Muscle-targeted adeno-associated virus capsid protein, adeno-associated virus containing same, vector and application

Abstract

The invention provides a muscle-targeted adeno-associated virus capsid protein, an adeno-associated virus containing the muscle-targeted adeno-associated virus capsid protein, a vector and application. The present disclosure provides an adeno-associated virus capsid protein, which comprises a targeting peptide, and the targeting peptide comprises any one of the following amino acid sequences: SEQ ID NO: 1-19. According to the invention, a reasonable design and directed evolution combined method is adopted, and on the basis of a receptor integrin-RGD polypeptide specific binding principle, random amino acids are carried to adapt to construction and screening of the AAV vector of a capsid structure, so that the problems of poor stability of the capsid structure, change of histocompatibility and the like caused by direct insertion of a sequence are avoided; therefore, efficient transduction of muscular tissues is realized, and a reliable platform is provided for gene therapy.

Description

Technical Field

[0001] This disclosure relates to the technical field of viral vectors, specifically to an adeno-associated virus capsid protein that targets muscle, an adeno-associated virus containing the capsid protein, a vector, and applications, and more specifically to a dominant mutant of the adeno-associated virus AAV5 capsid protein that specifically targets muscle based on integrin modification. Background Technology

[0002] Recombinant adeno-associated virus (rAAV) vectors are among the most promising technologies in in vivo gene therapy. AAV-mediated systemic gene delivery to muscle has been a major challenge in gene therapy for muscle diseases, with capillary vessels being a primary barrier limiting vector diffusion. To achieve optimal therapeutic effects, AAV-mediated gene therapy for muscle diseases typically requires the injection of high doses of virus (>1E+14 vg / kg). Serious adverse events and toxicities have been observed in some high-dose systemic delivery AAV gene therapy clinical trials, highlighting the need for more efficient AAV vectors to achieve efficacy at lower doses.

[0003] Recently, mutants modified with AAV9 capsids, such as myoAAV (Tabebordbar, Mohammadsharif et al. “Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.” Cell vol. 184,19 (2021): 4919-4938.e22. doi:10.1016 / j.cell.2021.08.028), have demonstrated significant muscle transduction effects in mice and non-human primates. These capsids contain exposed RGD motifs that can bind to integrins. However, the widespread presence of neutralizing antibodies (NAbs) in the AAV9 capsid in humans limits the conversion applications of related AAV serotypes. AAV5 is a unique serotype with lower levels of naturally occurring neutralizing antibodies compared to other serotypes in multiple populations, including Europeans, Asians, healthy individuals, hemophiliacs, and HIV-infected individuals. Furthermore, in some clinical trials, persistent levels of systemic coagulation factor VIII were detected even in hemophilia patients with pre-existing anti-AAV5-NAbs, suggesting that AAV5 has the potential to bypass existing neutralizing antibodies. Therefore, AAV5 is a promising scaffold for developing ideal serotypes suitable for large numbers of subjects. Summary of the Invention

[0004] The problem the invention aims to solve

[0005] Based on the binding of the RGD sequence to integrin, this invention inserts RGD / N / S / G and four random amino acids between amino acids 575 and 577 of the capsid protein of the AAV5 serotype. Through screening and modification, it is expected to obtain a novel adeno-associated virus vector with high cardiac and skeletal muscle transduction efficiency.

[0006] Solution for solving the problem

[0007] [1]. An adeno-associated virus capsid protein comprising a targeting peptide, said targeting peptide containing the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0008] X3 is D, S, N, or G;

[0009] X4 is L, G, K, T, R, M, V, or F;

[0010] X5 is S, R, P, I, A, E, T, G, or L;

[0011] X6 is S, N, V, Q, G, D, A, I, P, R or L;

[0012] X7 is L, G, S, K, H, N, R, T, A, or D;

[0013] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 1-19;

[0014] Optionally, adeno-associated viruses (AAVs) using capsid proteins containing the target peptide have improved transduction efficiency for cells expressing integrins compared to AAVs using wild-type capsid proteins.

[0015] [2]. According to the adeno-associated virus capsid protein described in [1], wherein the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0016] X3 is D, S, N, or G;

[0017] X4 is L, G, K, T, R, M, V, or F;

[0018] X5 is S, R, P, I, A, T, G, or L;

[0019] X6 is S, N, V, Q, G, D, P, R, or L;

[0020] X7 is L, G, S, K, H, R, T, A, or D;

[0021] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 2-3, 7-19;

[0022] Optionally, adeno-associated viruses (AAVs) using capsid proteins containing the target peptide have improved transduction efficiency for cells expressing integrins compared to AAVs using wild-type capsid proteins.

[0023] [3]. According to the adeno-associated virus capsid protein described in [1], wherein the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0024] X3 is either D or S;

[0025] X4 is L, G, K, or T;

[0026] X5 is S, R, A, E, or P;

[0027] X6 is S, N, V, A, I, or Q;

[0028] X7 is L, G, S, H, N, K, or R;

[0029] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 1-5, 7 and 9;

[0030] Optionally, compared to adeno-associated viruses with wild-type capsid proteins, adeno-associated viruses with capsid proteins containing the target peptide have enhanced transduction efficacy for muscle and / or heart.

[0031] [4]. The adeno-associated virus capsid protein according to [1] or [3], wherein the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0032] X3 is D;

[0033] X4 is L, G, K, or T;

[0034] X5 is S, R, A, or P;

[0035] X6 is S, N, V, A, or Q;

[0036] X7 is L, G, S, H, N, or R;

[0037] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 1-4, 7 and 9;

[0038] Optionally, adeno-associated viruses having a capsid protein containing the target peptide have improved transduction efficiency in muscle compared to adeno-associated viruses having wild-type capsid proteins.

[0039] [5]. The adeno-associated virus capsid protein according to [1] or [3], wherein the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0040] X3 is either D or S;

[0041] X4 is L, G, K, or T;

[0042] X5 is S, R, A, E, or P;

[0043] X6 is S, N, V, A, I, or Q;

[0044] X7 is L, S, H, N, K, or R;

[0045] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 2-5, 7 and 9;

[0046] Optionally, adeno-associated viruses having a capsid protein containing the target peptide have improved transduction efficiency for the heart compared to adeno-associated viruses having wild-type capsid proteins.

[0047] [6]. The adeno-associated virus capsid protein according to [1] or [3], wherein the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0048] X3 is D;

[0049] X4 is L, G, K, or T;

[0050] X5 is S, R, A, or P;

[0051] X6 is S, N, V, A, or Q;

[0052] X7 is L, S, H, N, or R;

[0053] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 2-4, 7 and 9;

[0054] Optionally, adeno-associated viruses having a capsid protein containing the target peptide have improved transduction efficiency for muscle and heart compared to adeno-associated viruses having wild-type capsid proteins.

[0055] [7]. Adeno-associated virus capsid protein according to any one of [1] to [6], wherein the targeting peptide comprises the amino acid sequence shown in SEQ ID NO: 9.

[0056] [8]. Adeno-associated virus capsid protein according to any one of [1] to [7], wherein the adeno-associated virus is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43 and rh.74;

[0057] Preferably, AAV includes AAV5.

[0058] [9]. Adeno-associated virus capsid protein according to any one of [1] to [8], wherein AAV5 includes AAV5VP1 or a variant thereof;

[0059] Preferably, the amino acid sequence of AAV5 VP1 is shown in SEQ ID NO: 97.

[0060]

[10] . Adeno-associated virus capsid protein according to any one of [1] to [9], wherein the targeting peptide is inserted at the position between amino acids 575 and 577 of AAV5 VP1 or a variant thereof;

[0061] Preferably, the adeno-associated virus capsid protein comprises the amino acid sequence shown in any of the following: SEQ ID NO: 20-38, and an amino acid sequence having at least 80%, 85%, 90%, 95%, 97% or 99% identity with the amino acid sequence shown in any of SEQ ID NO: 20-38.

[0062]

[11] . A targeting peptide, said targeting peptide comprising the sequence RG-X3-X4-X5-X6-X7-X8, wherein,

[0063] in,

[0064] X3 is D, S, N, or G;

[0065] X4 is L, G, K, T, R, M, V, or F;

[0066] X5 is S, R, P, I, A, E, T, G, or L;

[0067] X6 is S, N, V, Q, G, D, A, I, P, R or L;

[0068] X7 is L, G, S, K, H, N, R, T, A, or D;

[0069] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO:1-19;

[0070] Optionally, compared with adeno-associated viruses using wild-type capsid proteins, adeno-associated viruses using capsid proteins containing the target peptide have improved transduction efficiency for cells expressing integrins.

[0071] The targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein...

[0072] X3 is D, S, N, or G;

[0073] X4 is L, G, K, T, R, M, V, or F;

[0074] X5 is S, R, P, I, A, T, G, or L;

[0075] X6 is S, N, V, Q, G, D, P, R, or L;

[0076] X7 is L, G, S, K, H, R, T, A, or D;

[0077] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 2-3, 7-19;

[0078] Optionally, compared with adeno-associated viruses using wild-type capsid proteins, adeno-associated viruses using capsid proteins containing the target peptide have improved transduction efficiency for cells expressing integrins.

