Recombinant adeno-associated virus with modified AAV capsid polypeptide

Abstract

The present invention relates to modified adeno-associated virus (AAV) capsid polypeptides for delivery of therapeutic gene products and novel recombinant adeno-associated viruses (rAAV) comprising the modified AAV capsid polypeptides. The present invention also relates to pharmaceutical compositions comprising the rAAV of the present invention, and methods of treating retinal diseases comprising administering the rAAV or pharmaceutical compositions of the present invention to the eye of a subject in need of such treatment.

Description

Technical Field

[0001] The present invention relates to gene therapy. In particular, the present invention relates to modified adeno-associated virus (AAV) capsid polypeptides for delivering gene products for therapy and novel recombinant adeno-associated viruses (rAAVs) comprising the modified AAV capsid polypeptides.

Background Art

[0002] In recent years, gene therapy has been attracting increasing attention. Improved gene transfer techniques are of great help in the development of gene therapy.

[0003] Gene therapy was initially associated with introducing foreign genes into a patient's cells to correct congenital genetic errors such as loss-of-function mutations. Most currently approved gene therapy protocols involve introducing a functional copy of a gene that is deficient in a patient into the patient's somatic cells. However, recently, gene therapy has been broadly defined as the modification of a disease phenotype by introducing new genetic information into an organism that requires gene therapy.

[0004] In in vivo gene therapy, the transferred gene (transgene) is introduced in situ into the organs, tissues, and cells of a recipient organism such as muscle hematopoietic stem cells, arterial walls, the nervous system, the lungs, and the eyes.

[0005] In vivo gene therapy by introducing a transgene in situ into the eye has been used in the treatment of eye diseases (such as those causing blindness). Examples of such diseases are retinitis pigmentosa, macular diseases, Leber congenital amaurosis, Leber hereditary optic neuropathy, early-onset severe retinal dystrophy, achromatopsia, retinoschisis, vitreoretinopathy, vitreoretinopathy, glaucoma, Stargardt disease, choroid-free, age-related macular degeneration (AMD) (including Wet-AMD), spinocerebellar ataxia type 7 (SCAT), color blindness, and lysosomal storage diseases affecting the cornea (such as mucopolysaccharidosis (MPS) IV and MPS VII).

[0006] Adeno-associated virus (AAV) is a member of the Parvoviridae family. It is a simple single-stranded DNA virus that requires a helper virus (such as an adenovirus) for replication. The wild-type AAV genome contains approximately 4.7 kilobases (kb) and includes a cap gene and a rep gene between two reverse-ended repeat (ITR) sequences that have interrupted palindromic sequences that can fold into hairpin structures that function as primers during the initiation of DNA replication. The cap gene encodes the viral capsid protein, and the rep gene is involved in AAV replication and integration. AAV can infect a variety of cells, and its viral DNA can be integrated into human chromosome 19 in the presence of the rep product.

[0007] Most rAAV gene therapies currently in clinical development focus on the central nervous system (CNS), including the brain and eyes. There are currently over a dozen capsid serotypes used as vectors in clinical trials, with AAV2-based platforms being the most common for eye diseases.

[0008] In particular, Luxtana, the first rAAV gene therapy approved by the U.S. Food and Drug Administration (FDA), treats patients with genotyped vision loss caused by RPE65 gene mutations using AAV2. However, there is an increasing need for newer, modified, and more efficient capsids.

[0009] The manipulation of novel AAV capsids has been continuously pursued to obtain new properties and characteristics. The main limitations to a rational design approach are related to insufficient knowledge regarding AAV cell surface binding, internalization, transport, detachment, and gene expression.

[0010] While several modified AAV capsids have been developed (see, for example, International Publication Nos. 2012145601A2, 2016134375A1, and 2018022905A2), there remains a need to develop novel modified AAV capsids to improve gene therapy for ocular diseases, such as gene therapy for in situ delivery of polynucleotides encoding products to treat the disease. [Overview of the project] [Means for solving the problem]

[0011] In a first aspect, the present invention relates to a modified AAV capsid polypeptide comprising a peptide inserted into loop IV, compared to a parent AAV capsid polypeptide, wherein the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. This invention provides a modified AAV capsid polypeptide containing the amino acid sequence shown.

[0012] The present invention further provides a polynucleotide encoding the modified AAV capsid polypeptide of the present invention, a vector comprising the polynucleotide, and a host cell comprising the polynucleotide or the vector.

[0013] In a second aspect, the present invention provides a system or kit for packaging rAAV comprising the polynucleotide, vector, or host cell of the present invention.

[0014] In a third aspect, the present invention provides a recombinant adeno-associated virus (rAAV) comprising a modified AAV capsid polypeptide of the present invention and a genome encoding a gene product.

[0015] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the rAAV of the present invention.

[0016] The present invention provides a method for treating a retinal disease, comprising administering the rAAV or the pharmaceutical composition of the present invention to the eye of a subject in need of treatment for the retinal disease.

[0017] The rAAV or pharmaceutical composition of the present invention for use in treating a retinal disease is provided.

[0018] The present invention provides the use of the rAAV or pharmaceutical composition of the present invention in the preparation of a medicament for treating a retinal disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1A] Shows a helper plasmid for preparing rAAV. [Figure 1B] Shows a packaging plasmid for preparing rAAV. [Figure 1C] Shows a transgene plasmid for preparing rAAV. [Figure 1D] Shows a transgene plasmid for preparing rAAV. [Figure 2] Shows fluorescence microscopy of retinal cells transduced with rAAV containing a genome encoding GFP at various MOIs. [Figure 3] Shows the expression of Nb24 in retinal cells transduced with rAAV containing a genome encoding bivalent Nb24 and various capsid polypeptides. The Y-axis shows the "lg" value of the concentration, and the "0" point represents a concentration of "1". [Figure 4] Shows the workflow of a study in a laser-induced choroidal neovascularization (CNV) non-human primate (NHP) model. [Figure 5]Shows the mechanism of ELISA for the detection of Nb24. [Figure 6] Shows the Nb24 levels in eye tissues. [Figure 7] Shows the Nb24 levels in peripheral tissues and serum. [Figure 8] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with vehicle. [Figure 9] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with Eylea (registered trademark). [Figure 10] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.LAE-Nb24. [Figure 11] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.RH10-Nb24. [Figure 12] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.LGP-Nb24. [Figure 13] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.7m8-Nb24. [Figure 14] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.GKG-Nb24. [Figure 15] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.NSP-Nb24. [Figure 16] Shows fundus photographs (FP) and fundus fluorescein angiography (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV9-Nb24.

Mode for Carrying Out the Invention

[0020] 1. Definition Unless otherwise indicated, all terms used herein have the same meaning as those to those skilled in the art, and the implementation of the present invention utilizes the prior art of microbiology and recombinant DNA technology, which is within the scope of the knowledge of those skilled in the art.

[0021] The term “retinal cells” as used herein may refer to any cell type contained in the retina, such as retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, as well as photoreceptor cells including rods and cones, Müller glial cells, and retinal pigment epithelium.

[0022] "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives. Unless otherwise required, the term encompasses all subtypes and both naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also called recombinant AAV vector (or "rAAV vector"). The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" refers to AAVs that infect primates, "non-primate AAV" refers to AAVs that infect non-primate mammals, and "bovine AAV" refers to AAVs that infect bovine mammals, and so on.

[0023] The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeat (TR), Rep protein, and capsid subunit, are publicly known in the art. Such sequences can be found in public databases such as GenBank. See, for example, GenBank accession numbers NC_002077(AAV-1), AF063497(AAV-1), NC_001401(AAV-2), AF043303(AAV-2), NC_001729(AAV-3), NC_001829(AAV-4), U89790(AAV-4), NC_006152(AAV-5), AF513851(AAV-7), AF513852(AAV-8), and NC_006261(AAV-8); their disclosures are incorporated herein by reference to teach the AAV nucleic acid and amino acid sequences.

