Recombinant adeno-associated viruses having modified AAV capsid polypeptides

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 and novel recombinant adeno-associated viruses (rAAV) comprising the modified AAV capsid polypeptides for delivery of therapeutic gene products. [Background technology]

[0002] In recent years, gene therapy has attracted increasing attention. Improved gene transfer technology will greatly aid the development of gene therapy.

[0003] Gene therapy was initially associated with the introduction of foreign genes into a patient's cells to correct inherited genetic errors, such as loss-of-function mutations. Most currently approved gene therapy protocols involve the introduction of functional copies of genes that are missing in the patient into the patient's somatic cells. Recently, however, gene therapy has been broadly defined as the correction of a disease phenotype by introducing new genetic information into an organism in need of gene therapy.

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

[0005] In vivo gene therapy by introducing transgenes in situ into the eye has been used to treat ocular diseases, such as those that cause blindness. Examples of such diseases are retinitis pigmentosa, maculopathy, Leber congenital amaurosis, Leber hereditary optic neuropathy, early-onset severe retinal dystrophies, achromatopsia, retinal blepharophimosis, ocular albinism, ocular albinism, glaucoma, Stargardt's 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 adenovirus) for replication. The genome of wild-type AAV contains approximately 4.7 kilobases (kb) and contains the cap and rep genes between two inverted terminal repeat (ITR) sequences with interrupted palindromic sequences that can fold into a hairpin structure that functions as a primer during the initiation of DNA replication. The cap gene codes for the viral capsid protein, and the rep gene is involved in AAV replication and integration. AAV can infect a wide variety of cells, and viral DNA can integrate into human chromosome 19 in the presence of the rep product.

[0007] The majority of rAAV gene therapies in clinical development are focused on the CNS, including the brain and eye. There are roughly a dozen capsid serotypes currently used as vectors in clinical trials, with the most common being AAV2-based platforms for ocular diseases.

[0008] In particular, Luxturna, the first rAAV gene therapy approved by the U.S. Food and Drug Administration (FDA), uses AAV2 to treat patients with inherited vision loss caused by RPE65 gene mutations. However, newer modified and more efficient capsids are increasingly needed.

[0009] The engineering of novel AAV capsids has been constantly pursued to obtain new properties and characteristics. The main limitations to rational design approaches relate to the insufficient knowledge regarding AAV cell surface binding, internalization, trafficking, uncoating and gene expression.

[0010] Although several modified AAV capsids have been developed (see, e.g., WO2012145601A2, WO2016134375A1, and WO2018022905A2), 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 a polynucleotide encoding a product to treat a disease. Summary of the Invention [Means for solving the problem]

[0011] In a first aspect, the present invention provides a modified AAV capsid polypeptide comprising a peptide inserted into loop IV compared to a parent AAV capsid polypeptide, wherein the inserted peptide has the 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; X5 is selected from T, S and G; X6 is selected from T, R and K; X7 is selected from K, P and N. The present invention provides a modified AAV capsid polypeptide comprising the amino acid sequence shown in

[0012] The present invention further provides polynucleotides encoding the modified AAV capsid polypeptides of the present invention, vectors comprising the polynucleotides, and host cells comprising the polynucleotides or the vectors.

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

[0014] In a third aspect, the present invention provides a recombinant adeno-associated virus (rAAV) comprising a modified AAV capsid polypeptide of the 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 of treating a retinal disease comprising administering an rAAV or a pharmaceutical composition of the present invention to the eye of a subject in need of treatment for the retinal disease.

[0017] Provided is an rAAV or pharmaceutical composition of the invention for use in treating a retinal disease.

[0018] The invention provides the use of an rAAV or pharmaceutical composition of the invention in the preparation of a medicament for treating a retinal disease. [Brief description of the drawings]

[0019] [Figure 1A] 1 shows helper plasmids for preparing rAAV. [Figure 1B] 1 shows packaging plasmids for preparing rAAV. [Figure 1C] 1 shows a transgene plasmid for preparing rAAV. [Figure 1D] 1 shows a transgene plasmid for preparing rAAV. [Diagram 2] Fluorescence microscopy of retinal cells transduced with rAAV containing a genome encoding GFP at various MOIs. [Diagram 3] FIG. 1 shows the expression of Nb24 in retinal cells transduced with rAAV containing genomes encoding bivalent Nb24 and various capsid polypeptides, with the Y-axis showing the "lg" value of concentration and a "0" point representing a concentration of "1." [Figure 4] Shown is the workflow of a study in a laser-induced choroidal neovascularization (CNV) non-human primate (NHP) model. [Diagram 5]Figure 1 shows the mechanism of ELISA for detection of Nb24. [Figure 6] 1 shows Nb24 levels in ocular tissues. [Figure 7] Nb24 levels in peripheral tissues and serum are shown. [Figure 8] Fundus photograph (FP) and fundus aortic photograph (FFA) images of the left eye (OS) and right eye (OR) of vehicle-treated monkeys are shown. [Figure 9] 3 shows fundus photographs (FP) and fundus aortic photographs (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with Eylea®. [Figure 10] Shown are fundus photographs (FP) and fundus aortic photographs (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.LAE-Nb24. [Figure 11] Shown are fundus photographs (FP) and fundus aortic photographs (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.RH10-Nb24. [Figure 12] Shown are fundus photographs (FP) and fundus aortic photographs (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.LGP-Nb24. [Figure 13] Shown are fundus photograph (FP) and fundus aortic photograph (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.7m8-Nb24. [Figure 14] Shown are fundus photographs (FP) and fundus aortic photographs (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.GKG-Nb24. [Figure 15] Shown are fundus photographs (FP) and fundus aortic photographs (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV.NSP-Nb24. [Figure 16] Shown are fundus photograph (FP) and fundus aortic photograph (FFA) images of the left eye (OS) and right eye (OR) of monkeys treated with AAV9-Nb24. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] 1.Definition Unless otherwise indicated, all terms used herein have the same meaning as they would to one of ordinary skill in the art and the practice of the present invention employs conventional techniques of microbiology and recombinant DNA technology that are within the knowledge of those of ordinary skill in the art.

