Modified AAV Capsid Protein, and Uses in Neutralizing Antibody Evasion

Abstract

The present disclosure provides a modified AAV capsid protein with reduced binding of serum antibodies, an AAV capsid comprising such modified capsid protein, and an AAV vector comprising such AAV capsid. A method of using such AAV capsid is also provided.

Description

Technology Field

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

[0002] Recombinant AAVs possess unique advantages in gene therapy. For example, Zolgesma, one of the most commercially successful drugs developed by Novartis, is based on AAV. In fact, many gene therapy pipelines targeting genetic disorders of the nervous system or muscle tissue are based on AAV. However, the presence of neutralizing antibodies significantly limits the application of the therapy; consequently, the documentation for Zolgesma explicitly requires that the patient's serum antibody titer be less than 1:50. If AAV mutants capable of efficiently evading neutralizing antibodies are developed, the scope of application for related gene therapies will be greatly expanded.

[0003] However, efficiently overcoming the recognition of neutralizing antibodies against AAV has become a critical challenge. These antibodies rapidly recognize and bind to AAV vectors, preventing the effective delivery of therapeutic genes to target cells and potentially significantly reducing therapeutic efficacy.

[0004] There is still a need for methods to modify AAV mutants to efficiently evade recognition by multiple neutralizing antibodies during gene therapy.

[0005] In one embodiment, the present disclosure provides a modified AAV capsid protein comprising at least one mutated antigenic epitope comprising at least one amino acid mutation 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 numbering corresponds to the wild-type AAV9 VP1 protein (SEQ No. 1), and at least one amino acid mutation substantially reduces the binding of a serum antibody to the AAV capsid protein.

[0006] In one embodiment, the present disclosure provides a polynucleotide encoding a modified AAV capsid protein as disclosed herein. In one embodiment, the present disclosure provides an expression vector comprising a polynucleotide as disclosed herein. In one embodiment, the present disclosure provides a cell comprising a polynucleotide or an expression vector as disclosed herein.

[0007] In one embodiment, the present disclosure provides an AAV capsid comprising a modified AAV capsid protein as disclosed herein. In one embodiment, the present disclosure provides an AAV virus vector comprising an AAV capsid as disclosed herein.

[0008] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an AAV virus vector as disclosed herein and a pharmaceutically acceptable carrier.

[0009] In one embodiment, the present disclosure provides a method for delivering a cargo nucleic acid to a cell, comprising the step of bringing the cell into contact with a viral vector as disclosed herein.

[0010] In one embodiment, the present disclosure provides a method for delivering a cargo nucleic acid to a subject, comprising the step of administering to the subject an effective amount of a viral vector as disclosed herein or a pharmaceutical composition as disclosed herein.

[0011] In one embodiment, the present disclosure provides a method for treating a subject requiring treatment, comprising the step of administering to the subject a therapeutically effective amount of an AAV virus vector as disclosed herein or a pharmaceutical composition as disclosed herein.

[0012] In another aspect, the present disclosure provides a method for producing an AAV vector that evades neutralizing antibodies in a host, comprising: a) producing a library of modified AAV vectors by inserting short peptides into various sites within a VP3 protein; b) determining the binding of the modified AAV vectors to samples containing pooled serum of different subjects; and c) identifying modified AAV vectors with reduced binding to serum samples.

[0013] The foregoing summary is merely illustrative and is not intended to be limiting in any way. In addition to the exemplary aspects, embodiments, and features described above, additional aspects, embodiments, and features will become apparent by referring to the drawings and the following detailed description. Brief explanation of the drawing

[0014] Figure 1 shows the mapping of 122 selected insertion sites on the surface of an AAV9 capsid. This figure was created based on the disclosed AAV9 crystal structure (PDB #7MT0) using Pymol. Insertion sites are indicated in red. Figure 2 shows the results of serum dilution binding to ELISA plates coated with different concentrations of wild-type AAV9. Figure 3 shows the ELISA results of mixed serum dilutions binding to wild-type AAV9 and 122 AAV9 variants. Reads were sorted from highest to lowest, and wild-type AAV9 binding signals were labeled. In the screening, 20 mutants showed a reduction of more than 50% compared to wild-type AAV9. Figures 4a and 4b show wild-type AAV9 and 21 selected variants recognized by 48 individual serum samples at a 1:50 dilution. Figure 4a shows the ELISA results of binding of all serum antibodies to the AAV9 variant inserted at the indicated positions. Figure 4b shows the positive rate among 48 individual serum samples calculated for each AAV9 variant, where the ELISA cutoff is 0.9. Figures 5a and 5b show the mapping of 13 major residues that significantly reduce serum antibody binding upon insertion. Figure 5a shows the structure of the entire AAV9 capsid, and Figure 5b shows one subunit of the AAV9 capsid. These figures were constructed using Pymol based on the published AAV9 crystal structure (PDB #7MT0). Major residues are indicated by arrows. Figure 6 shows the inhibition of transduction of serum samples for wild-type AAV9 (AAV9-wt) and an AAV9 variant (509-ST) with a Strep-II tag inserted into 509 of VP1. Figure 7 shows the inhibition of transduction of serum samples for wild-type AAV9 (AAV9-wt) and AAV9 variants with different types of mutations at position 509 of VP1. 509-S2: 2-amino acid peptide insertion, 509-S4: 4-amino acid peptide insertion, 509-S6: 6-amino acid peptide insertion, 509-Myc: Myc-tag insertion, 509-GS4: GGGS insertion, 509-GS8: GGGSGGGS insertion, 509-RP8: Replacement of the original sequences at positions 506 to 513 of VP1 with the strep-II tag sequence WSHPQFES. Figure 8 shows the repression of transduction in serum samples for wild-type AAV9 (AAV9-wt) and AAV9 variants with different types of mutations at position 559 of VP1. 559-ST: Strep-II tag insertion at position 559 of VP1, 559-Myc: Myc-tag insertion at position 559 of VP1. Figure 9 shows the inhibition of transduction of serum samples for wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 512 of VP1. AAV6-ST512: Strep-II tag insertion at position 512 of VP1. Figure 10 shows the inhibition of transduction of serum samples for wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 513 of VP1. AAV6-ST513: Strep-II tag insertion at position 513 of VP1. Figure 11 shows the inhibition of transduction of serum samples for wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 556 of VP1. AAV6-ST556: Strep-II tag insertion at position 556 of VP1. Figure 12 shows the inhibition of transduction of serum samples for wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 557 of VP1. AAV6-ST557: Strep-II tag insertion at position 557 of VP1. Figure 13 shows the inhibition of transduction of serum samples for wild-type AAV6 (AAV6-WT) and an AAV6 variant with an insertion at position 559 of VP1. AAV6-ST559: Strep-II tag insertion at position 559 of VP1. Specific details for implementing the invention

[0015] In the following detailed description, reference is made to the drawings presented above, which are intended to explain the various embodiments provided herein and constitute part of the description of the embodiments. Unless otherwise indicated or suggested by the context, similar symbols in the drawings generally represent similar components in the description.

[0016] All descriptions provided herein are intended only to illustrate various embodiments of the invention provided herein and are not intended to be limiting. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of this disclosure, and such equivalent embodiments are understood to be incorporated herein.

[0017] It should be understood that, for clarity, certain features of the disclosure described in the context of separate embodiments may be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosure described in the context of a single embodiment may be provided individually or in any suitable sub-combination. For example, if one specific embodiment disclosed herein includes components A, B, and C, it should be understood that the disclosure is intended to include embodiments containing A, B, or C individually, or any combination of A, B, or C.

[0018] All references cited in this disclosure, including patent applications, registered patents, published papers or other publications, are incorporated by reference in their entirety to provide a methodology that may be used in connection with the description provided herein. Where any term presented in one or more publications is similar or identical to a term explicitly defined in this disclosure, the definition of the term explicitly provided in this disclosure shall prevail in all respects.

[0019] All technical and scientific terms used in this disclosure are to be deemed to have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains, unless otherwise explicitly defined.

[0020] I. definition

[0021] Before providing a detailed description of the invention in this disclosure, the following matters are specified and defined.

[0022] As used herein, that is, throughout the entire disclosure, the singular form is interpreted to mean "one or more" or "at least one" unless otherwise specified. For example, "gene" may mean one gene or more than one gene.

[0023] As used herein, unless otherwise explicitly indicated, all numerical ranges described herein may include each numerical value and each sub-range within such range.

[0024] As used herein, the terms “about,” “approximately,” “roughly,” or similar terms refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% with respect to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, when the terms “about” or “roughly” are used before a number, they indicate a value that is added to or subtracted from the value by a range of 15%, 10%, 5%, or 1%.

[0025] As used herein, the term “polypeptide” is used interchangeably with “peptide,” “protein,” or similar terms and is used to refer to a polymer of amino acid residues or an assembly of polymers of multiple amino acid residues. This term applies to both natural amino acid polymers and non-natural amino acid polymers, and is interpreted to include amino acid polymers in which one or more amino acid residues are artificial or synthetic chemical mimics of the corresponding natural amino acids. The term “protein” generally refers to large polypeptides. The term “peptide” generally refers to small polypeptides. A polypeptide sequence is generally described with the left end of the polypeptide sequence being the amino-terminus (N-terminus) and the right end of the polypeptide sequence being the carboxyl-terminus (C-terminus).

[0026] As used herein, the term “nucleic acid” refers to oligonucleotides or polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and further includes single-stranded (sense or antisense) and double-stranded polynucleotides as equivalents, derivatives, variants, and analogs of RNA or DNA prepared from nucleotide analogs. Since the term “nucleic acid” does not refer to or imply polynucleotide chains of a specific length, nucleotides, polynucleotides, and oligonucleotides are also understood to be included in this definition.

[0027] As used herein, terms such as "comprise," "include," "include," "contain," "contain," "contain," "have," "have," and "having" are synonyms and are used in an inclusive and open manner, without excluding additional elements, features, stages, actions, operations, etc.

[0028] As used herein, the term "or" is used in an inclusive sense (not an exclusive sense), and, for example, when used to link a list of elements, the term "or" means one, some, or all of the elements in the list.