[0079] Preferably, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0080] X3 is either D or S;

[0081] X4 is L, G, K, or T;

[0082] X5 is S, R, A, E, or P;

[0083] X6 is S, N, V, A, I, or Q;

[0084] X7 is L, G, S, H, N, K, or R;

[0085] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 1-5, 7 and 9;

[0086] Optionally, compared to adeno-associated viruses with wild-type capsid proteins, adeno-associated viruses with capsid proteins containing the target peptide have improved transduction efficiency for muscle and / or heart.

[0087] Preferably, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0088] X3 is D;

[0089] X4 is L, G, K, or T;

[0090] X5 is S, R, A, or P;

[0091] X6 is S, N, V, A, or Q;

[0092] X7 is L, G, S, H, N, or R;

[0093] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 1-4, 7 and 9;

[0094] Optionally, compared to adeno-associated viruses with wild-type capsid proteins, adeno-associated viruses with capsid proteins containing the target peptide have improved transduction efficiency in muscle.

[0095] Preferably, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0096] X3 is either D or S;

[0097] X4 is L, G, K, or T;

[0098] X5 is S, R, A, E, or P;

[0099] X6 is S, N, V, A, I, or Q;

[0100] X7 is L, S, H, N, K, or R;

[0101] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 2-5, 7 and 9;

[0102] Optionally, compared to adeno-associated viruses with wild-type capsid proteins, adeno-associated viruses with capsid proteins containing the target peptide have improved transduction efficiency for the heart.

[0103] Optionally, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0104] X3 is D;

[0105] X4 is L, G, K, or T;

[0106] X5 is S, R, A, or P;

[0107] X6 is S, N, V, A, or Q;

[0108] X7 is L, S, H, N, or R;

[0109] Optionally, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 2-4, 7 and 9;

[0110] Optionally, compared to adeno-associated viruses with wild-type capsid proteins, adeno-associated viruses with capsid proteins containing the target peptide have improved transduction efficiency for muscle and heart.

[0111] More preferably, the targeting peptide comprises an amino acid sequence as shown in SEQ ID NO:9.

[0112]

[12] . A polynucleotide encoding the adeno-associated virus capsid protein described in any one of [1] to

[10] , or encoding the target peptide described in

[11] .

[0113]

[13] . An adeno-associated virus comprising the adeno-associated virus capsid protein described in any one of [1] to

[10] .

[0114]

[14] . The adeno-associated virus according to

[13] further includes genetically modified organisms;

[0115] Optionally, the genetic modification is a therapeutic genetic modification, a preventive genetic modification, or a diagnostic genetic modification.

[0116]

[15] . A transgenic delivery vector comprising an adeno-associated virus as described in

[13] or

[14] .

[0117]

[16] . A pharmaceutical composition comprising:

[0118] (a) an adeno-associated virus as described in

[13] or

[14] , or a transgenic delivery vector as described in

[15] ; and, optionally,

[0119] (b) Pharmaceutically acceptable carriers.

[0120]

[17] . Use of adeno-associated viruses as described in

[13] or

[14] , transgene delivery vectors as described in

[15] , or pharmaceutical compositions as described in

[16] in the preparation of reagents for delivering transgenes into cells.

[0121]

[18] . According to the use described in

[17] , wherein the cells are derived from the subject;

[0122] Preferably, the subject is a mammalian subject;

[0123] More preferably, the subject is a human being.

[0124]

[19] . According to the use described in

[17] or

[18] , wherein the cells are derived from muscle and / or heart.

[0125]

[20] . According to the use described in

[18] or

[19] , wherein the subject suffers from muscle and / or heart disease.

[0126]

[21] . The use according to any one of

[17] to

[20] , wherein the adeno-associated virus, the transgenic delivery vector, or the pharmaceutical composition is administered to the subject by intravenous injection.

[0127]

[22] . Use of adeno-associated virus as described in

[13] or

[14] , transgenic delivery vector as described in

[15] , or pharmaceutical composition as described in

[16] in the preparation of a medicament for treating a disease;

[0128] Preferably, the disease includes muscle and / or heart disease.

[0129]

[23] . A method of treating a disease, comprising the step of administering an adeno-associated virus as described in

[13] or

[14] , a transgenic delivery vector as described in

[15] , or a pharmaceutical composition as described in

[16] to a subject;

[0130] Preferably, the disease includes muscle and / or heart disease.

[0131] The effects of the invention

[0132] This disclosure employs a combination of rational design and directed evolution, based on the specific binding principle of receptor integrin-RGD peptides, and constructs and screens AAV vectors carrying random amino acids adapted to the capsid structure. This avoids problems such as poor capsid structure stability and changes in tissue affinity caused by direct sequence insertion, thereby achieving efficient transduction to muscle tissue and providing a reliable platform for gene therapy. Attached Figure Description

[0133] Figure 1 The transduction efficiency of eight candidate AAVs (5020 / 5021 / 5022 / 5023 / 5024 / 5026 / 5027 / 5028) and wild-type AAV5 (AAV5-WT) infecting human immortalized skeletal muscle cells AC-16 was shown.

[0134] Figure 2 The transduction of human cardiac organoids infected with eight candidate AAVs (5020 / 5021 / 5022 / 5023 / 5024 / 5026 / 5027 / 5028) and wild-type AAV5 (AAV5-WT) is shown.

[0135] Figure 3The binding affinity of eight candidate AAVs (5020 / 5021 / 5022 / 5023 / 5024 / 5026 / 5027 / 5028) and wild-type AAV5 (AAV5-WT) to integrin αVβ6 / αVβ8 was shown.

[0136] Figure 4 The enrichment folds of candidate AAVs (5081 / 5082 / 5083 / 5084 / 5085 / 5086 / 5087 / 5088 / 5089 / 5090 / 5091) selected from the second round small library of AAV5-RGD / S / N / GXXXX (pIVB3197) in in vitro integrin-overexpressing cells were normalized to the 293T enrichment mean.

[0137] Figure 5A The in vivo transduction capacity of wild-type AAV5 (AAV5-WT), 5081 and 5083 mice is compared (in vivo).

[0138] Figure 5B for Figure 5A The statistical chart shows that 886 represents wild-type AAV5.

[0139] Figure 5C The in vivo transduction capacity of wild-type AAV5 (AAV5-WT) and 5081 mice is shown (tissue).

[0140] Figure 6A The enrichment folds of 5020, 5022, 5024, 5026, and 5081 in the liver, heart, brain, and muscle of cynomolgus monkeys are shown. The enrichment folds of wild-type AAV5 (AAV5-WT) are normalized to 1.

[0141] Figure 6B The enrichment folds of 5020, 5022, 5024, 5026, and 5081 in the left, right, middle, and caudate lobes of the cynomolgus monkey liver are compared. The enrichment folds of wild-type AAV5 (AAV5-WT) are normalized to 1.

[0142] Figure 6C The enrichment folds of 5020, 5022, 5024, 5026, and 5081 in the left and right atria and ventricles of cynomolgus monkeys are shown. The enrichment folds of wild-type AAV5 (AAV5-WT) were normalized to 1.

[0143] Figure 6D The enrichment folds of AAV5 in the bilateral deltoid, biceps brachii, wrist extensor, gluteus maximus, biceps femoris, and gastrocnemius muscles of cynomolgus monkeys are compared. Wild-type AAV5 enrichment folds were normalized to 1. Detailed Implementation

[0144] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0145] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0146] In this specification, the terms "substantially" or "truly" are used to indicate that the standard deviation from the theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%.

[0147] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0148] In this specification, "optional" or "optionally" means that the event or situation described below may or may not occur, and the description includes both the scenario in which the event occurs and the scenario in which the event does not occur.

[0149] The terms “comprising” and “having”, and any variations thereof, in this disclosure are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0150] In this disclosure, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0151] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0152] According to this disclosure, "Vg" refers to the viral genome; equivalent to a genome copy.

[0153] According to this disclosure, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein to refer to a polymeric form of amino acids of any length, including encoded and non-coding amino acids, chemically or biochemically modified or derived amino acids, and polypeptides having a similar peptide backbone.

[0154] According to this disclosure, the terms "nucleic acid molecule," "polynucleotide," "polynucleotide," and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0155] As used in this disclosure, the term "amino acid" can include natural amino acids, non-natural amino acids, amino acid analogs, and all their D and L stereoisomers. According to this disclosure, the three-letter and single-letter codes for amino acids are as described in J. biol. chem, 243, p3558 (1968). The amino acids and their abbreviations and English abbreviations in this disclosure are as follows: Histidine (His, H); Serine (Ser, S); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Threonine (Thr, T); Phenylalanine (Phe, F); Aspartic acid (Asp, D); Tyrosine (Tyr, Y); Leucine (Leu, L); Isoleucine (Ile, I); Arginine (Arg, R); Alanine (Ala, A); Valine (Val, V); Tryptophan (Trp, W); Methionine (Met, M); Asparagine (Asn, N); Cysteine ​​(Cys, C); Lysine (Lys, K); Proline (Pro, P).

[0156] According to this disclosure, an amino acid "addition" refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence. According to this disclosure, an amino acid "deletion" refers to the deletion of one, two, or three or more amino acids from an amino acid sequence. According to this disclosure, an amino acid "insertion" refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence; the inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids may be adjacent to each other. According to this disclosure, an amino acid "substitution" refers to the replacement of an amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein, "substitution" can be a conserved amino acid substitution.

