrix gel was injected into the axillae of NCG mice (obtained from Vital River Pharmaceuticals, Beijing) to construct an NSG-N87 tumor model. After approximately 10-14 days, once the tumor size reached 100-200 mm... 3 At that time, the constructed AAV-MMP-XTEN vector (under investigation) was injected via tail vein. In vivo imaging was performed at time points D3, D5, D7, D10, and D14 to observe the expression and systemic distribution of the vector in tumor tissue. After imaging was completed on day 14, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, muscle, and tumor tissues were ground and sonicated to quantify the expression of Luciferase in different tissues.

[0283] The results are as follows Figure 23 As shown, bioluminescence imaging results indicate that the enrichment capacity of AAV in tumor tissue was significantly enhanced by introducing the Affibody-XTEN domain. Furthermore, the cleavable linker can locally unmask the tumor tissue, effectively restoring the infectivity of AAV. In contrast, while unmodified AAV exhibits some gene transduction ability in tumor tissue, it also shows significant off-target effects in non-target tissues such as the liver. These results demonstrate that active targeting of Mask-AAV by fusing Affibody, which targets tumor cells, to the N-terminus of the XTEN protein can further improve the targeting effect in tumor tissue and increase the tumor-specific expression level.

[0284] Example 20: Mask AAV technology reduces AAV humoral immunity levels

[0285] Mask-AAV technology, while improving tissue targeting, also masks the AAV immune sites to some extent due to the masking effect of the mask module, which can further reduce humoral immunity levels in AAV gene therapy. In mice, intravenous injection of Mask-AAV effectively reduced the levels of total antibodies and neutralizing antibodies induced against AAV. Under ROS-responsive conditions, to detect whether site-specific modification of PEG could reduce the production of AAV neutralizing antibodies, in a mouse model of ischemia-reperfusion, intravenous injection of mPtet-modified AAV9 (AAV9...) was performed... mPtet ), and PEG-modified AAV vectors (AAV9-PEG) BRSeven days after intravenous injection, blood was collected from the orbital venous plexus of mice. After centrifugation at 2500 rpm, the serum was collected for analysis of total anti-AAV antibodies and neutralizing antibodies. Neutralizing antibodies were co-incubated with a wild-type AAV vector containing a luciferase reporter gene, and then transfected into 293T cells to verify the neutralizing antibody content. Specifically, the serum was serially diluted (1:200, 400, 800, 1600), then co-incubated with AAV9 at 37°C for 1 hour (MOI 1E5), added to 293T cells, and after 48 hours of infection, cell lysis buffer was added, followed by the addition of luciferase substrate for chemiluminescence detection. The detection of total anti-AAV antibodies was performed by ELISA, in which serum was serially diluted (1:200, 400, 800), wild-type AAV capsid particles were modified onto the substrate using NHS-amino reaction, mouse serum was added, and the substrate was incubated at 37 degrees for 1 hour. Secondary antibody against anti-MOUSE IgG was added, and HRP substrate was added. The absorbance value was detected at 450 nm.

[0286] The results are as follows Figure 24 As shown, the ELISA analysis results indicate that, compared with AAV9 mPTet Compared to the treatment group, AAV9-PEG BR The level of total anti-AAV IgG in mice in the treatment group was significantly reduced ( Figure 24 b); Simultaneously, the results of the cellular-level neutralization experiment also showed that viral infection of target cells increased, indicating a significant reduction in the production of neutralizing antibodies (NAbs). Figure 24 a).