[0029] As used herein, the expression “at least one” means one or more, namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. Expressions referring to “at least one” in a list of items are interpreted to refer to all combinations of said item, including a single member. For example, “at least one of A, B, or C” is intended to include A, B, C, A and B, A and C, B and C, and A, B, and C. Conjunction expressions such as “at least one of X, Y, and Z” are understood to convey that an item, term, etc., may be at least one of X, Y, or Z in the context of general use, unless otherwise specifically stated. Accordingly, such conjunction expressions are not generally intended to imply that a particular embodiment requires at least one of X, at least one of Y, and at least one of Z to exist, respectively.

[0030] As used herein, “reduce,” “reduce,” “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.

[0031] The “sequence identity percentage (%)” for an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in the reference sequence after aligning the sequences and introducing gaps if necessary to achieve maximum agreement. Alignment to determine the percentage of amino acid (or nucleic acid) sequence identity can be performed using publicly available tools such as, for example, BLASTN, BLASTp (available on the National Center for Biotechnology Information (NCBI) website, also see [Altschul SF et al, J. Mol. Biol., 215:403-410 (1990)]; [Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)]), ClustalW2 (available on the European Bioinformatics Institute website, also see [Higgins DG et al, Methods in Enzymology, 266:383-402 (1996)]; [Larkin MA et al, Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)]), and ALIGN or Megalign (DNASTAR) software. A person skilled in the art may use the default parameters provided by the tool or customize the parameters to be suitable for alignment by, for example, selecting a suitable algorithm. In certain embodiments, non-identical residue positions may differ due to conservative amino acid substitutions.

[0032] As used herein, “homologue sequence” and “homologous sequence” are used interchangeably and refer to a polynucleotide sequence (or its complementary strand) or amino acid sequence having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optionally aligned.

[0033] "Conservative amino acid substitution" is the substitution of an amino acid residue by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitution does not substantially alter the functional properties of a protein. If two or more amino acid sequences differ from each other due to conservative substitution, the percentage or degree of similarity may be upscaled to compensate for the conservative nature of the substitution. Means for performing such adjustment are well known to those skilled in the art. For example, refer to the literature incorporated herein by reference [Pearson (1994) Methods Mol. Biol. 24: 307-331].

[0034] As used herein, “one embodiment,” “one embodiment,” “specific embodiment,” “related embodiment,” “specific embodiment,” “additional embodiment,” or “additional embodiment,” or any combination thereof, should be understood to mean that a specific feature, structure, or characteristic described in relation to that specific embodiment is included in at least one embodiment of this disclosure. Accordingly, the presence or appearance of the foregoing phrases at various locations throughout this disclosure does not necessarily refer to the same embodiment. Furthermore, a specific feature, structure, or characteristic may be combined in any suitable manner in one or more embodiments.

[0035] Conditional language used herein, such as “may,” “may,” “may,” “for example,” etc., is intended to convey that, generally, certain embodiments include certain features, elements, and / or steps but not others, unless explicitly stated otherwise or understood otherwise in the context in which they are used. Accordingly, such conditional language does not imply that features, elements, and / or steps are in any way essential to one or more embodiments.

[0036] II. Modified adeno-associated virus (AAV) capsid protein

[0037] The present disclosure provides a modified AAV capsid protein in which binding to serum antibodies, particularly anti-AAV antibodies or anti-AAV neutralizing antibodies, is substantially reduced. The present disclosure also provides an AAV viral vector comprising the modified AAV capsid protein provided herein, in which binding to serum antibodies, particularly anti-AAV antibodies or anti-AAV neutralizing antibodies, is substantially reduced. The AAV viral vector provided herein is particularly useful, for example, for delivering or expressing cargo nucleic acids to a subject for therapeutic purposes.

[0038] In one aspect, the present disclosure provides a modified AAV capsid protein comprising at least one mutated antigenic epitope. In certain embodiments, the mutated antigenic epitope comprises at least one amino acid mutation (e.g., insertion, substitution, deletion, or any combination thereof). In certain 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 numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1). The AAV capsid protein can be any suitable serotype, and the corresponding location can be identified by aligning 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.

[0039] a. Advantages of the viral vector provided herein

[0040] The AAV virus vector provided herein is particularly useful and advantageous compared to other virus vectors in at least the following aspects.

[0041] The recombinant AAV or AAV viral vectors provided to this institution possess unique advantages in gene therapy. First, AAV is a safe vector choice as it is non-pathogenic to humans. Second, extensive research indicates that AAV genes are generally not integrated into the genome, significantly reducing potential risks associated with treatment. Furthermore, AAV can infect various cell types, including dividing and non-dividing cells, thereby expanding the scope of gene therapy. Additionally, the existence of numerous natural AAV serotypes facilitates the selection or design of gene vectors, allowing scientists to choose the most suitable serotype based on the type and location of the disease. Finally, compared to other viral vectors, AAV can induce a relatively weak immune response, reducing the likelihood of side effects and complications. Due to these unique characteristics, AAV is a widely used delivery tool in gene therapy.

[0042] The recombinant AAV or AAV virus vector provided herein is advantageous for overcoming existing immunity to AAV, which provides an important solution to a long-standing problem.

[0043] Because AAVs are widely present in nature, a key challenge in AAV gene therapy is the potential presence of neutralizing antibodies in the patient's body. These antibodies rapidly recognize and bind to AAV vectors, hindering the vector's effective delivery of cargo nucleic acids to target cells and consequently significantly reducing the efficacy of the treatment. This situation occurs in patients who have previously been exposed to the virus or already possess these antibodies due to a natural immune response. It is estimated that 40 to 70 percent of the population possesses pre-existing neutralizing antibodies against AAV in their serum. In these patients, even if an AAV vector introduces the gene of interest into the body, the gene cannot reach and exert its effect on target cells. The presence of neutralizing antibodies not only introduces unpredictable variables for potential gene therapy recipients but also severely impedes the widespread clinical application of AAV therapy. Therefore, before administering AAV treatment, physicians must perform detailed serological tests to determine the presence of neutralizing antibodies that may affect therapeutic efficacy and adjust treatment strategies accordingly.

[0044] The efficiency of neutralizing antibody recognition plays a crucial and decisive role in the implementation of AAV gene therapy. Therefore, reducing the recognition efficiency of AAV by neutralizing antibodies is clinically highly significant. If this recognition can be effectively reduced, more AAV vectors can evade attack by the immune system, allowing more therapeutic genes to successfully enter target cells, which will significantly enhance the efficacy of gene therapy. This not only helps existing patients achieve better treatment outcomes but also expands the scope of AAV gene therapy, enabling more patients to benefit from this innovative treatment. Consequently, effectively overcoming the recognition of neutralizing antibodies against AAV has been one of the biggest challenges in AAV gene therapy.

[0045] To address this problem, researchers have conducted a series of genetic engineering studies to introduce mutations into AAVs to enable them to evade recognition by neutralizing antibodies. These types of AAV mutants are receiving significant attention in the field of gene therapy because they can change the current treatment paradigm and offer new hope to patients who are unable to benefit from gene therapy due to the presence of neutralizing antibodies. While some early studies have made progress, most existing research is based on binding information between a single monoclonal antibody and AAV. This approach differs significantly from real-world scenarios: due to the highly diverse repertoire of immune responses in human B cell lineages, exposure to AAV can generate various anti-AAV polyclonal antibodies. Since different antibodies can recognize different regions on the AAV surface, strategies that modify antigenic epitopes to suit a single anti-AAV antibody are ineffective in achieving effective immune evasion. Due to the complexity of serum neutralizing antibodies, no AAV mutant capable of efficiently avoiding recognition by multiple neutralizing antibodies has been developed to date.

[0046] The modified AAV capsid protein provided herein addresses these problems. Generally, 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, which can reduce the recognition of existing anti-AAV antibodies in the host serum, e.g., human serum. In particular, the antigenic epitope regions provided in this disclosure are unique and effective because the modified capsid regions of this disclosure were discovered using human serum samples containing various natural neutralizing antibodies. The modification of the AAV capsid provided herein can effectively reduce the recognition of many natural neutralizing antibodies in human serum.

[0047] Using genetic engineering techniques, the inventors introduced peptide insertions into 122 different sites within the CAP9 gene sequence encoding the VP protein of the AAV9 capsid; according to crystal structures, these 122 insertion sites can effectively encompass all major structural features of the viral capsid surface. By introducing these mutations into the CAP9 gene, 122 distinct AAV9 variants were obtained, each having a unique peptide inserted into a specific site on the capsid surface. By comparing the recognition efficiency of existing antibodies in mixed serum from multiple adults against these AAV9 variants, the inventors identified 21 critical regions on the AAV9 capsid surface, where existing antibody binding was reduced by more than 50% compared to wild-type AAV9. Further analysis showed that mutations at 13 of the selected 21 sites could effectively evade recognition by existing antibodies from most individual serum samples tested. Structural changes at these key locations, caused by insertion, substitution, or deletion, can protect the transduction activity of AAV from being neutralized by serum antibodies.

[0048] b. AAV capsid protein

[0049] The present disclosure provides a modified AAV capsid protein in which binding to serum antibodies, particularly anti-AAV antibodies or anti-AAV neutralizing antibodies, is substantially reduced.

[0050] As used herein, the term "adeno-associated virus" (AAV) refers to a small virus that infects humans and some other primate species. AAV is a type of non-pathogenic single-stranded DNA virus commonly found in humans and other mammals. AAV belongs to the family Parvoviridae and the genus Dependoparvovirus.

[0051] As used herein, the term "capsid" refers to the protein envelope of a virus that surrounds viral nucleic acid. Generally, a capsid contains several oligomeric structural subunits (i.e., protomers).

