Novel aav variants

Abstract

The present disclosure provides a variant AAV capsid protein comprising one or more modifications comprising an insertion of 7 amino acids at a position between amino acid positions 587 and 588 of an AAV capsid protein, and a polynucleotide, host cell, vector, AAV virion, or pharmaceutical composition thereof. The present disclosure also provides a method of treating a disease comprising administering to a subject in need thereof an effective amount of a recombinant AAV virion.

Description

[0001] priority This application claims the benefit and priority of PCT application No. PCT / CN2024 / 086887, filed on April 9, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to variant AAV capsid proteins, particularly AAV2 capsid variants for intravitreal gene therapy. Background Technology

[0003] Adeno-associated virus (AAV) belongs to the Parvoviridae family and the Dependent Virus genus. Its members require co-infection with a helper virus, such as adenovirus, to promote replication, and AAV establishes latent infection in the absence of a helper virus. The virion consists of a 25 nm icosahedral capsid containing a 4.7 kb single-stranded DNA genome with two open reading frames: rep and cap. The non-structural rep gene encodes four regulatory proteins essential for viral replication, while cap encodes three structural proteins (VP1-3) that assemble into a 60-mer capsid shell. The viral capsid mediates the ability of AAV vectors to overcome many biological barriers to viral transduction, including cell surface receptor binding, endocytosis, intracellular transport, and uncoating in the nucleus. AAV-based gene therapy holds promise as a treatment for many diseases. However, low transduction efficiency in certain tissues is a technical obstacle to the clinical use of AAV-based gene therapy in many cases.

[0004] To date, there is still a need to develop AAV-mediated gene delivery systems with enhanced tissue transduction. Summary of the Invention

[0005] This invention provides a variant AAV capsid protein comprising one or more modifications, said modifications including an insertion sequence as shown in Formula I at position 587 and 588 of the parental AAV capsid protein. YX1X2X3X4X5X6X7Z (Formula I), where X is any amino acid, Y is non-existent, LA(n) or A(n), Z is A(n), and n is any integer from 1 to 5.

[0006] In one embodiment, the variant AAV capsid protein includes an insertion sequence as shown in Formula II at position 587 and 588 of the parental AAV capsid protein. X1X2X3X4X5X6X7 (Formula II), where X is any amino acid.

[0007] In one embodiment, the variant AAV capsid protein includes an insertion sequence located between amino acid positions 587 and 588 of the parental AAV capsid protein, the insertion sequence being as shown in Formula II (X1X2X3X4X5X6X7). 1-7 It is any amino acid; preferably, the inserted sequence is as shown in X1X2X3PX5RN, where X5 is D or E, and X... 1-3 It can be any amino acid, preferably, the inserted sequence is as shown in X1X2X3X4RPX7, X 1-4 X7 is any amino acid, and X7 is E, Q, G, or S. Preferably, the inserted sequence is as shown in X1X2DX4RPQ, X 1-2 X1 or X4 is any amino acid, X4 is H or T, preferably, the inserted sequence is as shown in X1TEX4RPX7, where X1 or X4 is any amino acid, and X7 is Q or S, or preferably, the inserted sequence is as shown in X1X2X3X4KPE, X 1-4 It can be any amino acid.

[0008] In one embodiment, the variant AAV capsid protein includes an insert sequence selected from the group consisting of SEQ ID NO: 1-467; or a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-467.

[0009] In one embodiment, the variant AAV capsid protein includes an insert sequence selected from the group consisting of SEQ ID NO: 524-558; or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 524-558.

[0010] In one embodiment, the variant AAV capsid protein comprises an insert sequence selected from the group consisting of SEQ ID NO: 23, 33, 36, 45, 57-67; or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 23, 33, 36, 45, 57-67.

[0011] In one embodiment, the AAV is selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and any AAV serotype or variant thereof isolated from humans and non-human mammals.

[0012] In one embodiment, the capsid protein further comprises any one of flanking substitution and point mutation modification.

[0013] In one embodiment, the capsid protein comprises a combination of modifications including flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and insertion sequences at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGT, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG.

[0014] In one embodiment, the capsid protein comprises a combination of modifications including flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and insertion sequences at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from GPR, QQN, KAA, GIP, RTP, RDP, WAP, TAA, GPS, LAP, QQK, RDN, GRP, RSP, SGN, RGY, QQT, QAP, or SSD.

[0015] In one embodiment, the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, and the insertion sequence is SEQ ID NO: 33.

[0016] In one embodiment, the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, RGG, and the insertion sequence is SEQ ID NO: 23.

[0017] In one embodiment, the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, RGG, and the insertion sequence is SEQ ID NO: 36.

[0018] In one embodiment, the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from KGG substitutions and the insertion sequence is selected from SEQ ID NO: 57-67.

[0019] In one embodiment, the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from RCN substitutions and the insertion sequence is selected from SEQ ID NO: 57-67.