[0287] Enzyme-responsive Mask-AAV also showed a good effect in reducing AAV immunogenicity, with different Mask units exhibiting varying effects. Similar to the above assays, in liver-responsive, bond-breaking Mask-AAV, intravenous injection of Mask-AAV (including mPtet-modified AAV-DJ) was performed. mPtetAfter constructing the Mask-AAV vectors described in Examples 13-15, blood was collected from the orbital sinus of mice, and serum was separated for the detection of total antibodies and neutralizing antibodies. For total antibodies, an ELISA method was used. Wild-type AAV-DJ capsid particles (1E9 / well) were coated onto the substrate. The serum was diluted 200-fold and incubated at room temperature for 2 hours. Secondary antibody against anti-mouse IgG (1:1500 dilution) was added, and finally, HRP substrate was added. The absorbance was measured at 450 nm. For neutralizing antibody detection, serum was serially diluted in serum-free DMEM and co-incubated with the wild-type AAV-DJ vector containing the luciferase reporter gene at 37°C for 1 hour. The mixture was then added to 293T cells (MOI=1000). After 6 hours of viral infection, the medium was changed to complete DMEM medium. The cells were detected using a luciferase assay kit after 48 hours. The percentage of transduction at different dilutions was calculated using DMEM without mouse serum as a control. The dilution at 50% neutralization was calculated from the fitted curve as the neutralizing titer.

[0288] The results are as follows Figure 25 As shown, the effects of different masking strategies on the immunogenicity of AAV are compared with those of unmodified AAV-DJ. mPTet AAV-DJ (fg) with different molecular weight PEG (ac), XTEN (de), and PKD masking was intravenously injected into BALB / c mice, and the total IgG level and neutralizing antibody (NAbs) levels were measured. The results showed that, in both ELISA and cell neutralization experiments, AAV modified with different masking strategies effectively reduced the levels of anti-AAV total IgG and neutralizing antibodies.

[0289] Example 21: Mask-AAV for Secondary AAV Injection

[0290] Since Mask-AAV effectively reduces total and neutralizing antibody levels against AAV, we further explored its potential for secondary injection. Typically, after AAV gene therapy, patients rapidly develop a strong immune response against AAV, making secondary AAV gene therapy impossible. In this experiment, AAV DJ-WT and AAV-DJ, packaged with the EGFP gene, were used... mPTet Different mask modules of AAV were intravenously injected into 6-7 week old Balb / C male rats at a dose of 2E10 / rat. 21 days later, each group was injected with the corresponding AAV packaged with the Fluc-T2A-EGFP gene at a dose of 2E10 / rat. 7 days later, in vivo imaging was performed to detect the expression of the luciferase gene in each group.

[0291] The results are as follows Figure 26As shown in ab, in vivo bioluminescence imaging results indicate that the pre-existing neutralizing antibody against AAV generated after the first dose can significantly inhibit the infectivity of unmodified AAV; in contrast, AAV modified with MASK can still maintain a high level of gene transduction activity in the presence of pre-existing neutralizing antibodies.

[0292] Example 22: Mask AAV infection under pre-stored neutralizing antibodies

[0293] A major burden of AAV-based gene therapy is the prevalence of neutralizing antibodies, with seropositivity rates ranging from 10% to over 90% depending on serotype and geographic location. To demonstrate that Mask AAV can achieve targeted gene delivery even with pre-existing neutralizing antibodies, the anti-neutralizing ability of Mask-AAV against IVIg (intravenous immunoglobulin) was verified in vitro and in vivo. For the in vitro experiments, AAV... mPTet Mask-AAV was incubated with IVIg and proteinG beads (obtained from Tiandi Renhe Biotechnology Co., Ltd.) at 37°C for 2 hours. The neutralized AAV-IVIg-ProteinG complex was then removed using a magnetic rack, and the supernatant was used for Q-PCR quantification of AAV. mPTet To assess the neutralization level of Mask-AAV, the supernatant was treated with H2O2 to break bonds before infecting 293T cells, and the expression of the target gene was measured after 48 hours. For in vivo experiments, BABL / C female mice were intravenously injected with IVIg (3 mg / mouse) one day prior to the experiment, followed by intravenous injection of Mask AAV virus (2E10 / mouse). Seven days after injection, in vivo chemiluminescence imaging was used to detect luciferase expression in mice.

