Adeno-associated virus capsid protein, adeno-associated virus containing the same, vectors and applications

Abstract

This disclosure provides adeno-associated virus capsid proteins, adeno-associated viruses containing them, vectors, and applications. Specifically, this disclosure provides adeno-associated virus capsid proteins containing a target peptide comprising (i) sequence AAX1X2X3X4X5X6X7X8AA and (ii) sequence NG, one or more of the above. This disclosure also provides target peptides, adeno-associated viruses, transgene delivery vectors, pharmaceutical compositions, etc. Compared to wild-type adeno-associated viruses, the adeno-associated virus capsid proteins and adeno-associated viruses provided in this disclosure have improved cell infection efficiency in vivo and / or in vitro, good targeting of the eye, and can be used as delivery vectors for the treatment of eye-related diseases.

Description

[Technical Field]

[0001] Priority rights and related applications

[0002] This disclosure claims priority to PCT / CN2023 / 095837, filed on 23 May 2023, with the title of the invention "Adeno-associated virus capsid protein, adeno-associated virus comprising the same, vector and application," the entire contents of said application, including its appendices, are incorporated herein by reference.

[0003] Technical field This disclosure relates to the technology of viral vectors, and more specifically to adeno-associated virus capsid proteins, adeno-associated viruses containing them, vectors, and applications. [Background technology]

[0004] Background technology As the number of approved gene therapies increases, improving the delivery efficiency and safety of gene delivery vectors has become a critical development consideration. Recombinant adeno-associated virus (rAAV) vectors are one such example. For instance, in 2017, Spark Therapeutics, Inc.'s adeno-associated virus type 2 (AAV2)-based gene therapy drug "Luxturna" received approval from the U.S. Food and Drug Administration (FDA) for the treatment of Leber's congenital amaurosis (LCA) caused by RPE65 gene mutations. Adeno-associated viruses (AAVs) have low pathogenicity and the ability to stably express proteins for long periods in various tissues and organs. However, wild-type AAV serotypes infect a wide range of tissues and organs in mammals, and because their target tissues are so broad, they can lead to gene delivery to untargeted tissues, exacerbating side effects.

[0005] The tissue-specificity and cell delivery efficiency of AAV vectors are primarily determined by their capsid. To obtain superior therapeutic effects, it is desirable to obtain organ-specific AAV vectors (e.g., eye) by appropriately modifying the AAV capsid protein (Cap). This is usually achieved by inserting a polypeptide of 7 to 20 amino acids into the AAV capsid protein through site-directed mutagenesis. For example, by inserting a 14-amino acid peptide fragment L14 (specifically QAGTFALRGDNPQG, SEQ ID NO: 66; containing the RGD structure) after the 587th amino acid in the coding sequence of the AAV2 capsid protein, tumor cells targeting β1 integrin-positive cells can be obtained (Girod et al. (1999), Nat. Med. 5, 1052-1056).

[0006] However, the effects are unpredictable depending on the polypeptide inserted into the AAV capsid protein. Furthermore, there remains a need in the industry to develop and apply modified novel AAV capsid proteins to AAV vectors in order to create effective gene delivery vectors. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Invention Description

[0008] Problems that the invention aims to solve

[0009] In view of the above-mentioned problems in the prior art, this disclosure aims to provide an eye-targeting peptide used in AAV capsid proteins, as well as an AAV capsid protein, AAV, and an AAV vector containing the targeting peptide, in order to improve the eye-targeting ability of AAV / AAV vectors as gene delivery vectors. [Means for solving the problem]

[0010] Means for Solving the Problem

[0011] To achieve a better therapeutic effect, the present disclosure designed 15 modifications by inserting several amino acids after the 587th amino acid of AAV2 or substituting the 586th and 587th amino acids, aiming to obtain a novel adeno-associated virus vector with high introduction efficiency into the retina.

[0012] The first aspect of the present disclosure is an adeno-associated virus capsid protein containing a target peptide, wherein the target peptide is

[0013] (i) the sequence AAX1X2X3X4X5X6X7X8AA,

[0014] (ii) the sequence NG including one or more of the above,

[0015] In the sequence AAX1X2X3X4X5X6X7X8AA,

[0016] (a) X1 is A,

[0017] (b) X2 is R or G,

[0018] (c) X3 is G or N,

[0019] (d) X4 is D, S or G,

[0020] (e) X5 is L or R,

[0021] (f) X6 is A, G, Q, or deleted,

[0022] (g) X7 is T, R, N, A, H, or deleted,

[0023] (h) X8 is I, L, P, A, or deleted, and provides an adeno-associated virus capsid protein. <00 In some embodiments, in the sequence AAX1X2X3X4X5X6X7X8AA,

[0025] (a) X1 is A,

[0026] (b) X2 is R,

[0027] (c) X3 is G,

[0028] (d) X4 is D or S,

[0029] (e) X5 is L,

[0030] (f) X6 is A, G, Q, or is deleted,

[0031] (g) X7 is T, R, N, A, or is deleted,

[0032] (h) X8 is I, L, P, or is deleted.

[0033] In some specific embodiments, the target peptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 1-14 and SEQ NG.

[0034] In some preferred embodiments, the target peptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 2, 8, 11, 12, 13, 14 and SEQ NG.

[0035] In some more preferred embodiments, the target peptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 8, 13, 14 and SEQ NG.

[0036] In some embodiments, the adeno-associated virus is one or more selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43 and rh.74.

[0037] In some preferred embodiments, the AAV includes the AAV2.

[0038] In some embodiments, AAV2 includes AAV2 VP1 or a variant thereof.

[0039] In some preferred embodiments, the amino acid sequence of AAV2 VP1 is as shown in SEQ ID NO: 60.

[0040] In some preferred embodiments, variants of AAV2 VP1 include a variant in which the 34th proline (P) is mutated to alanine (A) compared to the amino acid sequence shown in SEQ ID NO: 60.

[0041] In some specific embodiments, the target peptide is inserted at the 587th amino acid position of AAV2 VP1 or a variant thereof.

[0042] In some specific embodiments, the target peptide is substituted at the 586th and 587th amino acids of AAV2 VP1 or a variant thereof.

[0043] In some preferred embodiments, the adeno-associated virus capsid protein comprises an amino acid sequence represented by any one of SEQ ID NOs: 15-29 and 65, and an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% identity with the amino acid sequence represented by any one of SEQ ID NOs: 15-29 and 65.

[0044] The second phase of this disclosure is,

[0045] (i) Array AAX1X2X3X4X5X6X7X8AA,

[0046] (ii) Array NG A target peptide comprising one or more of the following:

[0047] In the array AAX1X2X3X4X5X6X7X8AA,

[0048] (a) X1 is A,

[0049] (b) X2 is R or G,

[0050] (c) X3 is G or N,

[0051] (d) X4 is D, S or G,

[0052] (e) X5 is either L or R,

[0053] (f) X6 is A, G, Q, or missing,

[0054] (g)X7 is T, R, N, A, H, or missing,

[0055] (h)X8 provides a target peptide that is I, L, P, A, or missing.

[0056] In some arbitrary embodiments, in the sequence AAX1X2X3X4X5X6X7X8AA,

[0057] (a) X1 is A,

[0058] (b) X2 is R,

[0059] (c)X3 is G,

[0060] (d) X4 is either D or S,

[0061] (e) X5 is L,

[0062] (f) X6 is A, G, Q, or missing,

[0063] (g)X7 is T, R, N, A, or missing.