[0052] The AAV capsid, useful for enclosing genetic material such as the viral genome, is a hollow icosahedral structure with a diameter of approximately 26 nm. It can be visualized as a regular icosahedron composed of 20 triangles, forming one pentagon for every five triangles. There are 5 rotation axes between each pair of opposite pentagons, 3 rotation axes between each pair of opposite triangle centers, and 2 rotation axes between each pair of opposite edge centers. The AAV capsid contains a total of 60 copies of capsid proteins VP1, VP2, and VP3, with the ratio of these three subunits in each capsid being 5:5:50. These subunits are encoded by overlapping sequences within the same open reading frame. Neutralizing antibodies recognize the antigenic epitope regions on the surface of the viral capsid and bind to the AAV.

[0053] In certain embodiments, the AAV capsid protein provided herein is modified so that binding to serum antibodies, e.g., without limitation, anti-AAV antibodies, or more specifically, anti-AAV neutralizing antibodies, is substantially reduced.

[0054] As used herein, a modified AAV capsid protein with “substantially reduced binding to serum antibodies” is interpreted to include a modified AAV capsid protein with binding to serum antibodies that is at least about 30% less, at least about 40% less, at least about 50% less, at least about 70% less, at least about 80% less, or at least about 90% less than the unmodified or corresponding wild-type AAV capsid protein under equivalent assay conditions.

[0055] In certain embodiments, the modified AAV capsid protein provided herein has a binding to a serum antibody in a suitable detection assay compared to the unmodified or corresponding wild-type AAV capsid protein under an equivalent assay setup of 20% or less, 15% or less, 10% or less, 8% or less, 5% or less, 2% or less, or 1% or less.

[0056] In some embodiments, the serum antibody comprises an anti-AAV antibody. The anti-AAV antibody may specifically bind to the AAV capsid. Additionally, in some embodiments, the serum antibody may bind to an antigenic epitope within the AAV capsid protein provided herein. In some embodiments, the serum antibody is an existing anti-AAV antibody present in the host's serum and may bind to an antigenic epitope within 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 may be a human, a dog, a cat, or a horse. In certain embodiments, the host is a human.

[0057] In some embodiments, the serum antibody comprises an anti-AAV neutralizing antibody. As used herein, the term "anti-AAV neutralizing antibody" refers to an antibody capable of effectively inhibiting AAV virus particles from infecting susceptible host cells (e.g., target cells for gene therapy drug delivery).

[0058] Various methods are suitable for evaluating how well the modified AAV capsid protein provided herein binds to serum antibodies. For example, this binding can be measured by exposing AAV virus particles containing the modified capsid protein to a serum sample containing serum antibodies. To assess the degree of reduced binding, a comparative test can be performed in parallel using control AAV virus particles presenting an equivalent but modified capsid protein. This parallel test provides a comparison between the antibody binding levels of the modified capsid protein and the original capsid protein.

[0059] As used herein, the term "capsid protein" refers to the structural proteins of the viral capsid. The AAV capsid contains three capsid proteins, including viral protein 1 (VP1), VP2, and VP3, which are assembled into an icosahedral capsid. In the AAV genome, VP1, VP2, and VP3 are all encoded by overlapping sequences within a single open reading frame. The entire sequence of VP3 is contained within VP2, and the entirety of VP2 is contained within VP1 (VP1u), which has a unique N-terminal domain. To date, only a common VP3 region is observed in the capsid structures of all AAV serotypes determined by cryo-electron microscopy (cryo-EM) and image reconstruction (cryo-reconstruction) or X-ray crystallography.

[0060] In certain embodiments, the modified AAV capsid protein provided herein comprises at least one mutated antigenic epitope comprising at least one amino acid mutation.

[0061] As used herein, the term "antigenic epitope" refers to a part of the AAV capsid protein that can be recognized and bound by antibodies (e.g., neutralizing antibodies). The antigenic epitope region can be linear or stereotyped (i.e., containing spaced amino acid residues). Detailed analysis of the AAV capsid protein has revealed a conserved structural motif characterized by an 8-strand β-barrel (strands βB through βI) and the associated α-helix (αA). Antigenic diversity among AAV serotypes arises primarily from structurally variable regions (VRs) located in the surface loops connecting these β-strands. These VRs play a pivotal role in shaping the unique topography of the capsid surface and result in serotype-specific variations in cell tropism, tissue transduction efficiency, and antigenic properties.

[0062] In a specific embodiment, at least one mutated antigenic epitope comprises at least one amino acid mutation 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, where the numbering corresponds to the wild-type AAV9 VP1 protein. In some embodiments, the antigenic epitope region is located at selected positions 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588 and 715, where the numbering corresponds to the wild-type AAV9 VP1 protein (Sequence No. 1).

[0063] As used herein, the expression “corresponds” with respect to an AAV capsid protein reflects that an amino acid residue of a reference capsid protein corresponds to a numbered amino acid residue of a standard AAV capsid protein (e.g., wild-type AAV9 VP1 protein) having specific homology with the reference capsid protein. The reference capsid protein and the standard capsid protein may be different AAV serotypes, and such correspondence can be obtained, for example, by sequence alignment.

[0064] 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 regard, mutations in corresponding residues in AAV serotypes other than AAV9 may have a similar evasion effect on transduction inhibition. Therefore, when listing the locations where mutations are introduced within the antigenic epitope region in this disclosure, only specific locations within a specific AAV9 VP1 protein (e.g., having the sequence of SEQ ID NO. 1) are used as references or examples, and this is not intended to specifically limit the mutation locations to the capsid protein of wild-type AAV9.

[0065] In other words, the amino acid mutation sites within the antigenic epitopes provided in the present disclosure (e.g., particularly the sites 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715 mentioned above) include not only specific sites within the AAV9 VP1 protein (having the amino acid sequence of SEQ ID NO. 1), but also corresponding sites within the capsid proteins of other AAV serotypes other than AAV9 VP1 that can be obtained, for example, by sequence alignment. Sequence alignment can be performed by aligning a selected AAV capsid sequence with a reference sequence, such as AAV9 or another AAV serotype, using any suitable computer program and technique such as Clustal W, CAP Sequence Assembly, MAP, MEME, and Vector NTI utilities.

[0066] 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. 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, and these are just a few examples.

[0067] In some embodiments, the wild-type AAV9 VP1 protein comprises or consists of the amino acid sequence presented in SEQ ID NO. 1.

[0068] Wild-type AAV9 VP1 protein (Sequence No. 1)

[0069]

[0070] The modified AAV capsid protein provided herein may be any suitable AAV serotype. In some embodiments, the modified AAV capsid protein provided herein is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV843, AAVbb2, AAVcyS, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64, AAVhu37, AAV3B, AAVhu48, AAVhu43, AAVhu44, 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, AAVhu1, AAVhu4, AAVhu2, AAVhu61, AAVrh62, AAVrh48, AAVrh54, AAVrh55, AAVcy2, AAVrh35, AAVrh37, AAVrh36, AAVcy6, AAVcy4, AAVcy3, AAVcy5, AAVrh13, AAVrh38, AAVhu66, AAVhu42, AAVhu67, AAVhu40, AAVhu41, AAVrh40, AAVrh2, AAVbb1, AAVhu17, AAVhu6, AAVrh25, AAVpi2, AAVpi3, AAVrh57, 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 AAV serotypes selected from bovine AAV.

[0071] Examples of AAV capsid gene sequences and protein sequences are available in the GenBank database; refer to the following 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, AY243015, 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, AY530595, AY530572, AY530588, AY530575, AY530565, AY530590, AY530602, AY530566, AY530587, AY530585, AY530564, AY530592, AY530623, AY530574, AY530593, AY530560, AY530594, AY530573, AF513852, AY530624, AY530561, AY242997, AY530625, AY530567, AY530556, AY530578, AY530568, AY530618, AY243020, AY530579, AY530619, AY530596, AY530612, AY243000, AY530597, AY530620, AY242998, AY530598, AY242999, AY530599, AY243016,NC 001729, NC 001401, AY243018, NC 001863, AY530608, AY243019, NC 001829, AY530610, AY243017, AY243001, AY530613, AY243013, AY243002, AY530614, AY243003, AY695378, AY530558, AY530626, AY695376, AY695375, AY530605, AY695374, AY530603, AY530627, AY695373, AY695372, AY530604, AY695371, AY530600, AY695370, AY530559, AY695377, AY243007, AY243023, AY186198, AY629583, NC 004828, AY530629, AY530576, AY243015, AY388617, AY530577, AY530582, AY530615, AY530621, AY530617, AY530557, AY530616 or AY530554.,

[0072] In some embodiments, the capsid protein may be modified, chimeric, or synthetic. A chimeric capsid comprises parts 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.

[0073] In some embodiments, the modified AAV capsid proteins provided herein are derived from wild-type or original capsid proteins of AAVs suitable for therapeutic use, and include, for example, without limitation, AAV1, AAV2, AAV3, AAV3B, AAV5, AAV6, AAV8, AAV9, AAV10, AAVrh.74, AAVrh.10, and AAVhu.37, etc. The foregoing Exemplary amino acid sequences of the wild-type or original VP1 protein of AAV are provided in the table below.

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] In some embodiments, the modified AAV capsid protein provided herein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 and 69 to 80, provided that the variant comprises at least one amino acid mutation at one or more position(s) specified herein. In some embodiments, except for at least one amino acid mutation at the position(s) specified herein, the modified AAV capsid protein provided herein has at least 90%, 95%, or at least 98% sequence identity with respect to SEQ ID NOs. 1 and 69 to 80 outside the region of at least one amino acid mutation.

[0084] In some embodiments, the modified AAV capsid protein provided herein has at least 90%, 95%, or at least 98% sequence identity with respect to SEQ ID NOs 1 and 69 to 80.

[0085] In some embodiments, amino acid mutations are selected from insertions, substitutions, deletions, or any combination thereof.