[0294] The results are as follows Figure 27 As shown, qPCR was used to quantify the residual virus after antibody neutralization. The results showed that unmodified AAV-DJ... mPTet Almost entirely captured by intravenously injected immunoglobulin (IVIG), while mask-modified AAV was effectively retained under the same conditions. Figure 27 (ab). Further viral transduction experiments showed that, under antibody-neutralizing conditions, the MASK-AAV-mediated transgene expression level was significantly higher than that of unmodified AAV-DJ. mPTet In in vivo experiments, bioluminescence imaging results showed that in the presence of neutralizing antibodies (NAb) + In mice, MASK-AAV maintained high transduction efficiency, while unmodified AAV-DJ... mPTet The transduction capacity is significantly reduced ( Figure 27c). The above results indicate that PEG modification can, to some extent, resist antibody-mediated neutralization, thereby mitigating the decline in transduction efficiency of unmodified AAV in the presence of neutralizing antibodies and enhancing its gene delivery capacity under complex immune background conditions.

[0295] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A modified adeno-associated virus (AAV) comprising a modified adeno-associated virus (AAV) capsid protein having at least one non-natural amino acid residue with a coupling moiety covalently linked to the non-natural amino acid residue via a cleavable linker, the non-natural amino acid residue being derived from a non-natural amino acid containing a tetrazine group; Preferably, the non-natural amino acid containing a tetrazine group has the chemical structure shown in Formula I: (Equation I) in, R1 is selected from H, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C6-C 10 Aromatic group, 5 to 10-membered heteroaryl group, preferably, R1 is selected from methyl, ethyl, isopropyl, n-butyl, phenyl, 4-pyridyl; L1 is selected from direct bonds, CH2, NH2, O, and S, and m is an integer selected from 1 to 6; preferably, L1 is CH2 and m is 1; L2 is selected from C6-C 10 Aromatic subunit, where n is an integer selected from 0 to 3; preferably, L2 is selected from 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, where n is 0 or 1; most preferably, L2 is 1,3-phenylene, where n is 0 or 1; More preferably, the non-natural amino acid is selected from any one of the following: ； More preferably, the non-natural amino acid is mPet.

2. The AAV according to claim 1, wherein, The non-natural amino acid residues with the coupling portion have the chemical structure shown in Formula II-1 or Formula II-2: (Formula II-1) (Formula II-2) Wherein, the non-natural amino acid residue is located at the Nth position of the capsid protein amino acid sequence, and the direction from label 1 to label 2 is the direction from the N end to the C end of the amino acid sequence. Label 1 represents the amino acid residues at positions 1 to N-1 of the capsid protein amino acid sequence, and label 2 represents the amino acid residues at positions N+1 to the C end of the capsid protein amino acid sequence. R1 is selected from H, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C6-C 10 Aromatic group, 5 to 10-membered heteroaryl group, preferably, R1 is selected from methyl, ethyl, isopropyl, n-butyl, phenyl, 4-pyridyl; L1 is selected from direct bonds, CH2, NH2, O, and S, and m is an integer selected from 1 to 6; preferably, L1 is CH2 and m is 1; L2 is selected from C6-C 10 Aromatic subunit, where n is an integer selected from 0 to 3; preferably, L2 is selected from 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, where n is 0 or 1; most preferably, L2 is 1,3-phenylene, where n is 0 or 1; X is selected from direct bond, ether bond, etc. , , , , Preferably, X is selected from... , , One of them is connected to a carbon atom on cyclooctane, and L3 is connected at position 2; L3 is a pyrolytic linker; M represents the coupling part.

