Modified aav capsid proteins and their use in escaping neutralizing antibodies

Abstract

The present disclosure provides modified AAV capsid proteins with reduced serum antibody binding, AAV capsids comprising such modified capsid proteins, and AAV vectors comprising such AAV capsids. The present disclosure also provides methods of using such AAV capsids.

Description

Technical Field

[0001] This disclosure relates generally to the field of gene therapy, and more specifically to modified adeno-associated virus (AAV) capsid proteins and their use in escape-neutralizing antibodies. Background Technology

[0002] Recombinant AAV has unique advantages for gene therapy. As an example, one of the most commercially successful drugs, Zolgensma, developed by Novartis, is based on AAV. In fact, many gene therapy pipelines targeting inherited disorders of the nervous system or muscle tissue are AAV-based. However, the presence of neutralizing antibodies greatly limits the application of this therapy; therefore, Zolgensma's instructions explicitly require patients to have a serum antibody titer of less than 1:50. If AAV mutants that effectively evade neutralizing antibodies could be developed, the scope of application for related gene therapy drugs would be greatly expanded.

[0003] However, effectively overcoming the recognition of AAV neutralizing antibodies has become a key challenge. These antibodies can rapidly recognize and bind to AAV vectors, preventing them from effectively delivering therapeutic genes to target cells, thereby significantly reducing the effectiveness of treatment.

[0004] Methods for modifying AAV mutants are still needed to effectively evade recognition by various neutralizing antibodies during gene therapy. Summary of the Invention

[0005] On one hand, this disclosure provides a modified AAV capsid protein comprising at least one mutated antigenic epitope, said at least one mutated antigenic epitope comprising at least one amino acid mutation located at a position selected from the group consisting of: 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588 and 715; wherein the number corresponds to wild-type AAV9 VP1 protein (SEQ ID NO: 1), and wherein said at least one amino acid mutation significantly reduces the binding of serum antibodies to said AAV capsid protein.

[0006] In one aspect, this disclosure provides a polynucleotide encoding an AAV-modified capsid protein as disclosed herein. In another aspect, this disclosure provides an expression vector comprising the polynucleotide as disclosed herein. In yet another aspect, this disclosure provides a cell comprising the polynucleotide or expression vector as disclosed herein.

[0007] On one hand, this disclosure provides an AAV capsid comprising modified AAV capsid proteins as disclosed herein. On the other hand, this disclosure provides an AAV viral vector comprising an AAV capsid as disclosed herein.

[0008] On the one hand, this disclosure provides a pharmaceutical composition comprising an AAV viral vector as disclosed herein and a pharmaceutically acceptable vector.

[0009] On one hand, this disclosure provides a method for delivering a payload nucleic acid into a cell, the method comprising contacting the cell with a viral vector as disclosed herein.

[0010] On one hand, this disclosure provides a method for delivering a payload nucleic acid to a subject, the method comprising administering to the subject an effective amount of a viral vector as disclosed herein or a pharmaceutical composition as disclosed herein.

[0011] On one hand, this disclosure provides a method for treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of an AAV viral vector as disclosed herein or a pharmaceutical composition as disclosed herein.

[0012] In another aspect, this disclosure provides a method for generating an AAV vector that produces neutralizing antibodies in an escape host, the method comprising: a) inserting a short peptide into multiple sites of a VP3 protein to generate a library of modified AAV vectors; b) determining the binding of the modified AAV vectors to a sample containing mixed serum from different subjects; and c) identifying the modified AAV vectors with reduced binding to the serum sample.

[0013] The foregoing description of the invention is merely exemplary and is not intended to limit the invention in any way. In addition to the exemplary aspects, embodiments, and features described above, other aspects, embodiments, and features will become apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0014] Figure 1 The diagram shows the locations of 122 selected insertion sites on the AAV9 capsid surface. This plot is based on the published AAV9 crystal structure (PDB # 7MT0) and was drawn using Pymol. Insertion sites are marked in red.

[0015] Figure 2 The results show the binding of serum dilutions to ELISA plates coated with different concentrations of wild-type AAV9.

[0016] Figure 3ELISA results for mixed serum dilutions binding to wild-type AAV9 and 122 AAV9 variants are shown. Readings are sorted from highest to lowest, and the binding signal of wild-type AAV9 is indicated by a label. Twenty mutants were screened with binding signals that were more than 50% lower than those of wild-type AAV9.

[0017] Figure 4A and 4B Wild-type AAV9 and 21 selected variants were identified in 48 individual serum samples at a 1:50 dilution. Figure 4A The ELISA results show the binding of all serum antibodies with the inserted AAV9 variant at the specified location. Figure 4B The positive rates for each AAV9 variant are shown in 48 individual serum samples, with an ELISA cutoff of 0.9.

[0018] Figure 5A and 5B The locations of 13 key residues that significantly reduce serum antibody binding are shown. Figure 5A The structure of the entire AAV9 capsid is shown, and Figure 5B A subunit of the AAV9 capsid is shown. The figure is based on the published AAV9 crystal structure (PDB # 7MT0) and was plotted using Pymol. Key residues are indicated by arrows.

[0019] Figure 6 The transduction inhibition of serum samples on wild-type AAV9 (AAV9-wt) and AAV9 variant with a Strep-II tag insertion at 509 of VP1 (509-ST) is shown.

[0020] Figure 7 The transduction inhibition of serum samples against wild-type AAV9 (AAV9-wt) and AAV9 variants with different types of mutations at position 509 of VP1 is shown. 509-S2: 2-amino acid insertion; 509-S4: 4-amino acid insertion; 509-S6: 6-amino acid insertion; 509-Myc: Myc-tag insertion; 509-GS4: GGGS insertion; 509-GS8: GGGSGGGS insertion. 509-RP8: The original sequence at positions 506-513 of VP1 was replaced with the strep-II tag sequence WSHPQFES.

[0021] Figure 8The transduction inhibition of serum samples against wild-type AAV9 (AAV9-wt) and AAV9 variants with different types of mutations at position 559 of VP1 is shown. 559-ST: A Strep-II tag is inserted at position 559 of VP1, and 559-Myc: A Myc tag is inserted at position 559 of VP1.

[0022] Figure 9 The transduction inhibition of serum samples against wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 512 of VP1 is shown. AAV6-ST512: Strep-II tag inserted at position 512 of VP1.

[0023] Figure 10 The transduction inhibition of serum samples against wild-type AAV6 (AAV6-WT) and AAV6 variants with an insertion at position 513 of VP1 is shown. AAV6-ST513: Strep-II tag inserted at position 513 of VP1.

[0024] Figure 11 The transduction inhibition of serum samples against wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 556 of VP1 is shown. AAV6-ST556: Strep-II tag inserted at position 556 of VP1.

[0025] Figure 12 The transduction inhibition of serum samples against wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 557 of VP1 is shown. AAV6-ST557: Strep-II tag inserted at position 557 of VP1.

[0026] Figure 13 The transduction inhibition of serum samples against wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 559 of VP1 is shown. AAV6-ST559: Strep-II tag inserted at position 559 of VP1. Detailed Implementation

[0027] In the following detailed description, reference is made to the accompanying drawings, which are intended to accompany the various embodiments provided herein and are intended to form a part of the description of those embodiments. Unless the context otherwise indicates or specifies, similar symbols in the drawings generally identify similar components in the description.

[0028] All descriptions provided herein are intended to illustrate various embodiments of the invention and are not intended to be limiting. Those skilled in the art will understand that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and that such equivalent embodiments will be included herein.

[0029] It should be understood that, for clarity, certain features of this disclosure described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of this disclosure described in the context of individual embodiments may also be provided individually or in any suitable sub-combination. For illustration, if a particular embodiment disclosed herein includes components A, B, and C, it should be understood that this disclosure is also intended to include embodiments that individually contain A, B, or C, or any combination of A, B, or C.

[0030] All references cited in this disclosure, including patent applications, granted patents, published articles, or other publications, are incorporated herein by reference in their entirety to provide methods that can be used in conjunction with the descriptions provided herein. Any terminology presented in one or more publications that is similar to or identical to terms expressly defined in this disclosure shall, in all respects, be subject to the definitions expressly provided in this disclosure.

[0031] Unless otherwise expressly defined, all technical and scientific terms used in this disclosure are generally considered to have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0032] I. definition Before providing a detailed description of the invention in this disclosure, note and define the following.

[0033] As used herein, and throughout this disclosure, unless otherwise stated, the articles “a / an” and “the” will be interpreted as meaning “one or more” or “at least one / a kind”. For example, “a gene” can mean one gene or more than one gene.

[0034] As used herein, unless otherwise expressly stated, any range of numbers described herein may include every number of the range and every subrange.

[0035] As used herein, the terms “about,” “approximately,” “around,” etc., refer to a quantity, level, value, number, frequency, percentage, size, dimensions, weight, or length that varies relative to a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length by as much as 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In certain embodiments, when the term “about” or “approximately” precedes a numerical value, it indicates a range of 15%, 10%, 5%, or 1% added to or subtracted from the value.

[0036] As used herein, the term "polypeptide" is used interchangeably with "peptide," "protein," etc., and refers to a polymer of amino acid residues or an assembly of multiple polymers of amino acid residues. The term is applicable to both naturally occurring and non-naturally occurring amino acid polymers, and is interpreted to also cover amino acid polymers in which one or more amino acid residues are artificial or synthetic chemical mimics of their corresponding naturally occurring amino acids. The term "protein" generally refers to a large polypeptide. The term "peptide" generally refers to a short polypeptide. A polypeptide sequence is generally described as having an amino terminus (N-terminus) on the left and a carboxyl terminus (C-terminus) on the right.

[0037] As used herein, the term "nucleic acid" refers to oligonucleotides or polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and further should be interpreted to include equivalents, derivatives, variants, and analogs of RNA or DNA made of nucleotide analogs, single-stranded (sense or antisense), and double-stranded polynucleotides. It should be understood that the term "nucleic acid" does not refer to or infer a polynucleotide chain of a specific length; therefore, nucleotides, polynucleotides, and oligonucleotides are also included in the definition.

[0038] As used herein, the terms “comprise”, “comprises”, “comprising”, “include”, “includes”, “including”, “contain”, “contains”, “containing”, “have”, “has”, “having”, etc., are synonymous and are used inclusively in an open-ended manner, without excluding other elements, features, steps, actions, operations, etc.

[0039] As used herein, the term “or” is used in its inclusive sense (rather than in its exclusive sense), so when used, for example, to connect lists of elements, the term “or” means one, some, or all of the elements in the list.