[0064] (h)X8 is I, L, P, or missing.

[0065] In some preferred embodiments, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-14 and sequence NG.

[0066] In some more preferred embodiments, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 8, 11, 12, 13, 14 and sequence NG.

[0067] In some more preferred embodiments, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 8, 13, and 14.

[0068] A third aspect of this disclosure provides polynucleotides encoding the adeno-associated virus capsid protein described in the first aspect of this disclosure, or the target peptide described in the second aspect of this disclosure.

[0069] The fourth aspect of this disclosure provides an adeno-associated virus comprising the adeno-associated virus capsid protein described in the first aspect of this disclosure.

[0070] In some embodiments, the adeno-associated virus further comprises a transgene.

[0071] In some optional embodiments, the transgene is a therapeutic transgene, a prophylactic transgene, or a diagnostic transgene.

[0072] A fifth aspect of this disclosure provides a transgene delivery vector containing the adeno-associated virus described in the fourth aspect of this disclosure.

[0073] The sixth phase of this disclosure is:

[0074] (a) an adeno-associated virus as described in Section 4 of this Disclosure or a transgene delivery vector as described in Section 5 of this Disclosure, and

[0075] (b) Provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier, in some cases.

[0076] Aspect VII of this disclosure provides the use of the adeno-associated virus described in Aspect V of this disclosure, the transgene delivery vector described in Aspect V of this disclosure, or the pharmaceutical composition described in Aspect V of this disclosure in the manufacture of a reagent for delivering transgenes to cells.

[0077] In some embodiments, the cells are derived from the subject.

[0078] In some preferred embodiments, the subject is a mammal.

[0079] In some more preferred embodiments, the subject is a human being.

[0080] In some embodiments, the cells are derived from the eye.

[0081] In some embodiments, the subject suffers from an eye disease.

[0082] In some embodiments, the adeno-associated virus, the transgene delivery vector, or the pharmaceutical composition is administered to the subject by subretinal or intravitreal injection.

[0083] The eighth aspect of this disclosure provides the use of the adeno-associated virus described in the fourth aspect of this disclosure, the transgene delivery vector described in the fifth aspect of this disclosure, or the pharmaceutical composition described in the sixth aspect of this disclosure in the manufacture of a drug for the treatment of a disease.

[0084] In some embodiments, the disease includes eye diseases.

[0085] The ninth aspect of this disclosure provides a method for treating a disease, comprising the step of administering to a subject an adeno-associated virus as described in the fourth aspect of this disclosure, a transgene delivery vector as described in the fifth aspect of this disclosure, or a pharmaceutical composition as described in the sixth aspect of this disclosure.

[0086] In some embodiments, the disease includes eye diseases. [Effects of the Invention]

[0087] Effects of the invention

[0088] The adeno-associated virus capsid protein and adeno-associated virus contained herein have improved in vivo and / or in vitro cell infection efficiency and good targeting properties for the eye compared to wild-type adeno-associated virus, and can be used as a delivery vector for the treatment of eye-related diseases. [Brief explanation of the drawing]

[0089] [Figure 1] Figure 1 shows the insertion sites of candidate AAV capsid proteins and a schematic diagram of the plasmid packaging system for the novel AAV capsid.

[0090] [Figure 2] Figure 2 shows the quantitative results for 15 candidate AAVs that infected human retinal epithelial cells (ARPE-19), using the proportion of GFP-positive ARPE-19 cells as a statistical indicator.

[0091] [Figure 3] Figure 3 shows the status of introduction of four candidate AAVs (AAV IVT8 / IVT13 / IVT14 / IVT15) and wild-type AAV2 into the mouse retina.

[0092] [Figure 4] Figure 4A shows the introduction status of three candidate AAVs (AAV IVT8 / IVT13 / IVT15) and wild-type AAV2 when infecting human retinal organoids, and Figure 4B shows the percentage of GFP in retinal cells.

[0093] [Figure 5A] Figure 5A shows the GFP expression signal in the retina of mice injected intravitreously with AAV IVT13 and wild-type AAV2 (2E+9vg / eye).

[0094] [Figure 5B] Figure 5B shows a section of the entire retina of a mouse into which AAV IVT13 and wild-type AAV2 were introduced.

[0095] [Figure 5C] Figure 5C shows sections of mouse retinas into which AAV IVT13 and wild-type AAV2 have been introduced, with GFP, DAPI, and the retinal ganglion cell marker RBPMS indicated.

[0096] [Figure 5D] Figure 5D shows sections of mouse retinas into which AAV IVT13 and wild-type AAV2 have been introduced, with GFP, DAPI, and the bipolar cell marker PKCα indicated.

[0097] [Figure 5E] Figure 5E shows a section of the retina of a mouse introduced with AAV IVT13 and wild-type AAV2, with GFP, DAPI, and the retinal pigment epithelial marker RPE65 indicated.

[0098] [Figure 6A] Figure 6A shows sections of monkey retinas into which AAV IVT13 and wild-type AAV2 have been introduced, with GFP, DAPI, and the retinal bipolar cell marker PKCα indicated.

[0099] [Figure 6B] Figure 6B shows sections of monkey retinas into which AAV IVT13 and wild-type AAV2 have been introduced, with GFP, DAPI, and the retinal photoreceptor layer marker RS1 indicated.

[0100] [Figure 7] Figure 7 shows the transduction efficiency when ARPE-19 cells were infected with AAV IVT13, AAV IVT13-P34A, and wild-type AAV2.

[0101] [Figure 8] Figure 8 shows the distribution of AAV IVT13, AAV IVT13-P34A, and wild-type AAV2 when used to infect retinal organoids. [Modes for carrying out the invention]

[0102] Specific implementation methods To facilitate understanding of this disclosure, technical and scientific terms are defined below. Unless otherwise specified herein, other technical and scientific terms used herein have meanings generally understood by those skilled in the art to which this disclosure pertains.

[0103] In this specification, a numerical range expressed as "Numerical A to Numerical B" means that the values ​​A and B at both ends are included within this range.

[0104] In this specification, “basically” or “substantially” means that the standard deviation from the theoretical model or theoretical data is within 5%, preferably within 3%, and more preferably within 1%.

[0105] In this specification, the term "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0106] In this specification, “any” or “optionally” means that the events or circumstances described below may or may not occur, and that this includes both cases in which such events occur and cases in which they do not occur.

[0107] The terms “including” and “having” in this disclosure, and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, article, or device comprising a set of steps may include, but is not limited to, any steps not listed, and may also include any other steps specific to those processes, methods, articles, or devices.

[0108] In this disclosure, "multiple" refers to two or more. "And / or" describes the relationship between the related objects and indicates that there can be three types of relationships. For example, "A and / or B" represents three situations: A exists alone, both A and B exist, and B exists alone. The " / " in the sentence generally indicates that the related objects on both sides are in an "or" relationship.

[0109] In this specification, “some specific / preferred embodiments,” “another specific / preferred embodiments,” “embodiments,” etc., mean that certain elements (e.g., features, structures, properties, and / or characteristics) described in relation to such embodiments are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it should be understood that such elements may be incorporated into various embodiments in any preferred form.

[0110] According to this disclosure, "Vg" refers to the viral genome and is equivalent to a genome copy.

