Controlled modification of adeno-associated virus (AAV) for enhancing gene therapy

Abstract

The present invention discloses a platform for chemically modifying AAV capsids with control over site and stoichiometry. An AAV packaging system is described that allows for site-specific introduction of natural and unnatural amino acid mutations into any subset of the three capsid proteins. These engineered residues can then be used to chemically functionalize the resulting capsid with precise control over site and stoichiometry. Such a controlled modification strategy can be used to attach a wide variety of entities to AAV capsids to manipulate their tropism, immunogenicity, etc.

Description

Technical Field

[0001] Government Support This invention was made with government support under Grant No. 1817893 awarded by the National Science Foundation. The government has certain rights in this invention.

[0002] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 018,573, filed May 1, 2,020. This provisional application is hereby incorporated by reference in its entirety.

[0003] Incorporation by Reference of Materials in ASCII Text File This application incorporates by reference the Sequence Listing contained in the following ASCII text file: 0342_0010WO1_SL.txt; created on May 3, 2021, size 76,879 bytes.

[0004] Field of the Invention The present invention is directed to the field of biotechnology focused on the development of recombinant (engineered) AAV-based gene therapy vectors by controlled chemical modification of viral capsids.

Background Art

[0005] Adeno-associated viruses (AAVs) have emerged as one of the most promising delivery vehicles for gene therapy. They are naturally replication-deficient, exhibit only a low innate immune response, efficiently infect both dividing and non-dividing cells, and provide stable, long-term in vivo expression of delivered therapeutic genes. Indeed, several AAV-based gene therapies have recently been approved, and many others are currently in clinical trials. Despite the potential of AAV-based gene therapies to offer cures for numerous diseases currently untreatable, several significant challenges remain. For example, almost all current approaches rely on the natural serotypes of AAVs exhibiting limited innate tissue affinity. Naturally, diseases associated with cell types efficiently infected by these vectors can be targeted by this approach. However, many other diseases occurring in tissues that do not readily infect with existing AAV vectors remain refractory. The ability to remanipulate the tissue affinity of existing AAV vectors would be extremely useful in creating custom vectors capable of delivering gene payloads to desired locations, substantially expanding the scope of AAV-based gene therapy. In addition, many patients have pre-existing immunity to existing AAV vectors. Furthermore, administration of therapeutic AAV vectors triggers a strong adaptive immune response, preventing a second dose of the same vector. The ability to manipulate existing AAV vectors to circumvent such immune recognition is crucial for the development of sustainable gene therapies. Directed evolution has been used to develop AAV capsids with more therapeutically desirable traits. However, this approach is often unreliable in yielding acceptable solutions, and the resulting mutant AAV capsids frequently exhibit packaging defects.

[0006] The ability to present regulatory entities on existing AAV capsids in a controlled manner offers an attractive modular approach to manipulating their properties. Numerous methods have been attempted toward this goal, including N-terminal extension of minor capsid proteins, insertion of peptide loops into acceptable sites on the capsid protein, and modification of existing amino acids such as lysine on the capsid surface. However, N-terminal extension and loop insertion often disrupt the assembly of the AAV capsid complex. Due to the delicacy of AAV capsids, peptide loop insertion is limited to a very small number of positions, and the resulting capsids often exhibit suboptimal packaging. These methods offer only limited flexibility in optimizing the placement of regulatory regions, the structural diversity of entities that can be presented, and the number of targeted regions presented on the capsid surface. [Overview of the project] [Means for solving the problem]

[0007] It is possible to produce AAV incorporating UAA having an azide functional group, which is then conjugated to a cyclooctin-modified cRGDFC via strain-enhanced azide-alkyne cycloaddition (SPAAC), thereby retargeting the virus to the αVβ3 integrin receptor overexpressed in certain cancer cells. However, the aforementioned UAA incorporation is limited to the incorporation of UAA into all 60 capsid proteins of AAV. As described herein, overmodification of all 60 capsid proteins of AAV virus particles (including minor capsid proteins VP1 and VP2 and major capsid protein VP3) has been demonstrated to be highly detrimental to their infectivity. The ability to control the number of amino acid mutations to generate variant capsids for incorporating / introducing a specific number of native amino acids and / or UAA modifications / mutations per capsid (compared to wild-type AAV) is a key feature of the present invention for enabling the synthesis of infectious genetically modified adeno-associated viruses containing variant (mutant) capsid proteins with mutated amino acid residues. These mutated residues in the variant capsid protein allow for the attachment (e.g., covalent bond) of biological and / or chemical entities (hereinafter also referred to as “groups”) to site-specific bioconjugation “handles” to AAV, resulting in the incorporation / introduction of a distinct number of activity modulators that enable optimal manipulation of AAV function.

[0008] The present invention encompasses genetically engineered / modified adeno-associated viruses (AAVs) having modifications / mutations at one or more site-specific locations of the capsid protein compared to the corresponding sites of wild-type AAV, and methods for producing these genetically modified viruses. Importantly, as described herein, the modifications may be limited to one or more specific chemoselective locations in a single capsid protein such as VP1, VP2, or VP3, or in multiple of these capsid proteins such as in both VP1 and VP2, without affecting the infectivity of the AAV particles (i.e., the infectivity of the genetically modified AAV is essentially comparable to, or only slightly altered from, the infectivity of wild-type AAV under similar biological conditions).

[0009] AAV is a small, non-enveloped parvovirus with a 4.5 kb single-stranded DNA genome encoding non-structural Rep and AAP proteins and structural Cap proteins VP1, VP2, and VP3, which share a common C-terminus and differ by N-terminal elongation. Unlike enveloped viruses, which have a lipid membrane decorated with surface proteins, AAV has a dense icosahedral capsid consisting of only 60 copies of proteins VP1, VP2, and VP3 in a stoichiometric ratio of approximately 1:1:10. AAV is replication-deficient and requires the presence of a helper virus, such as an adenovirus, for complete viral production and escape. AAV has several different serotypes with different innate tropisms (affinity). For example, one serotype described herein is AAV2 (AAV2), which targets cells expressing the heparan sulfate proteoglycan receptor.

[0010] Adeno-associated viruses are emerging as the most promising candidates for human gene therapy. These viruses boast desirable properties such as low immunogenicity and long-term gene expression, making them ideal for gene therapy vectors. However, many other characteristics of these viruses often do not align with therapeutic needs. For example, the natural serotypes of the viruses possess unique cell specificity, which limits which cells / tissues can be targeted for human gene therapy. The ability to functionalize existing AAV vectors with precise control over site and stoichiometry offers an attractive means of introducing therapeutically desirable traits such as cell specificity and immune evasion.

[0011] This specification describes techniques for precisely and site-specifically genetically engineering AAV by introducing amino acids having entities or groups, such as UAAs, to the minor capsid proteins of AAV, including bioconjugation entities or "handles" (e.g., the natural amino acid cysteine, or non-natural amino acids with broad bioconjugation chemistry). The minor capsid proteins VP1 and VP2 are present in 5 copies each per AAV capsid. As described herein, the constructs and methods of the present invention enable site-selective modification of any of the three capsid proteins, but specifically modify VP1 or VP2 or both VP1 and VP2, enabling the attachment of 5 or 10 groups per fully constructed AAV capsid (as used herein, “fully assembled” means a viral assembly that incorporates all three capsid proteins, the minor VP1 and VP2 capsid proteins and the major capsid protein, VP3, to form an infectious viral particle). This method further enables the incorporation of bioconjugation handles (up to two bioconjugation handles per capsid protein), allowing for the precise binding of 15 or 20 bioconjugation groups per capsid.

[0012] More specifically, the present invention encompasses a method for generating an AAV vector in which one or a combination thereof of the minor capsid proteins VP1 and VP2 can be site-specifically chemically modified. Since these three capsid proteins exist in different stoichiometric concentrations (5 copies, 5 copies, and 50 copies of VP1, VP2, and VP3 per capsid, respectively), the method provides the ability to introduce modifications to the capsid with precise control over both the modification site and the number of modifications, and to produce a homogeneous AAV conjugate that retains the biological activity of the virus, particularly its ability to infect target cells.