[0020] In one embodiment, the capsid protein further comprises a point mutation, wherein the point mutation comprises one or more amino acid substitutions corresponding to the amino acid sequence of the parent AAV, wherein the one or more amino acid substitutions are selected from Y737F, Y444F, Y500F, K556E, R595L, R595M, A597V, A597S, and Q596H.

[0021] In one embodiment, the point mutation comprises one of the following substitutions or combinations of substitutions: (i) Y737F; (ii) Y444F, Y500F, Y737F; (iii) K556E.

[0022] In one embodiment, the capsid protein comprises a combination of modifications including flanking substitutions of RAA at amino acid positions 585 to 587 of the parental AAV capsid protein, amino acid substitution Y737F, and insertions of the amino acid sequence as shown in SEQ ID NO: 33 between amino acid positions 587 and 588.

[0023] In one embodiment, the capsid protein comprises a combination of modifications including flanking substitutions of RAA at amino acid positions 585 to 587 of the parental AAV capsid protein, amino acid substitutions Y444F, Y500F, and Y737F, and insertions of the amino acid sequence as shown in SEQ ID NO: 33 between amino acid positions 587 and 588.

[0024] In one embodiment, the capsid protein comprises a combination of modifications including flanking substitutions of RAA at amino acid positions 585 to 587 of the parental AAV capsid protein, amino acid substitution K556E, and insertions of the amino acid sequence as shown in SEQ ID NO: 33 between amino acid positions 587 and 588.

[0025] In one embodiment, the capsid protein comprises a combination of modifications including the amino acid substitution Y737F of the parental AAV capsid protein and the insertion of an amino acid sequence as shown in SEQ ID NO: 23 between amino acid positions 587 and 588.

[0026] In one embodiment, the capsid protein comprises a combination of modifications including amino acid substitutions of the parental AAV capsid protein Y444F, Y500F, Y737F and insertions of the amino acid sequence as shown in SEQ ID NO: 23 between amino acid positions 587 and 588.

[0027] In one embodiment, the capsid protein comprises a combination of modifications comprising the amino acid substitution Y737F of the parental AAV capsid protein and the insertion of an amino acid sequence as shown in SEQ ID NO: 36 between amino acid positions 587 and 588.

[0028] In one embodiment, the capsid protein comprises a combination of modifications including amino acid substitutions of the parental AAV capsid protein Y444F, Y500F, Y737F and insertions of the amino acid sequence as shown in SEQ ID NO: 36 between amino acid positions 587 and 588.

[0029] The present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the above-described variant AAV capsid protein, or provides a vector comprising the above-described polynucleotide.

[0030] The present invention provides an isolated, genetically modified host cell containing the aforementioned polynucleotides.

[0031] The present invention provides a recombinant AAV toxic particle containing the above-mentioned variant AAV capsid protein.

[0032] In one specific embodiment, the present invention provides a pharmaceutical composition comprising: a) The recombinant adeno-associated virus particles disclosed in this invention; and b) Pharmaceutically acceptable excipients.

[0033] In another aspect, the present invention provides a recombinant AAV vector comprising a polynucleotide encoding a variant AAV capsid protein of the present invention, an AAV 5' inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional gene product, a regulatory sequence guiding the expression of the gene product in target cells, and an AAV 3' ITR.

[0034] In another aspect, the present invention also provides a method for delivering a nucleic acid vector encoding a functional gene product to cells and / or tissues using the recombinant AAV toxin or recombinant AAV vector of the present invention.

[0035] In one specific embodiment, the present invention also provides the use of the recombinant AAV virion or recombinant AAV vector of the present invention in the preparation of products for delivering nucleic acid vectors encoding functional gene products to cells and / or tissues.

[0036] In another aspect, the present invention also provides a method for treating a disease, the method comprising administering an effective amount of the recombinant AAV venom particles of the present invention to a subject in need, the recombinant AAV venom particles comprising a functional gene product.

[0037] In one specific embodiment, the present invention also provides the use of the recombinant AAV venom particles or recombinant AAV vector of the present invention in the preparation of a product for treating a disease in a subject in need; preferably, the disease is selected from liver disease, central nervous system disease and other diseases.

[0038] In one specific embodiment, the gene product is a polypeptide. Attached Figure Description

[0039] Figure 1 The AAV2 library screening workflow is shown.

[0040] Figure 2 The anatomy of the cynomolgus monkey's eye is shown. The retina is divided into five parts with three layers. The materials used for NGS are S1, S2, N1, N2, I1, I2, T1, T2, M1, M2, and RPE. The materials used for the second-round library packaging are RPE and PR. S: superior; N: nasal; I: inferior; T: temporal; M: macula.

[0041] Figure 3 It shows a motif with high retinal orientation and penetrability.

[0042] Figure 4 This demonstrates that AVT225 achieves whole-retinal transduction in the mouse retina. Compared with controls in 6-week-old mice, the vector mediated GFP expression after intravitreal injection of the novel capsid variant. A: AVT225; B: AAV7m8; C: AAVGL; D: AAV2.