[0086] As used herein, the term “insertion” refers to the introduction of one or more amino acids into a predetermined amino acid sequence. An insertion generally consists of inserting one or two amino acid residues, but this invention considers larger “peptide insertions,” e.g., the insertion of about 3 to about 5 or even up to about tens of amino acid residues. The inserted residue(s) may be natural or non-natural. The inserted residue or residues may be “natural amino acid residues” (i.e., encoded by the genetic code) and are 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). The insertion of one or more non-natural amino acid residues is also included in the definition of insertion herein. "Non-natural amino acid residue" refers to a residue other than the natural amino acid residues listed above, which may covalently bond to adjacent amino acid residue(s) in the polypeptide chain. Examples of non-natural amino acid residues include norleucine, ornithine, norvaline, homoserine, and [Ellman et al. Meth. Enzym Other amino acid residue analogs, such as those described in . 202:301-336 (1991)], are included. To generate these non-natural amino acid residues, the literature [Noren et al . Science 244:182 (1989)] and the procedures of the aforementioned Ellman et al. literature may be used ...is. Simply put, this procedure involves chemically activating repressor tRNA with non-natural amino acid residues and then the RNA in vitro It involves transcription and translation.

[0087] Unless otherwise defined, the insertion site in a predetermined amino acid sequence is between the carboxyl group (C-terminus) of the residue at the defined mutation position and the amino group (N-terminus) of the residue following the defined mutation position. For example, when the insertion mutation is defined as being at position 441 corresponding to the sequence of SEQ ID NO. 1, this means that the inserted amino acid(s) are inserted between the residue at position 441 and the residue at position 442 corresponding to SEQ ID NO. 1.

[0088] 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 “replacement” amino acid residue. The replacement residue(s) may be natural or non-natural as disclosed above.

[0089] As used herein, the term "deletion" refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.

[0090] In a specific embodiment, the antigenic epitope region into which at least one amino acid mutation is to be introduced is located at positions 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715, where the numbering corresponds to the wild-type AAV9 VP1 protein (Sequence No. 1). In some embodiments, the antigenic epitope region is located at selected positions 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588 and 715, where the numbering corresponds to the wild-type AAV9 VP1 protein (Sequence No. 1).

[0091] In some embodiments, the amino acid mutation comprises one or more mutations at a position selected from the group consisting of positions 509, 510, 512, 513, 514 or any combination thereof, where the numbering corresponds to the wild-type AAV9 VP1 protein (Sequence No. 1).

[0092] In some embodiments, the amino acid mutation comprises one or more mutations at a location selected from the group consisting of locations 559, 557, or any combination thereof, where the numbering corresponds to the wild-type AAV9 VP1 protein (Sequence No. 1).

[0093] In some embodiments, the amino acid mutation comprises one or more mutations at a position selected from the group consisting of positions 509, 510, 512, 513, 514, 559, 557 or any combination thereof, where the numbering corresponds to the wild-type AAV9 VP1 protein (Sequence No. 1).

[0094] In some embodiments, the amino acid mutations include a mutation at position 509, a mutation at position 510, and / or a mutation at position 559, where the numbering corresponds to the wild-type AAV9 VP1 protein (Sequence No. 1).

[0095] In some embodiments, the amino acid mutation comprises at least one amino acid insertion. In some embodiments, the amino acid mutation comprises 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 comprises an insertion of 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, or 2 to 10 amino acid residue lengths. In some embodiments, the insertion has low antigenicity that does not substantially bind to serum neutralizing antibodies.

[0096] In some embodiments, the mutated antigenic epitope comprises the amino acid sequence presented in Table 1. In certain embodiments, the modified AAV capsid protein provided herein comprises VP1 comprising a mutated antigenic epitope comprising the amino acid sequence presented in Table 1, wherein each X, Y, or Z independently represents an amino acid residue in each case; provided that each l, m, or n independently represents an integer from 0 to 20 in each case, where at least two of l, m, and n are present, they are not simultaneously 0, and where only one of l, m, and n is present, it is not 0. That is, (X) l can be null (when l is 0), a single amino acid residue (when l is 1), or a peptide fragment having a specific amino acid sequence (when l is an integer greater than 1). A similar interpretation is (Y) m and (Z) n It also applies to.

[0097]

[0098]

[0099] Each X, Y, and Z independently represents an amino acid residue in each case; each l, m, and n independently represents an integer from 0 to 20 in each case, provided that if at least two of l, m, and n exist, they are not simultaneously 0, and if only one of l, m, and n exists, it is not 0.

[0100] Underlined residues indicate residues at designated locations.

[0101] In a specific embodiment, the modified AAV capsid protein provided herein comprises VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 9, 81, 36, and 57 to 68, wherein one or more mutations are located at a position selected from the group consisting of positions 441, 442, and 444, or any combination thereof, wherein the numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1).

[0102] In a specific embodiment, the modified AAV capsid protein provided herein comprises a VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 16 to 18 and 37 to 39, wherein one or more mutations are located at a position selected from the group consisting of positions 509 and 510 or any combination thereof, wherein the numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1).

[0103] In a specific embodiment, the modified AAV capsid protein provided herein comprises a VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 19 to 25 and 40 to 46, wherein one or more mutations are located at a position selected from the group consisting of positions 512, 513 and 514, or any combination thereof, wherein the numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1).

[0104] In a specific embodiment, the modified AAV capsid protein provided herein comprises a VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 12, 26 and 47, wherein one or more mutations are at position 552, where the numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1).

[0105] In a specific embodiment, the modified AAV capsid protein provided herein comprises a VP1 comprising a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 13, 27 to 33, and 48 to 54, wherein one or more mutations are located at a position selected from the group consisting of positions 556, 557, and 559, or any combination thereof, wherein the numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1).

[0106] In a specific embodiment, the modified AAV capsid protein provided herein comprises VP1 containing a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 14, 34 and 55, wherein one or more mutations are at position 588, where the numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1).

[0107] In a specific embodiment, the modified AAV capsid protein provided herein comprises VP1 containing a mutated antigenic epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 15, 35 and 56, wherein one or more mutations are at position 715, where the numbering corresponds to the wild-type AAV9 VP1 protein (SEQ ID NO. 1).

[0108] In some embodiments, one or more mutations include at least one amino acid insertion. In certain embodiments, the modified AAV capsid protein provided herein is (Sequence No. 9), (Sequence No. 10), (Sequence No. 11), (Sequence No. 12), (Sequence No. 13), (Sequence No. 14), or VP1 comprising a mutated antigenic epitope comprising the amino acid sequence of (SEQ No. 15), wherein each X, Y, or Z independently represents the insertion of at least one amino acid residue in each case, and each l, m, or n independently represents an integer from 0 to 20 in each case, provided that if at least two of l, m, and n are present, they are not simultaneously 0, and if only one of l, m, and n is present, it is not 0. In certain embodiments, (X) l silver It may be null (if l is 0), a single amino acid residue (if l is 1), or a peptide fragment having a specific amino acid sequence (if l is an integer greater than 1). A similar interpretation is (Y) m and (Z) n Edo Applies. In certain embodiments, the mutated antigenic epitope comprises only a single insertion. In certain embodiments, the single 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 numbering corresponds to the wild-type AAV9 VP1 protein (SEQ No. 1). In certain embodiments, the insertion is at position 509. In certain embodiments, the insertion is at position 559. In certain embodiments, the insertion is at position 512, 513, 556, 557, or 559.

[0109] In some embodiments, the insert is a commonly used protein tag (e.g., a protein purification tag) having a length of 20 amino acid residues or less. In some embodiments, the protein tag is well-established and commercially available so that the generated mutant virus can be rapidly identified. In some embodiments, the protein tag has uniform polarity and charge characteristics.

[0110] In some embodiments, the insertion comprises the amino acid sequence of a Strep-II tag or a fragment thereof. In certain embodiments, the Strep-II tag comprises the amino acid sequence of WSHPQFES (SEQ No. 2). In some embodiments, the insertion comprises the amino acid sequence of a Myc tag or a fragment thereof. In certain embodiments, the Myc tag comprises the amino acid sequence of EQKLISEEDL (SEQ No. 3).

[0111] In a specific embodiment, the insertion comprises a peptide having two amino acid residues, e.g., PQ (SEQ No. 4). In a specific embodiment, the insertion comprises a peptide having four amino acid residues, e.g., HPQF (SEQ No. 5). In a specific embodiment, the insertion comprises a peptide having six amino acid residues, e.g., SHPQFE (SEQ No. 6). In a specific embodiment, the insertion comprises unrelated sequences including GGGS (SEQ No. 7) and GGGSGGGS (SEQ No. 8).

[0112] In some embodiments, the amino acid mutation may be a substitution of a region of the AAV capsid protein with a different peptide sequence. In some embodiments, the different peptide sequence for substitution comprises any one of the amino acid sequences of SEQ ID NOs 2 to 8 or a fragment thereof. In some embodiments, the different peptide sequence for substitution is a protein tag (e.g., a protein purification tag) having a length of 20 amino acid residues or less. In some embodiments, the protein tag is well-established and commercially available so that the generated mutant virus can be rapidly identified. In some embodiments, the protein tag has uniform polarity and charge characteristics. In certain embodiments, the protein tag is a Strep-II tag or a fragment thereof. In certain 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 certain embodiments, the Myc tag comprises the amino acid sequence of EQKLISEEDL (SEQ ID NO. 3).

[0113] In certain embodiments, the amino acid mutation comprises or corresponds to the insertion of a Strep-II tag, for example, containing the amino acid sequence of SEQ ID NO. 2, at selected positions 441, 442, 444, 507, 508, 509, 510, 511, 512, 513, 514, 515, 545, 552, 555, 556, 557, 559, 560, 588, and 715 of the wild-type AAV9 VP1 protein at selected positions. In certain embodiments, the amino acid mutation comprises or corresponds to the insertion of a Strep-II tag, for example, containing the amino acid sequence of SEQ ID NO. 2, at selected positions 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588 and 715 of the wild-type AAV9 VP1 protein SEQ ID NO. 1.

[0114] In certain embodiments, the amino acid mutation comprises or corresponds to the insertion of an amino acid sequence containing any one of SEQ ID NOs 2 to 9 at position 509 or 559 of wild-type AAV9 VP1 protein SEQ ID NO. 1.

[0115] In certain embodiments, the amino acid mutation comprises or corresponds to substituting positions 506 to 513 of wild-type AAV9 VP1 protein SEQ ID NO. 1 with a Strep-II tag, for example, a Strep-II tag comprising the amino acid sequence of SEQ ID NO. 2.