[0013] As described herein, it is possible to site-specifically incorporate native and non-native amino acids (UAAs) into capsid proteins using engineered aminoacyl-tRNA synthetase (aaRS) / tRNA pairs that suppress nonsense codons. Due to their small size, UAAs are well-accepted in AAV capsids at many positions.

[0014] The present invention encompasses genetically modified adeno-associated viruses (AAVs) in which the AAV capsid comprises at least one variant minor capsid protein VP1, VP2, or both VP1 and VP2, and these variant minor capsid proteins are mutated at one or more amino acid residue sites compared to wild-type AAV VP1 or VP2 capsid proteins, incorporating native or non-native amino acids (UAAs) that are not present in wild-type AAV. Importantly, the genetically modified AAVs described herein retain their infectivity compared to wild-type AAV under equivalent conditions.

[0015] In particular, the present invention relates to a genetically modified adeno-associated virus (AAV) comprising a variant minor capsid protein, wherein the capsid coding sequence (SEQ ID NO: 1) is mutated at the translation origin of the AAV VP1, VP2, or VP3 capsid protein open reading frame (ORF), preventing translation of VP1, VP2, or both VP1 and VP2, resulting in a genetically modified AAV in which VP1, VP2, VP3, or both VP1 and VP2 are deleted. The deleted / deficient capsid protein is then supplied in trans (i.e., expressed) from a second capsid coding sequence encoding the deleted minor capsid protein(s), resulting in a genetically modified AAV having one or more variant capsid proteins. In this specification, VP2 is also represented by SEQ ID NO: 2, and VP3 is also represented by SEQ ID NO: 3.

[0016] The genetically modified AAV capsid protein contains a sequence with sequence identity of at least approximately 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 90%-99% of sequence identity of sequence number 1. This variant capsid protein is mutated at specific sites to delete VP1, VP2, or both VP1 and VP2 (for example, as shown in Figure 2). For example, at position T454 of sequence number 1, a stop codon can be inserted using the techniques described herein. Such techniques are also described in PCT / US2020 / 055834, PCT / US2020 / 038766, or PCT / US2020 / 029567, the teachings of which are incorporated herein by reference in their entirety. More specifically, the stop codon incorporated into the variant capsid protein may be a TAG, TAA, or TGA suppressor stop codon.

[0017] The minor capsid proteins CMV-VP1-delVP23 (Figure 20, SEQ ID NO: 7); CMV-VP2-delVP3 (Figure 21, SEQ ID NO: 9); and CMV-VP1-VP2-delVP3 (Figure 22, SEQ ID NO: 10). While CMV promoter sequences are exemplified as described herein, any suitable promoter sequence can be incorporated. In addition, the minor capsid proteins can incorporate natural or non-natural amino acids as described herein.

[0018] It is important to note that a key advantage of the genetically modified AAV of the present invention is that the UAA incorporated into the minor capsid protein is at a low level, and therefore, their production efficiency is not impaired (or significantly affected) by the incorporation of UAA. Consequently, AAV packages / associates with an infectivity titer substantially comparable to that of wild-type viruses.

[0019] The variant capsid protein of the genetically modified AAV of the present invention may contain one or more mutant amino acid residues of VP1, VP2, or both VP1 and VP2, and this mutant amino acid residue site incorporates a non-natural amino acid (UAA). Examples of UAAs suitable for use in the present invention are shown in Figure 1, which shows formulas 1 to 12 (formulas 1 to 6 from left to right in the first row from the top, and formulas 7 to 12 from left to right in the second row, including the length of the carbon chain extension portion and substituents). More specifically, non-natural amino acids suitable for use in the present invention can be selected from the group consisting of phenylalanine analogs, tyrosyl analogs, tryptophanyl analogs, or lysyl analogs. In particular, the above analogues include p-benzoylphenylalanine (pBpA), O-methyltyrosine (OMeY), 5-azidotryptophan, 5-propargyloxyaminotryptophan, 5-aminotryptophan, 5-methoxytryptophan, 5-O-allyltryptophan, 5-bromotryptophan, azido-lysine (AzK), or N ε - Acetyllysine (AcK), C5Az, LCA, N ε - Acetyllysine (AcK), cyclopropene amino acid, N εThe following are selected from the group consisting of -(1-methylcyclopropa-2-enecarboxamide)-lysine (CpK), 5-hydroxytryptophan (5-HTP), LCALk, DiaazK, and LCKet.

[0020] Alternatively, mutations in the variant minor capsid protein of genetically modified AAV can be in native amino acid residues such as cysteine ​​or selenocysteine. Specifically, these native amino acid residues are different from the amino acid residues located at the same position in wild-type AAV.

[0021] The genetically modified AAV of the present invention may further include bioconjugation handles. Such bioconjugation handles can be covalently attached in a site-specific manner to mutated native or non-native amino acid residues of the variant minor capsid protein. As described herein, 5 to 10 bioconjugation handles can be specifically incorporated into the minor capsid protein, thus resulting in a genetically modified AAV having 5 to 10 bioconjugation handles per fully associated AAV capsid. In this case as well, a genetically modified AAV containing at least one variant minor capsid protein VP1, VP2, or both VP1 and VP2 retains target cell infectivity comparable to wild-type AAV under similar conditions, such as cell culture conditions. For example, the mutated amino acids may be conjugated with chemical or biological entities (for example, the biological entities may include proteins, peptides, nucleic acids, lipids, or carbohydrates).

[0022] Specifically included by the present invention is a genetically modified infectious AAV comprising a capsid containing a variant capsid protein. For example, this variant capsid protein can comprise the sequence of SEQ ID NO: 1, or a sequence having at least about 80% sequence identity with SEQ ID NO: 1, and this sequence is mutated to lack VP1, VP2, or both VP1 and VP2. The mutation of the capsid of the genetically modified AAV may be at one or more positions, such as positions 263, 454, 456, 587, and / or 588, and these mutated positions are relative to the wild-type VP1 sequence. It is also possible that the VP1 sequence is mutated at positions 263, 454, 456, and 588 and not mutated at position 587. Alternatively, it is possible that the VP1 sequence is mutated at positions 263, 454, 456 and not mutated at either position 585 or 588. The characteristics of the infectious genetically modified AAV of the present invention are an infectivity substantially comparable to that of wild-type AAV under equivalent conditions, the packaging of AAV having a titer substantially comparable to that of wild-type AAV under equivalent conditions, or both an infectivity and packaging comparable to wild-type AAV.

[0023] In one embodiment of the present invention, the mutated amino acid residue(s) of the modified AAV is / are functionalized or conjugated with a chemical entity or a protein entity (also referred to herein as a bioconjugation handle or group). As described herein, the chemical entity or protein entity is selected from the group consisting of a probe, a small molecule ligand, a peptide, a cyclic peptide, a nucleotide, a polymer, a protein, or a virus conjugate. In a particular embodiment, the genetically modified infectious AAV comprises a chemical entity or a protein entity, and this entity is the cyclic peptide cRGD polyethylene glycol (PEG).

[0024] In another embodiment of the present invention, the mutated amino acid residue site of the variant VP1 or VP2 incorporates a natural amino acid. In particular, this natural amino acid is cysteine or selenocysteine.

[0025] The genetically modified infectious AAV of the present invention, containing the above-mentioned variant capsid protein(s) (e.g., genetically modified AAV containing a mutant capsid protein functionalized with cRGD peptide), can be “retargeted” by binding to, recognizing, or interacting with its native congenital cell receptor, or by binding to, recognizing, or interacting with non-congenital target cells. As described herein, any number of retargeting groups bound per capsid dramatically affects the properties of the resulting AAV conjugate. This highlights the importance of controlling site and stoichiometry to alter the properties of AAV vectors through chemical modification. As described herein, precise and selective chemical modification of AAV capsids is now possible with unprecedented control over both site and stoichiometry (how many modifications are introduced per capsid). Such control will be crucial for the development of engineered AAV vectors with more therapeutically desirable properties.