[0043] Figure 5 The first round of NHP validation design is shown.

[0044] Figure 6 In vivo fundus GFP fluorescence is shown after intravitreal delivery of the rAAV.Sc-CB-GFP vector. In vivo fundus autofluorescence was performed on NHP retinas 3 weeks after intravitreal injection of 4E10 total vg of each sc-CB-GFP capsid.

[0045] Figure 7 Immunofluorescence of retinal cross sections and planar slices after 3 WPI is shown.

[0046] Figure 8 The comparison of the AVT capsid with AAV2 and AV2.7m8 in cynomolgus monkeys is shown. Detailed Implementation

[0047] The following description of this disclosure is intended only to illustrate various embodiments of this disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that such equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0048] The articles “a,” “one,” and “the” are used in this text to refer to one or more (i.e., at least one) grammatical object of the article.

[0049] As used herein, the term "about" or "approximately" refers to a quantity, level, value, quantity, frequency, frequency, percentage, size, size, amount, weight, or length that differs from a reference quantity, level, value, amount, frequency, percentage, size, weight, or length by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%. In certain embodiments, when preceding a numerical value, the term "about" or "approximately" indicates a range of 15%, 10%, 5%, or 1% added to or subtracted from that value.

[0050] Throughout this disclosure, unless the context otherwise requires, the word “comprising” will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but does not exclude any other steps or elements or groups of steps or elements. “consisting of” means to include, but is not limited to, anything following the phrase “consisting of.” Therefore, the phrase “consisting of” indicates that the listed elements are necessary or mandatory, and no other elements may be present. “Substantially consisting of” means to include any element listed following the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function of the listed elements as specified in this disclosure. Therefore, the phrase “substantially consisting of” indicates that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or function of the listed elements.

[0051] As used herein, the term “subject” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, including cynomolgus monkeys, non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Unless otherwise stated, the terms “patient” or “subject” are used interchangeably.

[0052] The terms “treatment” and “treatment method” refer to therapeutic therapies and preventative measures. Individuals requiring treatment may include those who already have a specific medical disorder and those who may eventually develop that disorder.

[0053] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. Unless otherwise required, the term covers all subtypes as well as naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector"). The term "AAV" includes AAV types 1 (AAV-1), 2 (AAV-2), 3 (AAV-3), 4 (AAV-4), 5 (AAV-5), 6 (AAV-6), 7 (AAV-7), 8 (AAV-8), 9 (AAV-9), 10 (AAV-10), 11 (AAV-11), 12 (AAV-12), 13 (AAV-13), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. See, for example, Mori et al. (2004) Virology 330:375. The term "AAV" also includes chimeric AAV. "Primate AAV" refers to AAVs that are derived from primates, "non-primate AAV" refers to AAVs that are derived from non-primate mammals, "bovine AAV" refers to AAVs that are derived from bovine mammals (e.g., cattle), and so on.

[0054] The AAV "rep" and "cap" genes are multinucleotide sequences encoding adeno-associated virus (AAV) replication and capsidation proteins. In this paper, AAV rep and cap are referred to as AAV "packaging genes".

[0055] The term "insertion sequence" is used below to describe peptides inserted into the capsid of parental AAV. In some cases, the length of the insertion peptide is 7 to 20 amino acids. In some cases, the length of the insertion peptide is 7 to 16 amino acids. In some cases, the length of the insertion peptide is 7 to 10 amino acids. In some cases, the length of the insertion peptide is 7 to 12 amino acids. The length of the insertion peptide is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some cases, the length of the insertion peptide is 7 amino acids. In some cases, the length of the insertion peptide is 8 amino acids. In some cases, the length of the insertion peptide is 9 amino acids. In some cases, the length of the insertion peptide is 10 amino acids. In some cases, the length of the insertion peptide is 11 amino acids. In some cases, the length of the insertion peptide is 12 amino acids. In some cases, the length of the insert peptide is 13 amino acids. In some cases, the length of the insert peptide is 14 amino acids. In some cases, the length of the insert peptide is 15 amino acids. In some cases, the length of the insert peptide is 16 amino acids.

[0056] Example Equipment and reagents Table 1. Equipment used in this invention

[0057] Table 2. Reagents and supplies used in this study

[0058] Table 3. Various oligonucleotides used in this study

[0059] Note: M represents A or C; N represents A, C, G or T; K represents G or T.

[0060] Example 1: Variant Generation 1.1 AAV Library Generation A DNA fragment library was generated using AAV2 Rep-Cap plasmid DNA template via PCR. The primers were Cap2PCR.

[0061] The purified PCR product was assembled with the GRK1 promoter p41, AAV2 (Cap2) VP1 and bovine growth hormone (bGH) poly A into a library backbone containing terminal inverted repeats (ITRs).