[0024] As used herein, “rAAV vector” refers to an AAV vector containing a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically the sequence of interest for genetic transformation of cells. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV reverse terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids (also called transgene plasmids). rAAV vectors can be either single-stranded (ssAAV) or self-complementary (scAAV).

[0025] An "AAV virus," "AAV virus particle," or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically all capsid proteins of wild-type AAV) and a capsidized polynucleotide rAAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than those in the wild-type AAV genome, such as transgenes delivered to mammalian cells), it is typically called an "rAAV vector particle" or simply an "rAAV vector." Therefore, the production of rAAV particles necessarily includes the production of rAAV vectors, since such vectors are contained within the rAAV particles.

[0026] "Packaging" refers to a series of intracellular events that result in the aggregation and encapsulation of AAV particles.

[0027] The AAV "rep" and "cap" genes refer to polynucleotide sequences encoding the replication and capsid-forming proteins of adeno-associated virus. The AAV rep and cap genes are referred to herein as the AAV "packaging genes." Plasmids or other expression vectors containing the rep and cap genes are referred herein as "packaging plasmids" or "packaging vectors."

[0028] The cap gene encodes three structural proteins, VP1, VP2, and VP3, which self-assemble into a 60-mer icosahedral capsid in a ratio of approximately 1:1:10. These three proteins are transcribed from the same open reading frame and share a C-terminal domain, but have different N-terminuses due to alternative start codons and alternative splicing (Esther J. Lee et al., Adeno-Associated Virus (AAV) Vectors: Rational Design Strategies for Capsid Engineering, Curr Opin Biomed Eng. 2018;7:58-63). For example, the wild-type AAV2 capsid may contain VP1 (SEQ ID NO: 1), VP2 (amino acids 138-735 of SEQ ID NO: 1), and VP3 (amino acids 203-735 of SEQ ID NO: 1).

[0029] A "helper virus" for AAV refers to a virus that enables AAV (e.g., wild-type AAV) to replicate and package in mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses, such as vaccinia. Adenoviruses encompass several different subgroups, but adenovirus type 5 of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositaries such as ATCC. Viruses of the Herpesviridae family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), which are also available from depositaries such as ATCC.

[0030] "Helper virus function" refers to a function encoded in the helper virus genome that enables the replication and packaging of AAV (in conjunction with other replication and packaging requirements described herein). As described herein, "Helper virus function" can be provided in several ways, including providing a helper virus or, for example, providing a polynucleotide sequence encoding the desired function in trans-producing cells. For example, a plasmid referred herein as a "helper plasmid," or another expression vector containing a nucleotide sequence encoding one or more adenovirus proteins, is transfected into producer cells together with the rAAV vector.

[0031] As used herein, the term “polynucleotide construct” refers to a single-stranded or double-stranded polynucleotide isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not exist naturally. If the polynucleotide construct contains a regulatory sequence necessary for expressing the coding sequence of the present invention, the polynucleotide construct includes an “expression cassette.”

[0032] As used herein, the term “polynucleotide” generally refers to nucleic acid molecules (e.g., 100 nucleotides long and up to 30 kilobases long) and sequences that are complementary (antisense) or identical (sense) to the sequences of messenger RNA (mRNA) or miRNA fragments or molecules. The term may also refer to DNA or RNA molecules that are either transcribed or untranscribed.

[0033] The term "exogenous polynucleotide" refers to a nucleotide sequence that does not originate from the host in which it is placed. It may be identical or heterologous to the host's DNA. An example is a target sequence inserted into a vector. Such exogenous DNA sequences can originate from a variety of sources, including DNA, cDNA, synthetic DNA, and RNA. Exogenous polynucleotides also include DNA sequences that encode antisense oligonucleotides.

[0034] "Heterogeneous" means that it originates from an entity that is genotypeically different from the rest of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector from a different species by genetic engineering is a heterogeneous polynucleotide. A promoter that has been removed from its natural coding sequence and operably ligated to a coding sequence that is not naturally ligated is a heterogeneous promoter. Therefore, for example, an rAAV containing heterogeneous nucleic acid encoding a heterogeneous gene product is an rAAV containing nucleic acid not normally found in naturally occurring wild-type AAV, and the encoded heterogeneous gene product is a gene product not normally encoded by naturally occurring wild-type AAV.

[0035] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids in the two sequences is the same when compared. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in packages from Genetics Computing Group (GCG), a wholly owned subsidiary of Oxford Molecular Group, Inc., located in Madison, Wisconsin, USA.

[0036] As used herein, the term “expression cassette” refers to a polynucleotide segment containing additional nucleotides provided for the expression of a polynucleotide, such as a polynucleotide encoding a polypeptide operably linked to a control sequence.

[0037] As used herein, the term “expression” includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0038] The “control sequence” comprises all elements necessary or beneficial for the expression of the polynucleotide encoding the polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or may be native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, an enhancer, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequence includes a promoter and signals for the termination of transcription and translation.

[0039] For example, the regulatory sequence may be a suitable promoter sequence, i.e., a nucleotide sequence recognized by the host cell for the expression of the polynucleotide encoding the polypeptide of the present invention. The promoter sequence includes a transcriptional regulatory sequence that mediates the expression of the polypeptide. The promoter may be any nucleotide sequence that exhibits transcriptional activity in a selected host cell, such as the lac operon of Escherichia coli (E. coli). Promoters also include mutant promoters, cleavage promoters, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to those of the host cell.

[0040] As used herein, the term “operably linked” refers to a configuration in which a control sequence is positioned appropriately relative to the coding sequence of a polynucleotide sequence, thereby directing the expression of the polypeptide coding sequence.

[0041] A "gene" refers to a polynucleotide that contains at least one open reading frame encoding a polynucleotide or polypeptide.

[0042] A "gene product" is a molecule that arises from the expression of a specific gene. Examples of gene products include polypeptides, aptamers, interfering RNA, and mRNA.

[0043] Small interfering RNA (siRNA), also known as "small interfering RNA," is an RNA double helix of nucleotides that targets a gene of interest ("target gene"). An "RNA double helix" refers to a structure formed by the complementary pairing of two regions of an RNA molecule. siRNA "targets" a gene in that the nucleotide sequence of the siRNA double helix is ​​complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the siRNA double helix is ​​less than 30 nucleotides. In some embodiments, the double helix may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides long. In some embodiments, the double helix is ​​19–25 nucleotides long. The RNA double helix portion of siRNA may be part of a hairpin structure. In addition to the double helix portion, the hairpin structure may include a loop portion positioned between the two sequences forming the double helix. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides long. The hairpin structure may also include a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides long.

[0044] Short hairpin RNA, or shRNA, is a polynucleotide construct capable of expressing interfering RNAs such as siRNA.

[0045] As used herein, the term “recombinant” refers to nucleic acids, vectors, polypeptides, or proteins produced using DNA recombination (cloning) methods, and is distinguishable from natural or wild-type nucleic acids, vectors, polypeptides, or proteins. The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymers of amino acids, including full-length proteins and their fragments.

[0046] As used herein, the term “host cell” refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of rAAV vectors. The term includes the offspring of the transduced original cell. Therefore, as used herein, “host cell” generally refers to a cell transduced with an exogenous DNA sequence. It is understood that the offspring of a single parental cell may not necessarily be morphologically or genomically or in terms of whole DNA complement with the original parent due to natural, accidental, or intentional mutations.

[0047] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and, typically, do not cause toxicity, allergies, or similar adverse reactions, such as stomach upset or dizziness, when administered to humans.

[0048] As used herein, the term “subjects” includes, but is not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; and laboratory animals including rodents such as mice, rats and guinea pigs. The term is not intended to indicate a specific age or sex. Therefore, adult and neonatal subjects, as well as fetuses, whether male or female, are intended to be covered.