[0157] According to this disclosure, "conservative modification," "conservative substitution," or "conservative replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), such that frequent changes can be made without altering the protein's biological activity. Those skilled in the art will appreciate that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, the substitution of structurally or functionally similar amino acids is unlikely to impair biological activity. Exemplary conserved substitutions are described in the following "Exemplary Conservative Amino Acid Substitutions."

[0158] Exemplary amino acid conservative substitution

[0159]

[0160]

[0161] According to this disclosure, "moderate to very high stringency conditions" includes "moderate stringency conditions," "moderate to high stringency conditions," "high stringency conditions," or "very high stringency conditions," which describe the conditions for nucleic acid hybridization and washing. For guidance on performing hybridization reactions, see Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in that literature, and either can be used. For example, specific hybridization conditions are as follows: (1) Low-toughness hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low-toughness conditions, the washing temperature can be increased to 55°C); (2) Medium-toughness hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High-toughness hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high-toughness hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0162] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of identity is obtained by aligning the two sequences.

[0163] In this disclosure, the terms “naturally occurring,” “unmodified,” or “wild-type” used to describe nucleic acids, peptides, cells, or organisms refer to nucleic acids, peptides, cells, or organisms that are present in nature. For example, a peptide or polynucleotide sequence present in an organism that can be isolated from a natural source is naturally occurring.

[0164] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic, preventative, or prophylactic measures, research, and diagnostic applications.

[0165] "Treatment" means administering an oral or topical therapeutic agent, such as an antibody comprising any of the present disclosure, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms.

[0166] In this specification, the term "prevention" refers to prophylactic treatment of subjects who do not have and have never had a disease but are at risk of developing a disease or who have had a disease in the past, do not have the disease currently, but are at risk of disease recurrence. In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of disease recurrence compared to the average healthy member of the subject population.

[0167] "Effective amount" includes an amount sufficient to ameliorate or prevent the symptoms or condition of a medical disorder. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the disorder to be treated, the overall health of the patient, the method of administration, the route and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0168] In this specification, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a disorder or sufficient to delay or minimize one or more symptoms associated with the disorder. A therapeutically effective amount refers to the amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a disorder. The term "therapeutically effective amount" can include an amount that improves the overall therapy; reduces or avoids the symptoms, signs or causes of a disorder; and / or enhances the therapeutic efficacy of another therapeutic agent.

[0169] In this specification, a "preventive effective amount" is an amount sufficient to prevent a disorder or one or more symptoms associated with the disorder or prevent its recurrence. A preventive effective amount refers to the amount of a therapeutic agent, alone or in combination with other agents, that provides a preventive benefit in preventing a disorder. The term "preventive effective amount" can include an amount that improves the overall prevention or enhances the preventive efficacy of another preventive agent.

[0170] In this specification, the term "subject" refers to a human (i.e., male or female of any age, e.g., pediatric subject (e.g., infant, child or adolescent) or adult subject (e.g., young, middle-aged or elderly)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g., cow, pig, horse, sheep, goat, cat or dog) or bird). The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic animal or a genetically engineered animal.

[0171] In this specification, the term "transduction efficiency" refers to the expression efficiency of the introduced target gene in target cells or target tissue organs after the delivery vector enters in vitro or in vivo. Detailed Description of the Invention

[0173] <AAV Capsid Protein and Targeting Peptide>

[0174] In some embodiments of this disclosure, an adeno-associated virus (AAV) capsid protein is provided, which includes a targeting peptide comprising the sequence RG-X3-X4-X5-X6-X7-X8;

[0175] in,

[0176] X3 is D, S, N, or G;

[0177] X4 is L, G, K, T, R, M, V, or F;

[0178] X5 is S, R, P, I, A, E, T, G, or L;

[0179] X6 is S, N, V, Q, G, D, A, I, P, R or L;

[0180] X7 is L, G, S, K, H, N, R, T, A, or D;

[0181] Compared to adeno-associated viruses with wild-type capsid proteins, adeno-associated viruses with capsid proteins containing the target peptide exhibit enhanced transduction efficiency (cell-mediated transgene expression ability) for cells expressing integrins.

[0182] In some specific embodiments, the targeting peptide comprises any of the following amino acid sequences: SEQ ID NO: 1-19.

[0183] In some preferred embodiments, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein,

[0184] X3 is D, S, N, or G;

[0185] X4 is L, G, K, T, R, M, V, or F;

[0186] X5 is S, R, P, I, A, T, G, or L;

[0187] X6 is S, N, V, Q, G, D, P, R, or L;

[0188] X7 is L, G, S, K, H, R, T, A, or D;

[0189] In some specific embodiments, the targeting peptide comprises the amino acid sequence shown in any of the following: SEQ ID NO: 2-3, 7-19.

[0190] In some implementations, adeno-associated viruses having capsid proteins containing the target peptide have improved transduction efficiency for muscle and / or heart compared to adeno-associated viruses having wild-type capsid proteins.

[0191] In some implementations, adeno-associated viruses having capsid proteins containing the target peptide have reduced transduction efficiency for the brain and / or liver compared to adeno-associated viruses having wild-type capsid proteins.

[0192] In some embodiments, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein

[0193] X3 is either D or S;

[0194] X4 is L, G, K, or T;

[0195] X5 is S, R, A, E, or P;

[0196] X6 is S, N, V, A, I, or Q;

[0197] X7 is L, G, S, H, N, K, or R.

[0198] In some specific embodiments, the targeting peptide comprises any of the following amino acid sequences: SEQ ID NO: 1-5, 7 and 9.

[0199] In some implementations, adeno-associated viruses having a capsid protein containing the target peptide have improved transduction efficiency in muscle compared to adeno-associated viruses having wild-type capsid proteins.

[0200] In some embodiments, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein

[0201] X3 is D;

[0202] X4 is L, G, K, or T;

[0203] X5 is S, R, A, or P;

[0204] X6 is S, N, V, A, or Q;

[0205] X7 is L, G, S, H, N, or R.

[0206] In some specific embodiments, the targeting peptide comprises any of the following amino acid sequences: SEQ ID NO: 1-4, 7 and 9.

[0207] In some implementations, adeno-associated viruses having a capsid protein containing the target peptide have improved transduction efficiency for the heart compared to adeno-associated viruses having wild-type capsid proteins.

[0208] In some embodiments, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein

[0209] X3 is either D or S;

[0210] X4 is L, G, K, or T;

[0211] X5 is S, R, A, E, or P;

[0212] X6 is S, N, V, A, I, or Q;

[0213] X7 is L, S, H, N, K, or R.

[0214] In some specific embodiments, the targeting peptide comprises any of the following amino acid sequences: SEQ ID NO: 2-5, 7, and 9.

[0215] In some implementations, adeno-associated viruses (AAVs) having capsid proteins containing the target peptide have improved transduction efficiency for muscle and heart compared to AAVs having wild-type capsid proteins.

[0216] In some embodiments, the targeting peptide comprises the sequence RG-X3-X4-X5-X6-X7-X8; wherein

[0217] X3 is D;

[0218] X4 is L, G, K, or T;

[0219] X5 is S, R, A, or P;

[0220] X6 is S, N, V, A, or Q;

[0221] X7 is L, S, H, N, or R.

[0222] In some specific embodiments, the targeting peptide comprises any of the following amino acid sequences: SEQ ID NO: 2-4, 7, and 9.

[0223] In some specific embodiments, the targeting peptide comprises amino acids as shown in SEQ ID NO: 9.

[0224] Some aspects of this disclosure also provide targeting peptides in the aforementioned capsid proteins.

[0225] In this disclosure, AAV is a tiny, non-enveloped virus with a 25 nm capsid. No disease is known or shown to be associated with wild-type viruses. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term, free-type transgenic expression. Vectors containing as few as 300 base pairs of AAV can be packaged and integrated. The spatial restriction of the exogenous DNA is approximately 4.7 kb. AAV vectors, such as those described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), can be used to introduce DNA into cells. AAV vectors have been used to introduce a variety of nucleic acids into different cell types (see, for example, Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993). Many alternative AAV variants are available (over 100 have been cloned), and AAV variants have been identified based on desired characteristics.

[0226] In some implementations, AAV is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, and rh.74.

[0227] In some preferred embodiments, AAV is AAV5.

[0228] The AAV capsid is an icosahedron composed of 60 VP capsid protein monomers, including 5 VP1 monomers, 5 VP2 monomers, and 50 VP3 monomers. VP1, VP2, and VP3 monomers are all transcribed and translated from the AAV's Cap gene. VP1 is the longest, containing approximately 735 amino acids. VP2 and VP3 are truncated versions of VP1, omitting the N-terminal amino acids of the VP1 protein. Conventionally, capsid protein modification sites are named according to the amino acid sequence of the VP1 protein. For example, inserting a targeting peptide between amino acids 575 and 577 of the AAV5 capsid / capsid protein means inserting the targeting peptide between amino acids 575 and 577 of the VP1 monomer of AAV5 (containing 724 amino acids), deleting the original amino acid 576 (or replacing it with the targeting peptide), and ensuring that the corresponding positions in AAV5 VP1, VP2, and VP3 all contain this targeting peptide. The amino acid sequence of the capsid / capsid protein of AAV, such as AAV5, is usually represented by the amino acid sequence of VP1.