[0026] A method for producing a genetically modified AAV with infectivity, wherein the AAV contains a variant AAV capsid protein, and VP1, VP2, or both VP1 and VP2 contain one or more mutated amino acid residue sites as compared to wild-type VP1, VP2, or both VP1 and VP2, is also encompassed by the present invention. Generally, the method includes the steps of providing competent host cells in culture, and transfecting the cultured cells with: 1) an AAV variant VP3 that does not express VP1 or VP2, or does not express both VP1 and VP2; 2) a variant VP1, VP2, or both VP1 and VP2 having an appropriate promoter that does not express VP3; 3) one or more plasmids containing additional factors necessary for AAV expression; 4) providing the necessary unnatural amino acids; and 5) providing a plasmid encoding an engineered aminoacyl-tRNA synthetase / tRNA pair that selectively introduces (charges) the unnatural amino acid in response to a stop codon. The above cells and plasmids are cultured under conditions sufficient for plasmid gene expression and AAV assembly, thereby producing a genetically modified AAV with infectivity containing a variant capsid protein, in which VP1, VP2, or both VP1 and VP2 are mutated at one or more amino acid residues as compared to wild-type AAV.

[0027] More specifically, the method includes the steps of providing competent host cells in culture, and co-transfecting the cultured cells with a plasmid / construct containing sequences necessary for the assembly and expression of the genetically modified infectious AAV. One mRNA encodes VP1, VP2, or VP3 having different start codon sites (see Figure 2). When the starting point is mutated, the expression of VP1, VP2, or VP3 can be controlled by expressing separate mRNAs for each capsid protein, and the capsid proteins can be selectively mutated, but the mutated minor capsid proteins VP1 and / or VP2 and the wild type can be assembled to obtain a genetically modified AAV with infectivity.

[0028] For example, as described herein, non-natural amino acids can be site-specifically incorporated into VP1 and / or VP2 by co-transfecting competent host cells with a plasmid encoding a pair of engineered tRNA synthetase and tRNA that introduces the non-natural amino acid. More specifically, an orthogonal tRNA / aaRS pair is provided, comprising an engineered amino-acylRNA synthetase (aaRS) and its corresponding tRNA, for co-translationally incorporating a non-natural amino acid (UAA) in response to a variant VP1, VP2, VP3, or both VP1 and VP2 mutation sites, and the non-natural amino acid required for incorporation into the mutation site is provided.

[0029] Along with plasmids encoding mutant VP1 and / or VP2 and wild-type VP3 transcripts, plasmids (multiple types) encoding further factors required for AAV capsid expression and complete capsid assembly (i.e., including VP1, VP2, and VP3) in cell culture are also provided (to be co-cultured). The cells, plasmids, and UAA are cultured / maintained under conditions sufficient for plasmid gene expression and AAV assembly, thereby producing infectious genetically modified AAV containing variant AAV capsid proteins in which VP1, VP2, or both VP1 and VP2 are mutated at one or more amino acid residue sites compared to wild-type AVV.

[0030] The manipulated aminoacyl-tRNA synthetase-tRNA pairs can be derived from E. coli leucyl pairs, E. coli tryptophanyl pairs, E. coli tyrosyl pairs, or archaeal pyrrolicyl pairs.

[0031] The infectious genetically modified AAV can be recovered / collected from cell cultures and its biological activity can be evaluated using techniques described herein and known to those skilled in the art.

[0032] The mutant amino acid residue sites of infectious genetically modified AAV can incorporate unnatural amino acid (UAA) analogs shown in Figure 1, formulas 1-12. More specifically, the unnatural amino acids are selected from the group consisting of phenylalanine analogs, tyrosyl analogs, tryptophanyl analogs, or lysyl analogs. These analogs include p-benzoylphenylalanine (pBpA), O-methyltyrosine (OMeY), 5-azidotryptophan, 5-propargyloxytryptophan, 5-aminotryptophan, 5-methoxytryptophan, 5-O-allyltryptophan, 5-bromotryptophan, azido-lysine (AzK), or N ε - Acetyllysine (AcK), C5Az, LCA, N ε - Acetyllysine (AcK), cyclopropene amino acid, N ε -(1-methylcyclopropa-2-enecarboxamide)-lysine (CpK), 5-hydroxytryptophan (5-HTP); selected from the group consisting of LCalk, DiaazK, and LCKet.

[0033] Alternatively, the variant capsid protein may incorporate a native amino acid, which may be cysteine ​​or selenocysteine.

[0034] The mutant amino acids of the above variant capsid protein can be functionalized by a chemical or protein entity, which is selected from the group consisting of probes, small molecule ligands, peptides, cyclic peptides, nucleotides, polymers, proteins, or viral conjugates. In one embodiment, the entity functionalizing the mutant amino acids of the variant capsid protein of the infectious genetically modified AAV is a cyclic peptide cRGD.

[0035] The infectious genetically modified AAV may contain a variant VP1 amino acid sequence that includes SEQ ID NO: 1, or a sequence having at least approximately 80% sequence identity with SEQ ID NO: 1, and this variant VP1 capsid protein is mutated at one or more positions located at 263, 454, 456, 587, and / or 588 of the VP1 sequence compared to the wild-type VP1 sequence. It is also possible that the VP1 sequence is mutated at positions 263, 454, 456, and 588, but not at position 587. Alternatively, the VP1 sequence is mutated at positions 263, 454, and 456, but not at positions 585 or 588.

[0036] The infectious genetically modified AAV may include a mutant VP2 amino acid sequence containing Sequence ID No. 2, or a sequence having at least approximately 80%, 85%, 90%, 95%, or 99% sequence identity with Sequence ID No. 2.

[0037] The infectious genetically modified AAV may include a VP3 amino acid sequence containing SEQ ID NO: 3, or a sequence having at least approximately 80%, 84%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 3.

[0038] The present invention also encompasses therapeutic or antigenic compositions comprising genetically modified AAVs as described herein, and further comprising one or more therapeutic or antigenic gene constructs suitable for gene therapy or vaccination (e.g., inducing an immune response in a subject). If the composition is a vaccine composition containing an antigen, the composition may further comprise an adjuvant.

[0039] Methods for treating diseases or conditions in subjects are also included in the present invention. For example, such a method may include the step of administering a therapeutic composition to a subject, the composition comprising a therapeutic amount of the AAV vector described herein and a gene construct encoding a protein or peptide, which can reduce or alleviate the disease or condition in the subject. The use of the gene-modified AAV vector of the present invention in therapeutic compositions may be particularly useful in treating diseases such as cancer or cystic fibrosis of sickle cell anemia, and providing a substitution gene encoding a functional protein in a targeted manner would reduce or completely alleviate the symptoms of cancer or disease.

[0040] Another example is a method for inducing an immune response in a subject, the method comprising the step of administering an antigenic / vaccine composition to the subject, the antigenic composition comprising the genetically modified AAV vector of the present invention and a gene construct encoding an antigenic protein or peptide that can induce an immune response in the subject. Such a method using the AAV vector of the present invention would specifically target immune cells that can initiate an immune response.

[0041] A kit containing the genetically modified AAV of the present invention is also included in the present invention. Such a kit may include a suitable vial containing the genetically modified AAV described herein, for example, a vial of AAV in a sterile diluent, and a vial / container of supplemental components for growing AAV in, for example, a cell culture. An explanatory brochure may also be included in the kit.

[0042] The above and other features of the present invention, including details of various novel configurations and combinations of components, as well as other advantages, will be described in more detail hereby with reference to the accompanying drawings and pointed out in the claims. It will be understood that specific methods and apparatus embodying the present invention are shown as examples and are not intended to limit the invention. The principles and features of the present invention may be employed in a variety of numerous embodiments without departing from the scope of the invention. [Brief explanation of the drawing]

[0043] In the attached drawings, reference numerals indicate the same part across different drawings. The drawings are not necessarily to scale. Instead, the emphasis is on illustrating the principles of the invention. The patent or application file includes at least one drawing performed in color. A copy of this patent or patent application publication with color drawings is available from the Office upon request and payment of the required fees.