[0049] The terms "genetic modification" and "genetic alteration" (and their grammatical variations) refer to mitosis or meiosis. The term is used interchangeably herein to refer to any process by which genetic elements (e.g., polynucleotides) are introduced into a cell by means other than those described above. The elements may be heterologous to the cell, or they may be additional copies or improved versions of elements already present in the cell. Genetic modification may be carried out by transfecting a cell with a recombinant plasmid or other polynucleotide via any process known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic modification may also be achieved, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, genetic elements are introduced into chromosomes or minichromosomes within a cell, but any modification that alters the phenotype and / or genotype of the cell and its offspring is included in this term.

[0050] Cells are said to be “stable” modified, transduced, genetically modified, or transformed by a gene sequence if the sequence is available to perform its function during long-term in vitro cell culture. Generally, such cells are “genetically” modified (genetically modified) in that a gene modification is introduced that can be inherited by the offspring of the modified cells.

[0051] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. This term also encompasses modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with labeling components. Polypeptides, e.g., anti-angiogenic polypeptides, neuroprotective polypeptides, etc., when discussed in relation to the delivery of gene products to mammalian subjects, and their compositions refer to the respective intact polypeptides, or any fragments or genetically modified derivatives thereof, which retain the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in the delivery of gene products to mammalian subjects (which may be called “transgenes” delivered to recipient cells) include polynucleotides encoding intact polypeptides or any fragments or genetically modified derivatives having the desired biochemical function.

[0052] "Isolated" plasmids, nucleic acids, vectors, viruses, virions, host cells, or other substances refer to preparations of a substance that lack at least some of other components that may be present in nature or when the substance or a similar substance is first prepared. Thus, for example, an isolated substance may be prepared by using purification techniques to concentrate it from a source mixture. Concentration can be measured on an absolute standard, such as weight per unit volume of solution, or with respect to any second potential interfering substances present in the source mixture. Increasing concentrations in embodiments of this disclosure are increasingly isolated. In some embodiments, isolated plasmids, nucleic acids, vectors, viruses, host cells, or other substances are purified to, for example, about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% or more pure.

[0053] As used herein, terms such as “treatment” and “treating” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures the disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, in particular humans, and includes: (a) preventing the development of a disease in subjects who are susceptible to the disease or at risk of acquiring the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., stopping its development; and (c) reducing the disease, i.e., causing regression of the disease.

[0054] As used herein, the term "ED50" means the median effective dose of a drug, i.e., the dose that can produce 50% of the maximum response. To deliver a transgene expressed intracellularly using a recombinant virus (e.g., rAAV), ED50 may be expressed as the MOI at which 50% of the transgene's maximum expression is achieved.

[0055] Where a range of values ​​is provided, it is understood that each intermediate value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless explicitly indicated in the context, and any other stated or intermediate values ​​within the stated range, are included in the invention. The upper and lower limits of these smaller ranges may independently be included within smaller ranges and are included in the invention, subject to any specifically excluded limits within the stated range. If a stated range includes one or both limits, the range excluding one or both of those limits is also included in the invention.

[0056] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. Thus, for example, a reference to “modified AAV capsid” includes multiple such capsids. It should also be noted that the claims may be drafted to exclude any element. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as “exclusively” or “only” in connection with the enumeration of elements of the claims or the use of “negative” limitations.

[0057] 2. Modified AAV capsid polypeptides and coding polynucleotides The present invention relates to a modified AAV capsid polypeptide comprising a peptide inserted into loop IV, compared to the parent AAV capsid polypeptide, wherein the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. This invention provides a modified AAV capsid polypeptide containing the amino acid sequence shown.

[0058] In some embodiments, spacer 1 and spacer 2 independently contain one or more amino acids. In some embodiments, spacer 1 and spacer 2 independently contain 1 to 10 amino acids. In some embodiments, spacer 1 and spacer 2 independently contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, spacer 1 and spacer 2 independently contain 1 to 3 amino acids. In some embodiments, spacer 1 and spacer 2 consist independently of 1 to 3 amino acids.

[0059] In some embodiments, the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. Spacer 1 and Spacer 2 independently contain one or more amino acids selected from A, L, and G. It contains the amino acid sequence shown.

[0060] In some embodiments, the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. Spacer 1 and Spacer 2 independently contain 1 to 10 amino acids selected from A, L, and G. It contains the amino acid sequence shown.

[0061] In some embodiments, the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. Spacer 1 and Spacer 2 independently contain 1 to 3 amino acids selected from A, L, and G. It contains the amino acid sequence shown.

[0062] In some embodiments, X1-X2-X3-X4-X5-X6-X7 is an amino acid sequence selected from SEQ ID NO: 6 (GKGPTTK), SEQ ID NO: 7 (LAEPSRP), SEQ ID NO: 8 (LGPPSKP), and SEQ ID NO: 9 (NSPTGRN).

[0063] In some embodiments, the inserted peptide is formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) The amino acid sequence includes the one shown in [wherein spacer 1 consists of amino acids Y1, Y2, and Y3, spacer 2 consists of amino acids Y4 and Y5, X1 is selected from G, L, and N, X2 is selected from K, A, G, and S, X3 is selected from G, E, and P, X4 is selected from P and T, X5 is selected from T, S, and G, X6 is selected from T, R, and K, and X7 is selected from K, P, and N].

[0064] In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G.

[0065] In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where Y1 is A, Y2 is A, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A.

[0066] In some embodiments, the inserted peptide comprises an amino acid sequence selected from SEQ ID NOs: 10, 11, 12, and 13.

[0067] In some embodiments, the inserted peptide is formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) The amino acid sequence includes the one shown in [wherein spacer 1 consists of amino acids Y1, Y2, and Y3, spacer 2 consists of amino acids Y4 and Y5, X1 is selected from G, L, and N, X2 is selected from K, A, G, and S, X3 is selected from G, E, and P, X4 is selected from P and T, X5 is selected from T, S, and G, X6 is selected from T, R, and K, and X7 is selected from K, P, and N].

[0068] In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present.

[0069] In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G.

[0070] In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A.

[0071] In some embodiments, the inserted peptide consists of an amino acid sequence selected from SEQ ID NOs: 10, 11, 12, and 13.

[0072] In some embodiments, the parent AAV capsid polypeptide is an AAV2 capsid polypeptide VP1, VP2, or VP3. In some embodiments, the parent AAV capsid polypeptide is an AAV2 capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1, amino acids 138-735 of SEQ ID NO: 1, or amino acids 203-735 of SEQ ID NO: 1 or a variant thereof. In some embodiments, the variant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical to SEQ ID NO: 1 in full length, amino acids 138-735, or amino acids 203-735. In some embodiments, the variant includes insertions, deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to SEQ ID NO: 1. Preferably, the amino acid insertion occurs at a site different from the inserted peptide of the present invention, and more preferably not in loop IV. Preferably, the amino acid substitution is a conserved substitution. In some embodiments, the parent AAV capsid polypeptide consists of the amino acid sequence of SEQ ID NO: 1, amino acids 138-735 of SEQ ID NO: 1, or amino acids 203-735 of SEQ ID NO: 1.

[0073] In the case of the AAV2 capsid polypeptide, loop IV corresponds to positions 570-611 of SEQ ID NO: 1. In other words, the modified AAV capsid polypeptide contains the peptide defined above, inserted into the region corresponding to positions 570-611 of SEQ ID NO: 1, compared to the parent AAV capsid polypeptide. In some embodiments, the peptide is inserted between the positions of the parent capsid polypeptide corresponding to positions 587 and 588 of SEQ ID NO: 1.

[0074] In some embodiments, the modified AAV capsid polypeptide of the present invention comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NOs: 2, 3, 4, or 5, or amino acids 203-747 of SEQ ID NOs: 2, 3, 4, or 5. In some embodiments, the modified AAV capsid polypeptide of the present invention consists of an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NOs: 2, 3, 4, or 5, or amino acids 203-747 of SEQ ID NOs: 2, 3, 4, or 5. In some embodiments, the modified AAV capsid polypeptide comprises or consists of a variant of SEQ ID NOs: 2, 3, 4, or 5. In some embodiments, the variant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical over its entire length to SEQ ID NOs. 2, 3, 4, or 5, amino acids 138-747 of SEQ ID NOs. 2, 3, 4, or 5. In some embodiments, the variant includes insertions, deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids compared to SEQ ID NOs. 2, 3, 4, or 5. Preferably, amino acid insertions occur at a different site from the inserted peptide of the present invention (SEQ ID NOs. 10, 11, 12, and 13), and more preferably not in loop IV. Preferably, amino acid substitutions are conserved substitutions.