[0021] The term "retinal cells," as used herein, can refer to any cell type contained in the retina, such as retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, and photoreceptor cells, including rods and cones, Muller glial cells, and the 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. The term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. 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 ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0023] The genomic sequences of various serotypes of AAV, as well as the sequences of natural terminal repeats (TRs), Rep proteins, and capsid subunits, are 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); the disclosures of which are incorporated herein by reference for teaching AAV nucleic acid and amino acid sequences.

[0024] As used herein, "rAAV vector" refers to an AAV vector that contains a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (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] "AAV virus" or "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 an encapsidated polynucleotide rAAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector." Thus, 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 lead to the assembly and encapsulation of AAV particles.

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

[0028] The cap gene encodes three structural proteins, VP1, VP2, and VP3, that 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-termini 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, a wild-type AAV2 capsid may include VP1 of SEQ ID NO:1, VP2 of amino acids 138-735 of SEQ ID NO:1, and VP3 of amino acids 203-735 of SEQ ID NO:1.

[0029] AAV "helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Many such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses, such as vaccinia. Adenoviruses encompass several different subgroups, with adenovirus type 5 of subgroup C being the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories 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 depositories such as ATCC.

[0030] "Helper virus functions" refers to functions encoded in the helper virus genome that enable replication and packaging of AAV (in conjunction with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in a number of ways, including by providing a helper virus or, for example, by providing a polynucleotide sequence encoding the necessary functions to the producer cell in trans. For example, a plasmid, referred to herein as a "helper plasmid," or other expression vector that contains a nucleotide sequence encoding one or more adenoviral proteins, is transfected into the producer cell along with the rAAV vector.

[0031] As used herein, the term "polynucleotide construct" refers to a single- or double-stranded polynucleotide isolated from a naturally occurring gene or modified to contain a non-naturally occurring nucleic acid segment. When the polynucleotide construct contains the control sequences necessary to express a coding sequence of the present invention, the polynucleotide construct comprises an "expression cassette."

[0032] As used herein, the term "polynucleotide" generally refers to a nucleic acid molecule (e.g., 100 bases in length and up to 30 kilobases in length) as well as a sequence that is complementary (antisense) or identical (sense) to a sequence of a messenger RNA (mRNA) or miRNA fragment or molecule. The term can also refer to DNA or RNA molecules that are either transcribed or non-transcribed.

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

[0034] "Heterologous" means derived from a genotypically different entity from the rest of the entity to which it is being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter that is removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally linked is a heterologous promoter. Thus, for example, a rAAV that contains a heterologous nucleic acid encoding a heterologous gene product is a rAAV that contains a nucleic acid that is not normally included in naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by naturally occurring wild-type AAV.

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

[0036] As used herein, the term "expression cassette" refers to a polynucleotide segment that includes additional nucleotides provided for expression of a polynucleotide, e.g., 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] "Control sequences" include all elements necessary or beneficial for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, enhancers, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and signals for the termination of transcription and translation.

[0039] For example, the control sequence may be an appropriate promoter sequence, i.e., a nucleotide sequence recognized by a host cell for expressing a polynucleotide encoding a polypeptide of the present invention. The promoter sequence includes transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, such as the lac operon of E. coli. Promoters also include mutant promoters, truncated promoters and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0040] As used herein, the term "operably linked" refers to a configuration in which a control sequence is positioned in appropriate relation to a coding sequence of a polynucleotide sequence such that the control sequence directs expression of the polypeptide coding sequence.

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

[0042] A "gene product" is a molecule that results from the expression of a particular gene. Gene products include, for example, polypeptides, aptamers, interfering RNA, mRNA, etc.

[0043] "Small interfering RNA" or "small interfering RNA" or siRNA is an RNA duplex of nucleotides that targets a gene of interest ("target gene"). "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. An siRNA "targets" a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the siRNA duplex is less than 30 nucleotides. In some embodiments, the duplex can 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 duplex is 19-25 nucleotides long. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure can include a loop portion positioned between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure can also include a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang that is 0, 1, 2, 3, 4, or 5 nucleotides in length.

[0044] "Short hairpin RNA," or shRNA, is a polynucleotide construct capable of directing the expression of an interfering RNA, such as an siRNA.

[0045] The term "recombinant" as used herein refers to a nucleic acid, vector, polypeptide, or protein that has been produced using DNA recombination (cloning) methods and is distinguishable from a naturally occurring or wild-type nucleic acid, vector, polypeptide, or protein. The terms "polypeptide" and "protein" are used interchangeably herein and refer to a polymer of amino acids, and include full-length proteins and fragments thereof.

[0046] As used herein, the term "host cell" refers to, for example, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of rAAV vectors. The term includes the progeny of the original cell that was transduced. Thus, as used herein, "host cell" generally refers to a cell that has been transduced with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical in morphology or genomic or total DNA complement to the original parent due to natural, accidental, or deliberate mutations.

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

[0048] The term "subject" as used herein includes, but is not limited to, humans, non-human primates, such as chimpanzees and other ape and monkey species; agricultural animals, such as cows, 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 does not denote a particular age or sex. Thus, adult and neonatal subjects, as well as fetuses, whether male or female, are intended to be covered.