[0229] In some implementations, AAV5 includes AAV5 VP1 or a variant thereof, AAV5 VP2 or a variant thereof, and / or AAV5 VP3 or a variant thereof.

[0230] For example, the amino acid sequence of AAV5 VP1 is shown below (SEQ ID NO: 97):

[0231] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIP AQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFT LPQYGYATLNRDNTENPTERSSFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYL EGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0232] (A total of 724 amino acids, with the target peptide insertion site between amino acids 575 and 577, and amino acid 576 deleted)

[0233] The amino acid sequence of AAV5 VP2 is shown below (SEQ ID NO: 98):

[0234] TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWS PRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPL VDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSST TAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0235] (A total of 588 amino acids, corresponding to amino acids 137-725 of AAV5 VP1. The target peptide insertion site is between amino acids 439 and 441, that is, between amino acids 575 and 577 of AAV5 VP1.)

[0236] The amino acid sequence of AAV5 VP3 is shown below (SEQ ID NO: 99):

[0237] MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIAN NLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYA NTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIV PGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL

[0238] (A total of 532 amino acids, corresponding to amino acids 193-725 of AAV5 VP1. The target peptide insertion site is between amino acids 383 and 385, that is, between amino acids 575 and 577 of AAV5 VP1.)

[0239] In some embodiments of this disclosure, the targeting peptide is inserted between amino acids 575 and 577 of AAV5 VP1 or a variant thereof. That is, the targeting peptide is inserted between amino acids 575 and 577 of the VP1 monomer of AAV5, and the original amino acid 576 is deleted, or it can be understood as replacing the original amino acid 576 with the targeting peptide.

[0240] In some preferred embodiments of this disclosure, the AAV capsid protein comprises any of the following amino acid sequences: SEQ ID NO:20-38.

[0241] In some specific embodiments, the sequence of AAV may have at least 80, 85, 90, 95, 97, or 99% identity with the exemplary AAV sequences described herein. For example, it may include variants, preferably without reducing the ability of AAV to mediate transgene expression in cells.

[0242] In some specific embodiments, the muscle includes skeletal muscle.

[0243] In some more specific embodiments, the muscle includes deltoid, biceps brachii, extensor carpi, gluteus maximus, biceps femoris, gastrocnemius, quadriceps femoris, and / or hamstring (the hamstring is composed of semitendinosus, semimembranosus, and biceps femoris).

[0244] In some more specific embodiments, the muscle includes gluteus maximus, biceps femoris, and / or biceps brachii.

[0245] In some specific embodiments, the heart includes left atrium, right atrium, left ventricle, and / or right ventricle.

[0246] <Polynucleotide>

[0247] In some embodiments of the present disclosure, a polynucleotide is provided that encodes the AAV capsid protein of the present disclosure or encodes the targeting peptide of the present disclosure.

[0248] The polynucleotide of the present disclosure can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0249] The polynucleotide encoding the AAV capsid protein of the present disclosure includes: a coding sequence encoding only the AAV capsid protein / targeting peptide; the coding sequence of the AAV capsid protein / targeting peptide and various additional coding sequences; the coding sequence of the AAV capsid protein / targeting peptide (and optionally additional coding sequences) and non-coding sequences.

[0250] The "polynucleotide encoding the AAV capsid protein / targeting peptide" can be a polynucleotide including the polynucleotide encoding this AAV capsid protein / targeting peptide, or can also be a polynucleotide further including additional coding and / or non-coding sequences.

[0251] The present disclosure also relates to a polynucleotide that hybridizes with the above sequence and has at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present disclosure particularly relates to a polynucleotide that can hybridize with the polynucleotide described in the present disclosure under stringent conditions. The stringent conditions are moderate stringent conditions, medium-high stringent conditions, high stringent conditions, or very high stringent conditions.

[0252] <AAV and Transgene Delivery Vector>

[0253] In some embodiments of this disclosure, an AAV is provided that comprises the AAV capsid protein described in this disclosure.

[0254] Some aspects of this disclosure also provide a transgenic delivery vector comprising the AAV described herein.

[0255] The AAV and transgenic delivery vector disclosed herein can be used to deliver any composition, such as a sequence of interest, to tissues, for example, to muscle and / or heart.

[0256] In some implementations, the AAV further includes genetically modified organisms.

[0257] In some specific embodiments, the transgene comprises a nucleotide sequence encoding a gene product. A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a specific protein after transcription and translation. A "gene product" is a molecule produced by the expression of a specific gene. Gene products include, for example, polypeptides, aptamers, interfering RNA, mRNA, etc.

[0258] In some specific implementations, the genetic modification is a therapeutic genetic modification, a preventative genetic modification, or a diagnostic genetic modification.

[0259] In some exemplary embodiments, the transgene is a therapeutic transgene that encodes a gene sequence for a therapeutic agent, such as a gene sequence encoding a treatment for muscle and / or heart disease.

[0260] In other exemplary embodiments, the transgene is a gene sequence encoding a reporter protein, such as a fluorescent protein (an enzyme that catalyzes the production of a detectable product).

[0261] <Pharmaceutical Compositions and Administration>

[0262] In some embodiments of this disclosure, a pharmaceutical composition is provided comprising:

[0263] (a) the aforementioned AAV, or the aforementioned transgenic delivery vector; and, optionally,

[0264] (b) Pharmaceutically acceptable carriers.

[0265] In some embodiments, the pharmaceutical compositions of this disclosure contain the aforementioned AAV or the aforementioned transgenic delivery vector as an active ingredient, the AAV or transgenic delivery vector comprising (i) a targeting peptide and (ii) a transgenic gene, such as a therapeutic transgenic gene, exemplary, a therapeutic agent for treating muscle and / or heart diseases.

[0266] As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents that are compatible with drug administration.

[0267] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, intravenous, intra-arterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion administration. Thus, delivery can be systemic or local.

[0268] The AAV or the aforementioned transgenic delivery vector provided in this disclosure has targeting specific muscles and / or the heart, and is more efficient in targeting specific muscles and / or the heart, thus allowing for administration at lower doses, thereby reducing adverse events and toxic reactions.

[0269] Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0270] <Uses and Methods>

[0271] In some embodiments of this disclosure, the use of the AAV, the transgenic delivery vector, and / or the pharmaceutical composition described herein is provided in the preparation of reagents for delivering transgenic genes into cells.

[0272] In other embodiments of this disclosure, a method for delivering a transgene to a cell is provided, the method comprising the step of contacting the cell with the AAV, the transgene delivery vector, and / or the pharmaceutical composition described in this disclosure.

[0273] The AAV described in this disclosure can be used to deliver genetically modified organisms (GMOs), such as therapeutic GMOs, to tissues, such as the muscle and / or heart of a subject. In some embodiments, the AAV described in this disclosure can deliver genetically modified / therapeutic GMOs to cells of the muscle and / or heart.

[0274] In some embodiments, the AAV described in this disclosure is used to deliver a nucleic acid sequence encoding a therapeutic agent to a subject suffering from muscle and / or heart disease.

[0275] In some specific embodiments of this disclosure, the use of the AAV, the transgenic delivery vector, and / or the pharmaceutical composition described herein is provided in the preparation of a medicament for treating a disease.

[0276] In other specific embodiments of this disclosure, a disease treatment method is provided, comprising the step of administering the AAV, the transgenic delivery vector, and / or the pharmaceutical composition described herein to a subject.

[0277] In this disclosure, the disease can be any disease that can be treated with AAV carrying the genetic material. In some preferred embodiments, the disease can be muscular and / or cardiac. In some specific implementations, muscle and / or cardiac diseases include, but are not limited to, Duchenne muscular dystrophy, alpha-1 antitrypsin deficiency, heart failure, and myocardial infarction (Tabebordbar, Mohammadsharif et al. “Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.” Cell vol. 184,19 (2021): 4919-4938.e22. doi:10.1016 / j.cell.2021.08.028; Rahaghi, Franck F. “Alpha-1 antitrypsin deficiency research and emerging treatment strategies: what's down the road?.” Therapeutic advances in chronic disease vol. 12_suppl 20406223211014025. 29 Jul. 2021, doi:10.1177 / 20406223211014025; Wang, Dan et al. “Adeno-associatedvirus vector as a platform for gene therapy delivery.” Nature reviews. Drugdiscovery vol. 18,5 (2019): 358-378. doi:10.1038 / s41573-019-0012-9; Bass-Stringer, Sebastian et al. “Adeno-Associated Virus Gene Therapy: Translational Progress and Future Prospects in the Treatment of HeartFailure.” Heart, lung & circulation vol. 27,11 (2018): 1285-1300. doi:10.1016 / j.hlc.2018.03.005).