3. The AAV according to any one of claims 1-2, wherein, The coupling part includes a mask moiety. Preferably, the masking portion is selected from polymers, such as synthetic polymers (e.g., PEG) or peptide polymers (e.g., XTEN protein). Preferably, the masking portion is selected from PEG; Preferably, the masking portion is selected from XTEN protein; preferably, the XTEN protein comprises the sequence shown in any one of SEQ ID NOs: 11-13; Preferably, the masking portion is selected from the extracellular domains of AAVR, such as PKD1 and / or PKD2; Preferably, the masking portion comprises an AAVR extracellular domain and an XTEN protein; Preferably, the coupling portion further includes a targeting portion (e.g., an antibody or its antigen-binding fragment, or an antibody mimic such as an affibody) connected to the masking portion; preferably, the coupling portion includes an XTEN protein with the targeting portion (e.g., an antibody or its antigen-binding fragment, or an antibody mimic such as an affibody) attached to its N-terminus.

4. The AAV according to any one of claims 1-3, wherein, The cleavable linker is cleaved by factors in the pathological microenvironment (such as metalloproteinases, cathepsins, reactive oxygen species (ROS)) or by exogenous stimuli (such as light, ultrasound). Preferably, the cleavable linker is cleaved by reactive oxygen species (ROS) and contains, for example, borate or borate ester groups; Preferably, the cleavable linker is photobleached and cleaved, for example, containing a fluorescent group, such as Cy7; Preferably, the cleavable linker is cleaved by a protease, for example, containing a restriction enzyme site sequence; preferably, the protease is a type II transmembrane serine protease (Matriptase-2), a Corin protease, a matrix metalloproteinase (MMP), or a urokinase-type plasminogen activator (UPA). Preferably, the restriction site sequence of Matriptase-2 includes the sequence shown in any one of SEQ ID NOs: 20-27; Preferably, the restriction enzyme site sequence of Corin includes the sequence shown in any one of SEQ ID NOs: 28-34; Preferably, the restriction enzyme site sequence of the MMP contains the sequence shown in any one of SEQ ID NOs: 35-36; Preferably, the restriction enzyme site sequence of UPA includes the sequence shown in SEQ ID NO: 42; Preferably, the cleavable linker is cleaved by a glycosidase (such as β-Glucuronidase) and contains, for example, a glucuronic acid group; Preferably, the pyrolytic linker is selected from: 、 、 , One link connects to X, and two links connect to M; CS is the restriction enzyme site sequence; Preferably, M-L3-X is selected from: 、 、 、 。 5. The AAV according to any one of claims 1-4, wherein, The non-natural amino acid residues are located at sites corresponding to the following positions: (a) The amino acid sequence of the AAV-DJ capsid protein shown in SEQ ID NO: 8, specifically S268, Q327, S448, T453, L462, T583, and L585, such as Q327, T453, and L585; (b) The amino acid sequence of the AAV9 capsid protein shown in SEQ ID NO: 7, specifically G453, G455, S499, A591, and H584, ​​for example, G455, S499, and H584; (c) H584 of the amino acid sequence of the AAV-PHP-eb capsid protein shown in SEQ ID NO: 9; (d) The amino acid sequence of the AAV8 capsid protein shown in SEQ ID NO: 10, specifically S266, Q328, S449, Q461, L463, D584, L586, Q588, T591, Q594; for example, D584, L586; or (e) The amino acid sequence of the AAV5 capsid protein shown in SEQ ID NO: 43, specifically D374, T444, A579, for example, A579.

6. The AAV according to any one of claims 1-5, wherein, The AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8.

7. The AAV according to any one of claims 1-6, further comprising a nucleic acid payload of interest. Preferably, the target nucleic acid payload is selected from therapeutic genes (e.g., antibodies, vaccines, cytokines such as angiogenic cytokines, hormones such as relaxin, coagulation factors, growth factors, enzymes, RNA interference agents such as shRNA, gene editing tools such as CRISPR / Cas systems) or reporter genes (e.g., fluorescent proteins or luciferases).