[0049] The terms "genetic modification" and "genetic modification" (and grammatical variations thereof) refer to the process by which genes are transformed into mitotic or meiotic divisions. It is used interchangeably herein to refer to the process by which a genetic element (e.g., a polynucleotide) is introduced into a cell by other than a recombinant DNA technique. The element may be heterologous to the cell, or may be an additional copy or improved version of an element already present in the cell. Genetic modification may be performed by transfecting the cell with a recombinant plasmid or other polynucleotide via any process known in the art, such as, for example, electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic modification may also be achieved by transduction or infection, for example, with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or minichromosome in the cell, but any modification that changes the phenotype and / or genotype of the cell and its progeny is included in the term.

[0050] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during long-term culture of the cells in vitro. Generally, such cells are "genetically" altered (genetically modified) in that a genetic modification has been introduced that is inheritable by progeny of the altered cell.

[0051] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The term also encompasses modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. When a polypeptide, such as an antiangiogenic polypeptide, a neuroprotective polypeptide, etc., is discussed in relation to delivering a gene product to a mammalian subject, and the composition thereof, it refers to the respective intact polypeptide, or any fragment or engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, reference to nucleic acids encoding antiangiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivering a gene product to a mammalian subject (which may be referred to as "transgenes" delivered to recipient cells) includes polynucleotides encoding intact polypeptides or any fragment or engineered derivative thereof having the desired biochemical function.

[0052] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell or other substance refers to a preparation of the substance that lacks at least some of the other components that may also be present when the substance or similar substance naturally occurs or is initially prepared. Thus, for example, an isolated substance may be prepared by using purification techniques to enrich it from a source mixture. Enrichment can be measured in absolute terms, such as weight per volume of solution, or can be measured with respect to secondary potential interfering substances present in the source mixture. Increasing enrichment of the embodiments of the present disclosure is increasingly isolated. An isolated plasmid, nucleic acid, vector, virus, host cell or other substance is, in some embodiments, purified, e.g., 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, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents the disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures the disease and / or the adverse effects caused by the disease. As used herein, "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to or at risk of acquiring the disease, but who has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., halting its development; (c) relieving the disease, i.e., causing regression of the disease.

[0054] As used herein, the term "ED50" refers to the median effective dose of a drug, i.e., the dose that can produce 50% of the maximum response. For the delivery of transgenes expressed in cells using recombinant viruses (e.g., rAAV), the ED50 can be expressed as the MOI at which 50% of the maximum expression of the transgene is achieved.

[0055] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, unless the context clearly dictates otherwise, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are 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" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "modified AAV capsid" includes a plurality of such capsids. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0057] 2. Modified AAV Capsid Polypeptides and Encoding Polynucleotides The present invention relates to a modified AAV capsid polypeptide comprising a peptide inserted into loop IV as compared to a parent AAV capsid polypeptide, wherein the inserted peptide has the 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; X5 is selected from T, S and G; X6 is selected from T, R and K; X7 is selected from K, P and N. The present invention provides a modified AAV capsid polypeptide comprising the amino acid sequence shown in

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

[0059] In some embodiments, the inserted peptide has 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; X5 is selected from T, S and G; X6 is selected from T, R and K; X7 is selected from K, P and N; spacer 1 and spacer 2 independently comprise one or more amino acids selected from A, L, and G. The amino acid sequence shown in

[0060] In some embodiments, the inserted peptide has 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; X5 is selected from T, S and G; 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. The amino acid sequence shown in

[0061] In some embodiments, the inserted peptide has 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; X5 is selected from T, S and G; 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. The amino acid sequence shown in

[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 has Formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) [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 an amino acid sequence of 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 an amino acid sequence of 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 an amino acid sequence of 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 an amino acid sequence of 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 of 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 of 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 of 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 has Formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) [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, if present, 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, if present, 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, if present, 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, if present, is independently selected from A, L, and G.

[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 over the full length, amino acids 138-735, or amino acids 203-735. In some embodiments, the variant comprises an insertion, deletion, substitution and / or addition 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 is at a site different from the inserted peptide of the invention, more preferably not in loop IV. Preferably, the amino acid substitution is a conservative substitution. In some embodiments, the parent AAV capsid polypeptide consists of the amino acid sequence of SEQ ID NO:1, amino acids 138 to 735 of SEQ ID NO:1, or amino acids 203 to 735 of SEQ ID NO:1.

[0073] In the case of an AAV2 capsid polypeptide, loop IV corresponds to positions 570 to 611 of SEQ ID NO: 1. In other words, the modified AAV capsid polypeptide thus comprises a peptide as defined above inserted, relative to the parent AAV capsid polypeptide, in a region corresponding to positions 570 to 611 of SEQ ID NO: 1. In some embodiments, the peptide is inserted between positions in the parent capsid polypeptide corresponding to positions 587 and 588 of SEQ ID NO: 1.

[0074] In some embodiments, a modified AAV capsid polypeptide of the invention comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NO: 2, 3, 4 or 5, or amino acids 203-747 of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, a modified AAV capsid polypeptide of the invention consists of an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NO: 2, 3, 4 or 5, or amino acids 203-747 of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, a modified AAV capsid polypeptide comprises or consists of a variant of SEQ ID NO: 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 the entire length to SEQ ID NO: 2, 3, 4 or 5, amino acids 138-747 of SEQ ID NO: 2, 3, 4 or 5, or amino acids 203-747 of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, the variant comprises an insertion, deletion, substitution and / or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to SEQ ID NO: 2, 3, 4 or 5. Preferably, the amino acid insertion is at a different site (SEQ ID NO: 10, 11, 12 and 13) than the inserted peptide of the invention, more preferably not in loop IV. Preferably, the amino acid substitution is a conservative substitution.