[0116] c. Polynucleotides, expression vectors, cells, and viral vectors

[0117] In one embodiment, the present disclosure provides a polynucleotide encoding the modified AAV capsid protein disclosed herein. Additionally, an expression vector comprising the polynucleotide, and a cell (in vivo or in culture medium) comprising the polynucleotide and / or the expression vector of the present disclosure are provided.

[0118] In one embodiment, the present disclosure provides an AAV capsid comprising a modified AAV capsid protein provided herein.

[0119] In one aspect, the present disclosure provides a viral vector comprising an AAV capsid provided herein. In a specific embodiment, the viral vector provided herein further comprises a nucleic acid comprising at least one terminal repeat sequence, wherein the nucleic acid is capsidized by the AAV capsid provided herein.

[0120] As used herein, "viral vector" refers to a nucleic acid vector comprising a nucleic acid having a 5' viral terminal repeat (TR) and / or a 3' viral terminal repeat sequence. A viral vector may comprise a pair of TRs or a single TR.

[0121] The terms “terminal repeat sequence” or “TR” include 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’ viral terminal repeat sequences and 3’ viral terminal repeat sequences may contain replication origins, and DNA synthesis may be initiated at one of the viral terminal repeat sequences and proceed to the other viral terminal repeat sequence. Examples of viral terminal repeat sequences include, but are not limited to, inversion terminal repeat sequences (ITRs) (e.g., those found in adeno-associated viruses (AAVs)) and long terminal repeat sequences (LTRs) (e.g., those found in retroviruses).

[0122] In certain embodiments, the viral vector comprises an AAV viral vector. In certain embodiments, the viral vector of the present disclosure comprises at least one ITR of AAV. The ITR sequence of AAV can induce replication and packaging of the viral vector genome.

[0123] The AAV ITR may be derived 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 all other AAVs currently known or to be discovered in the future. For further details, refer, for example, to the literature [BERNARD NF et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers)] and [Gao et al., (2004) J. Virol. 78:6381-6388]. The nucleotide sequence of the AAV ITR region is known. For example, see the literature [Kotin, RM (1994) Human Gene Therapy 5:793-801]; [Berns, KI "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (BN Fields and DM Knipe, eds.)].

[0124] AAV ITRs may be natural AAV ITRs or, alternatively, modified from natural AAV ITRs by mutation, deletion, or insertion, provided that the modified ITRs can still mediate desired biological functions such as replication, viral packaging, and integration. The 5' and 3' ITRs located on either side of a selected nucleotide sequence within the AAV vector do not necessarily have to be of the same AAV serotype or identical, provided that they function as intended, for example, enabling the excision and rescue of the sequence of interest and integration into the recipient cell genome.

[0125] In some embodiments, the viral vector comprises at least a portion of the AAV genome. In certain embodiments, the viral vector lacks the REP and CAP genes within the AAV genome.

[0126] In some embodiments, the nucleic acid further comprises a cargo nucleic acid operably linked to at least one terminal repeat sequence. In certain embodiments, the viral vector may comprise one or more cargo nucleic acids heterogeneous to the viral genome, at the 5' end of a TR or ITR and / or the 3' end of a TR or ITR, or between TRs or ITRs. In certain embodiments, the cargo nucleic acid replaces the REP and CAP genes originally present in the AAV genome.

[0127] In some embodiments, the cargo nucleic acid encodes a therapeutic protein or RNA.

[0128] The cargo nucleic acid sequence delivered within the viral vector of the present disclosure may be any heterogeneous nucleic acid sequence(s) of interest. The nucleic acid of interest comprises nucleic acids encoding polypeptides, which include therapeutic (e.g., medical or veterinary) or immunogenic (e.g., vaccine) polypeptides or functional RNAs.

[0129] Optionally, the cargo nucleic acid encodes a secreted polypeptide (e.g., a polypeptide that is a polypeptide secreted in its natural state, or a polypeptide engineered to be secreted through operable binding with a secretion signal sequence as known in the art).

[0130] Alternatively, the cargo nucleic acids provided herein may encode antisense nucleic acids, ribozymes (e.g., as described in U.S. Patent No. 5,877,022), RNA causing splicosome-mediated / ram-splicing, interfering RNAs (RNAi) mediating gene silencing including siRNA, shRNA, or miRNA, and other non-coding RNAs such as guide RNAs. Exemplary non-coding RNAs include RNAi for PCSK9 mRNA, RNAi for transthyretin mRNA, RNAi for multidrug resistance (MDR) mRNA (e.g. for tumor treatment and / or prevention, or for administration to the heart to prevent damage caused by chemotherapy), RNAi for myostatin mRNA (e.g. for Duchenne muscular dystrophy), RNAi for VEGF mRNA (e.g. for tumor treatment and / or prevention), and RNAi for phosphoramban mRNA (e.g. for the treatment of cardiovascular disease); These include phosphoramban inhibitors or dominant-negative molecules such as phosphoramban S 16E (e.g., for the treatment of cardiovascular disease), RNAi against adenosine kinases (e.g., for liver disease), and RNAi against pathogenic organisms and viruses (e.g., hepatitis B and / or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).

[0131] In some embodiments, the cargo nucleic acid provided herein may contain a nucleic acid sequence directing gene editing. For example, the nucleic acid may encode a guide RNA. In some embodiments, the guide RNA is a single guide RNA (sgRNA) comprising a crRNA sequence and a tracrRNA sequence. In some embodiments, the cargo nucleic acid provided herein may encode a nuclease. In some embodiments, the nuclease is a zinc finger nuclease, a homing endonuclease, TALEN (transcriptional activating factor-like effector nuclease), NgAgo (agonut endonuclease), SGN (structure-guided endonuclease), RGN (RNA-guided nuclease), or a variant or cleavage variant thereof. In some embodiments, the RNA-guided nuclease is Cas9 nuclease, Cas12(a) nuclease (Cpf1), Cas12b nuclease, Cas12c nuclease, TrpB-like nuclease, Cas13a nuclease (C2c2), Cas13b nuclease, or a modification or cleavage variant thereof. In some embodiments, the Cas9 nuclease is S. piogenes ( S. pyogenes ) Or S. Aureus ( S. aureus from ) It is separated or originates.

[0132] In some embodiments, the cargo nucleic acid provided herein may encode an immunogenic polypeptide useful, for example, for vaccination. The immunogenic polypeptide may be any polypeptide suitable for inducing an immune response and / or protecting a subject from infections and / or diseases, including but not limited to microorganisms, bacteria, protozoa, parasites, fungi, and / or viral infections and diseases. Alternatively, the immunogenic polypeptide 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 immunogen of interest known in the art, including but not limited to human immunodeficiency virus (HIV), HPV, monkey immunodeficiency virus (SIV), influenza virus, HIV or SIV gag protein, tumor antigen, cancer antigen, bacterial antigen, viral antigen, etc.

[0133] AAV vectors can be constructed using methods known in the art. The general principles of constructing rAAV vectors are known in the art. For example, the literature [Carter, 1992 ,See [Current Opinion in Biotechnology, 3:533-539]; and [Muzyczka, 1992, Curr. Top. Microbiol. Immunol., 158:97-129]. For example, Cargo nucleic acid can be inserted directly between ITRs of an AAV genome in which the Rep gene and / or Cap gene has been deleted. Other parts of the AAV genome may also be deleted, but sufficient ITR portions must remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. For example, U.S. Patents No. 5,173,414 and No. 5,139,941; International Publication No. WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993); literature [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 Opinion in Biotechnology 3:533-539]; [Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 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) J. Exp. Med. 179:1867-1875]. Alternatively, an AAV ITR can be excised from the viral genome or an AAV vector containing it and fused to the 5' and 3' of the cargo nucleic acid using standard ligation techniques.

[0134] In certain embodiments, the AAV viral vector of the present disclosure may also include AAV viral particles. As used herein, “viral particle” means a viral genome packaged within a viral capsid. The viral genome within the viral particle may be a modified viral genome and thus may lack and / or contain some sequences that are heterogeneous to the natural viral genome. AAV viral particles may be produced by introducing an AAV expression vector into a suitable host cell using known techniques such as transfection, together with a plasmid encoding the AAV cap / rep gene and other necessary mechanisms, such as a helper gene provided by an adenovirus or herpesvirus (see, for example, the entirety of which is incorporated herein by reference [MF Naso et al, BioDrugs, 31(4): 317-334 (2017)]). The AAV expression vector may be expressed in a host cell and packaged into a viral particle.

[0135] The cargo nucleic acid may further comprise one or more regulatory sequences operably linked to the nucleic acid encoding a therapeutic protein or RNA. As used herein, the term “operably linked” means that the encoding sequence is linked directly or indirectly to one or more regulatory sequences in the exogenous nucleic acid in a manner that enables the expression of the protein of interest from the encoding sequence within a cell. The encoding sequence and the regulatory sequence together may be referred to herein as an expression cassette. In certain 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 one or more introns inserted between the exons of the polyadenylation sequence and / or protein encoding sequence.

[0136] As used herein, the term “regulatory sequence” refers to any nucleotide sequence that is necessary or advantageous for the expression of an encoding sequence. A regulatory sequence may include, but is not limited to, one or more promoters, enhancers, transcription termination factors, polyadenylation sequences, internal ribosome entry sites, and / or one or more introns inserted between exons of a protein-coding sequence.

[0137] d. Method of producing viral vectors

[0138] The present disclosure further provides a method for producing a virus vector provided herein.

[0139] In one embodiment, the present disclosure provides a method for producing a viral vector, the method comprising the step of 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 for replication of the nucleic acid template and encapsulation into an AAV capsid (e.g., an AAV rep sequence and an AAV cap sequence encoding the AAV capsid of the present disclosure). Optionally, the nucleic acid further comprises at least one cargo nucleic acid. In certain embodiments, the nucleic acid comprises two AAV ITR sequences located at the 5' and 3' of the cargo nucleic acid (if present), and these do not necessarily have to be directly adjacent to the cargo nucleic acid.