[0075] In some embodiments, the modified AAV capsid polypeptide is encoded by a nucleotide sequence selected from SEQ ID NOs: 14, 15, 16, and 17. In some embodiments, the modified AAV capsid polypeptide is encoded by a variant of SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the variant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical to SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the variant includes substitutions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides compared to SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the variant includes insertions and / or deletions of 3n nucleotides (where n is an integer and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compared to SEQ ID NOs: 14, 15, 16, or 17. Preferably, the nucleotide insertion occurs at a site different from the nucleotide sequence encoding the inserted peptide of the present invention (SEQ ID NOs: 10, 11, 12, and 13), and more preferably not at the nucleotide sequence encoding loop IV. Preferably, the variant is a degenerate variant, or the nucleotide substitutions result in conserved amino acid substitutions.

[0076] The present invention further provides polynucleotides encoding the modified AAV capsid polypeptide of the present invention.

[0077] In some embodiments, the polynucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 14, 15, 16, and 17. In some embodiments, the polynucleotide consists of a nucleotide sequence selected from SEQ ID NOs: 14, 15, 16, and 17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical to SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the polynucleotide consists of a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical to SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the nucleotide sequence includes substitutions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides compared to SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the nucleotide sequence includes insertions and / or deletions of 3n nucleotides (where n is an integer, and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compared to SEQ ID NOs: 14, 15, 16, or 17. Preferably, the nucleotide insertion occurs at a site different from the nucleotide sequence encoding the inserted peptide of the present invention (SEQ ID NOs: 10, 11, 12, and 13), and more preferably not at the nucleotide sequence encoding loop IV. Preferably, the polynucleotide comprises a degenerate variant of SEQ ID NO: 14, 15, 16, or 17, or the nucleotide substitution results in a conserved amino acid substitution. Preferably, the polynucleotide consists of a degenerate variant of SEQ ID NO: 14, 15, 16, or 17, or the nucleotide substitution results in a conserved amino acid substitution.

[0078] The present invention provides a vector comprising the polynucleotide of the present invention, and a host cell comprising the polynucleotide or vector of the present invention.

[0079] The modified AAV capsid polypeptide of the present invention enables improved transduction of AAV into the eye, particularly retinal cells, compared to the parent AAV capsid polypeptide.

[0080] 3. Packaging of rAAV It is known in the art that rAAV can be packaged in a system comprising three plasmids: i) a transgene plasmid containing the rAAV genome encoding a desired gene product; ii) a packaging plasmid encoding the REP and / or CAP proteins; and iii) a helper plasmid (see, for example, CrossonSM et al., Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient CentrifugationMol Ther Methods Clin Dev.2018;10:1-7). Methods for producing rAAV are also described, for example, in U.S. Patent Application Publication No. 2005 / 0053922 and U.S. Patent Application Publication No. 2009 / 0202490.

[0081] The present invention provides a method for packaging the rAAV of the present invention, which includes introducing the polynucleotide or vector of the present invention into a host cell.

[0082] In some embodiments, the vector is an expression vector in which the polynucleotides of the present invention are operably ligated to a promoter. In some embodiments, the vector of the present invention further comprises a rep gene operably ligated to a promoter.

[0083] In some embodiments, the host cells further include a helper plasmid and / or an rAAV vector. When the host cells of the present invention are used to generate the rAAV of the present invention, they are referred to as “packaging cells”.

[0084] The polynucleotides or vectors of the present invention can be stably or transiently introduced into host cells using defined techniques, including but not limited to electroporation, calcium phosphate precipitation, and liposome-mediated transfection. For stable transformation, the target nucleic acid is typically operably linked to a select marker, such as a neomycin resistance gene.

[0085] Host cells are a variety of cells, including mammalian cells such as mouse cells and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, and suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, and CHO cells. Non-limiting examples of suitable host cells include, for example, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, and others. AAV-producing bacterial cells, such as Sf9 cells, can also be used to prepare the host cells of the present invention (see, for example, U.S. Patent No. 7,271,002; U.S. Patent Application No. 12 / 297,958).

[0086] The present invention provides a system or kit for packaging rAAV comprising the polynucleotide, vector, or host cell of the present invention. In some embodiments, the system or kit further comprises a helper plasmid and / or an rAAV vector.

[0087] 4. Recombinant AAV The present invention relates to a recombinant adeno-associated virus (rAAV) comprising a modified AAV capsid polypeptide and a genome encoding a gene product, wherein the modified AAV capsid polypeptide comprises a peptide inserted into loop IV compared to the parent AAV capsid polypeptide, and the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. This provides recombinant adeno-associated virus (rAAV) containing the amino acid sequence shown.

[0088] In some embodiments, spacer 1 and spacer 2 independently contain one or more amino acids. In some embodiments, spacer 1 and spacer 2 independently contain 1 to 10 amino acids. In some embodiments, spacer 1 and spacer 2 independently contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, spacer 1 and spacer 2 independently contain 1 to 3 amino acids. In some embodiments, spacer 1 and spacer 2 consist independently of 1 to 3 amino acids.

[0089] In some embodiments, the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. Spacer 1 and Spacer 2 independently contain one or more amino acids selected from A, L, and G. It contains the amino acid sequence shown.

[0090] In some embodiments, the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. Spacer 1 and Spacer 2 independently contain 1 to 10 amino acids selected from A, L, and G. It contains the amino acid sequence shown.

[0091] In some embodiments, the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) [In the formula, X1 is selected from G, L, and N. X2 is selected from K, A, G, and S. X3 is selected from G, E, and P. X4 is selected from P and T. The X5 is available in T, S, and G configurations. X6 is selected from T, R, and K. X7 is selected from K, P, and N. Spacer 1 and Spacer 2 independently contain 1 to 3 amino acids selected from A, L, and G. It contains the amino acid sequence shown.

[0092] In some embodiments, X1-X2-X3-X4-X5-X6-X7 is an amino acid sequence selected from SEQ ID NO: 6 (GKGPTTK), SEQ ID NO: 7 (LAEPSRP), SEQ ID NO: 8 (LGPPSKP), and SEQ ID NO: 9 (NSPTGRN).

[0093] In some embodiments, the inserted peptide is formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) The amino acid sequence includes the one shown in [wherein spacer 1 consists of amino acids Y1, Y2, and Y3, spacer 2 consists of amino acids Y4 and Y5, X1 is selected from G, L, and N, X2 is selected from K, A, G, and S, X3 is selected from G, E, and P, X4 is selected from P and T, X5 is selected from T, S, and G, X6 is selected from T, R, and K, and X7 is selected from K, P, and N].

[0094] In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present.

[0095] In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G.

[0096] In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide comprises the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A.

[0097] In some embodiments, the inserted peptide comprises an amino acid sequence selected from SEQ ID NO: 10 (AAAGKGPTTKAA), SEQ ID NO: 11 (AAALAEPSRPAA), SEQ ID NO: 12 (ALALGPPSKPAA), and SEQ ID NO: 13 (AAGNSPTGRNAA).

[0098] In some embodiments, the inserted peptide is formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) The amino acid sequence includes the one shown in [wherein spacer 1 consists of amino acids Y1, Y2, and Y3, spacer 2 consists of amino acids Y4 and Y5, X1 is selected from G, L, and N, X2 is selected from K, A, G, and S, X3 is selected from G, E, and P, X4 is selected from P and T, X5 is selected from T, S, and G, X6 is selected from T, R, and K, and X7 is selected from K, P, and N].

[0099] In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G, if present.

[0100] In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where each of Y1-Y5 is independently selected from A, L, and G.