[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 NO: 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 NO: 14, 15, 16, or 17. In some embodiments, the variant comprises 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 NO: 14, 15, 16 or 17. In some embodiments, the variant comprises insertions and / or deletions of 3n (where n is an integer and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotides compared to SEQ ID NO: 14, 15, 16 or 17. Preferably, the insertion of nucleotides is made at a different site than the nucleotide sequence encoding the inserted peptide of the invention (SEQ ID NO: 10, 11, 12 and 13), more preferably not in the nucleotide sequence encoding loop IV. Preferably, the variant is a degenerate variant or the substitution of nucleotides results in a conservative substitution of an amino acid.

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

[0077] In some embodiments, the polynucleotide comprises a nucleotide sequence selected from SEQ ID NO: 14, 15, 16, and 17. In some embodiments, the polynucleotide consists of a nucleotide sequence selected from SEQ ID NO: 14, 15, 16, and 17. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7% or 99.8% identical to SEQ ID NO: 14, 15, 16, or 17. In some embodiments, the polynucleotide consists of a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7% or 99.8% identical to SEQ ID NO: 14, 15, 16, or 17. In some embodiments, the nucleotide sequence comprises 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 NO: 14, 15, 16 or 17. In some embodiments, the nucleotide sequence comprises insertions and / or deletions of 3n (where n is an integer and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotides compared to SEQ ID NO: 14, 15, 16 or 17. Preferably, the insertion of nucleotides is made at a different site than the nucleotide sequence encoding the inserted peptide of the invention (SEQ ID NO: 10, 11, 12 and 13), more preferably not in 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 substitutions result in conserved amino acid substitutions. Preferably, the polynucleotide consists of a degenerate variant of SEQ ID NO: 14, 15, 16 or 17, or the nucleotide substitutions result in conserved amino acid substitutions.

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

[0079] The modified AAV capsid polypeptides of the present invention allow for improved transduction of AAV into ocular, and particularly retinal, cells, as compared to the parent AAV capsid polypeptide.

[0080] 3. Packaging rAAV It is known in the art that rAAV is packaged in a system that includes three plasmids, including: i) a transgene plasmid that includes the genome of the rAAV encoding the desired gene product, ii) a packaging plasmid that encodes the REP and / or CAP proteins, and iii) a helper plasmid (see, e.g., Crosson SM et al., Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation Mol 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 invention provides a method for packaging a rAAV of the invention comprising introducing a polynucleotide or vector of the invention into a host cell.

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

[0083] In some embodiments, the host cell further comprises a helper plasmid, and / or a rAAV vector. When a host cell of the invention is used to produce a rAAV of the invention, it is referred to as a "packaging cell."

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

[0085] Host cells can be of many types, such as mammalian cells, including, for example, 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, CHO cells, and the like. 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 Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721) COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. Bacterial cells that produce AAV, such as Sf9 cells, can also be used to prepare host cells of the invention (see, e.g., U.S. Pat. No. 7,271,002; U.S. Patent Application Serial No. 12 / 297,958).

[0086] The invention provides systems or kits for packaging rAAV comprising a polynucleotide, vector, or host cell of the invention. In some embodiments, the system or kit further comprises a helper plasmid and / or a 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 a parent AAV capsid polypeptide, the inserted peptide having a structure represented by 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; X5 is selected from T, S and G; X6 is selected from T, R and K; X7 is selected from K, P and N. The present invention provides a recombinant adeno-associated virus (rAAV) comprising the amino acid sequence shown in

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

[0089] In some embodiments, the inserted peptide has 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; X5 is selected from T, S and G; X6 is selected from T, R and K; X7 is selected from K, P and N; spacer 1 and spacer 2 independently comprise one or more amino acids selected from A, L, and G. The amino acid sequence shown in

[0090] In some embodiments, the inserted peptide has 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; X5 is selected from T, S and G; 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. The amino acid sequence shown in

[0091] In some embodiments, the inserted peptide has 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; X5 is selected from T, S and G; 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. The amino acid sequence shown in

[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 has Formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) [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 an amino acid sequence of Y1-Y2-Y3-SEQ ID NO:6 (GKGPTTK)-Y4-Y5, where each of Y1-Y5, if present, is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises an amino acid sequence of Y1-Y2-Y3-SEQ ID NO:7 (LAEPSRP)-Y4-Y5, where each of Y1-Y5, if present, is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises an amino acid sequence of Y1-Y2-Y3-SEQ ID NO:8 (LGPPSKP)-Y4-Y5, where each of Y1-Y5, if present, is independently selected from A, L, and G. In some embodiments, the inserted peptide comprises an amino acid sequence of Y1-Y2-Y3-SEQ ID NO:9 (NSPTGRN)-Y4-Y5, where each of Y1-Y5, if present, is independently selected from A, L, and G.

[0095] In some embodiments, the inserted peptide comprises an amino acid sequence of 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 an amino acid sequence of 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 an amino acid sequence of 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 an amino acid sequence of 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 of 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 of 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 of 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 has Formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) [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, if present, 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, if present, 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, if present, 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, if present, is independently selected from A, L, and G.

[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 over the full length, amino acids 138-735, or amino acids 203-735. In some embodiments, the variant comprises an insertion, deletion, substitution and / or addition 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 is at a site different from the inserted peptide of the invention, more preferably not in loop IV. Preferably, the amino acid substitution is a conservative substitution. In some embodiments, the parent AAV capsid polypeptide consists of the amino acid sequence of SEQ ID NO:1, amino acids 138 to 735 of SEQ ID NO:1, or amino acids 203 to 735 of SEQ ID NO:1.

[0104] In some embodiments, the modified AAV capsid polypeptide comprises a peptide as defined above inserted, relative to the parent AAV capsid polypeptide, in a region corresponding to positions 570 to 611 of SEQ ID NO: 1. In some embodiments, the peptide is inserted between positions in the parent capsid polypeptide corresponding to positions 587 and 588 of SEQ ID NO: 1.