[0044] [Figure 1] Figure 1 shows examples of structures of native and unnatural amino acids that can be incorporated into the AAV capsid using controlled stoichiometry. In particular, UAA analogs containing formulas 1-12 (formulas 1-6 from left to right in the first row, and formulas 7-12 from left to right in the second row, including the length of the carbon chain extension and substitutions) are shown. [Figure 2] Figure 2 shows a scheme for selectively introducing mutations (natural or non-natural amino acids) into VP1, VP2, or VP1 and VP2. The three capsid proteins are expressed from the same open reading frame (Cap) via alternative splicing and start codon use frequency. Mutations are manipulated at translational origins (indicated by red ×s) that prevent the expression of VP1, VP2, or VP3 from this ORF. The deficient minor capsid protein can then be supplied trans, driven by a strong promoter such as CMV. By isolating the expression of VP1, VP2, or VP1+VP2 from the remaining capsid proteins, it becomes possible to selectively mutate them without affecting the other capsid proteins. [Figure 3] Figure 3 shows the packaging of AAV2 in HEK293T cells using the construct described in Figure 2. AAV with variant capsid protein yields comparable results to the original system. [Figure 4]Figure 4 shows the infectivity of the packaged titer viruses produced in Figure 3 at a constant titer, as measured by their ability to deliver and express the EGFP reporter gene in HEK293T cells. [Figure 5] Figure 5 shows the selective non-native amino acid mutagenesis of individual minor capsid proteins in AAV capsids, and their use for chemically binding fluorophores. [Figure 6] Figure 6 shows that the number of retargeting ligands bound to the AAV capsid dramatically affects its retargeting efficiency. [Figure 7A] Figures 7A–7C show the precise labeling of AAV at the manipulated cysteine ​​residues. [Figure 7B] Figures 7A–7C show the precise labeling of AAV at the manipulated cysteine ​​residues. [Figure 7C] Figures 7A–7C show the precise labeling of AAV at the manipulated cysteine ​​residues. [Figure 8A] Figure 8A shows the amino acid sequence of AAV isoform VP1 (SEQ ID NO: 1). [Figure 8B] Figure 8B discloses sequence number 2. [Figure 8C] Figure 8C discloses Sequence ID 3. [Figure 9] Figure 9 shows the amino acid sequence (SEQ ID NO: 2) of the AAV capsid protein isoform VP2. [Figure 10] Figure 10 shows the amino acid sequence (SEQ ID NO: 3) of the AAV capsid protein isoform VP3. [Figure 11(1)] Figures 11A–11C show the selective incorporation of UAA C5Az into either VP1, VP2, or VP1+VP2. [Figure 11(2)] Figures 11A–11C show the selective incorporation of UAA C5Az into either VP1, VP2, or VP1+VP2. [Figure 12(1)] Figures 12A to 12C show the results of the LCA incorporated into VP1. [Figure 12(2)]Figures 12A to 12C show the results of the LCA incorporated into VP1. [Figure 13(1)] Figures 13A to 13C show the results of incorporating CpK into VP1. [Figure 13(2)] Figures 13A to 13C show the results of incorporating CpK into VP1. [Figure 14(1)] Figures 14A to 14C show the results of integrating 5HTP into VP1. [Figure 14(2)] Figures 14A to 14C show the results of integrating 5HTP into VP1. [Figure 15] Figures 15A-15B show the incorporation of several other unnatural amino acids, LCalk, DiazK, and LCKet, into VP1. [Figure 16] Figures 16A-16B show the results of selective PEGylation of AAV at VPI site 454. [Figure 17(1)] Figure 17 shows the nucleic acid sequence (SEQ ID NO: 4) encoding RC2-VP1-del. The location of the mutation is indicated in red. [Figure 17(2)] This is a continuation of Figure 17(1). [Figure 18(1)] Figure 18 shows the nucleic acid sequence (SEQ ID NO: 5) encoding RC2-VP2-del. The location of the mutation is shown in red. [Figure 18(2)] This is a continuation of Figure 18(1). [Figure 19(1)] Figure 19 shows the nucleic acid sequence (SEQ ID NO: 6) encoding RC2-VP12-del. The mutation site is indicated in red. [Figure 19(2)] This is a continuation of Figure 19(1). [Figure 20] Figure 20 shows the nucleic acid sequence (SEQ ID NO: 7) encoding CMV-VP1-delVP23. The location of the mutation is indicated in red. [Figure 21] Figure 21 shows the nucleic acid sequence (SEQ ID NO: 8) encoding CMV-VP2-delVP3. The location of the mutation is shown in red. [Figure 22(1)]Figure 22 shows the nucleic acid sequence (SEQ ID NO: 9) of CMV-VP1-VP2-delVP3. The location of the mutation is shown in red. [Figure 22(2)] This is a continuation of Figure 22(1). [Figure 23A] Figure 23A is the plasmid map of pIDTsmart-ITR-GFP-4xEcLtR-LeuRS. [Figure 23B(1)] Figure 23B shows the nucleic acid sequence (SEQ ID NO: 10) of the plasmid described above. [Figure 23B(2)] This is a continuation of Figure 23B(1). [Figure 23B(3)] This is a continuation of Figure 23B(2). [Figure 23B(4)] This is a continuation of Figure 23B(3). [Figure 24A] Figure 24A shows the plasmid map of pIDTsmart-TrpRS-8xWtR-ITR-GFP. [Figure 24B(1)] Figure 24B shows the nucleic acid sequence (SEQ ID NO: 11) of the plasmid described above. [Figure 24B(2)] This is a continuation of Figure 24B(1). [Figure 24B(3)] This is a continuation of Figure 24B(2). [Figure 24B(4)] This is a continuation of Figure 24B(3). [Figure 24B(5)] This is a continuation of Figure 24B(4). [Modes for carrying out the invention]

[0045] The present invention is described more fully below with reference to the accompanying drawings illustrating exemplary embodiments of the invention. However, the invention may be embodied in many different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided to ensure that this disclosure is thorough and complete and fully conveys the scope of the invention to those skilled in the art.

[0046] As used herein, the term "and / or" includes any and all combinations of one or more of the enumerated items relating to it. Furthermore, the singular forms and the articles "a," "an," and "the" also include the plural forms unless otherwise explicitly stated. In addition, the following terms will be understood: includes, comprises, including, and / or comprising, as used herein, specify the presence of a described feature, integer, process, operation, element, and / or component, but do not exclude the presence or addition of one or more other features, integers, processes, operations, elements, components, and / or groups thereof. Furthermore, when an element containing a component or subsystem is mentioned and / or indicated as being connected to or combined with another element, it will be understood that the element may be directly connected to or combined with the other element, or that intervening elements may be present.

[0047] The terms "first" and "second," etc., are used in this specification to describe various elements, but it will be understood that these elements should not be limited by these terms. These terms are used solely to distinguish one element from another. Thus, the elements discussed below may be called second elements, and similarly, second elements may be called first elements, to the extent that they do not depart from the teachings of the present invention.

[0048] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless they are explicitly defined herein in an idealized or overly formal sense.

[0049] It should be noted that the description of the present invention is not limited to these specific methodologies, compositions, cell lines / biosystems, or any other standard protocols. The present invention discloses a general platform for producing AAV vectors that can be chemically functionalized using chemoselective reactions with control over the modification site and stoichiometry, and the cell type used for viral packaging, the characteristics (personalities) of the engineered aminoacyl-tRNA synthetase (aaRS) or tRNA or UAA or native amino acids, the chemical reactions used to introduce modifications, etc., may change with advances in the art and in this research.