[0140] The nucleic acid and AAV rep and cap sequences are provided under conditions that allow a viral vector containing nucleic acid packaged within an AAV capsid to be produced in cells. The method may further include the step of collecting the viral vector from cells. The viral vector may be collected from a culture medium and / or by lysing cells.

[0141] In one embodiment, the present disclosure provides a method for producing a viral vector that evades existing anti-AAV antibodies in host serum, comprising: a) introducing at least one amino acid mutation into an AAV capsid protein; b) detecting the binding of the mutated AAV capsid protein to a serum antibody in host serum; and c) selecting at least one amino acid mutation in the AAV capsid protein that reduces the binding affinity to the serum antibody compared to a non-mutated corresponding AAV capsid protein.

[0142] In some embodiments, the anti-AAV antibody comprises an anti-AAV neutralizing antibody, and the method further comprises: d) producing an AAV particle comprising a mutated AAV capsid protein selected in step (c); e) determining the infectivity of the AAV particle to a suitable host cell in the presence of a serum antibody; and f) selecting at least one amino acid mutation in the AAV capsid protein that increases the infectivity of the AAV particle to a host cell in the presence of a serum antibody compared to an AAV particle comprising an unmutated corresponding AAV capsid protein.

[0143] In some embodiments, step e) comprises: (i) contacting the AAV particles of (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 control AAV particles; (iii) contacting the AAV particles selected in (ii) with an anti-AAV neutralizing serum antibody and suitable host cells under conditions where infection and replication can occur; and (iv) selecting AAV particles that are not neutralized by the anti-AAV neutralizing serum antibody of (iii). This comprehensive approach presents a platform technology that can be applied to modify any AAV capsid. The application of this platform technology generates AAV antigen variants derived from the original AAV capsid template without loss of transduction efficiency. Exemplary methods are, for example, [Cecchini. S et al. (2008) Gene Ther. 15(11):823-30]; [Ayuso E et al. It can be found in [Kotin RM et al. (2010) Curr Gene Ther. 10(6):423-36]; [Kotin RM et al. (2011) Hum Mol Genet 20(R1):R2-6]; [Wright JF et al. (2009) Hum Gene Ther. 20(7):698-706].

[0144] III. Method for delivering cargo nucleic acids to cells and targets

[0145] The viral vector of the present disclosure is useful for delivering nucleic acids to cells in vitro, in vitro, and in vivo. In particular, the viral vector can be advantageously used to deliver or transfer nucleic acids to animal cells, including mammalian cells. Accordingly, in some embodiments, the nucleic acid ("cargo nucleic acid") may be encapsulated by the modified capsid protein of the present disclosure.

[0146] Generally, the viral vectors of the present disclosure may be used to deliver a cargo nucleic acid encoding a polypeptide or functional RNA to treat and / or prevent any disease condition where delivering a therapeutic polypeptide or functional RNA is beneficial. In some embodiments, the viral vectors of the present disclosure may be used to deliver a cargo nucleic acid encoding a polypeptide or functional RNA to treat and / or prevent a disease or disorder. Diseases or disorders include, but are not limited to, genetic diseases, blood diseases, infectious diseases, neurological diseases, autoimmune diseases, inflammatory diseases, metabolic diseases, muscular diseases, and diseases related to the pancreas, kidney, liver, heart, or other specific tissues.

[0147] A viral vector according to the present disclosure provides a means for delivering a cargo nucleic acid to a wide range of cells, including dividing and non-dividing cells, in vivo. This viral vector may also be used to deliver a nucleic acid of interest to cells in vitro, for example, to produce polypeptides in vitro or for gene therapy in vitro. This viral vector is further useful for a method of delivering nucleic acids to a subject who requires them, for example, to express immunogenic or therapeutic polypeptides or functional RNAs. In this way, polypeptides or functional RNAs may be produced in vivo in the subject. The subject may be deficient in polypeptides and require polypeptides. Additionally, this method may be carried out because the production of polypeptides or functional RNAs in the subject may confer several beneficial effects.

[0148] As used herein in the broadest sense, the terms “administer,” “administering,” or “administration” refer to providing or distributing a pharmaceutically active substance to a subject in a pharmacologically appropriate manner. The term is intended to include “causing to be administered,” which means causing, prompting, encouraging, assisting, inducing, or directing another party to administer the pharmaceutically active substance to the subject, directly or indirectly. Note that in certain embodiments, the term is interchangeable with “delivery,” “delivery,” “delivering,” or similar terms. As used herein, the pharmaceutically active substance comprises a vehicle (i.e., an inducing vehicle and / or a target vehicle).

[0149] Exemplary modes of administration include oral, rectal, transmucosal, nasal, inhalation (e.g., via aerosol), oral (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular (including administration to skeletal muscle, diaphragm and / or cardiac muscle), intradermal, intrapleural, intracerebral, and intra-articular), local (e.g. to both skin and mucosal surfaces including airway surfaces, and transdermal administration), intralymphatic, etc., as well as direct tissue or tracheal injection (e.g. to the liver, skeletal muscle, cardiac muscle, diaphragm muscle, or brain). Administration may also be directed to a tumor (e.g., into or near a tumor or lymph node). In any given case, the most suitable route will depend on the nature and severity of the disease being treated and / or prevented and the nature of the specific vector used.

[0150] The dosage of viral vector and / or capsid to be administered to a subject depends on the mode of administration, the disease or condition to be treated or / or prevented, the individual subject's condition, the specific viral vector or capsid, the nucleic acid to be delivered, etc., and may be determined in a conventional manner. An exemplary dose to achieve a therapeutic effect is at least about 10 per kilogram of body weight. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 Transduction unit, optionally about 10 per kilogram of body weight 8 to 10 13 It is the titer of the transduction unit.

[0151] In certain embodiments, more than one administration (e.g., two, three, four, or more administrations) may be used over various intervals, such as daily, weekly, monthly, or yearly, to achieve a desired level of gene expression.

[0152] As used herein, the term “subject” includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammalians, e.g., non-human primates, mice, rats, cats, rabbits, sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” and “subject” are used interchangeably herein. In certain embodiments, the subject is a human. In some embodiments, the subject is a non-human primate.

[0153] The viral vector provided herein is advantageous in that it expands the cohort of subjects eligible for gene therapy using AAV vectors. In particular, the viral vector provided herein possesses a relatively high level of anti-AAV serum antibodies (e.g., anti-AAV neutralizing antibodies), allowing it to be administered to subjects who are not eligible for gene therapy using AAV vectors.

[0154] In some embodiments, the subject has a serum antibody titer against AAV (e.g., AAV9) of 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, or 500 or less, wherein the antibody titer is defined as a dilution factor of the subject serum to indicate a specific binding threshold to the AAV (e.g., AAV9) capsid protein.

[0155] Binding to the AAV capsid protein can be optionally measured by enzyme-linked immunosorbent assay (ELISA). In some embodiments, a specific binding threshold is determined based on antibody binding in the presence of a predetermined amount of AAV capsid protein, wherein the specific binding threshold is considered to be an antibody binding level sufficient to indicate specific binding to the AAV capsid protein.

[0156] In some embodiments, the subject has a serum antibody titer against AAV9 of 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, or 500 or less, wherein the antibody titer is defined as a dilution factor of the subject serum to represent 50% of maximum neutralization against the AAV9 capsid protein. Neutralization of AAV9 may optionally be measured by a reporter assay.

[0157] The term "neutralizing antibody titer" refers to a value reflecting the neutralization efficiency of serum containing at least one neutralizing antibody. The neutralizing antibody titer of serum can be analyzed by evaluating the ability of serum antibodies to inhibit the transduction or infection of AAV virus particles into susceptible host cells, wherein AAV virus particles may express reporter genes such as beta-galactosidase or luciferase after transduction or infection. The neutralizing antibody titer can be determined as a dilution factor of serum to represent about 50% of maximum neutralization against AAV virus particles present in a predetermined amount, optionally.

[0158] IV. Pharmaceutical composition

[0159] In a further embodiment, a pharmaceutical composition is provided comprising a viral vector and / or capsid and / or capsid protein of the present disclosure among pharmaceutically acceptable carriers, and optionally other drugs, pharmaceutical preparations, stabilizers, buffers, carriers, adjuvants, diluents, etc.

[0160] As used herein in its broadest sense, the term “pharmaceutical composition” refers to a formulation comprising a therapeutically effective amount of a pharmaceutically active ingredient, selectively combined with pharmaceutically acceptable excipients and / or carriers, etc. As used herein, the term “pharmaceutically acceptable” means suitable for normal pharmaceutical use, that is, does not cause serious side effects, such as adverse reactions, to subjects. As used herein, the terms “therapeutically effective amount” or “effective amount” refer to a dosage sufficient to make the treatment of a patient effective compared to the case of no treatment. As used herein, “effective amount” refers to an amount that produces a desired effect, e.g., an amount capable of treating or improving a disease or condition, or an amount capable of producing an intended therapeutic effect (e.g., induction of immune tolerance).

[0161] A pharmaceutically active ingredient generally requires one or more pharmaceutically acceptable carriers or excipients to take on a suitable form (i.e., pharmaceutical composition) and formulation for effective administration to a subject. As used herein, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not impair the biological activity and properties of the pharmaceutically active ingredient administered without causing severe irritation to the organism. As used herein, 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.

[0162] In this invention, pharmaceutical compositions suitable for use comprise sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the compositions must be sterile and stable under manufacturing and storage conditions and must be protected from the action of microorganisms such as bacteria and fungi.

[0163] Pharmaceutically acceptable carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. For example, for intravenous administration, suitable carriers may include physiological saline, antimicrobial water, or phosphate-buffered saline (PBS). Suitable fluidity may be maintained by the use of a coating, for example, lecithin, the maintenance of the required particle size in the case of a dispersion, and the use of a surfactant. Prevention of microbial action may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be desirable to include isotonic substances, for example, sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Delayed absorption of the composition for in vivo administration may be achieved by including absorption-delaying agents, for example, aluminum monostearate and gelatin, in the composition.