[0040] As used herein, the phrase “at least one” refers to one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. A phrase referring to “at least one” of a series of items is interpreted as referring to any combination of those items, including a single item. As an example, “at least one of A, B, or C” is intended to cover: A, B, C, A and B, A and C, B and C, and A, B, and C. Unless otherwise specifically stated, connective terms (such as the phrase “at least one of X, Y, and Z”) are generally understood in context to indicate that an item, term, etc., can be at least one of X, Y, or Z. Therefore, such connective terms are generally not intended to imply that a particular implementation requires at least one X, at least one Y, and at least one Z to be present.

[0041] As used herein, the terms “reduce,” “reduces,” “reduction,” and similar terms mean a reduction of at least about 10%, about 15%, about 20%, about 25%, about 35%, about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, or more.

[0042] "Sequence identity percentage (%)" is defined, when used for amino acid sequences (or nucleic acid sequences), as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence after sequence alignment, and, where necessary, vacancies to achieve maximum correspondence. For example, publicly available tools such as BLASTN and BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)) and ClustalW2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21): 2947-8) can be used. (2007) and ALIGN or Megalign (DNASTAR) software are used to perform alignments for determining the percentage of identity in amino acid (or nucleic acid) sequences. Those skilled in the art can use the default parameters provided by these tools, or can customize parameters suitable for alignment, for example, by selecting a suitable algorithm. In some embodiments, the positions of dissimilar residues may differ due to conserved amino acid substitutions.

[0043] As used herein, the terms “homologue sequence” and “homologous sequence” are used interchangeably and refer to a polynucleotide sequence (or its complementary strand) or amino acid sequence that, on an optional alignment, has at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence.

[0044] "Conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue in a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conserved amino acid substitution does not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to conserved substitution, the percentage or degree of similarity can be increased to correct for the conservatism of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson, (1994) Methods in Molecular Biology 24:307-331, which is incorporated herein by reference.

[0045] As used herein, references to “one embodiment,” “an embodiment,” “a particular embodiment,” “related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment,” or combinations thereof, are understood to mean that a particular feature, structure, or characteristic described in connection with that particular embodiment is included in at least one embodiment of this disclosure. Therefore, the foregoing phrases present or appearing throughout this disclosure do not necessarily all refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0046] Unless otherwise expressly stated, or otherwise understood in the context in which they are used, the conditional language used herein, such as “can / could / might / may,” “e.g.,” etc., is generally intended to convey that certain embodiments include certain features, elements, and / or steps that are not included in other embodiments. Therefore, such conditional language is not generally intended to imply that a feature, element, and / or step is necessary in any way for one or more embodiments.

[0047] II. Modified adeno-associated virus (AAV) capsid protein This disclosure provides a modified AAV capsid protein that significantly reduces binding to serum antibodies, particularly anti-AAV antibodies or anti-AAV neutralizing antibodies. This disclosure further provides AAV viral vectors comprising the modified AAV capsid protein provided herein, such that the binding of the AAV viral vector to serum antibodies, particularly anti-AAV antibodies or anti-AAV neutralizing antibodies, is significantly reduced. Such AAV viral vectors provided herein are particularly useful for delivering or expressing payload nucleic acids to subjects, for example, for therapeutic purposes.

[0048] On one hand, this disclosure provides a modified AAV capsid protein comprising at least one mutated antigenic epitope. In some embodiments, the mutated antigenic epitope comprises at least one amino acid mutation (e.g., insertion, substitution, deletion, or any combination thereof). In some embodiments, the amino acid mutation is located at a position selected from the group consisting of: 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715, where the numbers correspond to wild-type AAV9 VP1 protein (SEQ ID NO: 1). The AAV capsid protein can be any suitable serotype, and the corresponding position can be identified by comparing the amino acid sequence of the VP1 protein of the AAV capsid protein with the amino acid sequence of the VP1 protein of wild-type AAV9.

[0049] a. Advantages of the viral vector provided in this article The AAV viral vector presented in this article is particularly useful and advantageous compared to other viral vectors in at least the following respects.

[0050] The recombinant AAV or AAV viral vectors presented in this article offer unique advantages for gene therapy. First, AAV is non-pathogenic to humans, making it a safe vector option. Second, extensive research has shown that AAV genes typically do not integrate into the genome, significantly reducing potential therapy-related risks. Furthermore, AAV can infect various cell types, including dividing and non-dividing cells, expanding the scope of gene therapy. Additionally, AAV has multiple naturally occurring serotypes, allowing scientists to select the most suitable serotype based on disease type and lesion site, thus facilitating the selection or design of gene vectors. Finally, compared to other viral vectors, AAV can induce a relatively weaker immune response, reducing the likelihood of adverse reactions and complications. These unique characteristics make AAV a widely used delivery tool in gene therapy.

[0051] The recombinant AAV or AAV viral vector presented in this paper is advantageous in overcoming pre-existing immunity to AAV, providing an important solution to a long-standing problem.

[0052] Because AAV is widely present in nature, a key challenge in AAV gene therapy is the potential presence of neutralizing antibodies in patients. These antibodies can rapidly recognize and bind to AAV vectors, preventing the vectors from effectively delivering the payload nucleic acids to target cells, thereby significantly reducing the effectiveness of treatment. This occurs in patients who already possess these antibodies due to prior viral exposure or innate immune responses. It is estimated that 40-70% of individuals in the population have pre-existing neutralizing antibodies against AAV in their serum. For these patients, even if the AAV vector can introduce the target gene into the body, the gene cannot reach and affect the target cells. The presence of neutralizing antibodies not only introduces unpredictable variables in potential gene therapy recipients but also severely hinders the widespread clinical application of AAV therapy. Therefore, physicians typically need to perform detailed serological testing before initiating AAV therapy to identify the presence of neutralizing antibodies that may affect the therapeutic effect and adjust the treatment strategy accordingly.

[0053] In the implementation of AAV gene therapy, the efficiency of neutralizing antibody recognition plays a crucial and decisive role. Therefore, reducing the efficiency of AAV neutralizing antibody recognition is clinically significant. If this recognition can be effectively reduced, more AAV vectors can evade the immune system's attack, allowing more therapeutic genes to successfully enter target cells and significantly improving the effectiveness of gene therapy. This can not only help existing patients achieve better treatment outcomes but also expand the applicability of AAV gene therapy, giving more patients the opportunity to benefit from this innovative treatment method. Therefore, effectively overcoming the recognition of neutralizing antibodies against AAV has always been one of the biggest challenges in AAV gene therapy.

[0054] To address this issue, researchers have conducted a series of engineered modifications to AAV, introducing mutations that allow it to evade recognition by neutralizing antibodies. These types of AAV mutants are highly sought after in the field of gene therapy because they could transform current treatment paradigms and offer new hope to patients who cannot benefit from gene therapy due to the presence of neutralizing antibodies. Although some early studies have made progress, most existing work is based on information about the binding between a single monoclonal antibody and AAV. This approach differs significantly from reality: due to the high diversity of immune response profiles in the human B-cell lineage, exposure to AAV can generate multiple anti-AAV polyclonal antibodies. Different antibodies can recognize different regions on the surface of AAV, rendering strategies that modify the antigenic epitopes of a single anti-AAV antibody ineffective in achieving robust immune evasion. Due to the complexity of serum neutralizing antibodies, no AAV mutants capable of effectively evading recognition by multiple neutralizing antibodies have yet been developed.

[0055] The modified AAV capsid protein provided herein addresses this problem. Typically, the modified AAV capsid protein provided herein contains one or more mutations introduced into one or more specific antigenic regions (i.e., antigenic epitope regions) on the surface of the AAV capsid protein, said one or more mutations that can reduce the recognition of pre-existing anti-AAV antibodies in host serum (e.g., human serum). Specifically, the antigenic epitope regions provided herein are unique and effective because the capsid modification sites of this disclosure were discovered using human serum samples containing a variety of naturally occurring neutralizing antibodies. The modification of the AAV capsid provided herein can effectively reduce the recognition of a variety of naturally occurring neutralizing antibodies in human serum.

[0056] Using genetic engineering techniques, the inventors introduced insert peptides at 122 different sites within the CAP9 gene sequence encoding the AAV9 capsid VP protein. Based on crystal structure, these 122 insertion sites effectively cover all significant structural features on the viral capsid surface. By introducing these mutations into the CAP gene, 122 different engineered AAV9 variants were obtained, each with a unique insert peptide at a specific site on the capsid surface. By comparing the recognition efficiency of pre-existing antibodies in mixed sera from multiple adults for these AAV9 mutants, the inventors identified 21 key regions on the AAV9 capsid surface where pre-existing antibody binding was reduced by more than 50% compared to wild-type AAV9. Further analysis showed that mutations at 13 of the 21 selected sites effectively evaded recognition by pre-existing antibodies from most tested individual serum samples. Through insertion, substitution, or deletion, these structural changes at key sites protect AAV transduction activity from neutralization by serum antibodies.

[0057] b. AAV capsid protein This disclosure provides a modified AAV capsid protein that significantly reduces the binding of the modified AAV capsid protein to serum antibodies, particularly anti-AAV antibodies or anti-AAV neutralizing antibodies.

[0058] As used in this article, the term "adeno-associated virus" (AAV) refers to small viruses that infect humans and some other primate species. AAVs are a class of non-pathogenic single-stranded DNA viruses commonly found in humans and other mammals. AAVs belong to the family Parvoviridae and the genus Parvovirus.

[0059] As used herein, the term "capsid" refers to the protein shell that encapsulates viral nucleic acid. Typically, the capsid contains several oligomeric subunits (i.e., protomers).

[0060] The AAV capsid, used to encapsulate genetic material such as viral genomes, is an icosahedral hollow structure with a diameter of approximately 26 nm. It can be imagined as an icosahedron composed of 20 triangles, with every five triangles forming a pentagon. There are five rotation axes between each pair of opposite pentagons, three rotation axes between the centers of each pair of opposite triangles, and two rotation axes between the midpoints of each pair of opposite sides. The AAV capsid contains a total of 60 copies of the capsid proteins VP1, VP2, and VP3, with the ratio of these three subunits on each capsid being 5:5:50. These subunits are encoded by overlapping sequences within the same open reading frame. Neutralizing antibodies recognize antigenic epitope regions on the surface of the viral capsid and bind to AAV.

[0061] In some embodiments, the AAV capsid protein provided herein is modified such that its binding to serum antibodies (e.g., but not limited to anti-AAV antibodies, or more specifically anti-AAV neutralizing antibodies) is significantly reduced.