[0050] Previously, UAA integration into AAV capsids was directed at all 60 capsid proteins. Since the three overlapping capsid proteins are expressed from the same open reading frame (ORF) Cap via alternative splicing and start codon use frequency, selective modification of a subset of these proteins was impossible. However, subsequent chemical modifications of all 60 capsid proteins disrupt viral infectivity (e.g., Figure 6, blue traces). While the mechanism of this disruption is not well understood, it is reasonable to assume that molecular processes associated with the complex viral entry pathway may be affected by capsid overmodification. Furthermore, it is desirable to be able to construct AAV vectors that can site-specifically introduce a distinct number of chemical modifications. Indeed, retargeting experiments demonstrate the existence of an optimal number of ligands per capsid required for efficient retargeting (Figure 6). The degree of modification of AAV capsids with 60 UAA handles can be controlled by controlling the concentration of the modifying reagent or the reaction time (Figure 6). However, this results in a heterogeneous mixture of capsids in different modification states. The ability to create homogeneous AAV conjugates with a distinct number of modifications introduced per capsid is crucial for gene therapy.

[0051] In the AAV capsid, the three capsid proteins VP1, VP2, and VP3 are incorporated in a stoichiometric ratio of approximately 1:1:10. As a result, for a total of 60 copies of capsid protein per viral particle, approximately 5 copies each of the two minor capsid proteins VP1 and VP2 are present in the capsid, and 10 copies of VP3 are present. The ability to selectively introduce modified, modified native or non-native amino acid residues into these minor capsid proteins provides a means to introduce a controlled number of handles per capsid. However, since all AAV capsid genes are encoded in the same ORF and expressed by alternative splicing and start codon use frequency, it is difficult to mutate a single capsid protein without affecting others. This invention describes a method for separately expressing minor capsid proteins (VP1 or VP2 or VP1+VP2) by introducing mutations into the origins of translation of VP1 and / or VP2 so that VP1 and / or VP2 are not expressed from a native Cap ORF. The deficient capsid proteins can then be expressed in trans from a strong promoter, e.g., the CMV promoter described herein. Expression of undesirable capsid proteins (e.g., VP3) from this second ORF can also be similarly eliminated by mutating the origin of translation (Figure 2). This platform provides the ability to selectively manipulate any subset of the three capsid proteins by expressing any combination of the three capsid proteins from a single ORF and isolating the expression of a selected third capsid protein from a second ORF.

[0052] Using the platform described herein, it is now possible to selectively introduce unique non-coding codons (such as nonsense codons, tetranucleotide codons, or codons containing non-natural base pairs) into any combination of three capsid proteins by site-directed mutagenesis. When such mutant Capsules are co-expressed with a suitable engineered aminoacyl-tRNA synthetase (aaRS) / tRNA pair capable of decoding the unique codons, UAA residues can be incorporated into these sites.

[0053] In one embodiment of the present invention, UAA AzK was incorporated into residue T454 (VP1 numbering) of VP1, VP2, or VP1+VP2 (Figure 3). A pyrrolysyl-tRNA synthetase / tRNA pair was used. The resulting virus was packaged with efficiency and infectivity comparable to that of the wild-type virus, demonstrating that these modifications are well tolerated (Figures 3 and 4). The importance of controlling the number of modifications per capsid was demonstrated by the differential behavior of AAVs in which UAA was incorporated at 5 copies per capsid (mutation in either VP1 or VP2 alone), 10 copies per capsid (mutation in both VP1 and VP2), or 60 copies per capsid (all three capsid proteins were mutated). Retargeting ligands were then bound to these UAA handles (Figure 6). AAVs with 60 binding handles allowed for efficient retargeting in intermediate modified states, but complete modification resulted in the loss of all infectivity. Five or ten modifications per capsid were well tolerated, but ten retargeting ligands per capsid were required for efficient retargeting (Figure 6).

[0054] It should be noted that any other combination of capsid proteins, and any site within these proteins, can be manipulated using this strategy. The N-terminus of the capsid protein is particularly well suited for mutation because this section of the capsid protein is essentially internal when folded into a functional capsid, while the C-terminus of the capsid protein is exposed.

[0055] In addition, any other non-natural amino acids (exemplary examples are shown in Figure 1 and described herein) can be incorporated using other aaRS / tRNA pairs (including, but not limited to, bacterial tyrosyl, tryptophanyl, and leucyl-tRNA synthetase / tRNA pairs). This technique can also be extended to any other natural serotype of AAV as well as engineered and evolved variants of AAV.

[0056] Cysteine ​​and selenocysteine ​​are native amino acid residues found in proteins. Due to their low abundance and unique reactivity, they can be used in site-selective bioconjugation reactions. In another embodiment of the present invention, engineered surface-exposed cysteine ​​residues can be introduced into minor capsid proteins (Figures 7A and 7B). When introduced into all 60 capsid proteins, the same mutation was not well tolerated, resulting in low titers and poor infectivity. However, when surface-exposed cysteine ​​residues were introduced only into VP1 or VP2 at the T454 site, robust viral packaging and infectivity were observed. The ability to selectively modify engineered cysteine ​​residues on minor capsid proteins was also demonstrated (Figure 7C).

[0057] Due to the complexity associated with the association and cell entry processes that AAV capsids have optimized throughout evolution, AAV capsids often resist attempts to manipulate capsid proteins through native / non-native amino acid mutagenesis, loop insertions, protein fusions, etc. However, manipulation of only minor capsid proteins is considerably more tolerable, as it introduces considerably less overall disruption to the entire capsid. This is demonstrated by the tolerance of manipulated cysteine ​​residues in minor capsid proteins, but not everywhere (Figure 7). Thus, the present invention offers an opportunity to introduce more aggressive manipulations to alter the properties of AAV capsids by manipulating minor capsid proteins. In addition to native and non-native amino acid mutagenesis, the present invention can also be used to introduce peptide and protein fusions and insertions into minor capsid proteins that can either directly provide beneficial traits (e.g., binding to a specific target) or selectively modify them via chemical or enzymatic reactions (e.g., biotinylation tags, SNAP or HALO tags, etc.).

[0058] The present invention enables the introduction of a distinct number of manipulated residues (natural or unnatural) per capsid by selectively mutagenerating VP1, VP2, or VP1+VP2. Further control of the number of manipulated sites can be achieved by introducing multiple manipulated residues into VP1, VP2, or VP1+VP2. As described herein, various sites of the VP1 capsid protein are selectively mutated, such as 263, 454, 456, 587, and 588 (this numbering corresponds to amino acid residues of wild-type VP1). The platform can be extended to any packaging platform, including but not limited to mammalian cells, insect cells, and cell-free translation / packaging systems.

[0059] The present invention enables the incorporation of numerous native and unnatural amino acid residues into AAVs with site and copy number control using a wide variety of different chemistry that can be used to chemoselectively bind various entities. In one embodiment of the present invention, an azide-containing UAA was selectively introduced into the minor capsid protein of an AAV and subsequently conjugated to a fluorophore or retargeting ligand using a strain-enhanced azide-alkyne click reaction (Figures 5 and 6). In another embodiment, an engineered cysteine ​​residue in the minor capsid protein was introduced and subsequently conjugated to a fluorophore using a cysteine-maleimide coupling reaction. Capsid modification can be applied to any other chemoselective conjugation reactions, including but not limited to the following: reverse electron-demanded Diels-Alder reactions between distorted alkenes and tetrazine, or between furan and maleimide; condensation reactions between aldehydes / ketones and amino-oxy / hydrazine groups; chemoselective fast azo coupling reactions (CRACR); oxidative coupling reactions and photocatalytic coupling reactions; and nucleophilic substitution / addition to various electrophiles by cysteine ​​or selenocysteine ​​residues.