8. A method for preparing a modified adeno-associated virus (AAV), comprising the following steps: - Provided: (1) an adeno-associated virus (AAV) incorporating non-natural amino acids, comprising a site-mutated capsid protein having at least one non-natural amino acid residue containing a tetrazine group; (2) a coupling portion connected to a trans-cyclooctene (TCO) group, wherein the coupling portion is connected to the TCO group via a cleavable linker. - Incubate (1) and (2) together, and couple the non-natural amino acid to the coupling part by IEDDA reaction; Preferably, the coupling portion is as defined in claim 3; Preferably, the detachable connector is as defined in claim 4; Preferably, the position of the non-natural amino acid residue is as defined in claim 5; Preferably, the AAV is selected from any serotype, such as AAV-DJ, AAV9, AAV-PHP-eb, AAV5, AAV2, or AAV8; Preferably, the non-natural amino acid containing a tetrazine group has the chemical structure shown in Formula I: (Equation I) R1 is selected from H, halogen, hydroxyl, cyano, C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C6-C 10 Aromatic group, 5 to 10-membered heteroaryl group, preferably, R1 is selected from methyl, ethyl, isopropyl, n-butyl, phenyl, 4-pyridyl; L1 is selected from direct bonds, CH2, NH2, O, and S, and m is an integer selected from 1 to 6; preferably, L1 is CH2 and m is 1; L2 is selected from C6-C 10 Aromatic subunit, where n is an integer selected from 0 to 3; preferably, L2 is selected from 1,2-phenylene, 1,3-phenylene, 1,4-phenylene, where n is 0 or 1; most preferably, L2 is 1,3-phenylene, where n is 0 or 1; Preferably, the non-natural amino acid is selected from any one of the following: ； Preferably, the non-natural amino acid is mPet.

9. The method of claim 8, wherein, The coupling moiety connected to the trans-cyclooctene (TCO) group has the chemical structure shown in Formula III: (Formula III) Where X is selected from direct bond, ether bond, , , , , ; Preferably, X is selected from , , One of them is connected to the 4th carbon atom of trans-cyclooctene, and the 2nd position is connected to L3; L3 is a pyrolytic linker; Preferably, L3 is selected from , , , One link connects to X, and two links connect to M; CS is the restriction enzyme site sequence; M represents the coupling part; Most preferably, Formula III is selected from: , , , , where CS is the restriction site sequence and M is the coupling part.

10. The method according to any one of claims 8-9, wherein, The AAV incorporating non-natural amino acids is provided by the following steps: (a) Provide: (i) an AAV packaging system comprising a first nucleic acid molecule encoding a nucleotide sequence of the site-directed mutant capsid protein, wherein the nucleotide sequence contains a stop codon (e.g., TAG or TAA) at the position corresponding to the unnatural amino acid; (ii) a second nucleic acid molecule encoding an orthogonal tRNA synthetase; and (iii) a third nucleic acid molecule encoding an orthogonal tRNA; preferably, the orthogonal tRNA synthetase amidates the orthogonal tRNA with an unnatural amino acid containing a tetrazine group; (b) Introduce the AAV packaging system, the second nucleic acid molecule, and the third nucleic acid molecule simultaneously or in any order into a host cell (e.g., transform, transduce, or transfect). (c) The host cells obtained in step (b) are cultured in a culture medium containing non-natural amino acids to obtain the AAV incorporating non-natural amino acids; Preferably, the AAV packaging system comprises: (i) a packaging plasmid containing a Rep gene and a Cap gene, wherein the Cap gene is a first nucleic acid molecule; (ii) an auxiliary plasmid containing adenovirus auxiliary genes (E2A, E4, VA RNA); and (iii) an exogenous gene expression plasmid containing a nucleic acid payload of interest. Preferably, the second nucleic acid molecule and the third nucleic acid molecule are present in the same vector, such as different expression cassettes, or in different vectors; preferably, the second nucleic acid molecule is optionally operably linked to a promoter, such as a constitutive promoter, such as CMV, EF1α, CAG, or UBC promoters; preferably, the third nucleic acid molecule is optionally operably linked to a promoter, such as an RNA polymerase III promoter, such as the U6 promoter or the H1 promoter. Preferably, the orthogonal tRNA synthetase comprises the sequence shown in SEQ ID NO: 1, and the orthogonal tRNA comprises the sequence shown in SEQ ID NO:

2.