[0062] As used herein, AAV capsid proteins with “significantly reduced binding to serum antibodies” are interpreted as encompassing AAV capsid proteins that, under equivalent assay conditions, bind to serum antibodies at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, or at least about 90% less than the unmodified or corresponding wild-type AAV capsid proteins.

[0063] In some embodiments, under equivalent assay settings, the modified AAV capsid protein provided herein has a binding rate of no more than 20%, no more than 15%, no more than 10%, no more than 8%, no more than 5%, no more than 2%, or no more than 1% with serum antibodies in a suitable assay compared to unmodified or corresponding wild-type AAV capsid protein.

[0064] In some embodiments, the serum antibody includes an anti-AAV antibody. The anti-AAV antibody can specifically bind to the AAV capsid. In some embodiments, the serum antibody is capable of binding to an antigenic epitope in the AAV capsid protein provided herein. In some embodiments, the serum antibody is a pre-existing anti-AAV antibody present in the host's serum and capable of binding to an antigenic epitope in the AAV capsid protein provided herein. In some embodiments, the serum antibody comprises IgG (including IgG1, IgG2a, IgG2b, IgG3), IgM, IgE, or IgA. In some embodiments, the host is a human, canine, feline, or equine animal. In some embodiments, the host is a human.

[0065] In some embodiments, serum antibodies include anti-AAV neutralizing antibodies. As used herein, the term "anti-AAV neutralizing antibody" means an antibody that can effectively inhibit AAV viral particles from infecting its susceptible host cells (e.g., those cells targeted for gene therapy drug delivery).

[0066] Various methods are applicable to assessing the extent to which the modified AAV capsid protein presented herein binds to serum antibodies. For example, this binding can be detected by exposing AAV viral particles displaying the modified capsid protein to a serum sample containing serum antibodies. To assess the degree of reduced binding, a parallel comparative assay can be performed using control AAV viral particles presenting an equivalent but modified capsid protein. This parallel assay provides a comparison between the antibody binding levels of the modified capsid protein and the original capsid protein.

[0067] As used herein, the term "capsid protein" refers to the structural proteins of the viral capsid. The AAV capsid has three capsid proteins, including viral protein 1 (VP1), VP2, and VP3, which assemble into an icosahedral capsid. In the AAV genome, VP1, VP2, and VP3 are all encoded by overlapping sequences within an open reading frame. The complete sequence of VP3 is contained within VP2, and the complete sequence of VP2 is contained within VP1, which has a unique N-terminal (VP1u) domain. The common VP3 region has been observed in the capsid structures of all AAV serotypes identified to date, either by cryo-electron microscopy (cyro-EM) and image reconstruction (cyro-reconstruction) or by X-ray crystallography.

[0068] In some embodiments, the modified AAV capsid protein provided herein comprises at least one mutated antigenic epitope, said at least one mutated antigenic epitope comprising at least one amino acid mutation.

[0069] As used herein, the term "antigenic epitope" is a portion of the AAV capsid protein that is recognized and bound by antibodies (e.g., neutralizing antibodies). Antigenic epitope regions can be linear or conformational (i.e., comprising spaced-apart amino acid residues). Detailed analysis of the AAV capsid protein has revealed conserved structural motifs characterized by eight-chain β-barrels (chains βB to βI) and accompanying α-helices (αA). Antigenic diversity among AAV serotypes primarily arises from structural variable regions (VRs) located on the surface loops connecting these β-chains. These VRs are crucial in forming the unique morphology of the capsid surface, resulting in serotype-specific variations in cell tropism, tissue transduction efficiency, and antigenic properties.

[0070] In some embodiments, the at least one mutated antigenic epitope comprises at least one amino acid mutation located at a position selected from the group consisting of: positions 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715, wherein the number corresponds to the wild-type AAV9 VP1 protein. In some embodiments, the antigenic epitope region is located at a position selected from the group consisting of: 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588, and 715, wherein the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0071] As used herein, the phrase “corresponds to…” for AAV capsid proteins reflects the correspondence between the amino acid residues of a reference capsid protein and the numbered amino acid residues of a standard AAV capsid protein (e.g., wild-type AAV9 VP1 protein), which shares some homology with the reference capsid protein. The reference and standard capsid proteins can be different AAV serotypes, and such correspondences can be obtained, for example, through sequence alignment.

[0072] Due to the high conservation of the capsid amino acid sequence, significant cross-reactivity of neutralizing antibodies between different AAV serotypes has been reported. In this sense, mutations in corresponding residues on AAV serotypes other than AAV9 may have a similar escape effect on transduction inhibition. Therefore, in this disclosure, when listing the locations where mutations are introduced within the antigenic epitope region, specific locations in a particular AAV9 VP1 protein (e.g., the sequence having SEQ ID NO: 1) are used only as references or examples, and are not intended to specifically limit the mutation locations to the capsid protein of wild-type AAV9.

[0073] In other words, the locations of amino acid mutations within the antigenic epitopes provided in this disclosure (e.g., positions 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715, etc., mentioned above) include not only specific positions in the AAV9 VP1 protein (having the amino acid sequence of SEQ ID NO: 1), but also those corresponding positions in the capsid proteins of AAV serotypes other than AAV9 VP1, which can be obtained, for example, by sequence alignment. Sequence alignment can be performed by comparing a selected AAV capsid sequence with a reference sequence (e.g., AAV9 or another AAV serotype) using any suitable computer program and technique such as Clustal W, CAP Sequence Assembly, MAP, MEME, and Vector NTI tools.

[0074] For example, position 441 of the AAV9 VP1 protein corresponds to position 440 of the AAV2 VP1 protein, position 431 of the AAV5 VP1 protein, position 441 of the AAV6 VP1 protein, and position 443 of the AAV8 VP1 protein. Position 512 of the AAV9 VP1 protein corresponds to position 511 of the AAV2 VP1 protein, position 498 of the AAV5 VP1 protein, position 512 of the AAV6 VP1 protein, and position 514 of the AAV8 VP1 protein. To name just a few, position 559 of the AAV9 VP1 protein corresponds to position 558 of the AAV2 VP1 protein, position 548 of the AAV5 VP1 protein, position 559 of the AAV6 VP1 protein, and position 561 of the AAV8 VP1 protein.

[0075] In some embodiments, the wild-type AAV9 VP1 protein comprises or is composed of the amino acid sequence shown in SEQ ID NO: 1.

[0076] Wild-type AAV9 VP1 protein (SEQ ID NO: 1) MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL The modified AAV capsid protein provided herein can be any suitable AAV serotype. In some embodiments, the modified AAV capsid protein provided herein is selected from the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV843, AAVbb2, AAVcyS, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64, AAVhu37, AAV3B, AAVhu48, AAVhu43, AAVh... u44, AAVhu46, AAVhu19, AAVhu20, AAVhu23, AAVhu22, AAVhu24, AAVhu21, AAVhu27, AAVhu28, AAVhu29, AAVhu63, AAVhu64, AAVhu13, AAVhu56, AAVhu57, AAVhu49, AAVhu58, AAVhu34, AAVhu45, AAVhu47, AAVhu51, AAVhu52, AAVhu T41, AAVhu S17, AAVhu T88, AAVhu T71, AAVhu T70, AAVhu T40, AAVhu T32, AAVhu T17, AAVhu LG15, AAVhu9, AAVhu10, AAVhu11, AAVhu53, AAVhu55, AAVhu54, AAVhu7, AAVhu18, AAVhu15, AAVhu16, AAVhu25, AAVhu60, AAVch5, AAVhu3, AAVhu 1. AAVhu4, AAVhu2, AAVhu61, AAVrh62, AAVrh48, AAVrh54, AAVrh55, AAVcy2, AAVrh35, AAVrh37, AAVrh36, AAVcy6, AAVcy4, AAVcy3, AAVcy5, AAV rh13, AAVrh38, AAVhu66, AAVhu42, AAVhu67, AAVhu40, AAVhu41, AAVrh40, AAVrh2, AAVbb1, AAVhu17, AAVhu6, AAVrh25, AAVpi2, AAVpi3, AAVrh5 7. AAVrh50, AAVrh49, AAVhu39, AAVrh58, AAVrh61, AAVrh52, AAVrh53, AAVrh51, AAVhu14, AAVhu31, AAVhu32, AAVrh34, AAVrh33, AAVrh32, avian AAV ATCC VR-865, avian AAV strain DA-1 or bovine AAV.

[0077] Examples of AAV capsid gene sequences and protein sequences can be found in the GenBank database, see GenBank accession numbers: AF043303, AF028705, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC 001358, NC 001540, AF513851, AF513852, AY530579, AY631965, AY631966; AF063497, AF085716, AF513852, AY530579, AAS99264.1, AY243022, Y243015,AY530560,AY530600,AY530611,AY530628,AY530553,AY530606,AY530583,AY530555,AY530607,AY530580,AY530569,NC 006263, NC 005889, NC 001862, AY530609, AY530581, AY530563, AY530591, AY530562, AY530584, AY530622, AY530601, AY530586, AY243021, AY530570, AY530589, AY5305 95. AY530572, AY530588, AY530575, AY530565, AY530590, AY530602, AY530566, AY530587, AY530585, AY530564, AY530592, AY530623, AY530574, AY 530593, AY530560, AY530594, AY530573, AF513852, AY530624, AY530561, AY242997, AY530625, AY530567, AY530556, AY530578, AY530568, AY5306 18. AY243020, AY530579, AY530619, AY530596, AY530612, AY243000, AY530597, AY530620, AY242998, AY530598, AY242999, AY530599, AY243016, NC 001729, NC 001401, AY243018, NC001863, AY530608, AY243019, NC001829, AY530610, AY243017, AY243001, AY530613, AY243013, AY243002, AY5306 14. AY243003, AY695378, AY530558, AY530626, AY695376, AY695375, AY530605, AY 695374, AY530603, AY530627, AY695373, AY695372, AY530604, AY695371, AY5306 00, AY695370, AY530559, AY695377, AY243007, AY243023, AY186198, AY629583, NC 004828, AY530629, AY530576, AY243015, AY388617, AY530577, AY530582, AY530615, AY530621, AY530617, AY530557, AY530616 or AY530554.

[0078] In some embodiments, the capsid protein may be modified, chimeric, or synthetic. A chimeric capsid comprises portions of two or more capsid sequences. A synthetic capsid comprises a synthetic or artificially designed sequence. In some embodiments, the cap gene or capsid protein is derived from two or more AAV serotypes.