[0060] The methods described herein, but are not limited, can be used to conjugate a wide variety of entities, including probes (fluorescent probes, radioactive probes, MRI probes, luminescent probes, etc.), small molecule ligands, peptides, cyclic peptides, nucleotides (DNA, RNA, LNA, PNA, etc.), polymers (PEG, etc.), carbohydrates (e.g., sialic acid, etc.), proteins (e.g., enzymes, nanobodies, antibodies, etc.), and other AAVs of the same or different serotypes. Such conjugations can provide AAV conjugates that efficiently retarget to user-defined receptors and thus alter tissue affinity. Immune-evading AAVs can also be produced by site-specifically conjugating a group that passively protects the capsid from the immune system (PEG, peptides, carbohydrates, or other polymers, etc.) or a ligand that actively binds to an inhibitory receptor on an immune cell to switch off the immune response (e.g., SIGLEC ligand). Such conjugations can also be used to conjugate enzymes to AAV capsids to produce capsids with a superior infectivity profile, or to conjugate two AAVs of the same or different serotypes to create a novel class of vectors with expanded cargo capacity and novel tropism.

[0061] The controlled AAV modification techniques described herein can be used in many applications, including, but are not limited to, targeting AAV vectors to desired cell types by conjugating retargeting ligands, DNA / RNA / PNA aptamers, etc., including small molecules, peptides, cyclic peptides, nanobodies, antibodies and antibody fragments; optimizing the properties of such conjugates by systematically controlling the binding site, the number of retargeting groups per capsid, and the chemical properties of the linker; and weakening the immune response produced by AAV vectors by controlled binding of immunomodulatory entities, including but not limited to polyethylene glycol and other polymers, carbohydrates (e.g., sialic acid, etc.), ligands that bind to suppressive receptors on immune cells (e.g., SIGLEC receptors).

[0062] In particular, the genetically modified AAV of the present invention can be used as an enhancement vector for gene therapy. Using the techniques described herein, AAV can be genetically modified to specifically target / direct the delivery of therapeutic or antigenic gene constructs to target cells with enhanced / increased efficacy compared to unmodified AAV vectors. Cells can be cultured in vitro, in vivo, or ex vivo and delivered to subjects requiring it. As used herein, the term “subject” can include any animal subject, and in particular mammalian subjects such as humans. Human subjects can be treated with the AAV gene vector described herein for medical purposes such as the treatment of an existing disease, disorder, or condition, or as a preventive measure to prevent the onset of a disease, disorder, or condition, or animal subjects can be treated for medical, veterinary, or development purposes. The methods and compositions of the present invention may be used to treat any type of cancerous tumor or cancer cell. In addition, the genetically modified AAV of the present invention can be used in vaccine compositions in which a nucleic acid sequence encoding an antigenic substance such as a protein or peptide is delivered to target cells together with further components such as an adjuvant that induces an immune response in the subject.

[0063] Gene constructs delivered by genetically modified AAVs are therapeutically effective. As used herein, “therapeutic effective” means a sufficient amount to produce a desired biological effect (e.g., a sufficient amount to inhibit tumor growth, induce an immune response to an antigen, or inhibit an autoimmune disease) in at least a subset of cells in a subject, with a reasonable effect / risk ratio applicable to any medical treatment. The therapeutic effective amount of a construct / agent used in the present invention can be readily determined by those skilled in the art, by the use of known techniques, and by observing results obtained under similar circumstances.

[0064] The technology can also be used to conjugate two separate AAV vectors of the same or different serotypes using a bifunctional linker to increase the overall size of the genetic cargo delivered per cell. Such novel conjugates between two different AAV vectors also have a unique tendency to conjugate external payloads (e.g., proteins, small molecules, nucleic acids, probes, etc.) onto viral capsids delivered to cells in vitro and in vivo for research or therapeutic purposes, either independently or in conjunction with the genetic cargo inside the AAV capsid.

[0065] Finally, this technology can be used to investigate the entry pathways of AAV capsids into mammalian cells by incorporating fluorescent probes, photocrosslinkers, and affinity handles (such as biotin).

[0066] Without further detail, those skilled in the art will likely be able to make the most of the present invention based on the above description. Therefore, the following specific embodiments and examples should be construed as merely illustrative and not to limit the remainder of this disclosure.

[0067] The integration of UAA into the AAV capsid requires the efficient expression of its capsid protein containing the desired UAA modification in competent cells (e.g., mammalian cells) that serve as hosts for viral amplification. As previously mentioned, the UAA integration site can be identified by a stop codon (TAG, etc.), and the desired UAA can be delivered by an engineered tRNA / aminoacyl-tRNA synthetase pair having a congeneral anticodon. Consequently, the production of UAA-modified viruses must be accompanied by the co-expression of genetic components necessary for viral amplification and the genetic components necessary for viral amplification.

[0068] To incorporate UAA into the AAV capsid protein, substitutions of several surface-exposed endogenous amino acid residues were targeted on the AAV capsid (guided by the crystalline structure of the viral particle; see, for example, Xie et al., The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy, PNAS, August 6, 2002, Vol. 99, No. 16, p. 10407). Since such capsid mutations can potentially disrupt viral infectivity, regions known to tolerate change, such as the region on the 3x proximal spike, were targeted. Substitutions of essential arginine residues were also targeted in the heparan sulfate receptor-binding region, which disrupts innate tropism and promotes retargeting. This "erases" the innate host cell preference of AAV, making it possible to rewrite it with targeted agents using precise labeling methodologies. For example, the targeted site can be located in a conserved domain among all three AAV capsid proteins, resulting in their substitution by UAA in all of them. AAV can be generated, for example, by transfecting HEK293T cells with a plasmid containing the required element in the presence of UAA azido-lysine ("AzK"). In a specific example, the AzK amino acid was incorporated into T454 in response to the stop codon TAG using pyrrolysyl-tRNA synthetase from Methanosarcina barkeri and pyrrolysyl-tRNA (TAG suppressor) from M. mazeii. [Examples]

[0069] The plasmid maps and sequences used in the examples described below can be found in Figures 17 to 24.

[0070] Example 1: Packaging of AAV2 Figure 3 shows the packaging of AAV2 in HEK293T cells to comparable yields compared to the original system, using the constructs described in Figure 2. The qPCR titers of the original (wild-type (WT)) AAV2 are shown, and the titers of the rest are shown relative to WT. ΔVP1 and ΔVP2 represent AAV2 packaged using the Cap gene with VP1 and VP2 expression excluded, respectively. The virus still efficiently associates in the absence of the minor capsid protein. ΔVP1-CMV-VP1 and ΔVP2-CMV-VP2 represent AAV2 packaged using the Cap gene with VP1 and VP2 expression excluded, respectively, and each protein expressed trans from the CMV promoter. In these systems, the T454 residue in VP1 or VP2 is mutated to a TAG and repressed using a pyrrolysyl-tRNA synthetase / tRNA pair to incorporate UAA(AzK), which are represented by ΔVP1-CMV-VP1-454AzK and ΔVP2-CMV-VP2-454AzK, respectively. The packaging yield of the virus in the presence or absence of UAA added to the culture medium is shown.

[0071] Example 2: Infectivity of packaged viruses Figure 4 shows the infectivity of the packaged titer viruses produced in Figure 3 at a constant titer, as measured by the ability of the virus to deliver and express the EGFP reporter gene in HEK293T cells. ΔVP1 and ΔVP2 viruses are well packaged, but they exhibit significantly attenuated infectivity. Trans-supply of VP1 and VP2 (ΔVP1-CMV-VP1 and ΔVP2-CMV-VP2) rescues infectivity. ΔVP1-CMV-VP1-454AzK and ΔVP2-CMV-VP2-454AzK viruses produced in the presence of UAA exhibit strong infectivity, but not in its absence. Note that in the absence of UAA, TAG variants of the minor capsid protein are not expressed.

[0072] Example 3: Selective fluorophore labeling of mutant AAV Figure 5 illustrates the selective non-natural amino acid mutagenesis of individual minor capsid proteins in AAV capsids and their use for chemically binding fluorophores. After purification, different recombinant AAV2 preparations were labeled with cyclooctin-fluorophores that selectively label azide groups present in UAA AzK. The upper panel shows SDS-PAGE analysis of the AAV2 preparations, and the lower panel shows fluorescence images of the same gel. As expected, wild-type AAV2 showed no labeling, T454AzK (AzK in all 60 capsid proteins) showed labeling of all capsid proteins, while ΔVP1-CMV-VP1-454AzK and ΔVP2-CMV-VP2-454AzK showed selective labeling of VP1 and VP2, respectively, thus demonstrating our ability to selectively label distinct minor capsid proteins.