[0062] AAV2 (Cap2)VP1 amino acid sequence (SEQ ID NO: 508) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFS PRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLI DQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQ RGNRQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

[0063] The p41 promoter is used to drive capsid gene expression for AAV library production only in the presence of the Ad5 helper gene product. The GRK1 promoter is designed to restore Cap2 mRNA expression in retinal tissue. The assembled library plasmid DNA was transformed into competent cells for amplification. The amplified cap2 library plasmid was then co-transfected with pHelper and pRep2-AAP in HEK293 cells for AAV library production as follows: HEK293T cells were purchased from the National Collection of Authenticated Cells (SCSP-502). HEK293 cells were rapidly thawed by gentle stirring in a 37°C water bath for 2 minutes. The thawed cells were added to preheated culture medium, centrifuged at low speed to remove the frozen medium, and then resuspended in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The cells were then grown in a humidified incubator at 37°C with 5% CO2 supplementation, with the growth medium changed every 2–3 days.

[0064] For cell transfection: a) Cell seeding: 24 hours before transfection, plate the cells and observe their condition. Transfection can proceed when the cell density is 70%-80% and the cells are under good, uncontaminated conditions. b) Preparation of plasmid-PEI transfection complex: plasmid:PEI=1:1. Add Helper plasmid, Rep-cap plasmid and GOI plasmid to each 150 culture dish with a total plasmid amount of 58 μg, prepare transfection complex and let stand for 15 min. c) Transfection was performed using a transfection complex: serum-containing medium = 1:10, and the transfection was completed by placing the transfection complex in a 150 mm dish; d) Cultivate for 72 hours and harvest the virus.

[0065] Table 4. Sequence Information

[0066] 1.2 In vivo screening The AAV formulation was purified by gradient ultracentrifugation with iodixanol and concentrated to a purity and titer sufficient for in vivo studies. The formulation was then aliquoted and stored at -80°C. Endotoxin levels were determined by droplet digital PCR (ddPCR) titration of AAV particles (modified from Lock et al., 2014) using primers and probes targeting ITR elements and an internal control of known titer. Chromogenic endotoxin assay based on amoeboid lysate was used to determine endotoxin levels. The purified AAV virus library (first-round randomized library and second- to third-round recovered libraries) was then injected intravitreally at 3E11 vg / eye into cynomolgus monkeys aged 3–6 years. Twenty-one days post-injection, the monkeys were euthanized and their eyes were collected for post-mortem dissection of the retinal sublayers to assess transgenic product levels. Dissection was performed according to the following steps: Wash the sterile covered culture dish twice with pre-chilled physiological saline and then place it on moist ice. Place the eyeball on the pre-chilled glass culture dish. Remove the anterior pole of the eye from the ciliary body, then cut the eye cup into several sections (superior, nasal, inferior, temporal, and macula). Immerse the retina with the sclera in a trypsin solution at pH 7.2 (0.1% trypsin (1:250) and 10 mM HEPES dissolved in Ca2+-free solution). 2+ / Mg 2+ Incubate the culture dish in Hank's solution at 37°C in a CO2 incubator (95% air / 5% CO2) for 10 minutes. After soaking, quickly remove the trypsin solution with a Pasteur pipette and wipe the culture dish dry with sterile filter paper.

[0067] The procedure for separating the retina is as follows: 1. Gently cover the surface of the flat retina with a sterile Millipore filter membrane. During this step, prevent air from entering between the tissue and the Millipore filter membrane.

[0068] 2. Separate the entire retina (including the Millipore covering) from the choroid with the RPE and sclera, and place the retina with the Millipore filter and the choroid with the RPE and sclera in two covered culture dishes.

[0069] 3. Flip the retina with the Millipore filter membrane so that the outer nuclear layer is facing upwards, cover the outer nuclear layer with filter paper, and then separate the outer nuclear layer by gently removing the filter paper. Transfer the outer nuclear layer with the filter paper attached and the remaining retinal tissue to a labeling tube and freeze in dry ice, then store in a freezer set to maintain ≤-60°C until transport.

[0070] 4. Cover the choroid membrane with RPE and the sclera with a Millipore filter membrane, then separate the RPE by gently removing the filter paper. Transfer the RPE with the Millipore filter membrane to a labeling tube and freeze it in dry ice, then store it in a freezer set to maintain ≤-60°C until transport.

[0071] Total RNA was isolated and treated with DNase I. Cap2-specific mRNA was reverse transcribed and amplified by PCR (RT-PCR) for next-generation sequencing (NGS). Enriched candidates were selected using a customized enrichment score analysis. A total of 2035 candidates, including variants predicted by artificial intelligence modeling, were synthesized for a second round of screening. Simultaneously, the top 58 variants were packaged separately for in vitro and in vivo characterization. These top variants were selected using an NSG enrichment score. The top variant candidates selected for validation are shown in Table 5.

[0072] Table 5. Top-ranked variant candidates selected for validation

[0073] 1.3 Flanking sequence optimization and point mutation Six candidates were selected for flanking optimization at the insertion site (R585G586N587). RGN was replaced with random amino acids using random primers.