[0079] In some embodiments, the modified AAV capsid protein provided herein is derived from the wild-type or original capsid protein of AAV suitable for therapeutic use, such as, but not limited to, AAV1, AAV2, AAV3, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV10, AAVrh.74, AAVrh.10, and AAVhu.37. Exemplary amino acid sequences of the wild-type or original VP1 protein of the AAVs mentioned above are provided in the table below. In some embodiments, the modified AAV capsid protein provided herein comprises a variant of the amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 69-80, differing in that the variant contains at least one amino acid mutation at one or more positions specified herein. In some embodiments, the modified AAV capsid protein provided herein, except for the at least one amino acid mutation at the position specified herein, has at least 90%, 95%, or at least 98% sequence identity with SEQ ID NO: 1 and 69-80 outside the region of the at least one amino acid mutation.

[0080] In some embodiments, the modified AAV capsid protein provided herein has at least 90%, 95%, or at least 98% sequence identity with SEQ ID NO: 1 and 69-80.

[0081] In some implementations, the amino acid mutation is selected from insertion, substitution, deletion, or any combination thereof.

[0082] As used herein, the term "insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence. While insertions typically consist of the insertion of one or two amino acid residues, this application considers larger "insertion peptides," such as insertions of about three to about five or even up to about several dozen amino acid residues. The inserted residues can be naturally occurring or non-naturally occurring. One or more inserted residues can be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine ​​(Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine (Val). Insertions of one or more non-naturally occurring amino acid residues are also covered in the definition of insertion herein. "Non-naturally occurring amino acid residues" refer to residues other than those listed above that can covalently bind to adjacent amino acid residues in a polypeptide chain. Examples of non-naturally occurring amino acid residues include leucine, ornithine, valine, homoserine, and other amino acid residue analogs, such as those described in Ellman et al.'s *Enzymatic Methods*. Meth. Enzym(.)》 202:301-336 (1991). To generate such non-naturally occurring amino acid residues, the methods of Noren et al. (Science) 244:182 (1989) and Ellman et al., ibid., can be used. In short, these methods involve chemically activating repressive tRNA with non-naturally occurring amino acid residues, followed by in vitro transcription and translation of RNA.

[0083] Unless otherwise defined, the insertion site in the predetermined amino acid sequence is located between the carboxyl group (C-terminus) of the residue at the defined mutation position and the amino group (N-terminus) of the next residue after the defined mutation position. For example, when the insertion mutation is defined at position 441 corresponding to the sequence of SEQ ID NO: 1, it means that the inserted amino acid is inserted between the residue at position 441 and the residue at position 442 corresponding to SEQ ID NO: 1.

[0084] As used herein, the term “substitution” refers to replacing at least one existing amino acid residue in a predetermined amino acid sequence with another different “alternative” amino acid residue. The alternative residue may be naturally occurring or non-naturally occurring as disclosed above.

[0085] As used herein, the term “deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0086] In some embodiments, the antigenic epitope region for which the at least one amino acid mutation is to be introduced is located at a position selected from the following locations: 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715, wherein the numbers correspond to the wild-type AAV9 VP1 protein (SEQ ID NO: 1). In some embodiments, the antigenic epitope region is located at a position selected from the following locations: 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588, and 715, wherein the numbers correspond to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0087] In some embodiments, the amino acid mutation includes one or more mutations located at positions selected from the group consisting of: positions 509, 510, 512, 513, 514 or any combination thereof, wherein the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0088] In some embodiments, the amino acid mutation includes one or more mutations located at positions selected from the group consisting of positions 559, 557, or any combination thereof, wherein the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0089] In some embodiments, the amino acid mutation includes one or more mutations located at positions selected from the group consisting of: positions 509, 510, 512, 513, 514, 559, 557 or any combination thereof, wherein the number corresponds to the wild-type AAV9VP1 protein (SEQ ID NO: 1).

[0090] In some embodiments, the amino acid mutations include mutations at position 509, at position 510, and / or at position 559, wherein the numbers correspond to wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0091] In some embodiments, the amino acid mutation includes an insertion of at least one amino acid. In some embodiments, the amino acid mutation includes an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the amino acid mutation includes an insertion of 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, or 2 to 10 amino acid residues in length. In some embodiments, the insertion is of low antigenicity and does not substantially bind to serum neutralizing antibodies.

[0092] In some embodiments, the mutated antigenic epitope comprises the amino acid sequence shown in Table 1. In some embodiments, the modified AAV capsid protein provided herein comprises VP1, which comprises a mutated antigenic epitope comprising the amino acid sequence shown in Table 1, wherein each X, Y, or Z independently represents an amino acid residue each time it occurs; each l, m, or n independently represents an integer from 0 to 20 each time it occurs, provided that it is not simultaneously 0 when at least two of l, m, and n are present, and is not 0 when only one of l, m, and n is present. In other words, (X) l It can be empty (if l is 0), a single amino acid residue (if l is 1), or a peptide fragment with a specific amino acid sequence (if l is an integer greater than 1). A similar interpretation applies to (Y). m and (Z) n .

[0093] Table 1. Exemplary sequences of mutated antigenic epitopes Each X, Y, or Z independently represents an amino acid residue each time it appears; each l, m, or n independently represents an integer from 0 to 20 each time it appears, provided that it is not 0 when at least two of l, m, and n are present, and is not 0 when only one of l, m, and n is present.

[0094] Underlined residues indicate residues located at a specified position.

[0095] In some embodiments, the modified AAV capsid protein provided herein comprises VP1, said VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 81, 36 and 57-68, wherein one or more mutations are located at positions selected from the group consisting of 441, 442 and 444 or any combination thereof, wherein the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0096] In some embodiments, the modified AAV capsid protein provided herein comprises VP1, the VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 16-18 and 37-39, wherein one or more mutations are located at positions selected from the group consisting of positions 509 and 510 or any combination thereof, wherein the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0097] In some embodiments, the modified AAV capsid protein provided herein comprises VP1, said VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19-25 and 40-46, wherein said one or more mutations are located at positions selected from the group consisting of 512, 513 and 514 or any combination thereof, wherein the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0098] In some embodiments, the modified AAV capsid protein provided herein comprises VP1, the VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 12, 26 and 47, wherein one or more mutations are located at position 552, the number corresponding to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0099] In some embodiments, the modified AAV capsid protein provided herein comprises VP1, said VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, 27-33 and 48-54, wherein one or more mutations are located at positions selected from the group consisting of 556, 557 and 559 or any combination thereof, wherein the numbers correspond to wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0100] In some embodiments, the modified AAV capsid protein provided herein comprises VP1, the VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 14, 34 and 55, wherein one or more mutations are located at position 588, the number corresponding to wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0101] In some embodiments, the modified AAV capsid protein provided herein comprises VP1, the VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 15, 35 and 56, wherein one or more mutations are located at position 715, wherein the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1).

[0102] In some embodiments, the one or more mutations include at least one amino acid insertion. In some embodiments, the modified AAV capsid protein provided herein comprises VP1, wherein VP1 comprises a mutated antigenic epitope comprising MNPLI. D (X) l Q (Y) m Y L (Z) n YYLS (SEQ ID NO: 9), PGASS W (X) l A (Y) m LNGRN (SEQ ID NO: 10), SWAL N (X) l G (Y) m R (Z) n NSLMN (SEQ ID NO: 11), GTGRD N (X) l VDADK (SEQ ID NO: 12), DNVDAD (X) l K (Y) m V M (Z) n ITNEE (SEQ ID NO: 13), NHQSA Q (X) l AQAQT (SEQ ID NO: 14) or NVEFA V (X) l The amino acid sequence of NTEGV (SEQ ID NO: 15), wherein X, Y, or Z independently represents the insertion of at least one amino acid residue each time it appears, and each l, m, or n independently represents an integer from 0 to 20 each time it appears, provided that it is not simultaneously 0 when at least two of l, m, and n are present, and is not 0 when only one of l, m, and n is present. In some embodiments, (X) l It can be empty (if l is 0), a single amino acid residue (if l is 1), or a peptide fragment with a specific amino acid sequence (if l is an integer greater than 1). A similar interpretation applies to (Y). m and (Z) n In some embodiments, the mutated antigenic epitope contains only one insertion. In some embodiments, only one insertion is located at a position selected from the group consisting of: 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715, where the number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1). In some embodiments, the insertion is located at position 509. In some embodiments, the insertion is located at position 559. In some embodiments, the insertion is located at positions 512, 513, 556, 557, or 559.

[0103] In some embodiments, the insertion is a common protein tag (e.g., a protein purification tag) with a length not exceeding 20 amino acid residues. In some embodiments, the protein tag is recognized and commercially available, allowing for rapid identification of the resulting mutant virus. In some embodiments, the protein tag has uniform polarity and charge properties.

[0104] In some embodiments, an amino acid sequence or fragment thereof containing a Strep-II tag is inserted. In some embodiments, the Strep-II tag contains the amino acid sequence WSHPQFES (SEQ ID NO: 2). In some embodiments, an amino acid sequence or fragment thereof containing a Myc tag is inserted. In some embodiments, the Myc tag contains the amino acid sequence EQKLISEEDL (SEQ ID NO: 3).

[0105] In some embodiments, the insertion comprises a peptide having 2 amino acid residues, such as PQ (SEQ ID NO: 4). In some embodiments, the insertion comprises a peptide having 4 amino acid residues, such as HPQF (SEQ ID NO: 5). In some embodiments, the insertion comprises a peptide having 6 amino acid residues, such as SHPQFE (SEQ ID NO: 6). In some embodiments, the insertion comprises unrelated sequences, including GGGS (SEQ ID NO: 7) and GGGSGGGS (SEQ ID NO: 8).

[0106] In some embodiments, the amino acid mutation may be the substitution of a region in the AAV capsid protein with a different peptide sequence. In some embodiments, the different peptide sequence used for substitution comprises the amino acid sequence of any one of SEQ ID NO: 2-8 or a fragment thereof. In some embodiments, the different peptide sequence used for substitution is a protein tag (e.g., a protein purification tag) of no more than 20 amino acid residues in length. In some embodiments, the protein tag is recognized and commercially available, allowing for rapid identification of the resulting mutant virus. In some embodiments, the protein tag has uniform polarity and charge properties. In some embodiments, the protein tag is a Strep-II tag or a fragment thereof. In some embodiments, the Strep-II tag comprises the amino acid sequence of WSHPQFES (SEQ ID NO: 2). In some embodiments, the protein tag is a Myc tag or a fragment thereof. In some embodiments, the Myc tag comprises the amino acid sequence of EQKLISEEDL (SEQ ID NO: 3).