[0111] According to this disclosure, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably herein and mean polymeric forms of amino acids of any length, which may include encoded and unencoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having similar peptide backbones.

[0112] According to this disclosure, the terms “nucleic acid molecule,” “polynucleotide,” “polynucleic acid,” and “nucleic acid” are used interchangeably and refer to polymeric forms of nucleotides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, regulatory regions, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acid molecules may be linear or circular.

[0113] As used in this disclosure, the term "amino acid" includes natural amino acids, unnatural amino acids, amino acid analogs, and all D and L forms thereof. According to this disclosure, the three-letter and one-letter codes for amino acids used are as defined in J. biol. chem, 243, p3558 (1968). The amino acids and their abbreviations, as well as their English abbreviations, used in this disclosure are as follows: Histidine (His,H), serine (Ser,S), glutamic acid (Glu,E), glutamine (Gln,Q), glycine (Gly,G), threonine (Thr,T), phenylalanine (Phe,F), aspartic acid (Asp,D), tyrosine (Tyr,Y), leucine (Leu,L), isoleucine (Ile,I), arginine (Arg,R), alanine (Ala,A), valine (Val,V), tryptophan (Trp,W), methionine (Met,M), asparagine (Asn,N), cysteine ​​(Cys,C), lysine (Lys,K), proline (Pro,P).

[0114] According to this disclosure, "addition" of an amino acid refers to the addition of an amino acid to the C-terminus or N-terminus of an amino acid sequence. According to this disclosure, "deletion" of an amino acid refers to the removal of one, two, or three or more amino acids from an amino acid sequence. According to this disclosure, "insertion" of an amino acid refers to the insertion of an amino acid residue at an appropriate position in an amino acid sequence. The inserted amino acid residues may be adjacent to each other in whole or in part, or they may not be adjacent to each other. According to this disclosure, "substitution" of an amino acid refers to the replacement of an amino acid residue at a certain position in an amino acid sequence with another amino acid residue, where the "substitution" may be a conservative amino acid substitution.

[0115] According to this disclosure, “conservative modification,” “conservative substitution,” or “conservative replacement” refers to the substitution of an amino acid in a protein with another amino acid having similar properties (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation and rigidity), thereby allowing frequent modifications without altering the protein’s biological activity. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). Furthermore, amino acid substitutions with similar structure or function are unlikely to impair biological activity. Exemplary conservative substitutions are described below in “Exemplary Conservative Amino Acid Substitutions.”

[0116] Exemplary Conservative Substitutions of Amino Acids [Table A]

[0117] According to this disclosure, “medium to very high stringency conditions” include “medium stringency conditions,” “medium to high stringency conditions,” “high stringency conditions,” or “very high stringency conditions,” and nucleic acid hybridization and washing conditions are described. Guidance for carrying out hybridization reactions is provided in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1–6.3.6, which is incorporated herein by reference. This document describes aqueous and non-aqueous methods, either of which can be used. For example, specific hybridization conditions are as follows: (1) Low stringency hybridization conditions consist of two washes with 6× sodium chloride / sodium citrate (SSC), approximately 45°C, followed by at least 50°C, 0.2× SSC, and 0.1% SDS (in the case of low stringency conditions, the wash temperature may be raised to 55°C); (2) Medium stringency hybridization conditions consist of one or more washes with 6× SSC, approximately 45°C, followed by 60°C, 0.2× SSC, and 0.1% SDS; preferably (3) High stringency hybridization conditions consist of one or more washes with 6× SSC, approximately 45°C, followed by 65°C, 0.2× SSC, and 0.1% SDS; (4) Ultra-high stringency hybridization conditions consist of one or more washes with 0.5M sodium phosphate, 7% SDS, 65°C, followed by 65°C, 0.2× SSC, and 1% SDS.

[0118] "Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit—for example, if each position in two DNA molecules is occupied by adenine—then those molecules are homologous at that position. The percentage of identity between two sequences is a function of multiplying the number of matching or homologous positions shared by the two sequences by the number of positions being compared, and then multiplying that by 100%. For example, if, when two sequences are optimally aligned, 6 out of 10 positions in the two sequences match or are homologous, then these two sequences have 60% homology. Generally, a comparison is made when the alignment of the two sequences yields the highest possible percentage of identity.

[0119] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biofluids, “administration,” “application,” and “treatment” refer to the contact of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with animals, humans, subjects, cells, tissues, organs, or biofluids. “Administration,” “application,” and “treatment” can also refer to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods, for example. Treatment of cells includes contacting reagents with cells and contacting reagents with liquids (where the liquid is in contact with the cells). “Administration,” “application,” and “treatment” further mean the in vitro and ex vivo treatment of cells with reagents, diagnostic agents, conjugation compositions, or other cells. When applied to humans, veterinary medicine, or research subjects, “treatment” refers to therapeutic, preventive or prophylactic measures, research, and diagnostic applications.

[0120] "Treatment" means administering, orally or topically, a therapeutic agent containing any of the antibodies of this disclosure to a patient having one or more disease symptoms for which such therapeutic agent is known to be therapeutic. Generally, the therapeutic agent is administered in a dose effective in alleviating one or more disease symptoms in the patient or population being treated, inducing regression of such symptoms or preventing progression to a clinically measurable extent. The dose of a therapeutic agent effective in alleviating specific disease symptoms (also called the "therapeutic dose") varies depending on a variety of factors, including the patient's condition, age, weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the symptoms of a disease have been alleviated can be assessed by clinical measurements commonly used by physicians or other professional healthcare providers to assess the severity or progression of those symptoms.

[0121] In this specification, the term "prevention" refers to preventive treatment for subjects who do not currently have a disease, or who have not had a disease in the past but are at risk of developing a disease, or for subjects who have had a disease in the past and are not currently ill but are at risk of disease recurrence. In some embodiments, subjects have a higher risk of developing a disease or disease recurrence compared to the average healthy person in the subject population.

[0122] An "effective dose" encompasses an amount sufficient to improve or prevent the symptoms or condition of a medical illness. It also means an amount sufficient to enable or facilitate a diagnosis. The effective dose for a particular patient or veterinary subject varies depending on factors such as the medical condition being treated, the patient's overall health, the route and dose of administration, and the severity of side effects. The effective dose may also be the maximum dose or administration plan that avoids significant side effects or toxic effects.

[0123] As used herein, "therapeutically effective amount" refers to an amount sufficient to produce a therapeutic effect in the treatment of a disease, or to delay or minimize the manifestation of one or more symptoms associated with the disease. A therapeutically effective amount refers to the amount of a therapeutic agent that produces a therapeutic effect in the treatment of a disease, either alone or in combination with other therapies. The term "therapeutically effective amount" may include an amount that improves the overall treatment, reduces or avoids the symptoms, signs, or causes of the disease, and / or enhances the therapeutic effect of other therapeutic agents.

[0124] As used herein, "preventively effective amount" refers to an amount sufficient to prevent a disease or one or more symptoms associated with the disease, or to prevent its recurrence. A preventively effective amount refers to the amount of a therapeutic agent that produces a preventive effect in the prevention of a disease, either alone or in combination with other drugs. The term "preventively effective amount" may include an amount that improves the overall prevention, or enhances the preventive effect of other preventive agents.