[0073] Example 4: Retargeting efficiency of mutant AAV Figure 6 shows that the number of retargeting ligands bound to the AAV capsid dramatically affects its retargeting efficiency. By binding the cRGD ligand to the detargeted AAV2 capsid (where the binding of the innate primary receptor, the heparin sulfate proteoglycan receptor HSPG, is removed by mutating key residues R588 and R587 (specified by the amino acid residue position of VP1) to Ala), the cRGD ligand becomes capable of selectively binding to and infecting cancer cell lines such as SK-OV-3 that overexpress the αVβ3 integrin receptor. Since cyclooctin-cRGD progressively functionalizes the AzK side chain with cRGD, these graphs show the infectivity of the detargeted AAV2 mutants shown when incubated with cyclooctin-cRGD over time. For 454AA (60 AzK / capsid), infectivity against SK-OV-3 cells initially increased, then decreased, suggesting the optimal number of cRGDs per capsid required for efficient retargeting. Overmodification of the capsid at later time points likely leads to loss of infectivity by disrupting the viral entry process. For VP1-454AA and VP2-454AA (5 AzK / capsid each), infectivity increased and plateaued during long-term incubation, suggesting that binding of 5 cRGDs per capsid is not detrimental to AAV2 infectivity. However, the maximum infectivity achieved by these variants was low, suggesting that 5 cRGDs per capsid may not be sufficient for efficient retargeting. VP1+2-454AA (10AzK / capsid) behaved similarly to VP1-454AA and VP2-454AA, but its infectivity during long-term incubation reached a level similar to the optimal infectivity observed with T454-AA at the optimal level of modification.

[0074] Example 5: Precise labeling of AAV-manipulated cysteine ​​residues Figures 7A–7C show the precise labeling of AAV at the manipulated cysteine ​​residues. (A) Packaging efficiency (qPCR) of AAV produced using wild-type Cap (WT), T454C mutant of Cap (T454C in all three capsid proteins), VP1-T454C (T454C mutant of trans-substituted VP1), and VP2-T454C (T454C mutant of trans-substituted VP2). (B) Infectivity (FACS titer) of these viruses, measured by their ability to deliver and express the EGFP gene in HEK293T cells. (C) Selective labeling of the manipulated 454-cysteine ​​residue on VP1 with fluorescein-maleimide on VP1-454C virus. FAM fluorescence images show the results of the labeling reaction for WT and VP1-454C viruses, while SYPRO stains all proteins.

[0075] Example 6: Selective integration of UAA into VP1, VP2, or VP1+VP2 As shown in Figures 11A-11C, C5Az was incorporated into either VP1, VP2, or VP1+VP2 by co-transfecting HEK293T cells with a plasmid encoding C5Az-transformed E. coli leucyl-tRNA synthetase (EcLeuRS) and a tRNA pair (pIDTsmart-ITR-GFP-4xEcLtR-LeuRS), as well as plasmids encoding [RC2-VP1-del+CMV-VP1-delVP23], [RC2-VP2-del+CMV-VP2-delVP3], or [RC2-VP12-del+CMV-VP1-VP2-delVP3], respectively. Position 454 (VP1 numbering) in the desired protein was replaced with a TAG stop codon. A) Structure of C5Az, B) qPCR titers of various preparations shown for wild-type AAV2 production in the presence or absence of C5Az. C) Selective fluorescent labeling of VP1 with DBCO-rhodamine for AAV2-VP1-454-C5Az.

[0076] Example 7: Selective integration of CpK into VP1 As shown in Figures 13A-C, CpK was incorporated into VP1 by co-transfecting HEK293T cells with a plasmid encoding a modified E. coli leucyl-tRNA synthetase (EcLeuRS) and tRNA pair (pIDTsmart-ITR-GFP-4xEcLtR-LeuRS) and a plasmid encoding [RC2-VP1-del+CMV-VP1-delVP23]. Various sites in VP1 (as shown; VP1 numbered) were replaced with TAG stop codons. A) Structure of CpK, B) Relative titers of various preparations compared to wild-type AAV2 production in the presence or absence of LCA, C) Selective fluorescent labeling of VP1 with tetrazine-FITC on AAV preparations in which LCA was incorporated at site 456 of VP1.

[0077] Example 8: Selective integration of 5-HTP into VP1 As shown in Figures 14A-C, 5HTP was incorporated into VP1 by co-transfecting HEK293T cells with a plasmid encoding engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS) and a tRNA pair (pIDTsmart-TrpRS-8xWtR-ITR-GFP) for introducing 5HTP, as well as a plasmid encoding [RC2-VP1-del+CMV-VP1-delVP23]. Various sites in VP1 (as shown; VP1 numbered) were replaced with TGA stop codons. A) Structure of 5HTP, B) Relative titers of various preparations compared to wild-type AAV2 production in the presence or absence of 5HTP, C) Selective fluorescent labeling of VP1 with fluoresceinamine and ferricyanide on AAV preparations incorporating 5HTP at site 454 of VP1.

[0078] Example 9: Integrating UAA into VPI As shown in Figures 15A-B, these non-natural amino acids were incorporated into VP1 by co-transfecting HEK293T cells with a plasmid encoding modified E. coli leucyl-tRNA synthetase (EcLeuRS) and tRNA pairs (pIDTsmart-ITR-GFP-4xEcLtR-LeuRS), as well as a plasmid encoding [RC2-VP1-del+CMV-VP1-delVP23], which introduced several other non-natural amino acids. Site 454 (VP1 numbering) of VP1 was replaced with a TAG stop codon. A) Structure of the non-natural amino acid, B) Relative titer of various mutant AAV2 preparations compared to wild-type AAV2 production, in the presence or absence of the indicated non-natural amino acid.

[0079] Example 10: Modification of the LCA site of AAV2-VP1-454-LCA As shown in Figures 16A-B, AAV2-VP1-454-LCA was selectively modified at the LCA site with a 20kDa polyethylene glycol (PEG) polymer using the corresponding PEG-DBCO conjugate. SDS-PAGE analysis showed selective labeling of VP1. Wild-type AAV2, AAV2-VP1-454-LCA, and PEG-modified AAV2-VP1-454-LCA exhibited similar infectivity. Equal genomic copies of each virus were added to HEK293 cells, and the expression of the encoded luciferase reporter was monitored by a standard luciferase assay.

[0080] Although the present invention has been specifically illustrated and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various modifications of form and detail may be made in the above embodiments without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A genetically modified adeno-associated virus (AAV) comprising an AAV capsid, wherein the AAV capsid comprises SEQ ID NO: 1 and comprises a wild-type VP3 major capsid protein and at least one variant minor capsid protein VP1, VP2, or both VP1 and VP2, wherein the variant minor capsid protein is mutated at one or more amino acid residue sites compared to the wild-type VP1 or VP2 minor capsid protein and incorporates a natural amino acid or a non-natural amino acid (UAA). The capsid coding sequence encoding Sequence ID No. 1 is mutated at the translation origin of the VP1, VP2, or both VP1 and VP2 capsid protein open reading frame (ORF), preventing translation of VP1, VP2, or both VP1 and VP2. The aforementioned natural amino acid is incorporated into the T454 site. A genetically modified adeno-associated virus (AAV) in which the variant minor capsid protein is mutated at the translation origin of the VP2 and VP3, VP1 and VP3, or VP3 capsid protein open reading frame (ORF), and is encoded by a capsid coding sequence in which translation of VP2 and VP3, VP1 and VP3, or VP3 is prevented, and so that only the variant minor capsid protein is expressed from an appropriate promoter.

2. The genetically modified AAV according to claim 1, wherein the stop codon is incorporated into the capsid coding sequence that encodes the capsid protein, and the stop codon is a TAG, TAA, or TGA codon.