[0101] In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 6(GKGPTTK)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 7(LAEPSRP)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 8(LGPPSKP)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A. In some embodiments, the inserted peptide consists of the amino acid sequence Y1-Y2-Y3-SEQ ID NO: 9(NSPTGRN)-Y4-Y5, where Y1 is A, Y2 is A or L, Y3 is A or G, Y4 is A, and Y5 is A.

[0102] In some embodiments, the inserted peptide consists of an amino acid sequence selected from SEQ ID NO: 10 (AAAGKGPTTKAA), SEQ ID NO: 11 (AAALAEPSRPAA), SEQ ID NO: 12 (ALALGPPSKPAA), and SEQ ID NO: 13 (AAGNSPTGRNAA).

[0103] In some embodiments, the parent AAV capsid polypeptide is an AAV2 capsid polypeptide VP1, VP2, or VP3. In some embodiments, the parent AAV capsid polypeptide is an AAV2 capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1, amino acids 138-735 of SEQ ID NO: 1, or amino acids 203-735 of SEQ ID NO: 1 or a variant thereof. In some embodiments, the variant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical to SEQ ID NO: 1 in full length, amino acids 138-735, or amino acids 203-735. In some embodiments, the variant includes insertions, deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to SEQ ID NO: 1. Preferably, the amino acid insertion occurs at a site different from the inserted peptide of the present invention, and more preferably not in loop IV. Preferably, the amino acid substitution is a conserved substitution. In some embodiments, the parent AAV capsid polypeptide consists of the amino acid sequence of SEQ ID NO: 1, amino acids 138-735 of SEQ ID NO: 1, or amino acids 203-735 of SEQ ID NO: 1.

[0104] In some embodiments, the modified AAV capsid polypeptide includes the peptide defined above, inserted into the region corresponding to positions 570-611 of SEQ ID NO: 1, compared to the parent AAV capsid polypeptide. In some embodiments, the peptide is inserted between the positions of the parent capsid polypeptide corresponding to positions 587 and 588 of SEQ ID NO: 1.

[0105] In some embodiments, the modified AAV capsid polypeptide of the present invention comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NOs: 2, 3, 4, or 5, or amino acids 203-747 of SEQ ID NOs: 2, 3, 4, or 5. In some embodiments, the modified AAV capsid polypeptide of the present invention consists of an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NOs: 2, 3, 4, or 5, or amino acids 203-747 of SEQ ID NOs: 2, 3, 4, or 5. In some embodiments, the modified AAV capsid polypeptide comprises or consists of a variant of SEQ ID NOs: 2, 3, 4, or 5. In some embodiments, the variant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical over its entire length to SEQ ID NOs. 2, 3, 4, or 5, amino acids 138-747 of SEQ ID NOs. 2, 3, 4, or 5. In some embodiments, the variant includes insertions, deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids compared to SEQ ID NOs. 2, 3, 4, or 5. Preferably, amino acid insertions occur at a different site from the inserted peptide of the present invention (SEQ ID NOs. 10, 11, 12, or 13), and more preferably not in loop IV. Preferably, amino acid substitutions are conserved substitutions.

[0106] In some embodiments, the modified AAV capsid polypeptide is encoded by a nucleotide sequence selected from SEQ ID NOs: 14, 15, 16, and 17. In some embodiments, the modified AAV capsid polypeptide is encoded by a variant of SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the variant is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, or 99.8% identical to SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the variant includes substitutions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides compared to SEQ ID NOs: 14, 15, 16, or 17. In some embodiments, the variant includes insertions and / or deletions of 3n nucleotides (where n is an integer and is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) compared to SEQ ID NOs: 14, 15, 16, or 17. Preferably, the nucleotide insertion occurs at a site different from the nucleotide sequence encoding the inserted peptide of the present invention (SEQ ID NOs: 10, 11, 12, or 13), and more preferably not at the nucleotide sequence encoding loop IV. Preferably, the variant is a degenerate variant, or the nucleotide substitutions result in conserved amino acid substitutions.

[0107] In some embodiments, the rAAV of the present invention, when administered preferably by intravitreal injection, exhibits improved infectivity against retinal cells compared to AAVs containing parent AAV capsid polypeptides.

[0108] The rAAV genome of the present invention comprises an expression cassette containing a nucleotide sequence encoding a gene product. In some embodiments, the gene product is interfering RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a site-specific nuclease.

[0109] When the gene product is interfering RNA (RNAi), appropriate RNAi include RNAi that reduce the levels of intracellular apoptotic or angiogenic factors. For example, RNAi may be shRNA or siRNA that reduces the levels of gene products that induce or promote apoptosis in cells, such as the Bax, Bid, Bak, and Bad gene products (see, for example, U.S. Patent No. 7,846,730). Interfering RNA may also be anti-angiogenic products such as shRNA or siRNA against VEGF, VEGFR1, or VEGFR2.

[0110] When the gene product is an aptamer, exemplary targets of the aptamer include, for example, VEGF and PDGF.

[0111] When the gene product is a polypeptide, polypeptides typically enhance the function of retinal cells such as rod-shaped or cone-shaped photoreceptor cells, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. Functional peptides. Exemplary polypeptides include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptors); VEGF-binding antibodies; VEGF-binding antibody fragments such as scFv and nanobodies; endostatins; tamstatins; angiostatins; pigment epithelial-derived factor (PEDF); soluble Tie-2 receptors, etc.; tissue inhibitors of metalloproteinase-3 (TIMP-3); photoreactive opsins (opsins), e.g., rhodopsin; and anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-X1), etc. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF); fibroblast growth factor 2; neurotrophin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); source neurotrophic factor (BDNF; for example, a continuous segment (SEQ ID NO: 11) containing approximately 200 to 247 amino acids of the amino acid sequence shown in Figure 11, containing at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 100% of the amino acid sequence polypeptide; epidermal growth factor; rhodopsin; X-linked apoptotic protein inhibitors; and sonic factor (Sonic) Hedgehog).

[0112] In some embodiments, the gene product of interest is a site-specific endonuclease that provides site-specific knockdown of a gene function associated with a retinal disease, for example, the gene being highly expressed in the disease.

[0113] In addition to knocking out defective alleles, site-specific nucleases can also be used to stimulate homologous recombination of donor DNA with a functional copy of the protein encoding the defective allele. Therefore, for example, the rAAV of the present invention can be used to deliver a site-specific endonuclease that knocks out a defective allele, or to deliver a functional copy of the defective allele, thereby causing functional copy repair and providing a functional retinal protein. In some embodiments, the site-specific endonuclease and the functional copy of the defective allele are delivered by separate rAAVs.

[0114] Site-specific endonucleases include, for example, zinc finger nucleases (ZFNs); activator-like effector nucleases (TALENs); and CRISPR / Cas nucleases.

[0115] In some embodiments, the gene product is an antibody against VEGF. For example, Novamab identified a nanobody against VEGF called "Nb24" having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the gene product is a polypeptide containing SEQ ID NO: 24. In some embodiments, the gene product is a bivalent Nb24 containing two Nb24s linked by a linker such as a (G4S)2 linker. In some embodiments, the rAAV genome includes an expression cassette encoding a bivalent Nb24 adjacent to an ITR, e.g., SEQ ID NO: 20.

[0116] The rAAV of the present invention provides improved transduction into retinal cells, enhanced gene product expression, reduced inflammatory response, desired safety, and robust therapeutic effects on ocular diseases compared to parental AAV capsid polypeptides.

[0117] 5. Pharmaceutical compositions and medical uses The present invention provides a pharmaceutical composition comprising a) the rAAV of the present invention and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in humans.

[0118] Such excipients, carriers, diluents, and buffers include any drugs that can be administered without unusual toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. These may include mineral salts such as hydrochlorides, hydrobroms, phosphates, and sulfates; and pharmaceutically acceptable salts such as salts of organic acids such as acetates, propions, malons, and benzoates. Furthermore, auxiliary substances such as wetting agents or emulsifiers and pH buffers may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and do not need to be discussed in detail herein. Pharmaceutically acceptable excipients are described in detail in various publications, including, for example, A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy," 20th Edition, Lippincott, Williams, & Wilkins; HC Ansel et al., 7th edition, Lippincott, Williams, & Wilkins (eds.), Pharmaceutical Dosage Forms and Drug Delivery Systems (1999); and Handbook of Pharmaceutical Excipients (2000), A.H. Kibbe et al., 3rd edition, American Pharmaceutical Association.