[0278] Example

[0279] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0280] Example 1: Construction of library plasmids and viral packaging vectors

[0281] 1. pIVB496:

[0282] The AAV5 Rep-AAP helper plasmid, which is used to package the AAV library virus, was constructed by General Biotechnology (Anhui) Co., Ltd. (order number: G0219024-1). A reverse AAV2_P5 promoter sequence with a TATA box sequence was inserted before the AAV2rep gene in the pAAV-Rep2 Cap5 plasmid (Cell Biolabs, catalog number: VPK-425). The codons at amino acid positions 6, 10, 142, 150, and 216 of the AAV5_cap gene were mutated to stop codons. This mutation does not affect AAP protein expression.

[0283] AAV2_P5 promoter sequence (SEQ ID NO: 39):

[0284] TTCAAACCTCCCGCTTCAAAATGGAGACCCTGCGTGCTCACTCGGGCCCCCCCCCCCAGCGTGACCACATGGTGTCGCAAAATGTCGCAAAACACTCACGTGACCTCTAATACAGGACCTCCCTAACCCTATGACGTAATTCACGTCACGACTCCACCCTCC

[0285] 2. pIVB499:

[0286] To construct a one-round AAV5-RGD / N / S / GXXXX library, random insert primers synthesized by Beijing Qingke Biotechnology Co., Ltd. were used. PCR amplification was performed using Q5 DNA polymerase (NEB; M0493) with IVB235 and LW499F1 as primers and LW499T as a template to obtain the insert fragment (the fragment inserts into the AAV5_cap gene at the amino acid position S575-RGD / N / S / GXXXX-T577). The PCR fragment (S575-insertion sequence-T577) was digested with AarI enzyme (NEB, R0745) and the AAV5-Cap ITR backbone vector pIVB620 (i.e., pAAV-ITR-CMV-intron-AAV5 cap (S575-ccdB-T577)-ITR) (constructed by General Biotechnology (Anhui) Co., Ltd., order number: G0225214). The resulting library plasmid, pssAAV-ITR-CMV-intron-AAV5 Cap (S575-RGD / S / N / GXXXX-T577)-hGH polyA-ITR, was obtained by ligation using T4 DNA ligase (NEB, M0202). The final library plasmid was analyzed by NGS sequencing at Novogene (Tianjin) Co., Ltd. to determine library coverage and abundance.

[0287] PCR primers

[0288] IVB235 (SEQ ID NO: 40): TTGATGACACCTGCACCCCAGTGG

[0289] LW499F1 (SEQ ID NO: 41):

[0290] TCGACGTGTCTCACCTGCCTCGGAGCCGAGG

[0291] PCR template

[0292] LW499T (SEQ ID NO: 42):

[0293] ACCTGCCTCGGAGCCGAGGCRRCNNKNNKNNKNNKACCACTGGGGTGCAGGTGTC

[0294] Where R represents: A or G; N represents: A, T, G or C; K represents: G or T.

[0295] 3. pIVB3197:

[0296] To construct the AAV5-RGD / N / S / GXXXX second-round library, a mixed primer library of 446 primers synthesized by Genscript Biotech Co., Ltd. (order number: C2115YQJG0) was used as a template. PCR amplification was performed using Q5 DNA polymerase (NEB; M0493) and primers LW3165R1 and LW3197F1 to obtain the insert fragment (the amino acid position of the AAV5 cap gene insertion sequence is S575-insert sequence-T577). The above PCR fragment (containing the Q568-Y585 fragment of S575-insert sequence-T577) and the AAV5-Cap ITR backbone vector pIVB3129 (i.e., pAAV-ITR-CBH-EGFP-polyA-P40-intron-AAV5) were digested with Esp3I enzyme (NEB, R0734). The cap(Q568-ccdB-Y585)-ITR (constructed with Innovative DNA, record number: IVB230951-009) was used to construct the library plasmid by ligation using T4 DNA ligase (NEB, M0202).

[0297] The pAAV-ITR-CBH-EGFP-polyA-P40-intron-AAV5cap(S575-RGD / S / N / GXXXX-T577)-ITR library plasmid was identified by NGS sequencing at Novogene (Tianjin).

[0298] PCR primers

[0299] LW3165R1 (SEQ ID NO: 43):

[0300] TTTCGTCTCTTGTATGTGCCAGTCGCAGGAGCAGTGGT

[0301] LW3197F1 (SEQ ID NO: 44):

[0302] TTTCGTCTCAGCAGATGGCCACCAACAATCAAAGCAGAGGT

[0303] Example 2: AAV virus packaging and purification

[0304] The novel AAV virus packaging system is a three-plasmid packaging system. In this embodiment, the recombinant AAV is prepared by co-transfection of the three plasmids. The three plasmids include the RC plasmid (capsid & replication plasmid; containing the nucleotide sequence of the capsid), the AAVHelper plasmid (or Ad Helper plasmid, which provides the auxiliary factors required for AAV generation), and the pAAV plasmid (or transgenic plasmid, which provides the nucleotide sequence to be delivered, often containing the promoter and cDNA required for protein translation).

[0305] The AAV5 Rep-AAP helper (pIVB496) plasmid required for viral library packaging was constructed in Example 1 and preserved by the applicant; the AAV pHelper plasmid was purchased from Cell Biolabs and preserved by our company, containing the genes E2, E4 and VA RNA of adenovirus AdV; the 499 library-pAAV plasmid psAAV-ITR-CMV-intron-AAV5 Cap(S575-RGD / S / N / GXXXX-T577)-hGH polyA-ITR was constructed in Example 1 and preserved by the applicant (pIVB499), wherein the capsid sequence contains the modified inserted RGD / S / N / G and 4 random amino acids, namely RGD / S / N / GXXXX. Library 3197 - pAAV plasmid pssAAV-ITR-CMV-intron-AAV5 Cap (S575-446-RGD / S / N / GXXXX-T577) -hGH polyA-ITR was constructed in Example 1 and preserved by the applicant (pIVB3197), wherein the capsid sequence contains 446 modified inserted RGD / S / N / GXXXX sequences. To prevent capsid mosaic and genome mispackaging, the viral library was packaged and transfected using a pAAV plasmid: AAV5 Rep-AAPhelper plasmid: AAV pHelper plasmid mass ratio of 0.1:1:2.

[0306] The RC plasmid pAAV_Rep2 / Cap5 required for packaging the single-capped virus was constructed and preserved by the applicant (see Example 8); the AAV pHelper plasmid was purchased from Cell Biolabs and preserved by the applicant, containing the genes E2, E4, and VARNA of adenovirus AdV; the pAAV plasmid pssAAV-ITR-CMV-fluc-P2A-EGFP-3flag-WPRE-SV40_polyA-ITR was preserved by the applicant, containing the reporter gene firefly luciferase gene fLuc and the fluorescent protein EGFP gene. During single-capped virus packaging, transfection was performed using a pAAV plasmid:RC plasmid:AAV Helper plasmid in a mass ratio of 1:1:2.

[0307] For AAV virus packaging production, HEK 293T cells were seeded in 150 mm culture dishes and grown to 70%-80% confluence for plasmid transfection. The corresponding three plasmids were co-transfected into the cells using polyethyleneimine (PEI) according to the transfection ratio described above for virus library packaging or single-capsid virus packaging. 72 hours post-transfection, the supernatant and cell pellet were separated by low-speed centrifugation. Benzonase (total concentration 10 U / ml) and 2 mM MgCl2 were added to the cell supernatant, and the cells were digested at 37°C for 2-4 hours. The cell pellet was lysed using lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2) containing 0.5% sodium deoxycholate (w / v), and Benzonase (50 U / ml) was added. The cells were incubated at 37°C for 4 hours to digest free DNA molecules, and the supernatant was obtained after low-speed centrifugation at 4°C for 30 minutes. The treated cell supernatant and lysis buffer supernatant were mixed and precipitated overnight on ice with a 1:5 volume ratio of 40% PEG8000 and 2.5M NaCl solution. The next day, the supernatant was collected by centrifugation and purified using iodixanol ultracentrifugation. The purified AAV titer was determined by ddPCR (Sinafone DQ24 digital PCR instrument), and the virus was aliquoted and stored at -80 °C.

[0308] The specific procedure for ddPCR was as follows: ITR-F and ITR-R or GFP-F and GFP-R were used as primers, and ITR-P or GFP-P was used as probes. The 5' end of the probe was labeled with FAM fluorescent protein, and the 3' end was linked to a BHQ1 quencher. The primers and probes were synthesized by Qingke Company (Tianjin). The 62 bp fragment in the ITR sequence was specifically amplified using ITR-F and ITR-R as primers, and the 114 bp fragment in the EGFP sequence was specifically amplified using GFP-F and GFP-R as primers. The TaqMan probe binding method was used, and the physical titer of the viral genome was detected using a digital quantitative PCR instrument (model: Sinaf DQ24 digital PCR instrument). The operation procedure is described in the instructions of the reagents used. The virus treatment method is described in the literature (Aurnhammer C, et al. Hum Gene Ther Methods. 2012; 23(1): 18-28.).