[0125] As used herein, the term "subject" refers to a human (i.e., male or female of any age, such as pediatric subjects (e.g., infants, children, or adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., primates (e.g., cynomolgus monkeys or rhesus monkeys), commercially relevant mammals (e.g., cows, pigs, horses, sheep, goats, cats, or dogs)), or birds. The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic animal or a genetically modified animal.

[0126] Detailed Description

[0127] <AAV Capsid Protein and Target Peptide>

[0128] In some embodiments of the present disclosure,

[0129] (i) the sequence AAX1X2X3X4X5X6X7X8AA,

[0130] (ii) Array NG The present invention provides an adeno-associated virus (AAV) capsid protein containing one or more target peptides.

[0131] In some specific embodiments of this disclosure, in sequence AAX1X2X3X4X5X6X7X8AA,

[0132] (a) X1 is A,

[0133] (b) X2 is R or G,

[0134] (c) X3 is G or N,

[0135] (d) X4 is D, S or G,

[0136] (e) X5 is either L or R,

[0137] (f) X6 is A, G, Q, or missing,

[0138] (g)X7 is T, R, N, A, H, or missing,

[0139] (h)X8 is I, L, P, A, or missing.

[0140] For example, the target peptide includes the amino acid sequence shown in any one of SEQ ID NOs: 1 to 14.

[0141] In some more specific embodiments of this disclosure, in sequence AAX1X2X3X4X5X6X7X8AA,

[0142] (a) X1 is A,

[0143] (b) X2 is R,

[0144] (c)X3 is G,

[0145] (d) X4 is either D or S,

[0146] (e) X5 is L,

[0147] (f) X6 is A, G, Q, or missing,

[0148] (g)X7 is T, R, N, A, or missing.

[0149] (h)X8 is I, L, P, or missing.

[0150] For example, the target peptide includes the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13.

[0151] In some preferred embodiments of this disclosure, in the sequence AAX1X2X3X4X5X6X7X8AA,

[0152] (a) X1 is A,

[0153] (b) X2 is R,

[0154] (c)X3 is G,

[0155] (d) X4 is either D or S,

[0156] (e) X5 is L,

[0157] (f)X6 is G, Q, or missing,

[0158] (g)X7 is R, N, or deleted.

[0159] (h)X8 is P, I, L, or missing.

[0160] For example, the target peptide includes the amino acid sequence shown in any one of SEQ ID NOs: 2, 8, 11, 12, or 13.

[0161] In some preferred embodiments of this disclosure, in the sequence AAX1X2X3X4X5X6X7X8AA,

[0162] (a) X1 is A,

[0163] (b) X2 is R,

[0164] (c)X3 is G,

[0165] (d) X4 is D

[0166] (e) X5 is L,

[0167] (f) X6 is G,

[0168] (g)X7 is R,

[0169] (h)X8 is either L or P.

[0170] For example, the target peptide includes the amino acid sequence shown in either SEQ ID NO: 2 or 8.

[0171] In another preferred embodiment of this disclosure, in sequence AAX1X2X3X4X5X6X7X8AA,

[0172] (a) X1 is A,

[0173] (b) X2 is R,

[0174] (c)X3 is G,

[0175] (d) X4 is S,

[0176] (e) X5 is L,

[0177] (f)X6 is Q or missing,

[0178] (g)X7 is N or missing,

[0179] (h)X8 is I, L, or missing.

[0180] For example, the target peptide includes the amino acid sequence shown in any one of SEQ ID NOs: 11, 12, or 13.

[0181] In another preferred embodiment of the present disclosure, the target peptide comprises the amino acid sequence shown in SEQ ID NO: 14.

[0182] In some preferred embodiments of this disclosure, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-14 and sequence NG.

[0183] In some more preferred embodiments of this disclosure, the target peptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 2, 8, 11, 12, 13, 14 and sequence NG.

[0184] In some more preferred embodiments of this disclosure, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 8, 13, 14 and sequence NG.

[0185] In some more preferred embodiments of this disclosure, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 8, 13, and 14.

[0186] In this disclosure, AAV is a tiny, non-enveloped virus with a 25 nm capsid. No diseases associated with or associated with the wild-type virus are known. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal expression. AAV vectors up to 300 base pairs can be packaged and incorporated. The size constraint for exogenous DNA is approximately 4.7 kb. For example, AAV vectors such as the AAV vector described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985) can be used for DNA delivery into cells. AAV vectors are used to introduce various nucleic acids into different cell types (see, for example, Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); Flotte et al., J. Biol. Chem. 268:3781-3790 (1993)). Numerous AAV variants are available (over 100 have been cloned), and these variants are identified based on desired characteristics.

[0187] In some embodiments, the AAV is one or more selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh.10, rh.39, rh.43, and rh.74.

[0188] In some preferred embodiments, the AAV is an AAV2.

[0189] The AAV capsid is an icosahedron composed of 60 VP capsid protein monomers (including 5 VP1 monomers, 5 VP2 monomers, and 50 VP3 monomers). All VP1, VP2, and VP3 monomers are transcribed and translated from the AAV Cap gene. VP1 is the longest, containing approximately 735 amino acids. VP2 and VP3 are "shortened" versions of VP1 and do not contain the N-terminal amino acids of the VP1 protein. By convention, modification sites in the capsid protein are named based on the amino acid sequence of the VP1 protein. For example, inserting a target peptide after the 587th amino acid of the AAV2 capsid / capsid protein means inserting the target peptide after the 587th amino acid of the AAV2 VP1 monomer (containing 735 amino acids), so that this target peptide is included in the corresponding positions of AAV2 VP1, VP2, and VP3. The amino acid sequence of the AAV2 capsid / capsid protein is usually represented by the amino acid sequence of VP1.

[0190] In some embodiments, AAV2 includes AAV2 VP1 or a variant thereof, AAV2 VP2 or a variant thereof, and / or AAV2 VP3 or a variant thereof.

[0191] For example, the amino acid sequence of AAV2 VP1 is as follows (SEQ ID NO: 60): MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL * (It consists of a total of 735 amino acids, and the insertion site of the target peptide is between position N587 and R588.)

[0192] For example, variants of AAV2 VP1 include a variant in which the 34th proline (P) is mutated to alanine (A) compared to the amino acid sequence shown in SEQ ID NO: 60.

[0193] The amino acid sequence of AAV2 VP2 is as follows (SEQ ID NO: 61): MAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNR FHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNP LIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQR GNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL * (This consists of a total of 598 amino acids, corresponding to amino acids 138 through 736 of AAV2 VP1. The insertion site of the target peptide is between N450 and R451, which corresponds to between N587 and R588 of AAV2 VP1.)

[0194] The amino acid sequence of AAV2 VP3 is as follows (SEQ ID NO: 62):

[0195] MATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANN LTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGAS DIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVL PGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL * (This consists of a total of 533 amino acids, corresponding to amino acids 203 through 736 of AAV2 VP1. The insertion site of the target peptide is between N385 and R386, which corresponds to between N587 and R588 of AAV2 VP1.)

[0196] In some embodiments of this disclosure, the target peptide is inserted at the 587th amino acid position of AAV2 VP1 or a variant thereof. That is, the target peptide lies between the 587th and 588th amino acids of AAV2 VP1 or a variant thereof. In some specific embodiments of this disclosure, when the target peptide is inserted at the 587th amino acid position of AAV2 VP1 or a variant thereof, the target peptide comprises the sequence AAX1X2X3X4X5X6X7X8AA.