[0119] The present invention further provides a method for delivering a gene product to a target eye, particularly to retinal cells, comprising administering the rAAV or pharmaceutical composition of the present invention into the eye. Delivery of the gene product to retinal cells can provide treatment for retinal diseases. Retinal cells may be photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. In some cases, retinal cells are photoreceptor cells such as rods or cones.

[0120] The present invention provides a method for treating retinal disease, comprising administering an effective amount of the rAAV or pharmaceutical composition of the present invention to a subject requiring treatment for retinal disease. In some embodiments, the rAAV or pharmaceutical composition is administered by intraocular or intravitreal injection. The rAAV or pharmaceutical composition is administered by a single-dose or multi-dose (e.g., two, three, four or more doses) scheme. In the multi-dose scheme, the rAAV or pharmaceutical composition may be administered at different intervals, such as daily, weekly, monthly, or yearly, to achieve a desired level of gene expression.

[0121] The rAAV or pharmaceutical composition of the present invention is also provided for use in delivering gene products to a target eye, particularly retinal cells, including administration of the rAAV or pharmaceutical composition of the present invention into the eye. Delivery of gene products to retinal cells can provide treatment for retinal diseases. Retinal cells may be photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. In some cases, retinal cells are photoreceptor cells such as rods or cones.

[0122] The present invention also provides rAAV or pharmaceutical compositions for use in the treatment of retinal diseases. In some embodiments, the rAAV or pharmaceutical composition is administered by intraocular or intravitreal injection. The rAAV or pharmaceutical composition is administered by a single-dose or multi-dose (e.g., two, three, four or more doses) scheme. In the case of a multi-dose scheme, the rAAV or pharmaceutical composition can be administered at different intervals, such as daily, weekly, monthly, or yearly, to achieve a desired level of gene expression.

[0123] The present invention provides the use of rAAV or pharmaceutical compositions of the present invention in the preparation of pharmaceuticals for delivering gene products to target eyes, particularly retinal cells.

[0124] The present invention provides the use of rAAV or pharmaceutical compositions of the present invention in the preparation of pharmaceuticals for treating retinal diseases in subjects requiring treatment of retinal diseases.

[0125] Ophthalmic conditions that can be treated with rAAV or pharmaceutical compositions of the present invention include acute macular degeneration; Behçet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, e.g., acute macular degeneration, non-exudative age-related macular degeneration and exudative age-related macular degeneration; edema, e.g., macular edema, cystoid macular edema and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior segment or position; ocular tumors; retinal disorders, e.g., central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, and retinitis of the Retinitis; sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; diffuse pigmentation; posterior segment symptoms caused or affected by laser treatment of the eye; posterior segment symptoms caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; retinal vein bifurcation; anterior ischemic optic neuropathy; non-retinal diabetic retinal dysfunction; retinal fistula (retinal separation); retinitis pigmentosa; glaucoma; Usher syndrome; cone-rod cell dystrophy; Stargardt disease (yellow spot of the fundus); hereditary macular degeneration; choroidal degeneration; Leber congenital amaurosis; congenital resting night blindness; achoroidal; Barbie syndrome (Valde-Beedl syndrome); macular telangiectasia; Leber hereditary optic neuropathy; retinopathy of prematurity; and color vision deficiencies, including total color blindness, red-red blindness, green-green blindness, and blue-blue blindness. [Examples]

[0126] The following embodiments are provided for illustrative purposes only and are not limitations of this application.

[0127] Example 1: Preparation of an rAVV vector containing a modified AAV capsid polypeptide The rAAV vector was prepared using different packaging plasmids and transgene plasmids, similar to the method described by Crosson SM et al., 2018. In short, 3E6 293VPC cells (Thermo, catalog number: A35347) in serum-free virus-producing medium OPM-293 CD05 (Shanghai OPM Biosciences Co. Ltd. catalog number: 81075-001) were triple-transfected with polyethyleneimine using the following helper plasmid, rep / cap encoding packaging plasmid, and transgene plasmid: - Helper plasmids ("pHelper" containing Ad E2A, E4, and VA RNA helper genes, as described by Crosson Sm et al.; their map is shown in Figure 1A); -The packaging plasmid, its map is shown in Figure 1B, where "AAV2 Cap" is the nucleotide sequence encoding the capsid polypeptide listed in Table 1 (second column); - A transgene plasmid containing the rAAV genome of SEQ ID NO: 19 encoding GFP or the rAAV genome of SEQ ID NO: 20 encoding the anti-VEGF antibody bivalent Nb24 (see Figures 1C and 1D, respectively), with elements assembled using the Gibson assembly methodology (NEBuilder HiFi DNA Assembly Master Mix, NEB, catalog number E2621).

[0128] Packaging plasmids encoding the capsid polypeptide of SEQ ID NOs. 1, 18, or 22 were synthesized at Genscript Inc. (Nanjing, Jiangxi Province, China). Packaging plasmids encoding the capsid of SEQ ID NOs. 2, 3, 4, 5, or 21 were constructed by starting from the packing plasmid encoding SEQ ID NOs. 1 and inserting the nucleotide sequence from positions 1762 to 1797 of SEQ ID NOs. 14, 15, 16, 17, or 26 between positions 1761 and 1762 of SEQ ID NOs. 25 using the Gibson assembly methodology (NEBuilder HiFi DNA Assembly Master Mix, NEB, catalog number E2621).

[0129] After incubation at 37°C for 72 hours, cells were harvested and virus particles were purified by an iodixanol gradient (see Crosson SM et al.).

[0130] When rAAV was tested for titer by ddPCR, all titers were 10. 13 The levels were at the viral genome (vg) / mL level. Specifically, ddPCR was performed using Bio-Rad's QXDx AutoDG ddPCR System and QXDx Universal Kit for AutoDG ddPCR System, following the manufacturer's instructions. The primers used for ddPCR were as follows:

[0131] For GFP amplification: GFP-F ACTACAACAGCCACAACGTCTATATCA GFP-R GGCGGATCTTGAAGTTCACC GFP-P 5'-6-FAM-CCGACAAGC-ZEN-AGAAGAACGGCATCA-Iowa Black FQ-3'

[0132] For Nb24 amplification: qPCR-AMD-24-5-F GAGTGCGAGCTGGTGAG qPCR-AMD-24-5-R GCGTAGTCCCTGCTGATG qPCR-AMD-24-5-P CAAGGACGGCAGCACCTACTACAC

[0133] The prepared rAAVs are listed in Table 1.

[0134] [Table 1]

[0135] Example 2. In vitro study of rAAV in retinal cell-modified AAV capsids This example was performed to verify the expression of the GFP marker contained in the rAAV prepared in Example 1, and the test results show enhanced expression of the GFP marker in retinal cells transduced with rAAV containing modified AAV capsid polypeptide.

[0136] Human retinal pigment epithelial cells (ARPE19 cells, ATCC:CRL-2302) were seeded in 96-well plates (Thermo Scientific® 165305). AAV2-GFP and AAV.LGP-GFP prepared in Example 1 were added to ARPE19 cells 4 hours after seeding at various MOIs (800, 4,000, 20,000, and 100,000). After incubation at 37°C in 5% CO2 for 72 hours, the culture medium was replaced with PBS (Thermo Scientific, catalog no. 10010072), and the transduced cells were then detected for GFP protein using an automated fluorescence microscope (Agilent), with 485 / 20 excitation and 508 / 20 emission filter settings.