3. The genetically modified AAV according to claim 1, wherein the variant minor capsid protein is mutated at one or more amino acid residue sites and incorporates the native amino acid.

4. The genetically modified AAV according to claim 1 or claim 3, wherein the aforementioned natural amino acid is either cysteine ​​or selenocysteine.

5. The genetically modified AAV according to claim 1, wherein the variant minor capsid protein is mutated at one or more amino acid residue sites and incorporates the UAA.

6. The genetically modified AAV according to claim 1 or 5, wherein the UAA comprises a formula selected from the following group. 【Chemistry 1】

7. The variant minor capsid protein comprises p-benzoylphenylalanine (pBpA), O-methyltyrosine (OMeY), 5-azidotryptophan, 5-propargyloxytryptophan, 5-aminotryptophan, 5-methoxytryptophan, 5-O-allyltryptophan, 5-bromotryptophan, azidolysine (AzK), C5Az, LCA, N ε - Acetyllysine (AcK), cyclopropene amino acid, N ε A genetically modified AAV according to claim 1 or 5, selected from the group consisting of -(1-methylcyclopropa-2-enecarboxamide)-lysine (CpK), 5-hydroxytryptophan (5-HTP), LCALk, DiaazK, and LCKet.

8. The genetically modified AAV according to claim 1, wherein the natural amino acid or UAA of the variant minor capsid protein incorporates a bioconjugation handle.

9. The genetically modified AAV according to claim 8, wherein the AAV capsid comprises 5 to 10 bioconjugation handles per capsid.

10. The aforementioned genetically modified AAV a) Infectivity of target cells comparable to wild-type AAV, b) Packaged with a potency comparable to wild-type AAV, or c) Both of features a) and b) A genetically modified AAV according to any one of claims 1 to 9, characterized by the above.

11. The genetically modified AAV according to any one of claims 1 to 10, wherein the mutated VP1 amino acid sequence includes the sequence having SEQ ID NO: 1, and the variant VP1 capsid protein is mutated at one or more locations of position 263, position 454, position 456, position 587, or position 588.

12. The genetically modified AAV according to any one of claims 1 to 11, wherein the mutated amino acid is conjugated with a chemical or biological entity (protein, nucleic acid, lipid, or carbohydrate).

13. The genetically modified AAV according to claim 12, comprising a chemical entity or a protein entity, wherein the entity is selected from the group consisting of probes, small molecule ligands, peptides, cyclic peptides, nucleotides, polymers, and protein conjugates.

14. The genetically modified AAV according to claim 13, comprising a chemical entity or a protein entity, wherein the entity is a cyclic peptide cRGD or polyethylene glycol.

15. A method for producing infectious genetically modified AAV, wherein the AAV comprises at least one variant minor capsid protein VP1, VP2, or both VP1 and VP2, and the variant minor capsid protein comprises one or more mutant amino acid residue sites compared to wild-type VP1, VP2, or both VP1 and VP2. a) A step of providing competent host cells in culture, b) The cultured cells, 1) A first capsid-coding sequence encoding Sequence ID No. 1, which is mutated at the translation origin of the VP1, VP2, or both VP1 and VP2 capsid protein open reading frame (ORF), thereby preventing translation of VP1, VP2, or both VP1 and VP2. 2) A second capsid-coding sequence that encodes the variant minor capsid protein expressed from an appropriate promoter, wherein the second capsid-coding sequence is mutated at the origin of translation of VP2 and VP3, VP1 and VP3, or VP3 capsid protein ORF, thereby preventing translation of VP2 and VP3, VP1 and VP3, or VP3, and thus expressing only the variant minor capsid protein; and 3) A coding sequence for encoding an engineered aminoacyl-tRNA synthetase / tRNA pair that selectively introduces a native amino acid or a non-native amino acid (UAA) in response to a suppressor stop codon, and for introducing the native amino acid to the T454 site. A step of transfecting one or more plasmids containing, c) A step of providing the necessary natural amino acids or UAA, d) A step of culturing the cells under conditions sufficient for the expression of the plasmid gene and the association of the AAV. Includes, This provides a method for producing infectious genetically modified AAVs containing variant minor capsid proteins in which VP1, VP2, or both VP1 and VP2 are mutated at one or more amino acid residue sites compared to wild-type VP1, VP2, or both VP1 and VP2.

16. The method according to claim 15, wherein the appropriate promoter is a cytomegalovirus (CMV) promoter.

17. The method according to claim 15 or claim 16, wherein the variant minor capsid protein is mutated with a natural amino acid.

18. The method according to claim 15 or claim 16, wherein the variant minor capsid protein is mutated in UAA.

19. The method according to claim 18, wherein the UAA includes a formula selected from the group consisting of the following. 【Chemistry 2】

20. The method according to claim 15 or claim 16, wherein the mutated amino acid residue site incorporates a natural amino acid.

21. The method according to claim 20, wherein the natural amino acid is cysteine ​​or selenocysteine.

22. The method according to any one of claims 15 to 21, wherein the mutated amino acid is conjugated with a chemical or biological entity.

23. The method according to claim 22, wherein the entity is selected from the group consisting of probes, small molecule ligands, peptides, cyclic peptides, nucleotides, polymers, proteins, and viral conjugates.

24. The method according to claim 23, wherein the entity is a cyclic peptide cRGD or polyethylene glycol.

25. A therapeutic or antigenic composition comprising a genetically modified AAV according to any one of claims 1 to 14, and further comprising one or more therapeutic or antigenic gene constructs.

26. The therapeutic or antigenic composition according to claim 25, which is an antigenic composition and further comprises an adjuvant.

27. A composition according to claim 25 or 26 for use in the treatment of a disease or condition in a subject, the composition comprising a therapeutic amount of a gene construct encoding a protein or peptide that can reduce or alleviate the disease or condition in the subject.

28. A composition according to claim 25 or 26 for use in inducing an immune response in a subject, wherein the antigenic composition comprises a gene construct encoding an antigen capable of inducing an immune response in the subject.

29. A kit comprising the genetically modified AAV described in any one of claims 1 to 14.

30. A genetically modified adeno-associated virus (AAV) comprising an AAV capsid, wherein the AAV capsid comprises SEQ ID NO: 1 and comprises at least one variant minor capsid protein VP1, VP2, or both VP1 and VP2, wherein the variant minor capsid protein is mutated at one or more amino acid residue sites compared to the wild-type VP1 or VP2 minor capsid protein and incorporates a natural amino acid or a non-natural amino acid (UAA). The capsid coding sequence encoding Sequence ID No. 1 is mutated at the translation origin of the VP1, VP2, or both VP1 and VP2 capsid protein open reading frame (ORF), preventing translation of VP1, VP2, or both VP1 and VP2. The aforementioned natural amino acid is incorporated into the T454 site. The variant minor capsid protein is mutated at the translation origin of the VP2 and VP3, VP1 and VP3, or VP3 capsid protein open reading frame (ORF), and is expressed by a capsid coding sequence in which translation of VP2 and VP3, VP1 and VP3, or VP3 is prevented, thereby resulting in a genetically modified adeno-associated virus (AAV) in which only the variant minor capsid protein is expressed from an appropriate promoter.

31. a) A first capsid coding sequence encoding Sequence ID No. 1, which is mutated at the translation origin of the VP1, VP2, or both VP1 and VP2 capsid protein open reading frame (ORF), thereby preventing translation of VP1, VP2, or both VP1 and VP2, b) A second capsid coding sequence encoding a variant minor capsid protein, wherein the second capsid coding sequence is mutated at the origin of translation of VP2 and VP3, VP1 and VP3, or VP3 capsid protein ORF, preventing translation of VP2 and VP3, VP1 and VP3, or VP3, so that only the variant minor capsid protein is expressed. Host cells, including those containing the host cell.

32. A host cell according to claim 31, comprising a programmed aminoacyl-tRNA synthetase that selectively introduces a natural amino acid or a non-natural amino acid (UAA) into tRNA, and further comprising a coding sequence for introducing the natural amino acid into the T454 site.

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