[0309] Table 1:

[0310]

[0311] Example 3: Screening of the cynomolgus monkey AAV5-RGD / S / N / GXXXX (pIVB499) library

[0312] Jiangsu Dingtai Pharmaceutical Research (Group) Co., Ltd. was commissioned to conduct a screening experiment on cynomolgus monkeys infected with the AAV5-RGD / S / N / GXXXX (pIVB499) library (Contract No. DT-22120-671N). After screening for AAV5 neutralizing antibodies, two male monkeys aged 3-5 years and weighing approximately 3.4 kg with relatively low AAV5 neutralizing antibody levels were selected. The pIVB499 library virus (5 mL, 2E+13 vg, i.e., 2×10⁻⁶) was injected intravenously into the forelimbs. 13 (vg) / monkey, before intravenous injection, 0.5 mg of dexamethasone was administered intramuscularly. Eight days after intravenous injection, the anesthetized animals were euthanized, and tissue samples were taken from the heart, liver, lungs, kidneys, muscles, brain, retina, optic nerve, etc.

[0313] To obtain the screening and enrichment results of the pIVB499 library in cynomolgus monkeys, RNA was extracted from various tissues (heart, liver, lung, kidney, muscle, brain, retina, and optic nerve) of cynomolgus monkeys using Trizol (RNAiso Plus 9108, TAKARA). Reverse transcription was performed using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (AE311-02, TransGold). Twelve random nucleotides were introduced as a unique molecular identifier (UMI) onto the reverse transcription primer NGS-1-R9. The first round of PCR was performed using NGS-1-F8 and NGS-A-R8. After recovering the PCR products using AMPure magnetic beads (A63881, Backmen Coulter), a second round of PCR was performed using Illumina adapter primers. The obtained library-specific PCR fragments were then subjected to NGS next-generation sequencing (Illumina NovaSeq 6000, Beijing Novogene Technology Co., Ltd.).

[0314] Table 2:

[0315]

[0316] Capsid enrichment fold = current proportion of capsid in tissue sample / current proportion of capsid in original viral library. Based on the enrichment fold of capsids in heart and muscle, eight single capsids (5020, 5021, 5022, 5023, 5024, 5026, 5027, and 5028) were selected. The top 446 capsids were then used to construct a second-round viral library (pIVB3197; construction method is described in Examples 1 and 2).

[0317] Example 4: In vitro cell transduction experiment of novel AAV

[0318] To test the in vitro transduction efficiency of the novel AAV (the capsid proteins expressed by the AAV capsid protein expression plasmids are 5020, 5021, 5022, 5023, 5024, 5026, 5027, 5028 and AAV5 wild-type) screened in Example 3, AAV viruses carrying the GFP gene were packaged using the single capsid virus production method in Example 2, and their transduction efficiency in human immortalized skeletal muscle cells was detected.

[0319] Human immortalized skeletal muscle cells (AC16, Mason, catalog number: CTCC-008-0124) culture conditions: DMEM-F12 complete medium (89% DMEM-F12, 10% fetal bovine serum, 1% penicillin-streptomycin), cells were cultured at a rate of 1E+5 (i.e., 10⁻⁵ cells per 10⁻⁶ ... 5 Cells were seeded in 24-well culture plates, with AAV viruses (AAV5-WT) containing the novel capsid and wild-type AAV5 capsid selected in Example 3 added at a ratio of 10,000 MOI. Each virus was used in triplicate. After virus infection, the culture plates were incubated in a cell culture incubator (5% CO2, 37°C) for 72 hours. Cells were then digested with 0.25% trypsin-EDTA (gibco, catalog number: 25200056), resuspended in PBS, and the GFP fluorescence ratio was detected by flow cytometry. Figure 1 ).

[0320] See Figure 1 The results showed that 5020, 5021, 5022, and 5023 had better skeletal muscle cell transduction efficiency than wild-type AAV5, and 5027 showed an ability to in vitro infect target cells similar to wild-type AAV5.

[0321] Example 5: In vitro human cardiac organoid transduction experiment of novel AAV

[0322] To test the efficacy of the novel AAV-targeted organoids, 1E+11vg(10) was added to human iPSC-derived cardiac organoids differentiated for 39 days (purchased from ACROBiosystems, catalog number: CIPO-HWL002K). 11 AAV viruses carrying the GFP gene (5020, 5021, 5022, 5023, 5024, 5026, 5027, 5028, and wild-type AAV5) were used in organoids, with a blank control that was not infected with the virus. Seven days after infection, images of the GFP signal were obtained using a Thermo Fisher inverted fluorescence microscope (EVOS-M5000).

[0323] See Figure 2The results showed that 5022 and 5024 had better cardiac organoid infection capabilities than wild-type AAV5, with 5024 exhibiting the highest efficiency in targeting cardiac organoids. Compared to wild-type AAV5, 5021, 5023, and 5027 showed slightly improved efficiency in transducing cardiac organoids.

[0324] Example 6: In vitro integrin αVβ6 / αVβ8 binding transduction experiment of novel AAV

[0325] To verify the affinity of the novel AAV for binding to integrin αVβ6 / αVβ8, HEK 293T cells were seeded in 100 mm culture dishes and grown to 70%-80% confluence. They were then transfected with polyethyleneimine (PEI) at a mass ratio of ITGAV / ITGB6 / ITGB8 of 2:1:1. Forty-eight hours later, cells transfected with 293T and 293T-integrin were simultaneously digested with 0.25% trypsin-EDTA (gibco, catalog number: 25200056). Cells were seeded in 24 wells and infected with novel AAV viruses (5020, 5021, 5022, 5023, 5024, 5026, 5027, 5028, and wild-type AAV5) at an MOI of 10000. After 48 hours of culture in a cell culture incubator (5% CO2, 37°C), cells were digested with 0.25% trypsin-EDTA, resuspended in PBS, and the GFP fluorescence ratio was detected by flow cytometry.

[0326] See Figure 3 The results showed that when αVβ6 / αVβ8 was expressed, the in vitro transduction efficiency of 5021, 5022 and 5027 increased by 3.54, 3.78 and 2.77 times, respectively, with 5028 showing the highest increase of about 7.74 times, indicating that these capsids enter cells through αVβ6 / αVβ8 receptors.

[0327] Table 3:

[0328]

[0329] ITGB6 amino acid sequence (SEQ ID NO: 100):

[0330] MGIELLCLFFLFLGRNDHVQGGCALGGAETCEDCLLIGPQCAWCAQENFTHPSGVGERCDTPANLLAKGCQLNFIENPVSQVEILKNKPLSVGRQKNSSDIVQIAPQSLILKLRPGGAQTLQVHVRQTEDYPVDLYYLMDLSASMDDDLNTIKELGSRLSKEMSKLTSNFRLGFGSFVEKPVSPFVKTTPEEIANPCSSIPYFCLPTFGFKHILPLTNDAERFNEIVKNQKISANIDTPEGGFDAIMQAAVCKEKIGWRNDSLHLLVFVSDADSHFGMDSKLAGIVIPNDGLCHLDSKNEYSMSTVLEYPTIGQLIDKLVQNNVLLIFAVTQEQVHLYENYAKLIPGATVGLLQKDSGNILQLIISAYEELRSEVELEVLGDTEGLNLSFTAICNNGTLFQHQKKCSHMKVGDTASFSVTVNIPHCERRSRHIIIKPVGLGDALELLVSPECNCDCQKEVEVNSSKCHHGNGSFQCGVCACHPGHMGPRCECGEDMLSTDSCKEAPDHPSCSGRGDCYCGQCICHLSPYGNIYGPYCQCDNFSCVRHKGLLCGGNGDCDCGECVCRSGWTGEYCNCTTSTDSCVSEDGVLCSGRGDCVCGKCVCTNPGASGPTCERCPTCGDPCNSKRSCIECHLSAAGQAREECVDKCKLAGATISEEEDFSKDGSVSCSLQGENECLITFLITTDNEGKTIIHSINEKDCPKPPNIPMIMLGVSLAILLIGVVLLCIWKLLVSFHDRKEVAKFEAERSKAKWQTGTNPLYRGSTSTFKNVTYKHREKQKVDLSTDC

[0331] ITGAV amino acid sequence (SEQ ID NO: 101):<\

[0332]

[0333] ITGB8 amino acid sequence (SEQ ID NO: 102):

[0334] MCGSALAFFTAAFVCLQNDRRGPASFLWAAWVFSLVLGLGQGEDNRCASSNAASCARCLALGPECGWCVQEDFISGGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEINTQVTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHNNIEKLNSVGNDLSRKMAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLSLTENITEFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIGWRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLGQLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQKLISEVKVQVENQVQGIYFNITAICPDGSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGGKNYAIIKPIGFNETAKIHIHRNCSCQCEDNRGPKGKCVDETFLDSKCFQCDENKCHFDEDQFSSESCKSHKDQPVCSGRGVCVCGKCSCHKIKLGKVYGKYCEKDDFSCPYHHGNLCAGHGECEAGRCQCFSGWEGDRCQCPSAAAQHCVNSKGQVCSGRGTCVCGRCECTDPRSIGRFCEHCPTCYTACKENWNCMQCLHPHNLSQAILDQCKTSCALMEQQHYVDQTSECFSSPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQWNSNKIKSSSDYRVSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLNAHETFRCNF

[0335] Example 7: Second-round small library screening of in vitro overexpressing cells AAV5-RGD / S / N / GXXXX (pIVB3197)

[0336] To screen for capsids with higher affinity for integrin αVβ6 / αVβ8, a capsid virus library containing 446 AAV5-RGD / S / N / GXXXX (pIVB3197) was used to infect 293T cells (100 mm) at MOI=50000. After incubation at 37°C for 6 h, the cell supernatant was evenly distributed into six-well plates seeded with 293T-αVβ6 / αVβ8 cells. Transfection of 293T cells with ITGAV / ITGB6 / ITGB8 is described in Example 6. Cells were harvested 72 h after infection for RNA extraction and NGS library construction (method described in Example 3, primers for library construction are listed in Table 4).