[0137] rAAVs with modified AAV2 capsids showed improved transduction rates compared to rAAVs with wild-type AAV2 capsids. As shown in Figure 2, cells transduced with AAV.LGP-GFP showed a much stronger fluorescence signal than cells transduced with AAV2-GFP. In particular, for AAV2-GFP, the fluorescence signal could be observed at least 4,000 MOI, while for AAV.LGP-GFP, the fluorescence signal could be observed at 800 MOI; cells transduced with AAV.LGP-GFP at 4,000 MOI showed a fluorescence signal equivalent to that of cells transduced with AAV2-GFP at 100,000 MOI.

[0138] Example 3. In vitro study of rAAV in retinal cells This example was performed to verify the expression of the target polypeptide contained in the rAAV prepared in Example 1. Compared to rAAV containing wild-type capsid polypeptide, retinal cells transduced with rAAV containing modified AAV capsid polypeptide showed enhanced expression of the target polypeptide.

[0139] ARPE19 cells were transduced with rAAV encoding bivalent Nb24, prepared in Example 1, at various MOIs (100,000, 30,000, 10,000, 3,000, 1,000, 300, 100, and 30) as described in Example 2. 72 hours after rAAV addition, the culture medium was collected by pipetting, and Nb24 expression in the cell culture medium was detected by direct anti-VEGF ELISA.

[0140] Briefly, VEGF polypeptide (human VEGFA165A, R&D Systems, catalog number 293-VE / CF) was coated onto an ELISA plate (Corning® 3690) overnight at 4°C, blocked with 3% BSA in PBST (Thermo Scientific, 37536), and then a collected cell culture medium containing bivalent Nb24 was added to the plate and incubated for 1 hour (RT). After adding each antibody, anti-Nb24 antibody (goat anti-Nb24 polyclonal, Novamb) and anti-goat HRP antibody (donkey anti-goat IgG HRP, Invitrogen, catalog number 34028) were added sequentially while incubating for 1 hour (RT). TMB solution (Thermo Scientific, catalog number 34028) was added for HRP color development, and then the reaction was stopped by adding a stop buffer (sulfo acid, Beyotime, catalog number P0215). To calculate the expression level of bivalent Nb24 in cell culture medium, absorbance OD450 was measured using a plate reader (SpectraMax i3x multimode microplate reader, molecular device). The expression levels of Nb24 were calculated based on standard curves prepared using recombinant bivalent Nb24 protein at a series of concentrations, and are shown in Figure 3. The ED50 value of rAAV (MOI at 50% maximum expression) was calculated using Graphpad Prism and is shown in Table 2.

[0141] [Table 2]

[0142] As shown in Figure 3, AAV.LGP-Nb24 achieved the highest expression of bivalent Nb24, AAV.NSP-Nb24 showed higher expression than AAV2-Nb24 (wild type) and comparable to AAV.7m8-Nb24 (reference), while AAV.GKG-Nb24 and AAV.LAE-Nb24 achieved Nb24 expression comparable to AAV2-Nb24. As shown in Table 2, AAV.LGP-Nb24 showed the lowest ED50, while the ED50 of AAV.NSP-Nb24 was comparable to that of AAV2-Nb24 and AAV.7m8-Nb24.

[0143] The rAAV containing the modified AAV capsid polypeptide of the present invention has been demonstrated to achieve enhanced expression of the polypeptide encoded by the rAAV (bivalent Nb24) compared to rAAV containing the wild-type capsid polypeptide or a reference capsid polypeptide.

[0144] Example 4. In vivo therapeutic effect of rAAV containing modified AAV capsid polypeptide This example was conducted to verify the expression of transgenes and the therapeutic efficacy of laser-induced choroidal neovascularization (CNV) in a non-human primate (NHP) model achieved by rAAV containing modified AAV capsid polypeptide.

[0145] This study was conducted by CRO (JOINN LABORATORIES, China). Briefly, ocular naive cynomolgus monkeys were selected by serum AAV neutralizing antibody (Nab) screening (the 20 monkeys with the lowest Nab levels). The selected monkeys were treated with a laser for modeling purposes (Pennesi et al., Animal models of age-related macular degeneration. Mol Aspects Med, 2012. 33(4): p.487-509).

[0146] The rAAVs (listed in Table 1) encoding divalent Nb24 prepared in Example 1 in a formulation buffer (dPBS + 200 mM NaCl + 0.005% PF68 (dPBS and PF68 were purchased from Thermo under catalog numbers 10010072 and 24040032, respectively)) were administered to the eyes of monkeys as described in Table 3.

[0147] [Table 3]

[0148] This study was conducted according to Figure 4. Briefly, the tested substance was intravitreal-injected into monkeys on day 1, and the eyes were imaged on days 0, 22, 29, and 43. The monkeys were modeled on day 15, and the monkey serum was collected on days 3, 7, 14, 22, 29, and 43. Monkeys #291-309 were sacrificed on day 43, and their tissues (including ocular tissues such as the retina, choroid, sclera, optic nerve, vitreous humor, aqueous humor, ciliary body, iris, cornea, and lens; as well as peripheral tissues including the brain, heart, liver, kidneys, lungs, skeletal muscle, and spleen) were collected separately. Monkey #310 was administered a second dose of AAV9-Nb24 on day 44, and its eyes were further imaged on day 51.

[0149] Samples containing bivalent Nb24 from tissue were prepared by mixing 30 mg of tissue and 60 μL of lysis buffer (T-PER® Tissue Protein Extraction Reagent, Thermo, catalog no. 78510) with a proteinase inhibitor (Pierce® Protease and Phosphatase Inhibitor Mini Tablets, Thermo, catalog no. A32959). The mixture was homogenized at 1,500 rpm for 2 minutes at 30-second intervals using a homogenizer (2010 Geno / Grinder® Automated Tissue Homogenizer and Cell Lysis Apparatus, Spex SamplePrp), followed by centrifugation at 12,000 rpm for 20 minutes, and the supernatant was collected. The total protein content in the samples was determined using a BCA kit (Thermo Fisher, catalog no. 23235).

[0150] Nb24 levels in tissues and serum were detected by ELISA (see Figure 5). Briefly, human VEGFA165A (R&D systems 293-VE / CF) was coated onto ELISA plates overnight at 4°C, blocked with blocking buffer (SuperBlock® T20 (TBS) Blocking Buffer, Thermo Scientific, catalog no. 37536), and then samples from tissues and serum containing equal amounts of total protein were added to the plates, followed by a 1-hour incubation (RT). After each antibody was added, an anti-Nb24 antibody (goat anti-Nb24 polyclonal, Novamb) and an anti-goat HRP antibody (donkey anti-goat IgG HRP, Invitrogen, catalog no. 34028) were sequentially added while incubating (RT) for 1 hour. TMB solution (Thermo Scientific, catalog number 34028) was added to induce HRP coloration, and then the reaction was stopped by adding a stop buffer (sulfo acid, Beyotime, catalog number P0215). As described in Example 3, the absorbance OD450 was measured to calculate the level of divalent Nb24 expression in the sample.

[0151] Figure 6 shows the expression of bivalent Nb24 in ocular tissue, indicating that administration of AAV.LGP-Nb24 achieved at least equivalent, and even higher, bivalent Nb24 expression compared to the reference (AAV.7m8-Nb24) across the entire ocular compartment, including the retina and choroid, where CNV occurs. As shown in Figure 7, Nb24 was not expressed in peripheral tissues (Figures 7A-G) and was not significantly expressed in serum (Figure 7H).

[0152] Fluorescein angiography (FFA) of the fundus was used as the primary in vivo endpoint for evaluating fluorescein leakage in RNVs (Li et al., 2018 and Cao et al., 2018). Slit-lamp microscopy and color fundus photography were performed to record the ocular response to the treatment. Images are shown in Figures 8-16. Compared to the clinical dose of Eylea® (2 mg / eye) (Figure 9), AAV2.LAE-Nb24 (Figure 10), AAV.RH10-Nb24 (Figure 11), and AAV2.LGP-Nb24 (Figure 12) provided partial or robust therapeutic efficacy in laser-induced NHP CNV models, with AAV2.LGP-Nb24 showing particularly robust therapeutic efficacy. Reference rAAV (AAV.7m8-Nb24) induced a clear ocular inflammatory response (evidenced by severe fluorescein leakage in the anterior chamber) in three of the four eyes treated (see Figure 13), which did not occur in monkeys treated with AAV.GKG-Nb24, AAV.LAE-Nb24, AAV.LGP-Nb24, or AAV.NSP-Nb24. AAV9-Nb24 induced widespread choroidal leakage 7–14 days after subretinal injection (see Figure 16).