[0107] In some embodiments, the amino acid mutation comprises or includes the insertion of a Strep-II tag (e.g., an amino acid sequence comprising SEQ ID NO: 2) at positions selected from: wild-type AAV9 VP1 protein SEQ ID NO: 1, positions 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715. In some embodiments, the amino acid mutation comprises or includes the insertion of a Strep-II tag (e.g., an amino acid sequence comprising SEQ ID NO: 1) at positions selected from: wild-type AAV9 VP1 protein SEQ ID NO: 1, positions 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588, and 715.

[0108] In some embodiments, the amino acid mutation comprises or includes the insertion of an amino acid sequence of any of SEQ ID NO: 2-9, located at position 509 or 559 of SEQ ID NO: 1 of the wild-type AAV9 VP1 protein.

[0109] In some embodiments, the amino acid mutation includes or replaces positions 506-513 of SEQ ID NO: 1 of the wild-type AAV9 VP1 protein with a Strep-II tag (e.g., containing the amino acid sequence of SEQ ID NO: 2).

[0110] c. Polynucleotides, expression vectors, cell and viral vectors On one hand, this disclosure provides a polynucleotide encoding the modified AAV capsid protein disclosed herein. Further, it provides an expression vector comprising the polynucleotide, and cells (in vivo or in culture) comprising the polynucleotide and / or the expression vector of this disclosure.

[0111] On the one hand, this disclosure provides an AAV capsid comprising the modified AAV capsid protein provided herein.

[0112] On one hand, this disclosure provides a viral vector comprising the AAV capsid provided herein. In some embodiments, the viral vector provided herein further comprises nucleic acid containing at least one terminal repeat sequence, wherein the nucleic acid is encapsulated by the AAV capsid provided herein.

[0113] As used herein, "viral vector" refers to a nucleic acid vector containing nucleic acid with a 5' viral terminal repeat (TR) and / or a 3' viral terminal repeat sequence. A viral vector may contain a pair of TRs or a single TR.

[0114] The term "terminal repeat sequence" or "TR" includes any viral terminal repeat sequence or synthetic sequence that forms a hairpin structure and mediates desired functions such as replication, viral packaging, integration, and / or proviral rescue. For example, 5' and 3' viral terminal repeat sequences may contain origins of replication and allow DNA synthesis to begin from one viral terminal repeat sequence and continue to another. Examples of viral terminal repeat sequences include, but are not limited to, inverted terminal repeat sequences (ITRs) (e.g., sequences included in adeno-associated virus (AAV), long terminal repeat sequences (LTRs) (e.g., sequences included in retroviruses), etc.

[0115] In some embodiments, the viral vector includes an AAV viral vector. In some embodiments, the viral vector of this disclosure contains at least one ITR of AAV. The ITR sequence of AAV is capable of guiding the replication and packaging of the viral vector genome.

[0116] AAV ITRs can originate from any AAV, including but not limited to AAV serotype 1 (AAV 1), AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, AAV 12, avian AAV, bovine AAV, canine AAV, equine AAV, and sheep AAV, as well as any other AAVs now known or to be discovered later. For more information, see, for example, BERNARD NF et al., *Virusology*, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers), and Gao et al., (2004) *Journal of Virology* 78:6381-6388. The nucleotide sequence of the AAV ITR region is known. See, for example, Kotin, RM (1994) Human Gene Therapy 5:793-801; Berns, KI “Parvoviridae and their Replication” Fundamental Virology, 2nd ed. (edited by BN Fields and DM Knipe).

[0117] AAV ITRs can be natural AAV ITRs, or alternatively, they can be modified from natural AAV ITRs, for example, through mutation, deletion, or insertion, as long as the modified ITR can still mediate the desired biological functions, such as replication, viral packaging, integration, etc. The 5' and 3' ITRs of the selected nucleotide sequence side-attached to the AAV vector do not need to be the same or derived from the same AAV serotype, as long as they function as intended, for example, allowing the target sequence to be excised and rescued from the recipient cell genome, and integrated into the recipient cell genome.

[0118] In some embodiments, the viral vector contains at least a portion of the AAV genome. In some embodiments, the viral vector lacks the REP and CAP genes within the AAV genome.

[0119] In some embodiments, the nucleic acid further comprises a payload nucleic acid operatively linked to at least one terminal repeat sequence. In some embodiments, the viral vector may contain one or more payload nucleic acids heterologous to the viral genome at the 5' end of the TR or ITR and / or at the 3' end of the TR or ITR or between the TR or ITR. In some embodiments, the payload nucleic acid replaces the REP and CAP genes originally present in the AAV genome.

[0120] In some implementations, the payload nucleic acid encodes a therapeutic protein or RNA.

[0121] The payload nucleic acid sequence delivered in the viral vector of this disclosure can be any target heterologous nucleic acid sequence. The target nucleic acid includes a nucleic acid encoding a polypeptide, which comprises a therapeutic (e.g., for medical or veterinary use) or immunogenic (e.g., for a vaccine) polypeptide or functional RNA.

[0122] Optionally, the payload nucleic acid encodes a secreted polypeptide (e.g., a polypeptide that is a secreted polypeptide in its natural state, or a polypeptide engineered to be secreted, for example, by operatively linking it to a secretion signal sequence, as is known in the art).

[0123] Alternatively, the payload nucleic acid provided herein may encode antisense nucleic acids, ribozymes (e.g., as described in U.S. Patent No. 5,877,022), RNAs that influence spliceosome-mediated / ram splicing, interfering RNAs (RNAi), including siRNAs, shRNAs, or miRNAs that mediate gene silencing, and other non-translated RNAs, such as guide RNAs. Exemplary nontranslated RNAs include RNAi targeting PCSK9 mRNA, RNAi targeting transthyretin mRNA, RNAi targeting multidrug resistance (MDR) mRNA (e.g., for treating and / or preventing tumors and / or for administration to the heart to prevent damage from chemotherapy), RNAi targeting myostatin mRNA (e.g., for Duchenne muscular dystrophy), RNAi targeting VEGF mRNA (e.g., for treating and / or preventing tumors), RNAi targeting phosphatase mRNA (e.g., for treating cardiovascular disease); RNAi targeting phosphatase inhibitory or dominant negative molecules, such as phosphatase S16E (e.g., for treating cardiovascular disease), RNAi targeting adenosine kinase (e.g., for epilepsy), and RNAi targeting pathogens and viruses (e.g., hepatitis B and / or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).

[0124] In some embodiments, the payload nucleic acid provided herein may contain a nucleic acid sequence that guides gene editing. For example, the nucleic acid may encode a guide RNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA) containing both a crRNA sequence and a tracrRNA sequence. In some embodiments, the payload nucleic acid provided herein may encode a nuclease. In some embodiments, the nuclease is a zinc finger nuclease, a homing endonuclease, TALEN (transcription activator-like effector nuclease), NgAgo (agronaute endonuclease), SGN (structure-guided endonuclease), RGN (RNA-guided nuclease), or a modified or truncated variant thereof. In some embodiments, the RNA-guided nuclease is a Cas9 nuclease, a Cas12(a) nuclease (Cpf1), a Cas12b nuclease, a Cas12c nuclease, a TrpB-like nuclease, a Cas13a nuclease (C2c2), a Cas13b nuclease, or a modified or truncated variant thereof. In some embodiments, the Cas9 nuclease is isolated from or derived from *Streptococcus pyogenes* (…). S. pyogenes ) or Staphylococcus aureus ( S. aureus ).

[0125] In some embodiments, the payload nucleic acid provided herein may encode an immunogenic peptide, for example, that can be used for vaccination. The immunogenic peptide may be any peptide suitable for evoking an immune response and / or protecting a subject from infection and / or disease, including but not limited to microbial, bacterial, protozoan, parasitic, fungal, and / or viral infections and diseases. Alternatively, the immunogenic peptide may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of cancer cells. The nucleic acid may encode any target immunogen known in the art, including but not limited to immunogens derived from human immunodeficiency virus (HIV), HPV, simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc.

[0126] AAV vectors can be constructed using methods known in the art. The general principles of rAAV vector construction are known in the art. See, for example, Carter, 1992, "Biotechnology: A Review (…)". Current Opinion in Biotechnology )》, 3:533-539; and Muzyczka, 1992, Current Issues in Microbiology and Immunology ( Curr. Top. Microbiol. Immunol(.)》, 158:97-129. For example, the payload nucleic acid can be directly inserted between the ITRs of the AAV genome where the Rep gene and / or Cap gene are missing. Other parts of the AAV genome can also be missing, as long as sufficient ITR portion is preserved to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patents 5,173,414 and 5,139,941; International Publications WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); Lebkowski et al. (1988), *Molec. Cell. Biol.*, 8:3988-3996; Vincent et al. (1990), *Vaccines*, 90 (Cold Spring Harbor Laboratory Press); Carter, BJ (1992), *Current Events in Biotechnology*, 3:533-539; Muzyczka, N. (1992), *Current Events in Microbiology and Immunology*, 158:97-129; Kotin, RM (1994), *Human Gene Therapy*. 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) Journal of Experimental Medicine 179:1867-1875. Alternatively, the AAV ITR can be excised from the viral genome or from an AAV vector containing the viral genome and fused to the 5' and 3' of the payload nucleic acid using standard ligation techniques.

[0127] In some embodiments, the AAV viral vector of this disclosure may also encompass AAV viral particles. As used herein, “viral particle” means a viral genome packaged within a viral capsid. The viral genome in a viral particle may be a modified viral genome, such that it may lack some natural viral sequences and / or may contain some sequences heterologous to the natural viral genome. AAV viral particles can be generated by introducing an AAV expression vector, along with other essential elements such as a plasmid encoding the AAV cap / rep gene and a helper gene provided by an adenovirus or herpesvirus, into a suitable host cell using known techniques, such as transfection (see, for example, MF Naso et al., BioDrugs, 31(4):317-334 (2017), which is incorporated herein by reference in its entirety). The AAV expression vector can be expressed in a host cell and packaged into a viral particle.