[0105] In some embodiments, a modified AAV capsid polypeptide of the invention comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NO: 2, 3, 4 or 5, or amino acids 203-747 of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, a modified AAV capsid polypeptide of the invention consists of an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, and 5, amino acids 138-747 of SEQ ID NO: 2, 3, 4 or 5, or amino acids 203-747 of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, a modified AAV capsid polypeptide comprises or consists of a variant of SEQ ID NO: 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 the entire length to SEQ ID NO: 2, 3, 4 or 5, amino acids 138-747 of SEQ ID NO: 2, 3, 4 or 5, or amino acids 203-747 of SEQ ID NO: 2, 3, 4 or 5. In some embodiments, the variant comprises an insertion, deletion, substitution and / or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids compared to SEQ ID NO: 2, 3, 4 or 5. Preferably, the amino acid insertion is at a different site (SEQ ID NO: 10, 11, 12 or 13) than the inserted peptide of the invention, more preferably not in loop IV. Preferably, the amino acid substitution is a conservative substitution.

[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 NO: 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 NO: 14, 15, 16, or 17. In some embodiments, the variant comprises 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 NO: 14, 15, 16 or 17. In some embodiments, the variant comprises insertions and / or deletions of 3n (where n is an integer and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotides compared to SEQ ID NO: 14, 15, 16 or 17. Preferably, the insertion of nucleotides is made at a different site than the nucleotide sequence encoding the inserted peptide of the invention (SEQ ID NO: 10, 11, 12 or 13), more preferably not in the nucleotide sequence encoding loop IV. Preferably, the variant is a degenerate variant or the substitution of nucleotides results in a conservative substitution of an amino acid.

[0107] In some embodiments, the rAAV of the present invention exhibit improved infectivity for retinal cells compared to AAV comprising a parent AAV capsid polypeptide, preferably when administered by intravitreal injection.

[0108] The genome of the rAAV of the present invention comprises an expression cassette comprising a nucleotide sequence encoding a gene product. In some embodiments, the gene product is an 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 gene product is interference RNA (RNAi), suitable RNAi includes RNAi that reduces the level of intracellular apoptosis or angiogenesis factors.For example, RNAi can be shRNA or siRNA that reduces the level of gene product that induces or promotes apoptosis in cells, including, for example, Bax, Bid, Bak and Bad gene product (see, for example, U.S. Patent No. 7,846,730).Interference RNA can also be anti-angiogenic product, such as shRNA or siRNA against VEGF, VEGFR1 or VEGFR2.

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

[0111] When the gene product is a polypeptide, the polypeptide typically enhances retinal cell function, such as rod or cone photoreceptor cells, retinal ganglion cells, Muller 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 scFvs and nanobodies; endostatin; tumstatin; angiostatin; pigment epithelium-derived factor (PEDF); soluble Tie-2 receptors, and the like; tissue inhibitor of metalloproteinases-3 (TIMP-3); photoreactive opsins (opsins), such as rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-X1), and the like. 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 contiguous segment comprising from about amino acid 200 to 247 of the amino acid sequence depicted in FIG. 11 (SEQ ID NO: 11) is at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% of the amino acid sequence polypeptide; epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis protein; and Sonic hedgehog).

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

[0113] In addition to knocking out the defective allele, a site-specific nuclease can also be used to stimulate the homologous recombination of donor DNA with a functional copy of the protein that encodes the defective allele. Thus, for example, the rAAV of the present invention can be used to deliver a site-specific endonuclease that knocks out the defective allele, and can be used to deliver a functional copy of the defective allele, causing functional copy repair, thereby 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); transcription activator-like effector nucleases (TALENs), and CRISPR / Cas nucleases.

[0115] In some embodiments, the gene product is an antibody against VEGF. For example, Novamab has 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 comprising SEQ ID NO: 24. In some embodiments, the gene product is a bivalent Nb24 comprising two Nb24s linked by a linker, such as a (G4S)2 linker. In some embodiments, the genome of the rAAV comprises an expression cassette encoding a bivalent Nb24 flanked by ITRs, e.g., SEQ ID NO: 20.

[0116] The rAAV of the present invention provide improved transduction of retinal cells, enhanced expression of gene products, reduced inflammatory responses, desirable safety and / or robust therapeutic effects on ocular diseases compared to the parental AAV capsid polypeptide.

[0117] 5. Pharmaceutical Compositions and Medical Uses The invention provides pharmaceutical compositions comprising: a) an rAAV of the invention; and b) a pharma- ceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharma- ceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in humans.

[0118] Such excipients, carriers, diluents and buffers include any agent that can be administered without unusual toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. They may include pharma-ceutically acceptable salts, such as mineral acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, and the like; and salts of organic acids, such as acetate, propionate, malonate, benzoate, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharma-ceutically acceptable excipients are known in the art and need not be discussed in detail here. 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; H. C. 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, Amer. Pharmaceutical Assoc.

[0119] The present invention further provides a method for delivering a gene product to a subject's eye, particularly a retinal cell, comprising administering the rAAV or pharmaceutical composition of the present invention to the eye. Delivery of the gene product to a retinal cell can provide treatment for a retinal disease. The retinal cell can be a photoreceptor, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell. In some cases, the retinal cell is a photoreceptor cell, such as a rod or a cone.

[0120] The present invention provides a method for treating a retinal disease, comprising administering an effective amount of a rAAV or pharmaceutical composition of the present invention to a subject in need of treatment of a 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 a multiple dose (e.g., 2, 3, 4 or more doses) scheme. In the case of a multiple dose scheme, the rAAV or pharmaceutical composition can be administered at different intervals, such as daily, weekly, monthly, yearly, etc., to achieve a desired level of gene expression.