[0074] Table 6. Replacing RGN with random amino acids using random primers

[0075] Library assembly, in vivo selection, and NGS were performed as described above. Following NGS, 16 additional top-ranked variants were selected using NSG enrichment scores and packaged for in vitro and in vivo characterization. Simultaneously, point mutations were performed on AVT225, AVT239, and AVT260.

[0076] Table 7. Candidates selected from flanking optimized libraries and point mutations

[0077] Note: " / " means that there is no substitution or mutation at the corresponding position in the AAV variant.

[0078] Table 8. NGS Data and Enrichment Scores

[0079] Note: " / " means that there is no substitution or mutation at the corresponding position in the AAV variant.

[0080] Based on NGS data and enrichment scores, our AI team discovered many motifs with high retinal tropism and penetrability, which form different groups and families. Figure 3 ).

[0081] Example 2: Verification 2.1 Construction and purification of AAV plasmids The variant's Rep-cap plasmid was constructed using overlap PCR as follows: a) The AAV2 Rep-cap plasmid was used as a template, and AAV2F and overlap R were used as primers to amplify fragment 1; the AAV2 Rep-cap plasmid was then used as a template and amplified with overlap F and AAV2R to obtain fragment 2. b) Fragments 1 and 2 were used as templates, and AAV2 F and AAV2 R were used as primers to amplify fragment 3. c) The AAV2 Rep-cap plasmid and fragment 3 were digested with BsiwI and SpeI, and fragment 3 was inserted between the BsiwI and SpeI in the AAV2 Rep-cap plasmid to complete the construction. After Sanger sequencing, the plasmid was used for AAV production.

[0082] Three variants (AVT225, AVT226, and AVT228) plus controls (AAV2, AAV7m8, and AAVGL) were packaged with green fluorescent protein (GFP) transgenes driven by the chicken β-actin (CB) promoter. All six AAV variants were produced by transfection of HEK293 cells with a PEIpro (Polyplus) triple plasmid. Three days post-transfection, cells were harvested for lysis, benzonase (Sigma, E1014-25KU) treatment, and purification via iodixanol gradient ultracentrifugation. AAVs were filtered through 0.22 μm PBS after buffer exchange with 0.001% F68. Titers were measured by ddPCR using ITR primers and probes. AAV purity was assessed by SDS-PAGE.

[0083] Table 9. AAV titers

[0084] 2.2 Validation of the novel capsid in C57BL / 6J mice Six-week-old C57BL / 6J mice were intravitreally injected with the six AAVs (1E9 vg / eye). Animals were anesthetized by intraperitoneal injection of Zoletil (60 mg / kg) and xylazine (10 mg / kg). Mydriasis was achieved with tropicamide eye drops (National Medicine Permit No. J20180051). The injection system was the Nanofil micro-injection system equipped with a 36G bevel needle (World Precision Instrument, Nanofil-10 μL). The entire field of view was operated under a stereomicroscope (OLYMPUS, SZ61). For injection, the needle was tilted downwards and inserted close to the sclera approximately 2 mm from the limbus, penetrating the sclera, choroid, and retina to reach the vitreous body. After confirming the correct needle position in the vitreous body under the microscope, 1 μL of AAV solution was slowly injected. Special care was taken to avoid damaging the lens during injection.

[0085] Three weeks after injection (3 wpi), the animals were anesthetized and their systemic blood was replaced by pre-cooled saline at 4°C via cardiac perfusion. The eyes were removed and fixed in FAS fixative solution for 4 hours. The cornea and lens were then removed under a stereomicroscope, transferred to 30% sucrose / PBS solution for dehydration overnight, and finally embedded in OCT embedding medium.

[0086] Before sectioning, the embedded tissue blocks were equilibrated at -20°C for at least 30 minutes in a cryostat incubator. The embedded blocks were then fixed to a chuck using a separate OCT apparatus, and sagittal sections of the eyeball with a thickness of 10 μm to 20 μm were cut and collected using positively charged adhesive slides. Finally, the slides were dried at room temperature for 1 h and stored at -20°C.

[0087] For staining, the slides were first removed from -20°C and allowed to dry at room temperature for 20 minutes. Sections were drawn on the slides using an IHC pen to create hydrophobic rings. The slides were then washed three times with 500 μl PBS for 5 minutes each time. Cells were incubated for 1 hour with a blocking solution of PBS containing 0.5% Triton X-100 and 10% goat serum. After removing the blocking solution, primary antibody incubation solution was added and the cells were incubated overnight at 4°C. The slides were then washed five times with PBS containing 0.5% Triton X-100 for 5 minutes each time. After removing the washing solution, secondary antibody incubation solution was added and the cells were incubated for 1 hour at room temperature. Finally, the plate was washed five times with PBS for 5 minutes each time and sealed with a drop of sealing agent containing DAPI dye.