[0128] The payload nucleic acid may further include one or more regulatory sequences operatively linked to a nucleic acid encoding a therapeutic protein or RNA. As used herein, the term "operatively linked" means that the coding sequence is directly or indirectly linked or associated with one or more regulatory sequences in the exogenous nucleic acid in a manner that allows expression of the target protein from the coding sequence in the cell. The coding sequence, together with the regulatory sequence, may be referred to herein as an expression cassette. In some embodiments, the exogenous nucleic acid may be in the form of an expression vector. Examples of transcriptional regulatory elements include one or more promoters and / or enhancers, and optionally polyadenylated sequences and / or one or more introns inserted between exons of the protein-coding sequence.

[0129] As used herein, the term "regulatory sequence" refers to any nucleotide sequence that is essential or beneficial for the expression of a coding sequence. Regulatory sequences may include, but are not limited to, one or more promoters, enhancers, transcription terminators, polyadenylated sequences, internal ribosome entry sites, and / or one or more introns inserted between exons of a protein-coding sequence.

[0130] d. Methods for generating viral vectors This disclosure further provides a method for generating the viral vector provided herein.

[0131] In one embodiment, this disclosure provides a method for generating a viral vector, the method comprising providing a cell with: (a) a nucleic acid comprising at least one TR sequence (e.g., an AAV TR sequence), and (b) an AAV sequence sufficient to cause the nucleic acid template to replicate and encapsulate within an AAV capsid (e.g., an AAV rep sequence and an AAV cap sequence encoding the AAV capsid of this disclosure). Optionally, the nucleic acid further comprises at least one payload nucleic acid. In a particular embodiment, the nucleic acid comprises two AAVITR sequences located at the 5' and 3' of the payload nucleic acid (if present), although they do not need to be directly adjacent to the payload nucleic acid.

[0132] The method provides nucleic acid, along with AAV rep and cap sequences, under certain conditions to generate a viral vector containing nucleic acid packaged within an AAV capsid in cells. The method may further include a step of collecting the viral vector from the cells. The viral vector can be collected from a culture medium and / or by lysing cells.

[0133] In one embodiment, this disclosure provides a method for generating a viral vector that escapes pre-existing anti-AAV antibodies in host serum, the method comprising: a) introducing at least one amino acid mutation into an AAV capsid protein; b) measuring the binding of the mutated AAV capsid protein to serum antibodies in host serum; and c) selecting at least one amino acid mutation in the AAV capsid protein such that, compared to the unmutated corresponding AAV capsid protein, the at least one amino acid mutation reduces its binding affinity to serum antibodies.

[0134] In some embodiments, the anti-AAV antibody comprises an anti-AAV neutralizing antibody, and the method further comprises: d) generating AAV particles comprising the mutated AAV capsid protein selected in step (c); e) determining the infectivity of the AAV particles for suitable host cells in the presence of serum antibodies; and f) selecting at least one amino acid mutation in the AAV capsid protein that increases the infectivity of the AAV particles for host cells in the presence of serum antibodies compared to AAV particles comprising the corresponding unmutated AAV capsid protein.

[0135] In some implementations, step e) includes: (i) contacting the AAV particle from (d) with suitable host cells under conditions where infection and replication can occur; (ii) selecting AAV particles capable of completing at least one infection cycle and replicating to a titer similar to that of the control AAV particle; (iii) contacting the AAV particle selected in (ii) with anti-AAV neutralizing serum antibodies and suitable host cells under conditions where infection and replication can occur; and (iv) selecting AAV particles that have not been neutralized by the anti-AAV neutralizing serum antibodies from (iii). This integrated approach presents a platform technology that can be used to modify any AAV capsid. The application of this platform technology produces AAV antigenic variants derived from the original AAV capsid template without loss of transduction efficiency. Exemplary methods can be found in, for example, the following literature: Cecchini S et al. (2008) Gene Therapy 15(11):823-30; Ayuso E et al. (2010) Curr Gene Therapy 10(6):423-36; Kotin RM et al. (2011) Human Molecular Genetics 20(R1):R2-6; Wright JF et al. (2009) Human Gene Therapy 20(7):698-706.

[0136] III. Methods for delivering nucleic acid payloads to cells and subjects The viral vectors disclosed herein can be used to deliver nucleic acids into cells in vitro, ex vivo, and in vivo. Specifically, the viral vectors can be advantageously used to deliver or transfer nucleic acids into animals, including mammalian cells. Thus, in some embodiments, the nucleic acid (“loaded nucleic acid”) can be encapsulated with the modified capsid proteins of this disclosure.

[0137] Generally, the viral vectors of this disclosure can be used to deliver a payload nucleic acid encoding a polypeptide or functional RNA to treat and / or prevent any disease state that would benefit from the delivery of a therapeutic polypeptide or functional RNA. In some embodiments, the viral vectors of this disclosure can be used to deliver a payload nucleic acid encoding a polypeptide or functional RNA to treat and / or prevent diseases or conditions. Diseases or conditions include, but are not limited to, genetic diseases, hematologic disorders, infectious diseases, neurological disorders, autoimmune diseases, inflammatory diseases, metabolic diseases, muscular disorders, and diseases related to the pancreas, kidneys, liver, heart, or other specific tissues.

[0138] The viral vectors disclosed herein provide means for delivering payload nucleic acids into a wide range of cells, including dividing and non-dividing cells in vivo. The viral vectors can also be used to deliver target nucleic acids into cells in vitro, for example, to generate peptides in vitro or for use in ex vivo gene therapy. The viral vectors can also be used in methods for delivering nucleic acids to subjects in need, for example, to express immunogenic or therapeutic peptides or functional RNA. In this way, peptides or functional RNA can be generated in the subject. The subject may need the peptide because the subject lacks peptides. Furthermore, the implementation of the methods also has some beneficial effects because the generation of peptides or functional RNA in the subject can bring about certain benefits.

[0139] As used herein and in the broadest sense, the terms “administer,” “administering,” or “administration” refer to the administration or dispensing of a pharmaceutically active material to a subject in a pharmacologically compatible manner. The terms are intended to include “causing administration,” which means directly or indirectly causing, urging, encouraging, assisting, inducing, or guiding another party to administer the pharmaceutically active material to a subject. It should be noted that in some embodiments, the terms may also be used interchangeably with “deliver,” “delivery,” “delivering,” etc. As used herein, pharmaceutically active materials include mediators (i.e., inducer mediators and / or target mediators).

[0140] Exemplary modes of administration include oral, rectal, mucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, percutaneous, intrauterine (or intraovarian), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to skeletal muscle, diaphragm, and / or myocardium), intradermal, intrapleural, intracerebral, and intra-articular), local (e.g., administration to both skin and mucosal surfaces, including airway surfaces and percutaneous administration), intralymphatic, and direct tissue or organ injection (e.g., administration to the liver, skeletal muscle, myocardium, diaphragm, or brain). Administration may also be directed to tumors (e.g., in or near tumors or lymph nodes). In any given situation, the most appropriate route will depend on the nature and severity of the symptom being treated and / or prevented, and the nature of the specific carrier used.

[0141] The dosage of the viral vector and / or capsid administered to the subject depends on the administration method, the disease or condition to be treated and / or prevented, the individual subject's condition, the specific viral vector or capsid, and the nucleic acid to be delivered, and can be determined in a conventional manner. An exemplary dosage for achieving a therapeutic effect is at least about 10 mg / kg body weight. 5 10 6 10 710 8 10 9 10 10 10 11 10 12 10 13 10 14 10 15 One transduction unit, optionally approximately 10 per kilogram of body weight. 8 -10 13 The titer is measured in transduction units.

[0142] In certain implementations, more than one application (e.g., two, three, four or more applications) can be used to achieve the desired gene expression level over various time intervals (e.g., daily, weekly, monthly, yearly, etc.).

[0143] As used herein, the term "subject" includes both humans and non-human animals. Non-human animals include all vertebrates, such as mammals and non-mammals, including non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms "patient" or "subject" are used interchangeably herein. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate.

[0144] The viral vector provided in this paper is advantageous because it expands the pool of subjects eligible for AAV vector gene therapy. Specifically, the viral vector provided in this paper can be administered to subjects with relatively high levels of anti-AAV serum antibodies (e.g., anti-AAV neutralizing antibodies) who would otherwise be ineligible for AAV vector gene therapy.

[0145] In some implementations, the subject’s serum antibody titer against AAV (e.g., AAV9) does not exceed 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, or 500, and wherein the antibody titer is defined as the dilution factor of the subject’s serum required to produce threshold-specific binding to the AAV (e.g., AAV9) capsid protein.

[0146] Binding to the AAV capsid protein can optionally be measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, threshold-specific binding is determined based on antibody binding in the presence of a given amount of AAV capsid protein, wherein threshold-specific binding is considered to be an antibody binding level sufficient to indicate specific binding to the AAV capsid protein.

[0147] In some embodiments, the serum antibody titer against AAV9 in the subject does not exceed 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, or 500, and wherein the antibody titer is defined as the dilution factor of the subject's serum that gives 50% maximum neutralization of the AAV9 capsid protein. Neutralization of AAV9 may optionally be determined by reporter gene assay.

[0148] The term "neutralizing antibody titer" refers to a value reflecting the neutralizing efficiency of serum containing at least one neutralizing antibody. Serum neutralizing antibody titers can be analyzed by assessing the ability of serum antibodies to inhibit the transduction or infection of AAV virus particles into susceptible host cells, where the AAV virus particles may express reporter genes, such as β-galactosidase or luciferase, after transduction or infection. Neutralizing antibody titers can be determined by the serum dilution factor required to produce approximately 50% neutralization of AAV virus particles (optionally present in a given amount).

[0149] IV. Pharmaceutical Composition In another aspect, a pharmaceutical composition is provided, the pharmaceutical composition comprising the viral vector and / or capsid and / or capsid protein of the present disclosure in a pharmaceutically acceptable carrier, and optionally other agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc.

[0150] As used herein and in the broadest sense, the term "pharmaceutical composition" means a formulation comprising a therapeutically effective amount of a pharmaceutically active ingredient and optionally, pharmaceutically acceptable excipients and / or carriers. As used herein, the term "pharmaceuticalally acceptable" means suitable for normal pharmaceutical use, i.e., not causing serious side effects such as adverse events in subjects. As used herein, the term "therapeutically effective amount" or "effective amount" means a dose sufficient to make a patient's treatment effective compared to no treatment. As used herein, "effective amount" means an amount that produces the desired effect, such as an amount capable of treating or improving a disease or symptom, or otherwise capable of producing the desired therapeutic effect (e.g., inducing immune tolerance).

[0151] Pharmaceutical active ingredients typically require one or more pharmaceutically acceptable carriers or excipients to take a suitable form (i.e., pharmaceutical composition) and dosage form for effective administration to a subject. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered pharmaceutically active ingredient. In this document, the term "pharmaceutically acceptable excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of the pharmaceutically active ingredient.