[0121] Also provided is the rAAV or pharmaceutical composition of the present invention for use in delivering a gene product to the eye of a subject, particularly to a retinal cell, comprising administering the rAAV or pharmaceutical composition of the present invention to the eye. The delivery of the gene product to a retinal cell can provide treatment for a retinal disease. The retinal cell can be a photoreceptor, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell. In some cases, the retinal cell is a photoreceptor cell, such as a rod or a cone.

[0122] Also provided is the rAAV or pharmaceutical composition of the present invention for use in treating 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 a multiple dose (e.g., 2, 3, 4 or more doses) scheme. In the case of a multiple dose scheme, the rAAV or pharmaceutical composition can be administered at different intervals, such as daily, weekly, monthly, yearly, etc., to achieve a desired level of gene expression.

[0123] The invention provides the use of an rAAV or pharmaceutical composition of the invention in the preparation of a medicament for delivering a gene product to the eye of a subject, in particular to retinal cells.

[0124] The invention provides the use of an rAAV or pharmaceutical composition of the invention in the preparation of a medicament for treating a retinal disease in a subject in need thereof.

[0125] Ophthalmopathies that can be treated with the rAAV or pharmaceutical compositions of the invention include acute macular degeneration; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; multifocal choroiditis; ocular trauma affecting the posterior segment or location; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreous retinopathy (PVR), retinal artery occlusive disease, retinal detachment, retinitis of the eye, and / or other conditions. retiniti), sympathetic ophthalmia; Vogt-Koyanagi-Harada (VKH) syndrome; diffuse ocular pigmentation; posterior ocular conditions caused or affected by ocular laser procedures; posterior ocular conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; preretinal membrane disorders; retinal vein bifurcation; anterior ischemic optic neuropathy; non-retinal diabetic retinal dysfunction; retinal fistula (retinal schisis); retinitis pigmentosa; glaucoma; Usher syndrome; cone-rod cell dystrophy; Stargardt disease (yellow spot on the fundus); hereditary macular degeneration; chorioretinal degeneration; Leber's congenital amaurosis; congenital stationary night blindness; achoroidea; Barbie syndrome (Bardet-Biedl syndrome); macular telangiectasia; Leber's hereditary optic neuropathy; retinopathy of prematurity; color vision deficiencies including total achromatopsia, protanopia, protanopia, and tritanopia. EXAMPLES

[0126] The following examples are provided for illustrative purposes only and are not intended to be limiting on the present application.

[0127] Example 1: Preparation of rAVV Vectors Containing Modified AAV Capsid Polypeptides rAAV vectors were prepared using different packaging and transgene plasmids in a similar manner to that described in Crosson SM et al., 2018. Briefly, 3E6 293VPC cells (Thermo, Cat. No.: A35347) in serum-free virus production medium OPM-293 CD05 (Shanghai OPM Biosciences Co. Ltd. Cat. No.: 81075-001) were triply transfected with the following helper plasmids, packaging plasmids encoding rep / cap, and transgene plasmids using polyethylenimine: - a helper plasmid ("pHelper" containing the Ad E2A, E4 and VA RNA helper genes as described in Crosson Sm et al., a map of which is shown in Figure 1A); - a packaging plasmid, the map of which is shown in Figure 1B, in which "AAV2 Cap" is the nucleotide sequence encoding the capsid polypeptide listed in Table 1 (column 2); -Transgene plasmids containing the rAAV genome of SEQ ID NO: 19 encoding GFP or the rAAV genome of SEQ ID NO: 20 encoding the bivalent anti-VEGF antibody Nb24 (see Figures 1C and 1D, respectively), and the elements were assembled using Gibson assembly methodology (NEBuilder HiFi DNA Assembly Master Mix, NEB, catalog number E2621).

[0128] Packaging plasmids encoding capsid polypeptides of SEQ ID NO:1, 18 or 22 were synthesized by Genscript Inc. (Nanjing, Jiangxi, China), and packaging plasmids encoding capsids of SEQ ID NO:2, 3, 4, 5 or 21 were constructed by starting from a packaging plasmid encoding SEQ ID NO:1 and inserting the nucleotide sequence from positions 1762 to 1797 of SEQ ID NO:14, 15, 16, 17 or 26 between positions 1761 and 1762 of SEQ ID NO:25 using Gibson assembly methodology (NEBuilder HiFi DNA Assembly Master Mix, NEB, catalog no. E2621).

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

[0130] The rAAVs were tested for titer by ddPCR and all were 10 13 The levels of viral genomes (vg) / mL were measured. In particular, ddPCR was performed using Bio-Rad's QXDx AutoDG ddPCR System and QXDx Universal Kit for AutoDG ddPCR System according to the manufacturer's instructions. The primers 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 amplification of Nb24: 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 testing of rAAV in retinal cells 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 a 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 at various MOIs (800, 4,000, 20,000, and 100,000) 4 hours after the cells were seeded. After 72 hours of incubation at 37° C. and 5% CO2, the culture medium was replaced with PBS (Thermo, Catalog No. 10010072), and then the transduced cells were detected for GFP protein under an automated fluorescence microscope (Agilent) with 485 / 20 excitation and 508 / 20 emission filter settings.

[0137] rAAV with modified AAV2 capsids showed improved transduction rates compared to rAAV with wild-type AAV2 capsids. As shown in Figure 2, cells transduced with AAV.LGP-GFP show much stronger fluorescent signals than cells transduced with AAV2-GFP. Notably, for AAV2-GFP, fluorescent signals can be observed at an MOI of at least 4,000, whereas for AAV.LGP-GFP, fluorescent signals can be observed at an MOI of 800; cells transduced with AAV.LGP-GFP at an MOI of 4,000 show comparable fluorescent signals to cells transduced with AAV2-GFP at an MOI of 100,000.