11. Use of the modified AAV according to any one of claims 1-7 in the preparation of a reagent for delivering an anucleic acid payload of interest to a subject; Preferably, the target nucleic acid payload is delivered to the subject's inflamed or infected tissue (e.g., liver tissue, such as liver fibrosis; or heart tissue, such as myocardial ischemia-reperfusion; or lung tissue, such as pulmonary fibrosis, pneumonia, or acute lung injury); preferably, the cleavable linker is cleaved by reactive oxygen species (ROS) and, for example, contains borate groups or borate ester groups; preferably, the AAV is AAV-DJ, AAV9, or AAV5; Preferably, the target nucleic acid payload is delivered to the liver tissue of the subject; preferably, the cleavable linker is cleaved by a protease, for example, containing a Matriptase2 protease cleavage site sequence; preferably, the AAV is AAV8 or AAV-DJ; Preferably, the target nucleic acid payload is delivered to the heart tissue of the subject; preferably, the cleavable linker is cleaved by a protease, for example, containing a Corin protease cleavage site sequence; preferably, the AAV is AAV9; Preferably, the target nucleic acid payload is delivered to the subject's tumor tissue; preferably, the cleavable linker is cleaved by a protease, for example, containing a matrix metalloproteinase (MMP) cleavage site sequence; preferably, the coupling portion contains an XTEN protein; preferably, the coupling portion contains a targeting portion (e.g., an affibody) that targets tumor cells; preferably, the coupling portion contains an XTEN protein and a targeting portion (e.g., an affibody) linked to its N-terminus; preferably, the AAV is AAV-DJ; Preferably, the target nucleic acid payload is delivered to the muscle tissue of the subject; preferably, the AAV is AAV9; preferably, the cleavable linker is photobleached and cleaved, for example, containing a fluorescent group, such as Cy7; Preferably, the target nucleic acid payload is delivered to the brain tissue of the subject; preferably, the AAV is PHP-eb; preferably, the cleavable linker is photobleached and cleaved, for example, containing a fluorescent group, such as Cy7; Preferably, the modified AAV exhibits reduced humoral immunity levels in the subjects; Preferably, the subject has pre-existing anti-AAV antibodies (e.g., neutralizing antibodies); Preferably, the subject has undergone AAV gene therapy; Preferably, the subject is a human being; Preferably, the modified AAV is administered via injection, intratracheal administration, intranasal administration, or inhalation.

12. Use of the modified AAV according to any one of claims 1-7 in the preparation of a medicament for treating a disease in a subject; Preferably, the target nucleic acid payload contained in the modified AAV is selected from therapeutic genes (e.g., antibodies, vaccines, cytokines such as angiogenic cytokines, hormones such as relaxin, coagulation factors, growth factors, enzymes, RNA interference agents such as shRNA, gene editing tools such as CRISPR / Cas systems); preferably, the therapeutic gene encodes a bioactive molecule for treating the disease.