[0337] Table 4:

[0338]

[0339] Capsid enrichment fold = percentage of capsid in cells / percentage of capsid in the original viral library. Integrin αVβ6 / αVβ8 binding fold = enrichment fold of capsid in 293T-αVβ6 / αVβ8 cells / enrichment fold of capsid in 293T cells. Ranked by integrin binding fold (see Table 5). Figure 4 The top-ranked AAV viruses were identified as: 5081, 5082, 5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090, and 5091.

[0340] Table 5:

[0341]

[0342] Example 8: Construction of a novel AAV capsid protein expression plasmid

[0343] 1. Intermediate plasmid pIVB886: Construction of pAAV_Rep2 / Cap5-p5 plasmid (i.e., wild-type AAV5-RC plasmid)

[0344] This embodiment uses the Gibson assembly method for construction. Specific steps are detailed in the Gibson Assembly® Chemical Transformation Protocol (E2611). The Cap5 PCR fragment obtained by PCR amplification using pAAV_Rep2 / Cap5 (Cell Biolabs, catalog number: VPK-425) as a template and the AgeI / HindIII-digested linearized backbone plasmid fragment pAAV2 / 8 (Plasmid #112864) are assembled using Gibson to obtain the target plasmid. The specific sequence is shown below:

[0345] pIVB886: pAAV_Rep2 / Cap5-p5 plasmid vector sequence (SEQ ID NO: 55)

[0346]

[0347] 2. Construction of intermediate plasmid pIVB3098: pAAV_Rep2 / Cap5-Bsu36i-p5

[0348] The intermediate plasmid pIVB3098, constructed in step 1, has a Bsu36i restriction endonuclease site inserted at position 1710 bp in the Cap5 sequence of the intermediate vector pIVB886. The plasmid was constructed by General Biotech (Anhui) Co., Ltd., and the specific sequence is shown below:

[0349] pIVB3098: pAAV_Rep2 / Cap5-Bsu36i-p5 plasmid vector sequence (SEQ ID NO: 56)

[0350]

[0351] Novel AAV capsid protein expression plasmids were constructed using the Gibson assembly method (see Gibson Assembly® Chemical Transformation Protocol, E2611 for details). The PCR fragment and the Bsu36i-digested linearized intermediate plasmid pIVB3098 fragment were assembled to obtain different novel AAV capsid protein expression plasmids. In obtaining the PCR fragment, no template was required for PCR; primers were self-pairing for PCR amplification. Primer sequences are shown in Table 6 below.

[0352] Table 6:

[0353]

[0354]

[0355]

[0356] Experimental Results: In this embodiment, the plasmid DNA to be constructed was identified by enzyme digestion and Sanger sequencing, proving that the expression plasmid of the corresponding capsid protein was successfully constructed. The amino acid sequence of the capsid protein in the obtained AAV capsid protein expression plasmid is shown in Table 7 below.

[0357] Table 7:

[0358]

[0359] Example 9: Detection of transgene expression in mice

[0360] To detect the transduction of the fluc-P2A-GFP gene in mice by 5081, 5083, and wild-type AAV5, nine 6-8 week old wild-type c57 mice were randomly divided into three groups of three. Each group received 2 × 10⁻⁶ mice. 11 The virus was administered via tail vein injection at a dose of vg / mouse. Two weeks after injection, in vivo imaging was performed on mice to detect the systemic expression level of the luciferase gene fluc (see [link to article]). Figure 5A , Figure 5B (See Table 8). The results showed that, compared with wild-type AAV5, the 5081 virus exhibited significant leg muscle transduction in mice.

[0361] Table 8:

[0362]

[0363] Four weeks after injection, 5081 and wild-type AAV5-injected mice were dissected, and samples were taken from the heart, liver, kidney, and quadriceps femoris and hamstring muscles of the hind legs to detect the expression level of the luciferase gene fluc (see...). Figure 5C The results showed that 5081 was significantly expressed in the quadriceps and hamstring muscles of the hind legs, but its expression in the liver was not significantly different from that of wild-type AAV5.

[0364] The specific process of mouse in vivo imaging:

[0365] In vivo imaging: First, prepare a 30 mg / ml fluorescent developing solution. Weigh 0.15 g of D-Luciferinpotassium (purchased from Dalian Meilun Biotechnology Co., Ltd.) and add it to 5 ml of DPBS. Shake for 15 seconds to dissolve, keeping the solution dark throughout the process. Inject 150 μl into each mouse. Five mice are grouped together. After all mice have been injected, they are placed in a gas anesthesia container and the timing begins. After complete anesthesia, the mice are placed sequentially into the in vivo detection instrument (model: Boluteng Aniview600 multi-modal animal in vivo imaging system) and photographed. The total time from the start of timing to photographing is 5 minutes.

[0366] Tissue imaging: First, a 30 mg / ml fluorescent contrast agent and a 26 mg / ml anesthetic (prepared from tribromoethanol and tert-amyl alcohol, both purchased from Shanghai McLean Pharmaceutical Co., Ltd.) were prepared. The injection volume of the fluorescent contrast agent for each mouse was 150 μl. The injection volume of the anesthetic was 20 μl / g. First, 150 μl of the fluorescent contrast agent was injected, and timing began. Two minutes later, the anesthetic was injected, with the injection volume varying according to the mouse's body weight at a ratio of 30 μl / g. Then, dissection and perfusion were initiated. After perfusion, tissue samples were collected in the following order: heart, liver, kidney, quadriceps femoris muscle of the hind limb, and hamstring muscle. The samples were placed in clean culture dishes and placed in a live biopsy system for examination. The entire process took 10-15 minutes, ensuring the contrast agent was not quenched.

[0367] Example 10: Verification Experiment of Cynomolgus Monkey Monocapsid Virus

[0368] To verify the transduction efficiency of the newly screened AAV virus in non-human primates, we commissioned Jiangsu Dingtai Pharmaceutical Research (Group) Co., Ltd. (Contract No.: HT-JS-2023-155) to conduct a validation experiment in cynomolgus monkeys with AAV5 types 5020, 5022, 5024, 5026, 5081, and wild-type AAV5. After screening for AAV5 neutralizing antibodies, three monkeys aged 3-6 years and weighing approximately 3.4 kg with relatively low AAV5 neutralizing antibody levels were selected and injected intravenously into their forelimbs with a mixed virus (5E+12vg, i.e., 5×10⁻⁶). 12 One monkey, aged 4.3 years and weighing 3.04 kg, was injected with 5081 virus (1 mg / kg / virus). Each virus was packaged using a double-stranded pscAAV-CMV-EGFP-barcode-ITR plasmid (with different 12 bp barcode sequences inserted into the pscAAV-CMV-EGFP-ITR plasmid), ensuring that each viral genome carried a unique sequence code. Prednisone was administered intramuscularly at 1 mg / kg / day for the first three days before intravenous injection and during the first week after administration. Prednisone was administered at 0.5 mg / kg / day during the second week of administration. Fourteen days after intravenous injection, the anesthetized animals were euthanized, and tissue samples were taken from the heart (left and right atria and ventricles), liver (left, middle, right and caudate lobes), muscles (bilateral deltoids, biceps brachii, extensor carpi radialis, gluteus maximus, biceps femoris and gastrocnemius), and brain (frontal cortex, temporal cortex, parietal cortex, occipital cortex, frontal medulla, temporal medulla, parietal medulla, occipital medulla, hippocampus, corpus callosum, thalamus and striatum).

[0369] To obtain the expression results of monocapsid virus in various tissues of cynomolgus monkeys, RNA was extracted from various tissues (heart, liver, muscle, and brain) using Trizol (RNAiso Plus 9108, TAKARA). Reverse transcription was performed using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (AE311-02, TransGold). Twelve random nucleotides were introduced as a molecular tag (UMI) onto the reverse transcription primer LW3398R1. The first round of PCR was performed using LW3398F1 and NGS-1-R8. After recovering the PCR products using AMPure magnetic beads (A63881, Backmen Coulter), a second round of PCR was performed using Illumina adapter primers. The obtained library-specific PCR fragments were then subjected to NGS next-generation sequencing (Illumina NovaSeq 6000, Beijing Novogene Technology Co., Ltd.).

[0370] Table 9:

[0371]

[0372] The enrichment fold of novel AAVs in various tissues = the proportion of the capsid corresponding to the barcode in the tissue sample / the proportion of the corresponding capsid in the original viral library. The enrichment fold of wild-type AAV5 was set as the standard, with a value of 1. The enrichment folds of other AAV viruses in various tissues compared to wild-type AAV5 are shown below. Figures 6A-6D The results showed that, compared with wild-type AAV5, the 5081 virus significantly transduced in the muscles of cynomolgus monkeys, especially in the gluteus maximus, biceps femoris, and biceps brachii muscles, at 68.76, 64.88, and 80.54 times that of wild-type AAV5, respectively. Figure 6D The 5081 virus has a transduction efficiency 1.5-2 times higher than wild-type AAV5 in the heart. Figure 6A and Figure 6C It is noteworthy that 5081 expressed only 0.3 times less AAV5 on average across all four hepatic lobes than wild-type AAV5. Figure 6A Therefore, the 5081 virus shows promise as a novel adeno-associated virus vector with low liver and high cardiac and skeletal muscle transduction efficiency.