[0164] Pharmaceutical compositions may include, but are not limited to, solutions, emulsions, and liposome-containing formulations. Such compositions may be produced from various components including, but not limited to, pre-formed liquids, self-emulsifying solids, and self-emulsifying semi-solids.

[0165] In some embodiments, the pharmaceutical composition provided herein comprises a therapeutically effective amount of virion mixed with a pharmaceutically acceptable carrier and / or excipient, e.g., brine, phosphate-buffered saline, phosphate and amino acid, polymer, polyol, sugar, buffer, preservative and other protein. Exemplary amino acids, polymers, and sugars, etc. are octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, 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 glycol. In certain embodiments, this formulation is stable at 4°C for at least 6 months.

[0166] In some cases, the unit dose of the pharmaceutical composition of the present disclosure may be measured in pfu (plaque-forming units). In some cases, the pfu of a unit dose of the pharmaceutical composition of the present disclosure containing modified AAV is 1 × 10⁻⁶ 8 More than or about 1×10 15 It could be pfu.

[0167] The compositions provided herein include 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, citrate or acetate salts, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10-nonylphenol, sodium deoxycholate), solution and / or lyophilization stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic pressure regulators (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymer stabilizers and viscosity modifiers (e.g., polyvinylpyrrolidone, It may include poloxamer 488, carboxymethylcellulose, and a cosolvent (e.g., glycerol, polyethylene glycol, ethanol).

[0168] The composition according to the present invention may include pharmaceutically acceptable excipients. The composition may be prepared into useful formulations using conventional pharmaceutical manufacturing and compounding techniques. Techniques suitable for practicing the present invention can be found in the 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 Ed. Edited by ME Auten, 2001, Churchill Livingstone]. In one embodiment, the composition is in the form of an injectable sterile saline solution containing a preservative.

[0169] Hereinafter, one embodiment is provided as a specific embodiment of the method and kit provided in the present disclosure. It should be noted that this embodiment is for illustrative purposes only and does not limit the scope of the present disclosure.

[0170] Examples

[0171] Example 1. Design and construction of an insertion mutation of the AAV9 capsid protein

[0172] The AAV capsid used to encapsulate recombinant genes is a hollow icosahedral structure with a diameter of approximately 26 nm. It can be visualized as a regular icosahedron composed of 20 triangles, forming one pentagon for every five triangles. There are 5 rotation axes between each pair of opposite pentagons, 3 rotation axes between each pair of opposite triangle centers, and 2 rotation axes between each pair of opposite edge centers. This capsid is composed of a total of 60 capsid proteins, VP1, VP2, and VP3, with the ratio of these three subunits in each capsid being 5:5:50. These subunits are encoded by overlapping sequences within the same open reading frame. Neutralizing antibodies bind to AAV by recognizing antigenic epitopes on the surface of the viral capsid. Meanwhile, antibody binding interferes with AAV recognition by cell surface receptors, thereby inhibiting AAV from infecting target cells. On the other hand, antibody binding induces the elimination of AAV particles by the body's immune system, further reducing the effectiveness of AAV gene therapy. In certain situations, neutralization can block AAV from entering the cell until the molting process, which hinders the successful expression of therapeutic genes.

[0173] The first challenge in modifying the AAV capsid to evade neutralizing antibodies is to identify the major antigenic epitopes recognized by these antibodies. To determine the major regions of the AAV capsid surface involved in serum antibody recognition, the inventors used recombinant DNA technology to introduce peptide insertions into 122 different sites within the CAP9 gene sequence encoding the AAV9 capsid VP protein. Considering that the VP1, VP2, and VP3 proteins are all encoded by overlapping sequences within the same open reading frame, that VP3 is contained within VP1 and VP2, and that VP3 is a major structure for assembling the icosahedral capsid, the inventors selected insertion mutation sites within the region encoding the VP3 protein. By introducing these mutations into the CAP gene, the inventors obtained 122 different engineered AAV9 variants, each having a peptide insertion unique to a specific site on the capsid surface. Based on the disclosed AAV9 crystal structure (PDB #7MT0), the insertion site selected by the inventors can effectively encompass all prominent structural features of the virus capsid surface and systematically destroy all potential antigenic epitopes (highlighted in red in Fig. 1).

[0174] Another challenge in modifying the AAV capsid to evade neutralizing antibodies is using appropriate mutations to disrupt the antigenic epitopes recognized by existing antibodies. Theoretically, substitutions or insertions within important antigenic epitopes can interfere with antibody recognition. However, insertion mutations are more suitable for systematic screening because they are simple to generate and have a significant impact on the capsid structure. In the initial screening, the inventors selected StrepII, a commonly used protein purification tag, as the peptide sequence to be inserted at 122 sites. This selection was based on two considerations: first, StrepII-tag related detection tools, including probes and antibodies, are well-established and commercially available, allowing for the rapid identification of generated mutant viruses; and second, compared to other commonly used peptide-protein tags, the StrepII tag is shorter in length and has relatively uniform polarity and charge characteristics. Theoretically, its insertion is expected to have a lower impact on the overall structure of the AAV capsid than other commonly used protein tags.

[0175] Example 2. Screening of mutations that evade serum antibody binding

[0176] Because the immune response spectrum of human B cell lineages is highly diverse, different individuals produce various anti-AAV antibodies when exposed to the virus. To identify engineered AAV mutants capable of evading broad-spectrum neutralizing antibodies, the inventors prepared a serum mixture by mixing the peripheral blood serums of four adult individuals in equal proportions. Using this mixture as a screening sample, binding to 122 AAV9 mutants with peptide insertions at different sites was tested.

[0177] Various methods are available to measure AAV neutralizing antibodies in serum. To achieve both sensitivity and throughput, the inventors used an enzyme-linked immunosorbent assay (ELISA) to detect the binding of AAV9 variants to human IgG antibodies in mixed serum. To establish appropriate ELISA reaction conditions, the inventors first coated ELISA plates with wild-type AAV9 at different concentrations and then incubated these plates with mixed serums diluted to various concentrations. To evaluate human serum antibodies bound to wild-type AAV9, detection was performed using an HRP-labeled anti-human IgG secondary antibody (Fig. 2).

[0178] According to the results, when the concentration of AAV9 coated on the ELISA plate was between 5E9 and 2E10 vg / ml, it had a negligible effect on the ELISA measurements, suggesting that AAV within that concentration range effectively covered the bottom surface of the Nunc MaxiSorp ELISA plate used. Even at higher viral concentrations, antibody binding did not significantly increase. Based on these preliminary experimental results, the inventors determined the AAV9 concentration (5E9 vg / ml) and serum dilution factor (50x) for formal screening experiments. Under these conditions, the inventors screened for AAV9 variants capable of evading neutralizing antibodies in mixed serum.

[0179] In the screening experiment, the inventors coated wild-type AAV9 and 122 different AAV9 variants onto ELISA plates at a concentration of 5E9 vg / ml each, and then incubated them with a serum mixture diluted 50-fold. The binding of existing antibodies was detected using an HRP-labeled anti-human IgG secondary antibody. The screening results are shown in Figure 3.

[0180] According to the results, serum binding was reduced by more than 50% in 21 of the 122 insertions compared to the ELISA measurements of wild-type AAV9. This suggests that these 21 sites significantly affect recognition by existing antibodies in mixed serum. The insertion sites of the 21 clones and their ELISA signals are listed in Table 2.

[0181]

[0182] The insertion site is named according to its position in the amino acid sequence of the VP1 protein.

[0183] Example 3. Binding test of selected AAV9 variants on serum samples from various populations

[0184] To further investigate how antibodies from various populations recognize selected AAV variants, the inventors collected 48 adult peripheral blood serum samples from various countries, including East Asia, Africa, and Eastern Europe. These serum samples represent a fairly broad population. The inventors individually tested ELISA binding to 21 AAV9 variants with insertions at the sites listed in Table 1 for each of the 48 serum samples. By comparing this with wild-type AAV9 recognition in these serum samples under the same conditions, the inventors evaluated the effect of engineered modifications to these sites on existing antibody recognition in the representative population.

[0185] As a result, compared to 48 ELISA readings of wild-type AAV9 for 48 individual serum samples, peptide insertions at 13 positions, including residues 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588, and 715 according to the amino acid sequence of the VP1 protein, showed a significant decrease in binding with most of the 48 individual serum samples (Fig. 4). Specifically, 34 of the 48 serum dilutions showed ELISA readings exceeding 0.9 for wild-type AAV binding, with an estimated positive rate of 70.8%. In contrast, under the same conditions, for 13 AAV9 variants, only 3 to 16 samples reached an ELISA reading of 0.9, with positive rates ranging from 6.3% to 33.3%. In summary, compared to binding data of wild-type AAV9, more than 50% of serum samples showed a significant decrease in binding to AAV9 variants with insertions at each of the 13 selected positions, including residues 441, 442, 444, 509, 510, 512, 513, 514, 552, 557, 559, 588, and 715 according to the protein sequence of VP1. The positions of the 13 major residues are indicated in red on the AAV9 crystal structure (Fig. 5a). The inventors also indicated their positions in a representative VP3 subunit (Fig. 5b).

[0186] Example 4. Transduction inhibition test for AAV9 variants evading serum neutralizing antibodies

[0187] The decrease in ELISA signal demonstrated that AAV mutations can interfere with direct binding to existing anti-AAV antibodies. Anti-AAV antibodies in serum can be neutralizing or non-neutralizing antibodies. Neutralizing antibodies not only bind to the AAV capsid but also inhibit vector transduction. To test whether the AAV variant discovered by the inventors could evade the neutralizing effect of serum antibodies, a transduction inhibition assay was performed to measure the extent to which serum antibodies inhibit AAV-mediated reporter gene expression.