[0088] Image acquisition was performed using an optical microscope (Nikon, TS2R) with 4x, 10x, and 20x objectives. Images were captured using bright-field, DAPI, GFP, and Alexa Fluor 555 channels for the bright-field, DAPI, GFP, and Cy3 signaling pathways, respectively. For images requiring stitching into larger images, sequential multi-channel image acquisition with approximately 10% overlap was performed.

[0089] The results showed that, compared with the control, AVT225 exhibited very extensive infection and good tissue penetration. No signals from AVT226 and AVT228 were detected. Figure 4 This may be due to differences between species, as candidates were screened from monkeys and validated in mice.

[0090] Example 3: Intravitreal injection of NHP Six variants (AVT225, AVT235, AVT239, AVT248, AVT260, AVT264) plus controls (AAV2, AAV7m8, and R100) were packaged with either a green fluorescent protein (GFP) transgene driven by the chicken β-actin (CB) promoter or an mCherry or FXN-HA tag. All AAVs were produced by transfection of HEK293 cells with a PEIpro (Polyplus) triple plasmid.

[0091] Seven adult cynomolgus macaques (4-5 years old, ~3 kg) were intravitreally injected with 8E10 vg / eye (4E10 / eye / capsule). Each eye was injected with two capsid packages containing different marker genes, as shown below: Table 10. First Round NHP Validation Design

[0092] Example 4: Terminal, fundus autofluorescence and tissue collection Animals were anesthetized by intramuscular injection of Zoletil (50 mg / kg) and celazine (10 mg / kg), and topical administration of promecaine hydrochloride and tropicamide was given to relieve pain or dilate the pupil. A 31g insulin syringe was used as the injection system, inserted into the sclera (approximately 3 mm behind the limbus), and two separate 50 μL intravitreal injections were administered to each eye, for a total injection volume of 100 μL / eye. The needle was tilted downwards, and its position was confirmed under an operating microscope. The AAV solution was delivered to the intermediate vitreous body by slow injection. After the bolus injection, the needle was held steady for at least 2 minutes to minimize backflow of the injected solution. Additionally, 40 to 80 mg of methylprednisolone was administered weekly post-injection.

[0093] All animals underwent ophthalmic examinations and imaging by a committee-certified veterinary ophthalmologist at various time points before and after administration. Ophthalmic examinations included indirect ophthalmoscopy and slit-lamp biomicroscopy. Fundus imaging was performed using a confocal scanning laser fundus imaging system (Spectralis HRA + OCT, Heidelberg Engineering). Recorded images included infrared reflectance (IR), blue autofluorescence (BAF), and optical coherence tomography (OCT). During BAF imaging, multiple regions of the central and peripheral retina were recorded to reflect the distribution of GFP expression, with the sensitivity set to 100. OCT imaging was performed by scanning multiple rows of horizontal high-resolution images from the optic disc (ONH) to the fovea.

[0094] The novel capsids AVT225, AVT239, AVT248, and AVT260 produced very strong and widely distributed GFP signals in the NHP retina after 3 WPI. This signal from AVT225, AVT248, and AVT260 was significantly stronger than that from the positive control AAV7m8. Figure 6 ).

[0095] Example 5: Retinal sections, flat slides, and immunofluorescence For staining, the slides were first removed from -20°C and dried at room temperature for 20 min. Sections were drawn on the slides using an IHC pen to create hydrophobic rings. The slides were then washed three times with 500 μl PBS for 5 min each time. Cells were incubated for 1 h with a blocking solution of PBS containing 0.5% Triton X-100 and 10% goat serum. After removing the blocking solution, primary antibody incubation solution was added and the cells were incubated overnight at 4°C. The slides were then washed five times with PBS containing 0.5% Triton X-100 for 5 min each time. After removing the washing solution, secondary antibody incubation solution was added and the cells were incubated at room temperature for 1 h. Finally, the plate was washed five times with PBS for 5 min each time and sealed with a drop of sealing agent containing DAPI dye.

[0096] Retinal slide preparation: After cryosectioning, the remaining embedding blocks were thawed at room temperature. The retina was carefully dissected under a microscope and washed five times with PBS to remove residual OCT embedding agent. The retina was then blocked in PBS solution containing 2% Triton X-100 and 10% normal bovine serum for 1 hour, followed by overnight incubation with rabbit anti-mCherry primary antibody or anti-HA primary antibody at a 1:100 dilution. The retina was washed five times and incubated with secondary antibody solution at room temperature for 2 hours. After washing, the sample was prepared into flat glass slides and sealed with mounting medium containing DAPI dye drops.

[0097] Image acquisition was performed using an optical microscope (Nikon, TS2R) with 4x, 10x, and 20x objectives. Images were captured using bright-field, DAPI, GFP, and Alexa Fluor 555 channels for the bright-field, DAPI, GFP, and Cy3 signaling pathways, respectively. For images requiring stitching into larger images, sequential multi-channel image acquisition with approximately 10% overlap was performed.