[0152] In this document, applicable pharmaceutical compositions comprise sterile aqueous solutions or dispersions and sterile powders for immediate reconstitution of sterile injectable solutions or dispersions. In all cases, the compositions must be sterile and stable under the conditions of manufacture and storage, and must be protected against contamination by microorganisms such as bacteria and fungi.

[0153] Pharmaceutically acceptable carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For intravenous administration, suitable carriers may include, for example, physiological saline, antibacterial water, or phosphate-buffered saline (PBS). Proper flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. Prevention of microbial action can be achieved using various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride. Prolonged absorption of the internal composition can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0154] Pharmaceutical compositions may comprise, but are not limited to, solutions, emulsions, and formulations containing liposomes. These compositions may be generated from a variety of components, including but not limited to preformed liquids, self-emulsifying solids, and self-emulsifying semi-solids.

[0155] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of virions mixed with a pharmaceutically acceptable carrier and / or excipient, such as saline, phosphate-buffered saline, phosphates and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Exemplary amino acids, polymers, and sugars include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextran, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and ethylene glycol. In some embodiments, this formulation is stable at 4°C for at least six months.

[0156] In some cases, the unit dose of the pharmaceutical composition of this disclosure can be measured as pfu (plaque forming units). In some cases, the pfu per unit dose of the pharmaceutical composition of this disclosure containing modified AAV can be higher than 1 × 10⁻⁶. 8 pfu to approximately 1 × 1015 pfu.

[0157] The compositions provided herein may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphates, carbonates, acetates, or citrates) and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, citrates or acetates, amino acids and their salts), antioxidants (e.g., ascorbic acid, α-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solution stabilizers, and / or low-temperature / lyophilization stabilizers). Stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic pressure regulators (e.g., salt or sugar), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), defoamers (e.g., polydimethylsiloxane), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers and viscosity modifiers (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethyl cellulose), and solubilizers (e.g., glycerol, polyethylene glycol, ethanol).

[0158] The compositions according to the invention may contain pharmaceutically acceptable excipients. The compositions can be prepared using conventional pharmaceutical manufacturing and formulation techniques to obtain useful dosage forms. Techniques suitable for practicing the invention can be found in the following literature: *Handbook of Industrial Mixing: Science and Practice*, edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004, John Wiley & Sons, Inc.; and *Pharmaceutics: The Science of Dosage Form Design*, 2nd edition, edited by ME Auten, 2001, Churchill Livingstone. In one embodiment, the composition is present with a preservative in sterile saline solution for injection.

[0159] The following provides an example as a specific implementation of the methods and kits provided in this disclosure. It should be noted that the example is for illustrative purposes only and does not limit the scope of this disclosure.

[0160] Example Example 1. Design and construction of insertion mutations in AAV9 capsid protein The AAV capsid used to encapsulate recombinant genes is an icosahedral hollow structure with a diameter of approximately 26 nm. It can be imagined as an icosahedron composed of 20 triangles, with every five triangles forming a pentagon. There is a pentagonal rotation axis between each pair of opposite pentagons, a triple rotation axis between the centers of each pair of opposite triangles, and a double rotation axis between the midpoints of each pair of opposite sides. This capsid contains a total of 60 copies of the capsid proteins VP1, VP2, and VP3, with the ratio of these three subunits on each capsid being 5:5:50. These subunits are encoded by overlapping sequences within the same open reading frame. Neutralizing antibodies recognize antigenic epitopes on the surface of the viral capsid and bind to AAV. On one hand, antibody binding disrupts the recognition of AAV by cell surface receptors, inhibiting AAV infection of target cells. On the other hand, antibody binding allows AAV particles to be cleared by the body's immune system, further reducing the effectiveness of AAV gene therapy. In some cases, neutralization can even block the uncoating process after AAV enters the cell, hindering successful expression of the therapeutic gene.

[0161] The primary challenge in modifying the AAV capsid to evade neutralizing antibodies is identifying the major antigenic epitopes recognized by these antibodies. To determine key regions on the AAV capsid surface involved in serum antibody recognition, recombinant gene technology was used to introduce insert peptides at 122 different sites within the CAP9 gene sequence encoding the AAV9 capsid VP protein. Given that VP1, VP2, and VP3 proteins are all encoded by overlapping sequences within the same open reading frame, with VP3 contained within VP1 and VP2, and that VP3 is the major structure responsible for assembling the icosahedral capsid, insertion mutation sites were selected within the region encoding the VP3 protein. By introducing these mutations into the CAP gene, 122 different engineered AAV9 variants were obtained, each with a unique insert peptide at a specific site on the capsid surface. Based on the published AAV9 crystal structure (PDB #7MT0), the selected insertion sites effectively cover all salient structural features on the viral capsid surface, enabling the systematic disruption of all potential antigenic epitopes (in...). Figure 1 (Highlighted in red).

[0162] Another challenge in modifying the AAV capsid to evade neutralizing antibodies is employing appropriate mutations to disrupt the antigenic epitopes recognized by pre-existing antibodies. Theoretically, substitution or insertion within key antigenic epitopes could disrupt antibody recognition. However, insertion mutations are more suitable for systematic screening because they are simple to generate and have a significant impact on capsid structure. In the initial screening, the commonly used protein purification tag StrepII was chosen as the peptide sequence to be inserted at position 122. This choice was based on two considerations: first, StrepII-tag-related detection tools, including probes and antibodies, are well-known and commercially available, allowing for rapid identification of the resulting mutant virus; second, compared to other commonly used peptide protein tags, the StrepII-tag is shorter and has relatively uniform polarity and charge properties. Theoretically, its insertion is expected to have a less significant impact on the overall structure of the AAV capsid than other commonly used protein tags.

[0163] Example 2. Screening for mutations that escape serum antibody binding Due to the high diversity of immune response profiles in the human B-cell lineage, different individuals produce different anti-AAV antibodies upon exposure to the virus. To identify engineered AAV mutants capable of evading a wide range of neutralizing antibodies, a serum mixture was generated by mixing equal volumes of peripheral blood serum from four adult individuals. This mixture was used as a screening sample and tested for binding to 122 AAV9 mutants with insert peptides at different sites.

[0164] Several methods can be used to measure AAV neutralizing antibodies in serum. To balance detection sensitivity and throughput, enzyme-linked immunosorbent assay (ELISA) was used to detect the binding of AAV9 variants to human IgG antibodies in mixed serum. To establish appropriate ELISA reaction conditions, ELISA plates were first coated with different concentrations of wild-type AAV9, and then incubated with different concentrations of diluted mixed serum. HRP-labeled anti-human IgG secondary antibody was used for detection to evaluate human serum antibodies binding to wild-type AAV9. Figure 2 ).

[0165] The results showed that AAV9 concentrations between 5E9 and 2E10 vg / ml coated on the ELISA plate had minimal impact on ELISA readings, indicating that AAV9 in this concentration range effectively covered the bottom of the Nunc MaxiSorp ELISA plate used. Higher viral concentrations did not significantly increase antibody binding. Based on the results of this preliminary experiment, the AAV9 concentration (5E9 vg / ml) and serum dilution factor (50-fold) were determined for formal screening experiments. Under these conditions, AAV9 mutants capable of escaping neutralizing antibodies from mixed serum were screened.

[0166] In the screening experiments, ELISA plates were coated with wild-type AAV9 and 122 different AAV9 variants at a concentration of 5E9 vg / ml, and then incubated with a 50-fold diluted serum mixture. Binding of the pre-existing antibodies was detected using HRP-labeled anti-human IgG secondary antibodies. Screening results were... Figure 3 As shown in the image.

[0167] The results showed that, compared with the ELISA readings of wild-type AAV9, 21 of the 122 insertion sites resulted in a reduction of serum binding by more than 50%. This indicates that these 21 sites have a significant impact on the recognition of pre-existing antibodies from mixed serum. The insertion sites of the 21 clones and their ELISA signals are listed in Table 2.

[0168] Table 2. Insertion sites and ELISA signals of 21 clones The insertion site is named according to its position in the amino acid sequence of the VP1 protein.

[0169] Example 3. Binding test of selected AAV9 mutant individuals with serum samples from different populations. To further investigate how antibodies from different populations recognize selected AAV mutants, 48 ​​peripheral blood serum samples from adults in various countries, including East Asia, Africa, and Eastern Europe, were collected. These serum samples represent a broad population to some extent. Each of the 48 serum samples was individually tested for binding to an ELISA with 21 AAV9 variants inserted at the sites listed in Table 1. The impact of engineered modifications at these sites on the recognition ability of pre-existing antibodies in a representative population was evaluated by comparing the recognition of wild-type AAV9 by these serum samples under the same conditions.

[0170] The results showed that, compared with the 48 ELISA reads of wild-type AAV9 against 48 individual serum samples, the inserted peptides at 13 positions (including residues 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588, and 715 based on the amino acid sequence of the VP1 protein) exhibited significantly reduced binding to most of the 48 individual serum samples (Figure 4). Specifically, among the 48 serum dilutions, 34 samples had ELISA reads exceeding 0.9 for wild-type AAV binding, with a putative positive rate of 70.8%. Under the same conditions, only 3–16 samples achieved ELISA reads of 0.9 for these 13 AAV9 variants, exhibiting a positive rate ranging from 6.3% to 33.3%. In summary, compared with wild-type AAV9 binding data, over 50% of serum samples showed significantly reduced binding for AAV9 variants with insertions at each of the 13 selected sites. These 13 sites included residues 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588, and 715 based on the VP1 protein sequence. The positions of these 13 key residues are marked in red on the AAV9 crystal structure. Figure 5A Their positions were also marked in a VP3 subunit as representatives (). Figure 5B ).

[0171] Example 4. Transduction inhibition assay of AAV9 variants of escaping serum neutralizing antibodies The decreased ELISA signal demonstrates that AAV mutations can interfere with direct binding to pre-existing anti-AAV antibodies. Serum anti-AAV antibodies can be neutralizing or non-neutralizing. Neutralizing antibodies not only bind to the AAV capsid but also inhibit vector transduction. To test whether the AAV variants identified here can escape the neutralizing effect of serum antibodies, a transduction inhibition assay was performed to detect the extent to which serum antibodies inhibit AAV-mediated reporter gene expression.