[0188] For the transduction inhibition assay, an AAV variant with ST inserted at residue 509 was used as a proof of concept. To facilitate detection, the inventors produced an AAV containing a reporter gene. Briefly, the Nano-Luciferase reporter gene was packaged into the capsid used in the previous ELISA screening, including the wild-type AAV9 (AAV9-wt) and an AAV9 mutant (509-ST) with a Strep-II tag inserted at 509. These novel AAV constructs containing the Luciferase reporter gene were produced and purified in a manner similar to the AAV used in the ELISA screening. The serum mixture used in the previous screening (Fig. 3) was serially diluted, pre-incubated with the AAV reporter vector, and then inoculated into 293T cells. After incubation for 72 hours to allow cell transduction to occur, the expression of the Nano-Luciferase gene was measured using a commercial NanoGlo Luciferase kit. The inhibitory effect of the serum mixture on transduction was evaluated by measuring the decrease in Luciferase gene expression (Fig. 6).

[0189] As shown in the top panel of Figure 6, the presence of serum diluted to 1:810 reduced wild-type AAV9 transduction by more than 90%, indicating significant inhibition of transduction by existing neutralizing antibodies in the serum sample. However, no inhibition was detected for 509-ST at the same serum concentration. Indeed, even with serum pre-incubation at 1:10, the 509-ST variant was still able to transduce target cells with an efficiency of more than 90%, indicating significant evasion of neutralizing antibodies. These results indicate that AAV variants selected in previous screenings can evade the neutralizing effect of serum antibodies.

[0190] Example 5. Different types of mutations in the core site induce neutralizing antibody evasion

[0191] Screening of AAV mutants with StrepII tag insertion allowed us to identify a group of key sites affecting the recognition of serum neutralizing antibodies. It is important to clarify whether the disruption of antibody recognition is due to specific characteristics of the StrepII tag, or whether different mutations in these key AAV residues will exhibit similar evasion effects. To answer this question, the inventors replaced the octapeptide WSHPQFES of the StrepII tag inserted at position 509 with a number of mutation strategies, including a 2-amino acid peptide insertion of the PQ sequence (clone 509-S2), a 4-amino acid peptide of the HPQF sequence (clone 509-S4), and a 6-amino acid peptide of the SHPQFE sequence (clone 509-S6), as well as completely unrelated sequences such as the Myc-tag EQKLISEEDL (clone 509-Myc), the 4-peptide sequence GGGS (clone 509-GS4), and the 8-peptide sequence GGGSGGGS (clone 509-GS8). Additionally, the inventors replaced the original sequence on the surface of AAV9 with different peptides of the same length. For site 509, the inventors replaced the DKVMITNE sequence at positions 506 to 513 of VP1 with the Strep-II tag sequence WSHPQFES (clone name 509-RP8). An AAV variant containing a Nano-Luciferase reporter gene was produced with this labeled capsid.

[0192] Repression of gene transduction by serum was tested on different AAV9 variants and compared to inhibition on wild-type AAV9 in parallel experiments (Fig. 7). Consistent with previous experiments, the transduction efficiency of wild-type AAV9 was significantly inhibited by serum dilution. However, all insertions with different lengths / sequences at site 509, or substitutions around residue 509, were able to evade the neutralizing effect of serum antibodies. These results demonstrated that the site / location of the mutation, rather than the type of mutation, is important in interfering with serum antibody recognition.

[0193] The inventors expect that similar characteristics exist in other key residues that affect neutralizing antibody recognition. As a simple test, the inventors replaced the strepII tag of clone 559-ST with a Myc tag. Then, they generated an AAV vector containing a luciferase reporter gene for these two capsids. Consistent with the results for site 509, clone 559-Myc evaded transduction inhibition as efficiently as 559-ST (Fig. 8). This result further demonstrated that the mutation site, rather than the mutation type, plays a significant role in evading neutralizing antibody recognition.

[0194] Due to the high conservation of capsid amino acid sequences and 3D structures, significant cross-reactivity of neutralizing antibodies has been reported among different AAV serotypes. To test whether mutations at corresponding positions in AAV serotypes other than AAV9 would have a similar evasion effect against serum neutralizing antibodies, the inventors generated AAV6 mutants with Strep-II tags inserted at positions 512, 513, 556, 557, or 559 of the capsid protein VP1. These selected positions correspond to the same numbered positions on the AAV9 VP1 protein where mutations showed evasion effects against serum neutralizing antibodies in previous AAV9 mutant screenings. Since AAV6 and AAV9 have the same VP1 length, their numbering is also identical. The inventors tested the transduction efficiency of these five mutants in the presence of the mixed human serum used in previous experiments. As shown in Figures 9 through 13, all of these mutants successfully evaded the transduction inhibitory effect of serum dilution compared to wild-type AAV6. These results suggested that the neutralizing antibody evasion site identified in AAV9 may have a similar evasion effect in other AAV serotypes.

Claims

Claim 1 A modified AAV capsid protein comprising at least one mutated antigenic epitope comprising at least one amino acid mutation 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 numbering corresponds to the wild-type AAV9 VP1 protein (SEQ No. 1), and the at least one amino acid mutation substantially reduces the binding of a serum antibody to the AAV capsid protein. Claim 2 In claim 1, the serum antibody comprises a modified AAV capsid protein containing an existing anti-AAV antibody present in the host's serum. Claim 3 In paragraph 2, the existing anti-AAV antibody is a modified AAV capsid protein comprising an anti-AAV neutralizing antibody. Claim 4 Paragraph 2 or 3, a modified AAV capsid protein in which the host is a human. Claim 5 In any one of claims 1 to 4, the amino acid mutation is a modified AAV capsid protein selected from substitution, insertion, deletion, or any combination thereof. Claim 6 In claim 5, the amino acid mutation comprises the insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues, a modified AAV capsid protein. Claim 7 In claim 5 or 6, the insertion is a modified AAV capsid protein having a length of 2 to 20, 2 to 18, 2 to 16, 2 to 14, 2 to 12, or 2 to 10 amino acid residues. Claim 8 A modified AAV capsid protein according to any one of claims 1 to 7, wherein at least one amino acid mutation comprises one or more mutations at a position selected from the group consisting of positions 509, 510, 512, 513, 514, or any combination thereof. Claim 9 A modified AAV capsid protein according to any one of claims 1 to 8, wherein at least one amino acid mutation comprises one or more mutations at a location selected from the group consisting of locations 559, 557, or any combination thereof. Claim 10 A modified AAV capsid protein according to any one of claims 1 to 9, wherein at least one amino acid mutation comprises one or more mutations at a position selected from the group consisting of positions 509, 510, 512, 513, 514, 559, 557, or any combination thereof. Claim 11 A modified AAV capsid protein according to any one of claims 1 to 10, wherein at least one amino acid mutation comprises a mutation at position 509, a mutation at position 510, and / or a mutation at position 559. Claim 12 A modified AAV capsid protein according to any one of claims 8 to 11, wherein one or more mutations comprise at least one amino acid insertion. Claim 13 제1항 수제 제12한 중 자이 한 한로다다, AAV 구시드 레레은 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV12, AAV843, AAVbb2, AAVcyS, AAVrh10, AAVrh20, AAVrh39, AAVrh43, AAVrh64, AAVhu37, AAV3B, AAVhu48, AAVhu43, AAVhu44, 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, AAVhu1, AAVhu4, AAVhu2, AAVhu61, AAVrh62, AAVrh48, AAVrh54, AAVrh55, AAVcy2, AAVrh35, AAVrh37, AAVrh36, AAVcy6, AAVcy4, AAVcy3, AAVcy5, AAVrh13, AAVrh38, AAVhu66, AAVhu42, AAVhu67, AAVhu40, AAVhu41, AAVrh40, AAVrh2, AAVbb1, AAVhu17, AAVhu6, AAVrh25, AAVpi2, AAVpi3, AAVrh57, AAVrh50, AAVrh49, AAVhu39, AAVrh58, AAVrh61, AAVrh52, AAVrh53, AAVrh51, AAVhu14, AAVhu31,Modified AAV capsid protein of AAVhu32, AAVrh34, AAVrh33, AAVrh32, avian AAV ATCC VR-865, avian AAV strain DA-1, or an AAV serotype selected from bovine AAV. Claim 14 A modified AAV capsid protein according to any one of claims 1 to 13, wherein the mutated antigenic epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 9 to 68. Claim 15 A polynucleotide encoding a modified AAV capsid protein of any one of claims 1 to 14. Claim 16 An expression vector comprising the polynucleotide of claim 15. Claim 17 A cell comprising the polynucleotide of claim 15 or the expression vector of claim 16. Claim 18 An AAV capsid comprising a modified AAV capsid protein according to any one of claims 1 to 14. Claim 19 An AAV virus vector comprising the AAV capsid of claim 18. Claim 20 A viral vector according to claim 19, further comprising a nucleic acid comprising at least one terminal repeat sequence, wherein the nucleic acid is capsidized by an AAV capsid. Claim 21 In paragraph 20, the viral vector further comprises a cargo nucleic acid operably linked to at least one terminal repeat sequence. Claim 22 In paragraph 20, the cargo nucleic acid is a viral vector encoding a therapeutic protein or RNA. Claim 23 A pharmaceutical composition comprising an AAV virus vector of any one of claims 19 to 22 and a pharmaceutically acceptable carrier. Claim 24 A method for delivering a cargo nucleic acid to a cell, comprising the step of contacting the cell with the virus vector of claim 21 or 22. Claim 25 A method for delivering a cargo nucleic acid to a subject, comprising the step of administering an effective amount of the viral vector of claim 21 or 22 or the pharmaceutical composition of claim 23 to the subject. Claim 26 A method for treating a subject requiring treatment, comprising the step of administering a therapeutically effective amount of the AAV virus vector of claim 21 or 22 or the pharmaceutical composition of claim 23 to said subject. Claim 27 A method according to claim 25 or 26, wherein the subject has a serum antibody titer against AAV9 of 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, or 500 or less. Claim 28 A method for producing an AAV vector that evades neutralizing antibodies in a host, comprising: a) producing a library of modified AAV vectors by inserting short peptides into various sites within a VP3 protein; b) determining the binding of the modified AAV vectors to samples containing pooled serum of different subjects; and c) identifying the modified AAV vectors that have reduced binding to the serum samples.

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