[0098] In the foveal region where AAV2 was not infected, immunolabeled retinal cross-sections were stained with GFP and DAPI. AVT264 showed a weak infection, better than AAV2 but weaker than AAV7m8, while pores AVT260, AVT225, and AVT248 showed stronger infection than the positive control AAV7m8. The fovea is the thinnest part of the retina, and outside the fovea, AAV has a harder time infecting and penetrating the retinal layer. AAV2 showed no foveal infection, while pores AVT260, AVT225, and AVT248 showed better infection and penetration than AAV7m8. Retinal tilings showed extensive NHP retinal infection with AVT260, AVT225, and AVT248. Figure 7 ).

[0099] AAV-CB-GFP was injected intravitreally into adult cynomolgus monkeys at a dose of 1E12 vg / eye. Three weeks post-injection, in vivo fundus autofluorescence examination was performed, and immunolabeled retinal cross-sections were stained with GFP and DAPI. AAV2 was uninfected; AVT260, AVT277, and AVT279 showed stronger infection than the positive control AAV7m8. Figure 8 ).

[0100] Example 6: AAV variant enhances the ocular tropism of AAV8 Peptides selected from the AAV2 library were synthesized on a chip. Subsequently, Q588 to P593 of AAV8 were replaced with RGNRQA, and an AAV2 library was inserted after RGN to generate an AAV8 library. The AAV8 library was then injected into the eyes of non-human primates (NHPs) at a dose of 1E12 vg / eye. Three weeks later, DNA and RNA were extracted and subjected to next-generation sequencing (NGS). Top-ranked variant candidates enhanced the eye tropism of both AAV2 and AAV8.

[0101] Table 11. Top-ranked variant candidates validated in AAV8

[0102] The embodiments of the present invention have been described above, but the present invention is not limited thereto, and those skilled in the art will understand that modifications and changes can be made within the claimed scope of the present invention. Such modifications and changes should fall within the protection scope of the present invention.

Claims

1. A variant AAV capsid protein comprising one or more modifications, said modifications including an insertion sequence as shown in Formula I at position 587 and 588 of the parental AAV capsid protein. YX1X2X3X4X5X6X7Z (Formula I). Where X is any amino acid, Y is non-existent, LA(n) or A(n), Z is A(n), and n is any integer from 1 to 5.

2. The variant AAV capsid protein of claim 1, wherein the capsid protein comprises an insertion sequence at position 587 and 588 of the parental AAV capsid protein, wherein... The insertion sequence is shown as X1X2X3X4X5X6X7 (Equation II), X 1-7 It can be any amino acid; Preferably, the insertion sequence is as shown in X1X2X3PX5RN, where X5 is D or E, and X... 1-3 It can be any amino acid, preferably, the inserted sequence is as shown in X1X2X3X4RPX7, X 1-4 X7 is any amino acid, and X7 is E, Q, G, or S. Preferably, the inserted sequence is as shown in X1X2DX4RPQ, X 1-2 X1 or X4 is any amino acid, X4 is H or T, preferably, the inserted sequence is as shown in X1TEX4RPX7, where X1 or X4 is any amino acid, and X7 is Q or S, or preferably, the inserted sequence is as shown in X1X2X3X4KPE, X 1-4 It can be any amino acid.

3. The variant AAV capsid protein according to any one of claims 1 to 2, wherein the inserted sequence is selected from the group consisting of SEQ ID NO: 1-467; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-467.

4. The variant AAV capsid protein according to any one of claims 1 to 2, wherein the inserted sequence is selected from the group consisting of SEQ ID NO: 524-558; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 524-558.

5. The variant AAV capsid protein according to claim 3, wherein the insert peptide is selected from the group consisting of SEQ ID NO: 23, 33, 36, 45, 57-67; or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 23, 33, 36, 45, 57-67.

6. The variant AAV capsid protein of claim 1, wherein the AAV is selected from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and any AAV serotype or variant thereof isolated from humans and non-human mammals.

7. The variant AAV capsid protein according to any one of claims 1 to 6, wherein the capsid protein further comprises modification of either flanking substitution or point mutation.

8. The variant AAV capsid protein of claim 7, wherein the capsid protein comprises a combination of modifications comprising flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at a position between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG.

9. The variant AAV capsid protein of claim 7, wherein the capsid protein comprises a combination of modifications comprising flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at a position between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of GPR, QQN, KAA, GIP, RTP, RDP, WAP, TAA, GPS, LAP, QQK, RDN, GRP, RSP, SGN, RGY, QQT, QAP, or SSD.

10. The variant AAV capsid protein of claim 8, wherein the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, and the insertion sequence is SEQ ID NO:

33.

11. The variant AAV capsid protein of claim 8, wherein the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, and the insertion sequence is SEQ ID NO:

23.