[0138] Example 3. In vitro testing of rAAV in retinal cells This example was performed to verify the expression of a polypeptide of interest contained in the rAAV prepared in Example 1, and enhanced expression of the polypeptide of interest was observed in retinal cells transduced with an rAAV containing a modified AAV capsid polypeptide compared to an rAAV containing a wild-type capsid 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 addition of rAAV, the culture medium was collected by pipetting and then detected for Nb24 expression in the cell culture medium by direct anti-VEGF ELISA.

[0140] Briefly, VEGF polypeptide (human VEGFA165A, R&D Systems, Cat. No. 293-VE / CF) was coated on ELISA plates (Corning™ 3690) overnight at 4° C., and after blocking with 3% BSA in PBST (Thermo, 37536), harvested cell culture medium containing bivalent Nb24 was added to the plate, which was then incubated for 1 hour (RT). Anti-Nb24 antibody (goat anti-Nb24 polyclonal, Novamb) and anti-goat HRP antibody (donkey anti-goat IgG HRP, Invitrogen, Cat. No. 34028) were added sequentially, with 1 hour incubation (RT) after each antibody addition. TMB solution (Thermo Scientific, Cat. No. 34028) was added for HRP color development, which was then stopped by adding stop buffer (sulfoacid, Beyotime, Cat. No. P0215). To calculate the bivalent Nb24 expression level in the cell culture medium, the absorbance OD450 was measured with a plate reader (SpectraMax i3x multimode microplate reader, Molecular Devices). The expression levels of Nb24 were calculated based on a standard curve prepared with recombinant bivalent Nb24 protein at a range of concentrations and are shown in Figure 3, and the ED50 values ​​of rAAV (MOI for 50% maximal expression) were calculated using Graphpad Prism and are shown in Table 2.

[0141] [Table 2]

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

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

[0144] Example 4. In vivo therapeutic efficacy of rAAV containing modified AAV capsid polypeptides This example was performed to verify transgene expression and therapeutic efficacy in a laser-induced choroidal neovascularization (CNV) non-human primate (NHP) model achieved by rAAV containing a 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 (20 monkeys with the lowest Nab levels). The selected monkeys were treated with laser for modeling (Pennesi et al., Animal models of age-related macular degeneration. Mol Aspects Med, 2012. 33(4): p. 487-509).

[0146] The bivalent Nb24-encoding rAAV (listed in Table 1) prepared in Example 1 in formulation buffer (dPBS+200 mM NaCl+0.005% PF68 (dPBS and PF68 were purchased from Thermo under catalog numbers 10010072 and 24040032, respectively)) was administered to monkey eyes as described in Table 3.

[0147] [Table 3]

[0148] This study was performed according to FIG. 4. Briefly, the tested article was intravitreally injected into monkeys on day 1, 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 (ocular tissues including retina, choroid, sclera, optic nerve, vitreous body, aqueous humor, ciliary body, iris, cornea and lens; and peripheral tissues including brain, heart, liver, kidney, lung, skeletal muscle and spleen) were collected separately. Monkey #310 received a second dose of AAV9-Nb24 on day 44 and its eye was further imaged on day 51.

[0149] Samples containing bivalent Nb24 from tissues were prepared by mixing 30 mg of tissue and 60 μL of lysis buffer (T-PER™ Tissue Protein Extraction Reagent, Thermo, Cat. No. 78510) with proteinase inhibitors (Pierce™ Protease and Phosphatase Inhibitor Mini Tablets, Thermo, Cat. No. A32959), homogenizing the mixture at 1,500 rpm for 2 min with 30 s intervals using a homogenizer (2010 Geno / Grinder®-Automated Tissue Homogenizer and Cell Lysing Device, Spex SampleProp), centrifuging at 12,000 rpm for 20 min, and collecting the supernatant. Total protein content in the samples was determined using a BCA kit (Thermo Fisher, Cat. No. 23235).

[0150] Nb24 levels in tissues and serum were detected by ELISA (see FIG. 5). Briefly, human VEGFA165A (R&D systems 293-VE / CF) was coated on ELISA plates overnight at 4° C. and blocked with blocking buffer (SuperBlock™ T20 (TBS) Blocking Buffer, Thermo Scientific, Cat. No. 37536), followed by 1 hour incubation (RT) of samples from tissues and serum with equal content of total protein added to the plate. Anti-Nb24 antibody (goat anti-Nb24 polyclonal, Novamb) and anti-goat HRP antibody (donkey anti-goat IgG HRP, Invitrogen, Cat. No. 34028) were added sequentially, with 1 hour incubation (RT) after each antibody addition. TMB solution (Thermo Scientific, Cat. No. 34028) was added for HRP color development, and then stopped by adding stop buffer (sulfoacid, Beyotime, Cat. No. P0215). The absorbance OD450 was measured to calculate the bivalent Nb24 expression level in the samples, as described in Example 3.

[0151] Expression of bivalent Nb24 in ocular tissues is shown in Figure 6, which indicates that administration of AAV.LGP-Nb24 achieved at least equivalent, and even higher, expression of bivalent Nb24 compared to the reference (AAV.7m8-Nb24) throughout 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] Fundus fluorescein angiography (FFA) was used as the primary in vivo endpoint for the assessment of fluorescein leakage of RNV (Li et al., 2018 and Cao et al., 2018). Slit lamp microscopy and color fundus photography were also performed to document the ocular response to 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 the laser-induced NHP CNV model, among which AAV2.LGP-Nb24 provided highly robust therapeutic efficacy. The reference rAAV (AAV.7m8-Nb24) caused a clear ocular inflammatory response (evidenced by severe fluorescein leakage in the anterior chamber) in three of four treated eyes (see FIG. 13), but not in monkeys treated with AAV.GKG-Nb24, AAV.LAE-Nb24, AAV.LGP-Nb24, or AAV.NSP-Nb24. AAV9-Nb24 induced extensive choroidal leakage 7-14 days after subretinal injection (see FIG. 16).