[0172] For the transduction inhibition assay, an AAV variant with an ST insertion at residue 509 was used as a proof-of-concept. AAVs containing a reporter gene were generated for easy detection. In short, the nanoluciferase reporter gene was packaged with a capsid used in previous ELISA screenings, including the AAV9 wild-type (AAV9-wt) and the AAV9 mutant with a Strep-II tag insertion at residue 509 (509-ST). These new AAV constructs with the luciferase reporter gene were generated and purified in a manner similar to that used in ELISA screening. The serum mixture used in previous screenings ( Figure 3The serum mixture was serially diluted and pre-incubated with the AAV reporter vector, followed by seeding into 293T cells. After 72 hours of incubation for cell transduction, nanoluciferase gene expression was measured using the commercial NanoGlo luciferase kit. The transduction inhibition effect of the serum mixture was assessed by measuring the reduction in luciferase gene expression. Figure 6 ).

[0173] like Figure 6 As shown in the inset above, the presence of a 1:810 serum dilution reduced wild-type AAV9 transduction by over 90%, indicating a significant inhibition of transduction by pre-existing neutralizing antibodies in the serum sample. However, no inhibition was detected for 509-ST at the same serum concentration. In fact, even with pre-incubation using a 1:10 serum dilution, the 509-ST variant could still transduce target cells with over 90% efficiency, demonstrating significant escape from neutralizing antibodies. These results suggest that AAV variants selected from previous screenings can escape the neutralizing effect of serum antibodies.

[0174] Example 5. Different types of mutations at key sites allow neutralizing antibodies to escape. By screening AAV mutants with StrepII tag insertions, a group of key sites affecting serum neutralizing antibody recognition were identified. It is crucial to clarify whether the disruption of antibody recognition is due to the specific properties of the StrepII tag, or whether different mutations on these key AAV residues produce similar escape effects. To answer this question, the octapeptide WSHPQFES with the StrepII tag inserted at position 509 was replaced with various mutation strategies, including the 2-amino acid peptide sequence PQ (clone name 509-S2), the 4-amino acid peptide sequence HPQF (clone name 509-S4), and the 6-amino acid peptide sequence SHPQFE (clone name 509-S6), as well as completely unrelated sequences, including the Myc tag EQKLISEEDL (clone name 509-Myc), the tetrapeptide sequence GGGS (clone name 509-GS4), and the octapeptide sequence GGGSGGGS (clone name 509-GS8). Additionally, the original sequence on the AAV9 surface was replaced with different peptides of the same length. For site 509, the DKVMITNE sequence located at positions 506-513 of VP1 was replaced with the strep-II tag sequence WSHPQFES (clone named 509-RP8). Using these designated capsids, AAV variants containing the nanoluciferase reporter gene were generated. Serum inhibition of gene transduction was tested on different AAV9 variants and compared with inhibition of wild-type AAV9 in parallel experiments. Figure 7Consistent with previous experiments, the transduction efficiency of wild-type AAV9 was significantly inhibited by serum dilution. However, all insertions of varying lengths / sequences located at site 509, or substitutions near residue 509, escaped the neutralizing effect of serum antibodies. These results suggest that the site / location of the mutation, rather than the type of mutation, is crucial for disrupting serum antibody recognition.

[0175] Other key residues affecting neutralizing antibody recognition are expected to have similar properties. As a rapid test, the strepII tag of clone 559-ST was replaced with the Myc tag. This resulted in an AAV vector containing both capsids of the luciferase reporter gene. Consistent with the results at site 509, clone 559-Myc escaped transduction inhibition just as effectively as 559-ST. Figure 8 The results further demonstrate that the mutation site, rather than the mutation type, is key to the recognition of escape-neutralizing antibodies.

[0176] Due to the high conservation of capsid amino acid sequences and 3D structures, significant cross-reactivity with neutralizing antibodies has been reported among different AAV serotypes. To test whether mutations at corresponding positions on AAV serotypes other than AAV9 would have a similar escape effect against serum neutralizing antibodies, AAV6 mutants were constructed with Strep-II tags inserted at positions 512, 513, 556, 557, or 559 on the VP1 of the capsid protein. These selected positions correspond to the same numbered positions on the VP1 protein of AAV9, where mutations showed an escape effect against serum neutralizing antibodies in previous AAV9 mutant screenings. AAV6 and AAV9 have the same VP1 length and are therefore numbered the same. The transduction efficiency of these five mutants was tested in the presence of mixed human serum used in previous experiments. Figure 9-13 As shown, all these mutants successfully escaped the transduction inhibition effect of serum dilution compared to wild-type AAV6. These results suggest that the neutralizing antibody escape sites identified on AAV9 may have similar escape effects on other AAV serotypes.

Claims

1. A modified AAV capsid protein comprising at least one mutated antigenic epitope, said at least one mutated antigenic epitope comprising at least one amino acid mutation located at a position selected from the group consisting of: 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588 and 715; The number corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO: 1), and the mutation of at least one amino acid therein significantly reduces the binding of serum antibodies to the AAV capsid protein.

2. The modified AAV capsid protein according to claim 1, wherein the serum antibody comprises an anti-AAV antibody pre-existing in the host serum.

3. The modified AAV capsid protein according to claim 2, wherein the pre-existing anti-AAV antibody comprises an anti-AAV neutralizing antibody.

4. The modified AAV capsid protein according to claim 2 or 3, wherein the host is a human.

5. The modified AAV capsid protein according to any one of the preceding claims, wherein the amino acid mutation is selected from substitution, insertion, deletion or any combination thereof.

6. The modified AAV capsid protein according to claim 5, wherein the amino acid mutation comprises the insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues.

7. The modified AAV capsid protein according to claim 5 or 6, wherein the length of the insertion is 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, or 2 to 10 amino acid residues.

8. The modified AAV capsid protein according to any one of the preceding claims, wherein the at least one amino acid mutation comprises one or more mutations located at a position selected from the group consisting of: positions 509, 510, 512, 513, 514 or any combination thereof.

9. The modified AAV capsid protein according to any one of the preceding claims, wherein the at least one amino acid mutation comprises one or more mutations located at a position selected from positions 559, 557, or any combination thereof.

10. The modified AAV capsid protein according to any one of the preceding claims, wherein the at least one amino acid mutation comprises one or more mutations located at a position selected from the group consisting of: positions 509, 510, 512, 513, 514, 559, 557 or any combination thereof.

11. The modified AAV capsid protein according to any one of the preceding claims, wherein the at least one amino acid mutation includes a mutation at position 509, a mutation at position 510, and / or a mutation at position 559.

12. The modified AAV capsid protein according to any one of claims 8 to 11, wherein the one or more mutations comprise at least one amino acid insertion.

13. The modified AAV capsid protein according to any one of the preceding claims, wherein the AAV capsid protein has an AAV serotype selected from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV843, AAVbb2, AAVcyS, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64, AAVhu37, AAV3B, AAVhu48, AAVh u43, AAVhu44, AAVhu46, AAVhu19, AAVhu20, AAVhu23, AAVhu22, AAVhu24, AAVhu21, AAVhu27, AAVhu28, AAVhu29, AAVh u63, AAVhu64, AAVhu13, AAVhu56, AAVhu57, AAVhu49, AAVhu58, AAVhu34, AAVhu45, AAVhu47, AAVhu51, AAVhu52, AAVhu T41, AAVhu S17, AAVhu T88, AAVhu T71, AAVhu T70, AAVhu T40, AAVhu T32, AAVhu T17, AAVhu LG15, AAVhu9, AAVhu10, AAVhu11, AAVhu53, AAVhu55, AAVhu54, AAVhu7, AAVhu18, AAVhu15, AAVhu16, AAVhu25, AAVhu60, AAVch5, AAVhu3, AAVhu 1. AAVhu4, AAVhu2, AAVhu61, AAVrh62, AAVrh48, AAVrh54, AAVrh55, AAVcy2, AAVrh35, AAVrh37, AAVrh36, AAVcy6, AAVcy4, AAVcy3, AAVcy5, AAV rh13, AAVrh38, AAVhu66, AAVhu42, AAVhu67, AAVhu40, AAVhu41, AAVrh40, AAVrh2, AAVbb1, AAVhu17, AAVhu6, AAVrh25, AAVpi2, AAVpi3, AAVrh5 7. AAVrh50, AAVrh49, AAVhu39, AAVrh58, AAVrh61, AAVrh52, AAVrh53, AAVrh51, AAVhu14, AAVhu31, AAVhu32, AAVrh34, AAVrh33, AAVrh32, avian AAV ATCC VR-865, avian AAV strain DA-1 or bovine AAV.

14. The modified AAV capsid protein according to any one of the preceding claims, wherein the mutated antigenic epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9-68.

15. A polynucleotide encoding an AAV-modified capsid protein according to any one of claims 1 to 14.

16. An expression vector comprising the polynucleotide according to claim 15.

17. A cell comprising the polynucleotide of claim 15 or the expression vector of claim 16.

18. An AAV capsid comprising an AAV capsid protein modified according to any one of claims 1 to 14.

19. An AAV virus vector comprising the AAV capsid according to claim 18.

20. The viral vector of claim 19, further comprising a nucleic acid, the nucleic acid comprising at least one terminal repeat sequence, wherein the nucleic acid is encapsulated by the AAV capsid.

21. The viral vector of claim 20, wherein the nucleic acid further comprises a payload nucleic acid operatively linked to the at least one terminal repeat sequence.

22. The viral vector of claim 20, wherein the payload nucleic acid encodes a therapeutic protein or RNA.

23. A pharmaceutical composition comprising an AAV viral vector according to any one of claims 19 to 22 and a pharmaceutically acceptable vector.

24. A method for delivering a payload nucleic acid into a cell, the method comprising contacting the cell with a viral vector according to claim 21 or 22.

25. A method for delivering a payload nucleic acid to a subject, the method comprising administering to the subject an effective amount of a viral vector according to claim 21 or 22 or a pharmaceutical composition according to claim 23.

26. A method of treating a subject in need, the method comprising administering to the subject a therapeutically effective amount of the AAV viral vector according to claim 21 or 22 or the pharmaceutical composition according to claim 23.

27. The method of claim 25 or 26, wherein the subject's serum antibody titer against AAV9 does not exceed 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400 or 500.

28. A method for generating an AAV vector that produces neutralizing antibodies that escape from the host, the method comprising: a) inserting a short peptide into multiple sites of a VP3 protein to generate a library of modified AAV vectors; b) determining the binding of the modified AAV vectors to a sample containing mixed serum from different subjects; and c) identifying the modified AAV vectors with reduced binding to the serum sample.

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