12. The variant AAV capsid protein of claim 8, wherein the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at positions between amino acid positions 587 and 588, wherein the flanking substitutions are selected from substitutions of RAA, KGG, RAP, RGM, RGP, KAG, RCN, RGT, KAG, RGV, RGL, KGS, RAT, RAN, KSG, RDM, RGI, KAV, TAG, RAG, RSF, KVG, IAG, RCM, RGC, RAD, RSG, NAG, RGG, RPS, KGS, RVN, RPA, KCS, or RGG, and the insertion sequence is SEQ ID NO:

36.

13. The variant AAV capsid protein of claim 8, wherein the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at a position between amino acid positions 587 and 588, wherein the flanking substitutions are selected from KGG substitutions and the insertion sequence is selected from SEQ ID NO: 57-67.

14. The variant AAV capsid protein of claim 8, wherein the capsid protein comprises flanking substitutions at amino acid positions 585 to 587 of the parental AAV capsid protein and an insertion sequence at a position between amino acid positions 587 and 588, wherein the flanking substitutions are selected from RCN substitutions and the insertion sequence is selected from SEQ ID NO: 57-67.

15. The variant AAV capsid protein according to any one of claims 7-14, wherein the point mutation comprises one or more amino acid substitutions corresponding to the amino acid sequence of the parental AAV, wherein the one or more amino acid substitutions are selected from Y737F, Y444F, Y500F, K556E, R595L, R595M, A597V, A597S and Q596H.

16. The variant AAV capsid protein of claim 15, wherein the point mutation comprises one of the following substitutions or combinations thereof: (i) Y737F; (ii) Y444F, Y500F, Y737F; (iii) K556E.

17. The variant AAV capsid protein of claim 16, wherein the capsid protein comprises a combination of modifications including flanking substitutions of RAA at amino acid positions 585 to 587 of the parental AAV capsid protein, amino acid substitution Y737F, and insertion of an amino acid sequence as shown in SEQ ID NO: 33 between amino acid positions 587 and 588.

18. The variant AAV capsid protein of claim 16, wherein the capsid protein comprises a combination of modifications including flanking substitutions of RAA at amino acid positions 585 to 587 of the parental AAV capsid protein, amino acid substitutions Y444F, Y500F, and Y737F, and insertions of the amino acid sequence as shown in SEQ ID NO: 33 between amino acid positions 587 and 588.

19. The variant AAV capsid protein of claim 16, wherein the capsid protein comprises a combination of modifications including flanking substitutions of RAA at amino acid positions 585 to 587 of the parental AAV capsid protein, amino acid substitution K556E, and insertions of the amino acid sequence as shown in SEQ ID NO: 33 between amino acid positions 587 and 588.

20. The variant AAV capsid protein of claim 16, wherein the capsid protein comprises a combination of modifications comprising the amino acid substitution Y737F of the parental AAV capsid protein and the insertion of the amino acid sequence as shown in SEQ ID NO: 23 between amino acid positions 587 and 588.

21. The variant AAV capsid protein of claim 16, wherein the capsid protein comprises a combination of modifications comprising amino acid substitutions of the parental AAV capsid protein Y444F, Y500F, Y737F and an insertion of the amino acid sequence as shown in SEQ ID NO: 23 between amino acid positions 587 and 588.

22. The variant AAV capsid protein of claim 16, wherein the capsid protein comprises a combination of modifications comprising the amino acid substitution Y737F of the parental AAV capsid protein and the insertion of the amino acid sequence as shown in SEQ ID NO: 36 between amino acid positions 587 and 588.

23. The variant AAV capsid protein of claim 16, wherein the capsid protein comprises a combination of modifications comprising amino acid substitutions of the parental AAV capsid protein Y444F, Y500F, Y737F and an insertion of the amino acid sequence as shown in SEQ ID NO: 36 between amino acid positions 587 and 588.

24. An isolated polynucleotide comprising a nucleotide sequence encoding a variant AAV capsid protein as described in any one of claims 1 to 23.

25. A vector comprising the isolated polynucleotide of claim 24.

26. An isolated, genetically modified host cell comprising the polynucleotide of claim 24.

27. A recombinant AAV toxic particle comprising the variant AAV capsid protein of any one of claims 1 to 23.

28. A pharmaceutical composition comprising: a) the recombinant adeno-associated virus particle of claim 27; and b) a pharmaceutically acceptable excipient.

29. A recombinant AAV vector comprising a polynucleotide encoding a variant AAV capsid protein as described in any one of claims 1 to 23, an AAV 5' inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional gene product, a regulatory sequence guiding the expression of said gene product in target cells, and an AAV 3' ITR.

30. A method for delivering a nucleic acid vector encoding a functional gene product to cells and / or tissues using the recombinant AAV virion particle of claim 27 or the recombinant AAV vector of claim 29.

31. A method of treating a disease, the method comprising administering to a subject in need an effective amount of the recombinant AAV venom particle of claim 27, the recombinant AAV venom particle comprising a functional gene product.

32. The method according to any one of claims 30 or 31, wherein the gene product is a polypeptide.

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