[0197] In some embodiments of this disclosure, the target peptide substitutes the 586th and 587th amino acids of AAV2 VP1 or a variant thereof. In some specific embodiments of this disclosure, when the target peptide substitutes the 586th and 587th amino acids of AAV2 VP1 or a variant thereof, the target peptide includes the sequence NG described above.

[0198] In some preferred embodiments of this disclosure, the AAV capsid protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 15-29 and 65.

[0199] In some specific embodiments, the AAV sequence may have, for example, at least 80%, 85%, 90%, 95%, 97%, or 99% identity with the exemplary AAV sequences described herein. For example, variants may be included, preferably without reducing the AAV's ability to mediate transgene expression in cells.

[0200] <Polynucleotides>

[0201] In some embodiments of this disclosure, polynucleotides encoding the AAV capsid protein of this disclosure or the target peptide of this disclosure are provided.

[0202] Polynucleotides in this disclosure may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be coding or non-coding strands.

[0203] Encoding an AAV capsid protein as disclosed herein includes coding sequences that encode only an AAV capsid protein / target peptide, coding sequences of an AAV capsid protein / target peptide and various additional coding sequences, coding sequences of an AAV capsid protein / target peptide (and any additional coding sequences) and non-coding sequences.

[0204] The "polynucleotide encoding an AAV capsid protein / target peptide" may include a polynucleotide encoding this AAV capsid protein / target peptide, and may further include an additional coding sequence and / or a non-coding sequence.

[0205] The present disclosure also relates to polynucleotides that hybridize to the aforementioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present disclosure particularly relates to polynucleotides that can hybridize to the polynucleotides described in the present disclosure under stringent conditions. The stringent conditions are medium stringency conditions, medium to high stringency conditions, high stringency conditions, or ultra-high stringency conditions.

[0206] <AAV and Transgene Delivery Vector>

[0207] In some embodiments of the present disclosure, an AAV containing the AAV capsid protein described in the present disclosure is provided.

[0208] In some aspects of the present disclosure, a transgene delivery vector containing the AAV described in the present disclosure is further provided.

[0209] The AAV and transgene delivery vector of the present disclosure can be used to deliver any composition, such as a sequence of interest, to tissues such as the retina.

[0210] In some embodiments, the AAV further includes a transgene.

[0211] In some specific embodiments, the transgene includes a nucleotide sequence encoding a gene product. “Genes” refers to polynucleotides containing at least one open reading frame capable of encoding a specific protein after transcription and translation. “Genetic products” are molecules produced by the expression of a particular gene. Genetic products include, for example, polypeptides, aptamers, interfering RNA, mRNA, and the like.

[0212] In some specific embodiments, the transgene is a therapeutic transgene, a prophylactic transgene, or a diagnostic transgene.

[0213] In some exemplary embodiments, the transgene is a therapeutic transgene that encodes a therapeutic gene sequence (for example, a gene sequence that treats an eye disease).

[0214] In another exemplary embodiment, the transgene is a gene sequence encoding a reporter protein, such as a fluorescent protein (an enzyme that catalyzes the production of a detectable product).

[0215] <Pharmaceutical composition and method of administration>

[0216] In some embodiments of this disclosure,

[0217] (a) the above AAV or the above transgene delivery vector, and

[0218] (b) Provide a pharmaceutical composition comprising a pharmaceutically acceptable carrier, in some cases.

[0219] In some embodiments, the pharmaceutical compositions described herein include (i) a target peptide and (ii) a transgene, such as a therapeutic transgene (exemplary, a therapeutic agent for treating an eye disease), as an active ingredient, the aforementioned AAV or the aforementioned transgene delivery vector.

[0220] As used herein, the term "pharmaceutically acceptable carrier" includes salines, solvents, dispersions, coatings, antimicrobial / antifungal agents, isotonic agents, and absorption retarders that are suitable for pharmaceutically acceptable administration.

[0221] Pharmaceutical compositions are typically formulated to suit the intended route of administration. Examples of administration routes include parenteral administration, such as intravenous, intra-arterial, subcutaneous, intraperitoneal, intrathecal, and intramuscular injection or infusion. Therefore, delivery can be systemic or local. For example, delivery to the retina is performed via subretinal or intravitreous injection (see, e.g., Ochakovski et al., Front Neurosci. 2017; 11: 174; Xue et al., Eye (Lond). 2017 Sep; 31(9): 1308-1316).

[0222] The methods for formulating appropriate pharmaceutical compositions are publicly known in the industry. For example, see Remington: The Science and Practice of Pharmacy, 21st ed, 2005; and the Drugs and the Pharmaceutical Sciences: A Series of Textbooks and Monographs (Dekker, NY).

[0223] <Uses and Methods>

[0224] In some embodiments of this disclosure, the use of the AAV described herein, the transgene delivery vector described herein, and / or the pharmaceutical composition described herein is provided in the preparation of reagents for delivering transgenes to cells.

[0225] In another subset of the present disclosure, a method for delivering transgenes to cells is provided, comprising the step of contacting the aforementioned cells with the AAV described in the present disclosure, the transgene delivery vector described in the present disclosure, and / or the pharmaceutical composition described in the present disclosure.

[0226] The AAVs described herein can be used to deliver transgenes, such as therapeutic transgenes, to tissues such as the eye of a subject. In some embodiments, the AAVs described herein can deliver transgenes / therapeutic transgenes to retinal cells. Examples of retinal cells include retinal photoreceptors, interneurons, retinal ganglion cells, or retinal pigment epithelial cells (RPEs).

[0227] In some embodiments, the AAV described herein is used to deliver a nucleic acid sequence encoding a therapeutic agent to a subject suffering from an eye disease.

[0228] In some specific embodiments of this disclosure, the use of the AAV described herein, the transgene delivery vector described herein, and / or the pharmaceutical composition described herein is provided in the manufacture of a drug for the treatment of a disease.

[0229] In another specific embodiment of the present disclosure, a method for treating a disease is provided, comprising the step of administering to a subject an AAV described in the present disclosure, a transgene delivery vector described in the present disclosure, and / or a pharmaceutical composition described in the present disclosure.

[0230] In this disclosure, the disease may be any disease treatable by an AAV carrying a transgene. In some preferred embodiments, the disease may be an ocular disease. In some specific embodiments, ocular diseases include, but are not limited to, Wet Age-related Macular Degeneration (wetAMD), Bietti Crystalline Corneoretinal Dystrophy (BCD), X-linked juvenile retinoschisis (XLRS), Leber's Hereditary Optic Neuropathy (LHON), and Leber's congenital amaurosis (LCA). [Examples]

[0231] Examples

[0232] The Disclosure will be described in more detail below based on specific embodiments. The following embodiments are for illustrative purposes only and do not limit the scope of the Disclosure. The embodiments provided below are intended to guide those skilled in the art for further improvement and do not limit the Disclosure in any way.

[0233] Unless otherwise specified, the experimental methods in the following examples are standard procedures and are carried out in accordance with the techniques or conditions described in the literature of this industry, or in accordance with the product instructions. Unless otherwise specified, all materials, reagents, etc. used in the following examples are commercially available products.