[0373] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0374] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. An adeno-associated viral capsid protein comprising a targeting peptide comprising the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D, S, N, or G; X4 is L, G, K, T, R, M, V, or F; X5 is S, R, P, I, A, E, T, G, or L; X6 is S, N, V, Q, G, D, A, I, P, R, or L; X7 is L, G, S, K, H, N, R, T, A, or D; optionally, the targeting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 1-19; optionally, an adeno-associated virus employing a capsid protein comprising the targeting peptide has an increased transduction efficiency for cells expressing integrin as compared to an adeno-associated virus employing a wild-type capsid protein.

2. The adeno-associated virus capsid protein of claim 1, wherein, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D, S, N, or G; X4 is L, G, K, T, R, M, V, or F; X5 is S, R, P, I, A, T, G, or L; X6 is S, N, V, Q, G, D, P, R, or L; X7 is L, G, S, K, H, R, T, A, or D; optionally, the targeting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 2-3, 7-19; optionally, an adeno-associated virus employing a capsid protein comprising the targeting peptide has an increased transduction efficiency for cells expressing integrin as compared to an adeno-associated virus employing a wild-type capsid protein.

3. The adeno-associated virus capsid protein of claim 1, wherein, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D or S; X4 is L, G, K, or T; X5 is S, R, A, E, or P; X6 is S, N, V, A, I, or Q; X7 is L, G, S, H, N, K, or R; optionally, the targeting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 1-5, 7, and 9; optionally, an adeno-associated virus having a capsid protein comprising the targeting peptide has an increased transduction efficiency for muscle and / or heart as compared to an adeno-associated virus having a wild-type capsid protein.

4. The adeno-associated virus capsid protein of claim 1 or 3, wherein, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D; X4 is L, G, K, or T; X5 is S, R, A, or P; X6 is S, N, V, A, or Q; X7 is L, G, S, H, N, or R; optionally, the targeting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOS: 1-4, 7, and 9; optionally, an adeno-associated virus having a capsid protein comprising the targeting peptide has an increased transduction efficiency for muscle as compared to an adeno-associated virus having a wild-type capsid protein.

5. The adeno-associated virus capsid protein of claim 1 or 3, wherein, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D or S; X4 is L, G, K, or T; X5 is S, R, A, E, or P; X6 is S, N, V, A, I, or Q; X7 is L, S, H, N, K, or R; Optionally, the targeting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-5, 7, and 9; Optionally, the adeno-associated virus having the capsid protein comprising the targeting peptide has an improved transduction efficiency for the heart compared to the adeno-associated virus having a wild-type capsid protein.

6. The adeno-associated virus capsid protein of claim 1 or 3, wherein, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D; X4 is L, G, K, or T; X5 is S, R, A, or P; X6 is S, N, V, A, or Q; X7 is L, G, S, K, H, N, R, T, A, or D; Optionally, the targeting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-4, 7, and 9; Optionally, the adeno-associated virus having the capsid protein comprising the targeting peptide has an improved transduction efficiency for the muscle and heart compared to the adeno-associated virus having a wild-type capsid protein.

7. The adeno-associated virus capsid protein of any one of claims 1-6, wherein, the targeting peptide comprises an amino acid sequence set forth in SEQ ID NO:

9.

8. The adeno-associated virus capsid protein of any one of claims 1-7, wherein, the adeno-associated virus is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, and rh.74; Preferably, the AAV comprises AAV5.

9. The adeno-associated virus capsid protein of any one of claims 1-8, wherein, the AAV5 comprises AAV5 VP1 or a variant thereof; Preferably, the amino acid sequence of the AAV5 VP1 is set forth in SEQ ID NO:

97.

10. The adeno-associated virus capsid protein of any one of claims 1-9, wherein, the targeting peptide is inserted at a position between amino acids 575 and 577 of the AAV5 VP1 or a variant thereof; Preferably, the adeno-associated virus capsid protein comprises an amino acid sequence set forth in any one of SEQ ID NOs: 20-38, and an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 20-38.

11. A targeting peptide comprising the sequence R-G-X3-X4-X5-X6-X7-X8, wherein, wherein, X3 is D, S, N, or G; X4 is L, G, K, T, R, M, V, or F; X5 is S, R, P, I, A, T, G, or L; X6 is S, N, V, Q, G, D, P, R, or L; X7 is L, G, S, K, H, R, T, A, or D; Optionally, the targeting peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-19; Optionally, the adeno-associated virus having the capsid protein comprising the targeting peptide has an improved transduction efficiency for the muscle and heart compared to the adeno-associated virus having a wild-type capsid protein. the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D, S, N, or G; X4 is L, G, K, T, R, M, V, or F; X5 is S, R, P, I, A, T, G, or L; X6 is S, N, V, Q, G, D, P, R, or L; X7 is L, G, S, K, H, R, T, A, or D; Optionally, the targeting peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2-3, 7-19; Optionally, an adeno-associated virus having a capsid protein comprising the targeting peptide has an increased transduction efficiency for cells expressing integrin compared to an adeno-associated virus having a wild-type capsid protein; Preferably, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D or S; X4 is L, G, K, or T; X5 is S, R, A, or P; X6 is S, N, V, A, or Q; X7 is L, G, S, H, N, K, or R; Optionally, the targeting peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-5, 7, and 9; Optionally, an adeno-associated virus having a capsid protein comprising the targeting peptide has an increased transduction efficiency for muscle and / or heart compared to an adeno-associated virus having a wild-type capsid protein; Preferably, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D; X4 is L, G, K, or T; X5 is S, R, A, or P; X6 is S, N, V, A, or Q; X7 is L, G, S, H, N, K, or R; Optionally, the targeting peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4, 7, and 9; Optionally, an adeno-associated virus having a capsid protein comprising the targeting peptide has an increased transduction efficiency for muscle compared to an adeno-associated virus having a wild-type capsid protein; Preferably, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D or S; X4 is L, G, K, or T; X5 is S, R, A, E, or P; X6 is S, N, V, A, I, or Q; X7 is L, S, H, N, K, or R; Optionally, the targeting peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2-5, 7, and 9; Optionally, an adeno-associated virus having a capsid protein comprising the targeting peptide has an increased transduction efficiency for heart compared to an adeno-associated virus having a wild-type capsid protein; Preferably, the targeting peptide comprises the sequence R-G-X3-X4-X5-X6-X7-X8; wherein, X3 is D; X4 is L, G, K, or T; X5 is S, R, A, or P; X6 is S, N, V, A, or Q; X7 is L, S, H, N, K, or R; Optionally, the targeting peptide comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 2-4, 7, and 9; Optionally, an adeno-associated virus having a capsid protein comprising the targeting peptide has an increased transduction efficiency for muscle and heart compared to an adeno-associated virus having a wild-type capsid protein; More preferably, the targeting peptide comprises an amino acid sequence as set forth in SEQ ID NO:

9.

12. A polynucleotide encoding the adeno-associated virus capsid protein of any one of claims 1-10, or encoding the targeting peptide of claim 11.

13. An adeno-associated virus comprising the adeno-associated virus capsid protein of any one of claims 1-10.

14. The adeno-associated virus of claim 13, further comprising a transgene; Optionally, the transgene is a therapeutic transgene, a prophylactic transgene, or a diagnostic transgene.

15. A transgene delivery vehicle comprising the adeno-associated virus of claim 13 or 14.

16. A pharmaceutical composition comprising: (a) the adeno-associated virus of claim 13 or 14, or the transgene delivery vehicle of claim 15; and, optionally, (b) a pharmaceutically acceptable carrier.

17. Use of the adeno-associated virus of claim 13 or 14, the transgene delivery vehicle of claim 15, or the pharmaceutical composition of claim 16 in the manufacture of a medicament for delivering a transgene to a cell.

18. The use according to claim 17, wherein, The cell is from a subject; Preferably, the subject is a mammalian subject; More preferably, the subject is a human.

19. Use according to claim 17 or 18, wherein, The cell is from muscle and / or heart.

20. Use according to claim 18 or 19, wherein, The subject has a muscle and / or heart disease.

21. Use according to any one of claims 17 to 20, wherein, The adeno-associated virus, the transgene delivery vehicle, or the pharmaceutical composition is administered to the subject by intravenous injection.

22. Use of the adeno-associated virus of claim 13 or 14, the transgene delivery vehicle of claim 15, or the pharmaceutical composition of claim 16 in the manufacture of a medicament for treating a disease; Preferably, the disease comprises a muscle and / or heart disease.

23. A method of treating a disease, comprising the step of administering the adeno-associated virus of claim 13 or 14, the transgene delivery vehicle of claim 15, or the pharmaceutical composition of claim 16 to a subject; Preferably, the disease comprises a muscle and / or heart disease.

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