13. A method for screening sites in adeno-associated virus (AAV) capsid proteins into which non-natural amino acids can be incorporated, the method comprising: (a) Provide: (i) a first modified AAV having a non-natural amino acid residue at a predetermined site in the capsid protein; (ii) a second modified AAV having a non-natural amino acid residue with a blocking group (such as PEG) attached to the predetermined site in the capsid protein; (iii) a third modified AAV having a non-natural amino acid residue with an amino group attached to the predetermined site in the capsid protein; wherein the non-natural amino acid in (i)-(iii) is a non-natural amino acid containing a tetrazine group; (b) The first modified AAV, the second modified AAV, and the third modified AAV were incubated with cells (e.g., HEK-293T cells) and their infectious activity was detected. When the infectious activity of the first modified AAV and the third modified AAV were comparable, and the second modified AAV showed weak or undetectable infectious activity, the preset site was selected as the site for incorporating non-natural amino acids. Preferably, the ratio of the infectivity of the third modified AAV to the infectivity of the second modified AAV is not less than 10. 3 ; Preferably, the AAV contains a reporter gene, such as a fluorescent protein or luciferase, thereby generating a detectable signal from the reporter gene that is proportional to the infectious activity; Preferably, in the second modified AAV, the residue at the preset site is mutated to a non-natural amino acid containing a tetrazine group, and the blocking group is connected to a TCO group (e.g., TCO-PEG20K), thereby being linked to the non-natural amino acid via an IEDDA reaction between the TCO group and the tetrazine group. Preferably, in the third modified AAV, the residue at the preset site is mutated to a non-natural amino acid containing a tetrazine group, wherein the amino group is attached to a TCO group (e.g., TCO-NH2), thereby being linked to the non-natural amino acid via an IEDDA reaction between the TCO group and the tetrazine group.

14. A mutant aminoacyl-tRNA synthetase (aaRS) comprising the sequence shown in SEQ ID NO:

1.

15. An isolated nucleic acid molecule or vector comprising a nucleotide sequence encoding the mutant aaRS of claim 14.

16. A composition comprising: (a) the mutated aaRS of claim 14 or the first nucleic acid molecule encoding said mutated aaRS; and (b) tRNA or a second nucleic acid molecule encoding said tRNA, wherein, The tRNA contains the sequence shown in SEQ ID NO: 2; Preferably, the composition comprises one or more carriers, the one or more carriers comprising the first nucleic acid molecule and the second nucleic acid molecule; Preferably, the vector is a plasmid or a viral vector; Preferably, the first nucleic acid molecule is optionally operably linked to a first regulatory element, such as a promoter, like a constitutive promoter, such as the CMV, EF1α, CAG, or UBC promoter; Preferably, the second nucleic acid molecule is optionally operably linked to a second regulatory element, such as a promoter, such as an RNA polymerase III promoter, like the U6 promoter or the H1 promoter; Preferably, the composition further comprises a third nucleic acid molecule encoding a target protein, the third nucleic acid molecule containing a stop codon recognized by the tRNA; Preferably, the target protein contains non-natural amino acids, and the stop codon corresponds to the position of the non-natural amino acid in the target protein.

17. A method for producing a target protein, said target protein having at least one non-natural amino acid residue containing a tetrazine group, said method comprising: (a) Provide: (i) a first nucleic acid molecule comprising a nucleotide sequence encoding a target protein, wherein the codon at the position corresponding to the non-natural amino acid is a stop codon (e.g., TAG or TAA); (ii) a second nucleic acid molecule encoding the mutant aaRS of claim 14; and (iii) a third nucleic acid molecule encoding tRNA comprising the sequence shown in SEQ ID NO:

2. (b) Introduce the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule simultaneously or in any order into a host cell (e.g., transform, transduce, or transfect). (c) The host cells obtained in step (b) are cultured in a medium containing non-natural amino acids to allow the non-natural amino acids to be incorporated into the target protein being expressed in the cells; wherein the aaRS is capable of loading the non-natural amino acids onto tRNA and subsequently incorporating the non-natural amino acids into the protein at a position corresponding to the select codon. Preferably, the first nucleic acid molecule, the second nucleic acid molecule, and the third nucleic acid molecule are present on one or more vectors; preferably, the vector is a plasmid or a viral vector. Preferably, the target protein is selected from protein or polypeptide drugs (e.g., antibodies, vaccines, hormones, coagulation factors, growth factors or enzymes), detection or diagnostic reagents, or industrial proteins (e.g., industrial enzymes).

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