[0234] Materials and methods

[0235] HEK 293T cell line (ATCC, CRL-3216)

[0236] Human retinal epithelial cells (ARPE-19, Wuhan Procell, catalog number CL-0026)

[0237] Fetal bovine serum (Gibco, A3161002C)

[0238] DMEM medium (Dulbecco's Modified Eagle Medium, Gibco, 11965092)

[0239] DMEM / F12 medium (Dulbecco's Modified Eagle Medium, F-12, Gibco, 11320-033)

[0240] Penicillin-Streptomycin (Gibco, 15140122)

[0241] 0.05% Trypsin-EDTA (Gibco, 25300062)

[0242] Opti-MEM (Gibco, 31985070)

[0243] Polyethyleneimine Linear (PEI) MW40000 (YEASEN, 40816ES03)

[0244] Ca 2+ Mg 2+ D-PBS (Corning, 21-030-CV) containing [unclear].

[0245] 24-well TC-treated culture plate (Costar, 3524)

[0246] 100mm TC-treated culture dish (Jet Bio-Filtration, TCD010100)

[0247] 15 mL Centrifuge Tube (Axygen, SCT-15ML-25-S)

[0248] Pipettes (Serological Pipets, Jet Bio-Filtration)

[0249] Benzonase nuclease (Sigma-Aldrich, E8263-25KU)

[0250] Q5 High-Fidelity 2X Master Mix (Q5® High-Fidelity 2X Master Mix, NEB, M0492L).

[0251] Example 1: Target Analysis and Design

[0252] In this example, we design peptide fragments suitable for broadly targeting retinal cells. Fifteen possible target peptides were designed (see Table 1 below).

[0253] [Table 1]

[0254] Example 2: Design and construction of an AAV capsid protein expression plasmid encoding a capsid protein of interest.

[0255] 1. Construction of the intermediate plasmid RC2_IVB-NotI

[0256] In this example, a reverse P5 promoter sequence was added upstream of the Rep sequence of pAAV-RC2 (purchased from Cell Biolabs, catalog number: VPK-410-SER2), and a NotI restriction enzyme cleavage site was inserted at 1752 bp in the Cap2 sequence. This intermediate plasmid, RC2_IVB-NotI, was constructed by General BIOL. The specific sequence is as follows. RC2_IVB-NotI plasmid vector sequence (SEQ ID NO: 63)

[0257] 2. Construction of an AAV capsid protein expression plasmid encoding the capsid protein of interest.

[0258] AAV capsid protein expression plasmids encoding capsid proteins of interest were constructed using the Gibson Assembly method (see Gibson Assembly® Chemical Transformation Protocol (E2611) for specific procedures). Gibson assembly was performed on PCR fragments and fragments of the intermediate plasmid RC2_IVB-NotI constructed in Step 1, which involved linearization using NotI, to obtain various AAV capsid plasmids encoding capsid proteins of interest. During the process of obtaining PCR fragments, PCR amplification was performed by primer self-pairing without a PCR template. The primer sequences are shown in Table 2 below.

[0259] [Table 2-1] [Table 2-2] [Table 2-3]

[0260] Experimental results In this example, the plasmid DNA to be constructed was subjected to enzymatic digestion verification and Sanger sequencing analysis, and it was confirmed that the corresponding capsid protein expression plasmid was successfully constructed. The amino acid sequence of the AAV capsid protein expressed by the obtained AAV capsid protein expression plasmid is shown in Table 3 below.

[0261] [Table 3-1] [Table 3-2]

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

[0262] Example 3: Production of AAV virus

[0263] The novel AAV virus packaging system is a three-plasmid packaging system (Figure 1). In this example, recombinant AAV was produced by co-transfection of three types of plasmids. The three types of plasmids include an RC plasmid (a capsid and replication plasmid, which contains the nucleotide sequence of capsid Cap. That is, it is the AAV capsid protein expression plasmid constructed in Example 2), a helper plasmid (also called an Ad helper plasmid or an auxiliary plasmid, which supplies the auxiliary factors necessary for AAV production), and a pAAV plasmid (also called a transgenic plasmid or a target gene plasmid, which supplies the nucleotide sequence to be delivered and often contains a promoter and cDNA necessary for protein translation). The serotype of AAV is determined by the RC plasmid. For example, RC2 is used for packaging and production of AAV type 2.

[0264] HEK 293T cells were seeded in 100 mm TC-treated petri dishes and grown to 70%-80% confluence for plasmid transfection. AAV packaging was performed as follows: 5 μg of AAV_GFP plasmid (GFP expression pAAV plasmid, purchased from Cell Biolabs, catalog number: VPK-410-SER2), 5 μg of various AAV capsid protein expression plasmids encoding capsid proteins of interest constructed in Example 2 (and AAV capsid protein expression plasmids encoding wild-type capsid protein), and 10 μg of helper plasmid (purchased from Cell Biolabs, catalog number: VPK-410-SER2) were co-transfected into HEK 293T cells using polyethyleneimine (PEI). 72 hours after transfection, the cells and supernatant were collected. The cells were precipitated by slow centrifugation and lysed with 0.5% sodium deoxycholate (w / v). 50 U / ml of benzonase and 22 mM of MgCl were added, and the mixture was incubated at 37°C for 2 hours to degrade the free DNA molecules. Simultaneously, the supernatant was precipitated on ice with 40% PEG8000 and 2.5 M NaCl solution (1:5 volume). The cell lysate and supernatant were mixed and centrifuged. The supernatant was purified by iodixanol ultracentrifugation. The titer of the purified AAV was measured by RT-qPCR and stored in a refrigerator at -80°C.

[0265] Example 4: In vitro cell introduction experiment of novel AAV

[0266] To measure the in vitro introduction efficiency of the novel AAV (or novel AAV2) constructed in Example 3, various AAVs carrying the GFP gene constructed in Example 3 were used (the capsid proteins expressed by the AAV capsid protein expression plasmid within the AAV are IVT1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 / 12 / 13 / 14 / 15 and the wild-type capsid protein, respectively).

[0267] Human retinal epithelial cells (ARPE-19, Wuhan Procell, catalog number CL-0026) were cultured under the following conditions: Cells were placed in 24-well TC-treated culture plates in DMEM-F12 complete medium (89% DMEM-F12, 10% fetal bovine serum, 1% penicillin-streptomycin) for 1E+5 (i.e., 10) 5 Cells were seeded in 3 wells. The novel capsid AAV virus and wild-type AAV2 capsid AAV virus (AAV2WT) packaged in Example 3 were added at a multiple of infection (MOI) of 3000. Each virus was added to 3 wells. After infecting the cells with the virus, the culture plates were placed in a cell culture incubator (5% CO2, 37°C) and cultured for 72 hours. Subsequently, the cells were digested with 0.25% trypsin-EDTA (Gibco, catalog number: 25200056), resuspended in PBS, and the percentage of GFP fluorescence was detected by flow cytometry (Figure 2).

[0268] As shown in Figure 2, the results indicate that AAVIVT2 / 8 / 11 / 12 / 13 / 14 / 15 have a similar ability to infect target cells in vitro to AAV2WT.

[0269] Example 5: In vivo introduction experiment of novel AAV into mice

[0270] To measure the in vivo introduction efficiency of novel AAVs, 1 μL of AAV (AAV IVT8 / 13 / 14 / 15) and wild-type AAV2 (AAV2 WT) virus particles (all viruses prepared in Example 3) carrying the GFP gene were introduced at a concentration of 1E+12 vg / mL (1E+9 vg / eye, i.e., 10 9 The virus was injected intravitreously into 6-week-old C57BL / 6 male mice (purchased from SPF (Beijing) Biotechnology) using a microinjector (Hamilton, 7633-01) at a titer of vg / eye. Two weeks after viral injection, fundus images of the mice's retinas were acquired using the Phoenix Micron IV retinal microscopy imaging system, and images of the GFP signal were obtained (Figure 3).