[0153] rAAV containing modified capsid polypeptides demonstrated superior ocular transduction, robust transgene expression (evidence of high levels of bivalent Nb24 in the eye and surrounding tissues, signifying robust occlusion of retinal neovascularization), safety (evidence of minimal peripheral Nb24 expression), and therapeutic efficacy (evidence of reduced leakage in the retina).

[0154] array Capsid AAV2 of Sequence ID No. 1 [ka] The capsid AAV2_GKG of sequence number 2 was obtained by inserting sequence number 10 between positions 587 and 588 of sequence number 1. [ka] The capsid AAV2_LAE of sequence number 3 was obtained by inserting sequence number 11 between positions 587 and 588 of sequence number 1. [ka] The capsid AAV2_LGP of sequence number 4 was obtained by inserting sequence number 12 between positions 587 and 588 of sequence number 1. [ka] The capsid AAV2_NSP of sequence number 5 was obtained by inserting sequence number 13 between positions 587 and 588 of sequence number 1. [ka] Peptide GKG with SEQ ID NO: 6 inserted GKGPTTK Peptide with SEQ ID NO: 7 inserted_LAE LAEPSRP Peptide LGP with SEQ ID NO: 8 inserted LGPPSKP Sequence ID 9 Inserted Peptide_NSP NSPTGRN Peptide with insertion of sequence number 10_GKG full AAAGKGPTTKAA Peptide with SEQ ID NO: 11 inserted_LAE full AAALAEPSRPAA Peptide with SEQ ID NO: 12 inserted_LGP full ALALGPPSKPAA The inserted peptide of sequence number 13_NSP full AAGNSPTGRNAA Code array for sequence number 14: _AAV2_GKG [ka] Code array for sequence number 15: _AAV2_LAE [ka] Code array _AAV2_LGP for sequence number 16 [ka] Code array _AAV2_NSP for sequence number 17 [ka] AAV9 capsid of sequence number 18 [ka] The rAAV genome encoding GFP, sequence number 19. [ka] The rAAV genome encoding the bivalent Nb24 construct of Sequence ID No. 20 [ka] [ka] Amino acid sequence of AAV2_7m8 in SEQ ID NO: 21 [ka] AAV_RH10 amino acid sequence of SEQ ID NO: 22 [ka] The nucleotide sequence encoding AAV2 REP in Sequence ID No. 23 [ka] Sequence ID 24 (Nb amino acid sequence) EVQLQESGGGLVQPGGSLRLSCTASGFTFDDPDVGWFRQAPGNECELVSTISKDGSTYYTDSVKGRFTISRDYAKNTVYLQMNSLRAEDTAVYYCAADSNPIAPIRTCLGWYNYWGQGTLVTVSS Code sequence AAV2 capsid of sequence number 25 [ka] Code array AAV2_7m8 for sequence number 26 [ka] Code array AAV_RH10 for sequence number 27 [ka]

Claims

1. A modified adeno-associated virus (AAV) capsid polypeptide comprising a peptide inserted into loop IV compared to the parent AAV capsid polypeptide, wherein the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) The amino acid sequence shown in the formula, The spacers 1 and 2 each independently contain one or more amino acids selected from A, L, and G, preferably 1 to 10, more preferably 1 to 3, and the amino acid sequence X1-X2-X3-X4-X5-X6-X7 is an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, comprising a modified adeno-associated virus (AAV) capsid polypeptide.

2. The inserted peptide is given by formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) The amino acid sequence shown in the formula, Spacer 1 consists of amino acids Y1, Y2 and Y3, and Spacer 2 consists of amino acids Y4 and Y5, amino acid sequence A modified AAV capsid polypeptide according to claim 1, comprising:

3. The modified AAV capsid polypeptide according to claim 1, wherein the inserted peptide comprises an amino acid sequence selected from SEQ ID NO: 10 (AAAGKGPTTKAA), SEQ ID NO: 11 (AAALAEPSPAA), SEQ ID NO: 12 (ALALGPPSKPAA), and SEQ ID NO: 13 (AAGNSPTGRNAA).

4. The modified AAV capsid polypeptide according to claim 1, wherein the parent AAV capsid polypeptide is an AAV2 capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1, amino acids 138-735 of SEQ ID NO: 1, or amino acids 203-735 of SEQ ID NO: 1, or a variant thereof that is at least 90% identical to SEQ ID NO: 1 in terms of its entire length, amino acids 138-735, or amino acids 203-735.

5. The modified AAV capsid polypeptide according to claim 1, wherein the peptide is inserted between the positions corresponding to positions 587 and 588 of SEQ ID NO:

1.

6. The modified AAV capsid polypeptide according to claim 1, comprising an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138 to 747 of SEQ ID NOs: 2, 3, 4, or 5, or amino acids 203 to 747 of SEQ ID NOs: 2, 3, 4, or 5.

7. A polynucleotide encoding the modified AAV capsid polypeptide described in claim 1.

8. A vector comprising the polynucleotide described in claim 7.

9. A host cell comprising the polynucleotide described in claim 7 or the vector described in claim 8.

10. A system or kit for packaging rAAV containing the polynucleotide described in claim 7.

11. A recombinant adeno-associated virus (rAAV) comprising a modified AAV capsid polypeptide and a genome encoding a gene product, wherein the modified AAV capsid polypeptide comprises a peptide inserted into loop IV compared to the parent AAV capsid polypeptide, and the inserted peptide is of formula I: Spacer 1 - X1 - X2 - X3 - X4 - X5 - X6 - X7 - Spacer 2 (I) The amino acid sequence shown in the formula, The spacers 1 and 2 each independently contain one or more amino acids selected from A, L, and G, and the amino acid sequence X1-X2-X3-X4-X5-X6-X7 is an amino acid sequence selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, comprising a recombinant adeno-associated virus (rAAV).

12. The inserted peptide is given by formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) The amino acid sequence shown in the formula, Spacer 1 consists of amino acids Y1, Y2 and Y3, and Spacer 2 consists of amino acids Y4 and Y5, amino acid sequence The rAAV according to claim 11, including the rAAV described in claim 11.

13. The rAAV according to claim 11, wherein the inserted peptide comprises an amino acid sequence selected from SEQ ID NO: 10 (AAAGKGPTTKAA), SEQ ID NO: 11 (AAALAEPSPAA), SEQ ID NO: 12 (ALALGPPSKPAA), and SEQ ID NO: 13 (AAGNSPTGRNAA).

14. The rAAV according to claim 11, wherein the parent AAV capsid polypeptide is an AAV2 capsid polypeptide comprising the amino acid sequence of SEQ ID NO: 1, amino acids 138-735 of SEQ ID NO: 1, or amino acids 203-735 of SEQ ID NO: 1, or a variant thereof that is at least 90% identical to SEQ ID NO: 1 in terms of its entire length, amino acids 138-735, or amino acids 203-735.

15. The rAAV according to claim 11, wherein the peptide is inserted between the positions corresponding to positions 587 and 588 of Sequence ID No.

1.

16. The rAAV according to claim 11, wherein the modified AAV capsid polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138 to 747 of SEQ ID NOs: 2, 3, 4, or 5, or amino acids 203 to 747 of SEQ ID NOs: 2, 3, 4, or 5.

17. A pharmaceutical composition comprising rAAV as described in claim 11.

18. A pharmaceutical composition according to claim 11 or claim 17, for use in the treatment of retinal diseases.

19. Use of rAAV according to claim 11 or the pharmaceutical composition according to claim 17 in the preparation of a pharmaceutical for treating retinal diseases.

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