[0271] As shown in Figure 3, the results indicate that AAV IVT8 / 13 / 15 has superior mouse retinal infection ability compared to wild-type AAV2.

[0272] Example 6: In vitro introduction experiment of novel AAV into human retinal organoids

[0273] To measure the effectiveness of novel AAVs targeting organoids, we used GFP-carrying AAV (AAV IVT8 / 13 / 15) and wild-type AAV2 (AAV2 WT) virus particles in 1E+10vg(10 10 The GFP signal was added to human retinal organoids (see Method 1 in Chichagova V, Hilgen G, Ghareeb A, Georgiou M, Carter M, Sernagor E, Lako M, Armstrong L. Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent. Stem Cells. 2020 Feb;38(2):195-201. doi: 10.1002 / stem.3116. Epub 2019 Dec 30. Erratum in: Stem Cells. 2020 Oct 1;38(10):E4. PMID: 31721366; PMCID: PMC7383896.) 72 hours after viral infection, images of the GFP signal were obtained using a fluorescence microscope (Figure 4A). Organoids were digested with 0.25% trypsin-EDTA (Gibco, catalog number: 25200056), and the percentage of GFP fluorescence was measured by flow cytometry (Figure 4B). Null represents untransfected tissue, and wt represents transfection with wild-type AAV2.

[0274] As shown in Figure 4, the results indicate that AAV IVT8 / 13 / 15 has superior retinal organoid infection ability compared to wild-type AAV2.

[0275] Example 7: In Vivo Introduction Experiment of Novel AAV into Mice

[0276] To measure the in vivo introduction efficiency of novel AAV IVT13 into mice, 1 μL of AAV IVT13 carrying the GFP gene and wild-type AAV2 (AAV2 WT) virus particles were injected into the vitreous body of 6-week-old C57BL / 6 male mice (purchased from GemPharmatech LLC.) at a titer of 2E+12 vg / mL (2E+9 vg / eye, that is, 2×10 9 vg / eye) using a microinjector (model 7633-01, manufactured by Hamilton). Three weeks after the virus injection, fundus images of the mouse retina were taken using a Phoenix Micron IV retinal microscopy imaging system, and images of the GFP signal were obtained (Figure 5A).

[0277] To detect the introduction sites of AAV IVT13 in the mouse retina, frozen sections of the mouse retina were prepared, and immunofluorescence staining was performed using antibodies against the retinal ganglion marker RBPMS (manufactured by Abcam, ab152101, rabbit-derived antibody, 1:500), bipolar cell marker PKCα (manufactured by Abcam, ab32376, rabbit-derived antibody, 1:500), retinal pigment epithelium marker RPE65 (manufactured by Abcam, ab231782, rabbit-derived antibody, 1:500), and GFP (manufactured by Invitrogen, A10262, chicken-derived antibody, 1:500) respectively (Figure 5B - Figure 5E), and images were taken using a super-resolution confocal microscope ZEISS LSM880+ELYRAS.1 manufactured by Carl Zeiss or an inverted fluorescence microscope EVOS-M5000 manufactured by Thermo Fisher Scientific.

[0278] As a result, AAV IVT13 showed significantly higher retinal GFP fluorescence levels compared to wild-type AAV2 (Figure 5A). Furthermore, immunofluorescence staining revealed that, despite intravitreal administration, the novel AAV IVT13 was able to diffuse from the ganglion layer to the photoreceptor layer and ultimately to the retinal pigment epithelium (RPE) (Figures 5B-5E), demonstrating far superiority over wild-type AAV2. Thus, serotype AAV IVT13 exhibits superior retinal affinity and delivery efficiency.

[0279] Example 8: In vivo introduction experiment of novel AAV into cynomolgus monkeys

[0280] To measure the efficiency of introducing the novel AAV IVT13 into monkeys, two 5.7-year-old cynomolgus monkeys were anesthetized and injected with AAV IVT13 carrying the GFP gene and wild-type AAV2 (AAV2 WT) virus particles (the same virus, 1E+12vg / eye (i.e., 10)). 12 The virus was injected into both eyes of the same monkey using vg / eye, and then injected into the vitreous humor of the monkey using a syringe (Yeso med, specification: 0.3 ml). This injection experiment was performed at TRIAPEX. Three weeks after virus injection, frozen sections of the monkey's retina were prepared and immunostained with antibodies for the retinal bipolar cell marker PKCα (Abcam, ab32376, rabbit-derived antibody 1:500), the retinal photoreceptor layer marker RS1 (rabbit-derived antibody 1:500, Abcam, ab314231), and GFP (Invitrogen, A10262, chicken-derived antibody 1:500) (Figures 6A-6B). The sections were then imaged using a Carl Zeiss LSM880+ELYRAS.1 super-resolution confocal microscope or a Thermo Fisher Scientific EVOS-M5000 inverted fluorescence microscope.

[0281] As a result, the novel AAV IVT13 showed high affinity for the monkey macula. Furthermore, the IVT13 capsid was able to efficiently deliver genes to the ganglion layer, bipolar cells of the inner granular layer, and photoreceptor cells of the outer granular layer of the retina (Figures 6A-6B), with significantly higher delivery efficiency compared to wild-type AAV2. Therefore, serotype AAV IVT13 exhibits excellent affinity and delivery efficiency in the retinas of both mice and cynomolgus monkeys, enabling gene delivery to all layers of the retina.

[0282] Example 9: Modification of AAV IVT13 capsid protein

[0283] In this example, the RC2_IVT13 plasmid backbone was modified by mutating the 34th amino acid of the AAV2 capsid protein within the plasmid, proline (P), to alanine (A). This plasmid was constructed by General BIOL. The specific sequence is as follows (SEQ ID NO: 64):

[0284] The amino acid sequence of AAV capsid protein IVT13 - P34A expressed by the obtained AAV capsid protein expression plasmid is as follows (SEQ ID NO: 65):

Chemical formula

[0285] Here, those indicated in bold and underlined are the mutation sites of IVT13 - P34A compared with the IVT13 capsid protein, and those indicated in italics and underlined are the target peptide 13.

[0286] Example 10: In vitro cell transduction experiment of AAV IVT13 - P34A

[0287] To measure the in vitro transduction efficiency of the IVT13 - P34A capsid protein constructed in Example 9, AAVs (AAV2 WT, AAV IVT13, AAV IVT13 - P34A) carrying the GFP gene were prepared by the method described in Example 3. Then, human retinal epithelial cells (ARPE - 19) were infected with AAVs carrying the GFP gene (AAV2 WT, AAV IVT13, AAV IVT13 - P34A), and their transduction efficiency was detected.

[0288] Human retinal epithelial cells (ARPE-19, Wuhan Procell, catalog number CL-0026) were cultured under the following conditions: Cells were seeded at a rate of 1E+5 / well in 24-well TC-treated culture plates using DMEM-F12 complete medium (89% DMEM-F12, 10% fetal bovine serum, 1% penicillin-streptomycin). Novel capsid (IVT13 and IVT13-P34A) AAV viruses and wild-type AAV2 capsid AAV viruses (AAV2WT), packaged according to the method of Example 3, were added at multiples of infection (MOI) of 100 / 500 / 1000. Each virus was added to 3 wells. After infecting the cells with the viruses, the culture plates were placed in a cell culture incubator (5% CO2, 37°C) and cultured for 48 hours. Subsequently, the cells were digested with 0.25% trypsin-EDTA (Gibco, catalog number: 25200056), resuspended in PBS, and the percentage of GFP fluorescence was detected by flow cytometry (Figure 7).

[0289] As shown in Figure 7 and Table 4 below, when ARPE cells were infected with different MOIs, the modified AAV IVT13-P34A showed significantly improved transduction efficiency compared to wild-type AAV2, and also showed improved transduction efficiency compared to AAV IVT13.

[0290] [Table 4]

[0291] In Table 4 above, the numerical values ​​represent the percentage of GFP-positive cells, and the unit is %.

[0292] Example 11: In vitro introduction experiment of AAV IVT13-P34A into human retinal organoids

[0293] To measure the effect of AAV IVT13-P34A targeting organoids, human retinal organoids differentiated at day 79 were treated with 1E+10vg / organoid of AAV virus (AAV2 WT, AAV IVT13, AAV IVT13-P34A) carrying the GFP gene. (Construction method: Chichagova V, Hilgen G, Ghareeb A, Georgiou M, Carter M, Sernagor E, Lako M, Armstrong L. Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent. Stem Cells. 2020 Feb;38(2):195-201. doi: 10.1002 / stem.3116. Epub 2019 Dec 30. Erratum in: Stem Cells. 2020 Oct 1;38(10):E4. PMID:) It was added to method 1 described in 31721366; PMCID: PMC7383896. The blank control was not infected with the virus. Eight days after viral infection, images of the GFP signal were obtained using a Thermo Fisher Scientific EVOS-M5000 inverted microscope (Figure 8).

[0294] As shown in Figure 8, AAV IVT13-P34A clearly has superior ability to infect retinal organoids compared to wild-type AAV2 (AAV2 WT) and AAV IVT13.

Claims

1. An adeno-associated virus capsid protein containing a target peptide, wherein the target peptide is (i) Array AAX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ AA, (ii) Array NG This includes one or more types of, Array AAX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ In AA, (a) X １ A is, (b) X ２ is R or G, (c) X ３ is G or N, (d) X ４ is D, S, or G, (e) X ５ It is either L or R, (f) X ６ It is A, G, Q, or missing. (g)X ７ It is T, R, N, A, H, or missing. (h)X ８ These are adeno-associated virus capsid proteins, either I, L, P, A, or deleted.

2. Array AAX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ In AA, (a) X １ A is, (b) X ２ R is, (c) X ３ G is, (d) X ４ is either D or S, (e) X ５ L is, (f) X ６ It is A, G, Q, or missing. (g)X ７ It is T, R, N, A, or missing. (h)X ８ The adeno-associated virus capsid protein according to claim 1, wherein it is I, L, P, or missing.

3. The target peptide comprises the amino acid sequence shown in any one of Sequence IDs 1 to 14 and sequence NG. Preferably, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 8, 11, 12, 13, 14 and sequence NG. More preferably, the adeno-associated virus capsid protein according to claim 1 or 2, wherein the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 8, 13, 14 and sequence NG.

4. Adeno-associated viruses are one or more selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh. 10, rh. 39, rh. 43, and rh.

74. Preferably, the adeno-associated virus capsid protein according to any one of claims 1 to 3, wherein the AAV comprises AAV2.

5. AAV2 includes AAV2 VP1 or its variants. Preferably, the amino acid sequence of AAV2 VP1 is shown in SEQ ID NO: 60, Preferably, the adeno-associated virus capsid protein according to any one of claims 1 to 4, wherein the AAV2 VP1 variant includes a variant in which the 34th proline (P) is mutated to alanine (A) compared to the amino acid sequence shown in SEQ ID NO:

60.

6. The target peptide is inserted at the 587th amino acid position of AAV2 VP1 or its variant, or The target peptide is obtained by substituting the 586th and 587th amino acids of AAV2 VP1 or its variant. Preferably, the adeno-associated virus capsid protein according to any one of claims 1 to 5, comprising an amino acid sequence shown in any one of SEQ ID NOs: 15-29 and 65, and an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 15-29 and 65.

7. (i) Array AAX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ AA, (ii) Array NG, A target peptide comprising one or more of the following: Array AAX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ In AA, (a) X １ A is, (b) X ２ is R or G, (c) X ３ is G or N, (d) X ４ is D, S, or G, (e) X ５ It is either L or R, (f) X ６ It is A, G, Q, or missing. (g)X ７ It is T, R, N, A, H, or missing. (h)X ８ It is I, L, P, A, or missing. Depending on the case, the sequence AAX １ X ２ X ３ X ４ X ５ X ６ X ７ X ８ In AA, (a) X １ A is, (b) X ２ R is, (c) X ３ G is, (d) X ４ is either D or S, (e) X ５ L is, (f) X ６ It is A, G, Q, or missing. (g)X ７ It is T, R, N, A, or missing. (h)X ８ It is I, L, P, or missing. Preferably, it comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 14 and sequence NG, More preferably, the amino acid sequence comprises one of the sequences shown in SEQ ID NOs: 2, 8, 11, 12, 13, 14 and sequence NG. More preferably, the target peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 8, 13, and 14.

8. A polynucleotide encoding an adeno-associated virus capsid protein according to any one of claims 1 to 6, or encoding a target peptide according to claim 7.

9. An adeno-associated virus comprising the adeno-associated virus capsid protein according to any one of claims 1 to 6.

10. The adeno-associated virus according to claim 9, further comprising a transgene, The adeno-associated virus, wherein the transgene is optionally a therapeutic transgene, a prophylactic transgene, or a diagnostic transgene.

11. A transgene delivery vector comprising adeno-associated virus according to claim 9 or 10.

12. (a) an adeno-associated virus according to claim 9 or 10 or a transgene delivery vector according to claim 11, and Depending on the circumstances, a pharmaceutical composition comprising (b) a pharmaceutically acceptable carrier.

13. Use of the adeno-associated virus according to claim 9 or 10, the transgene delivery vector according to claim 11, or the pharmaceutical composition according to claim 12 in the manufacture of a reagent for introducing a transgene into cells.

14. The aforementioned cells are derived from the subject, Preferably, the subject is a mammal, More preferably, the subject is a human being, according to claim 13.

15. The use according to claim 13 or 14, wherein the cells are derived from the eye.

16. The use according to claim 14 or 15, wherein the subject suffers from an eye disease.

17. The use according to any one of claims 13 to 16, wherein the adeno-associated virus, the transgene delivery vector, or the pharmaceutical composition is administered to a subject by subretinal injection or intravitreous injection.

18. The use of the adeno-associated virus according to claim 9 or 10, the transgene delivery vector according to claim 11, or the pharmaceutical composition according to claim 12 in the manufacture of a drug for the treatment of a disease, Preferably, the disease includes eye diseases.

19. The process includes administering to a subject the adeno-associated virus described in claim 9 or 10, the transgene delivery vector described in claim 11, or the pharmaceutical composition described in claim 12. Preferably, the disease includes eye diseases, a method for treating a disease.

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