[0153] It can be seen that rAAV containing modified capsid polypeptides achieved superior ocular transduction, robust transgene expression (as evidenced by high levels of bivalent Nb24 in the eye and surrounding tissues signifying robust blockage of retinal neovascularization), safety (as evidenced by minimal peripheral Nb24 expression), and therapeutic efficacy (as evidenced by reduced leakage in the retina).

[0154] array Capsid AAV2 of SEQ ID NO:1 [ka] Capsid AAV2_GKG of SEQ ID NO:2 obtained by inserting SEQ ID NO:10 between positions 587 and 588 of SEQ ID NO:1 [ka] Capsid AAV2_LAE of SEQ ID NO:3 obtained by inserting SEQ ID NO:11 between positions 587 and 588 of SEQ ID NO:1 [ka] Capsid AAV2_LGP of SEQ ID NO:4 obtained by inserting SEQ ID NO:12 between positions 587 and 588 of SEQ ID NO:1 [ka] Capsid AAV2_NSP of SEQ ID NO:5 obtained by inserting SEQ ID NO:13 between positions 587 and 588 of SEQ ID NO:1 [ka] Inserted peptide of SEQ ID NO:6_GKG GKGPTTK Inserted peptide LAE of SEQ ID NO:7 LAEPSRP Inserted peptide of SEQ ID NO: 8_LGP LGPPSKP SEQ ID NO: 9 Inserted peptide_NSP NSPTGRN Inserted peptide of SEQ ID NO: 10_GKG full AAAGKGPTTKAA Inserted peptide of SEQ ID NO: 11_LAE full AAALAEPSRPAA Inserted peptide of SEQ ID NO: 12_LGP full ALALGPPSKPAA Inserted peptide of SEQ ID NO: 13_NSP full AAGNSPTGNAA Coding sequence of SEQ ID NO: 14_AAV2_GKG [ka] Coding sequence of SEQ ID NO: 15_AAV2_LAE [ka] Coding sequence of SEQ ID NO: 16_AAV2_LGP [ka] Coding sequence of SEQ ID NO: 17_AAV2_NSP [ka] AAV9 capsid of SEQ ID NO:18 [ka] rAAV genome encoding GFP of SEQ ID NO: 19 [ka] rAAV genome encoding the bivalent Nb24 construct of SEQ ID NO: 20 [ka] [ka] AAV2_7m8 amino acid sequence of SEQ ID NO: 21 [ka] AAV_RH10 amino acid sequence of SEQ ID NO: 22 [ka] Nucleotide sequence encoding AAV2 REP of SEQ ID NO:23 [ka] SEQ ID NO: 24 (Nb amino acid sequence) EVQLQESGGGLVQPGGSLRLSCTASGFTFDDPDVGWFRQAPGNECELVSTISKDGSTYYTDSVKGRFTISRDYAKNTVYLQMNSLRAEDTAVYYCAADSNPIAPIRTCLGWYNYWGQGTLVTVSS Coding sequence AAV2 capsid of SEQ ID NO:25 [ka] Coding sequence AAV2_7m8 of SEQ ID NO: 26 [ka] Coding sequence AAV_RH10 of SEQ ID NO: 27 [ka]

Claims

1. 1. A modified adeno-associated virus (AAV) capsid polypeptide, comprising an inserted peptide in loop IV compared to a parent AAV capsid polypeptide, said inserted peptide having the formula I: spacer 1-X1-X2-X3-X4-X5-X6-X7-spacer 2 (I) wherein: 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; X7 is selected from K, P and N; The spacers 1 and 2 each independently comprise one or more, preferably 1 to 10, more preferably 1 to 3 amino acids selected from A, L and G. A modified adeno-associated virus (AAV) capsid polypeptide comprising:

2. The inserted peptide has the formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) wherein: Spacer 1 consists of the amino acids Y1, Y2 and Y3, spacer 2 consists of the 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; X7 is selected from K, P and N; The modified AAV capsid polypeptide of claim 1, comprising:

3. 3. The modified AAV capsid polypeptide of claim 1 or 2, wherein 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).

4. 2. The modified AAV capsid polypeptide of claim 1, wherein 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).

5. 5. The modified AAV capsid polypeptide of any one of claims 1 to 4, wherein the parent AAV capsid polypeptide is an AAV2 capsid polypeptide comprising the amino acid sequence of SEQ ID NO:1, amino acids 138 to 735 of SEQ ID NO:1, or amino acids 203 to 735 of SEQ ID NO:1, or a variant thereof that is at least 90% identical to SEQ ID NO:1 over the full length, amino acids 138 to 735, or amino acids 203 to 735.

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

1.

7. 7. The modified AAV capsid polypeptide of any one of claims 1 to 6, comprising an amino acid sequence selected from SEQ ID NOs: 2, 3, 4 and 5, amino acids 138 to 747 of SEQ ID NO: 2, 3, 4 or 5, or amino acids 203 to 747 of SEQ ID NO: 2, 3, 4 or 5.

8. A polynucleotide encoding a modified AAV capsid polypeptide according to any one of claims 1 to 7.

9. A vector comprising the polynucleotide of claim 8.

10. A host cell comprising the polynucleotide of claim 8 or the vector of claim 9.

11. 11. A system or kit for packaging rAAV comprising the polynucleotide of claim 8, the vector of claim 9, or the host cell of claim 10.

12. 1. A recombinant adeno-associated virus (rAAV) comprising a modified AAV capsid polypeptide and a genome encoding a gene product, said modified AAV capsid polypeptide comprising an inserted peptide in loop IV compared to a parent AAV capsid polypeptide, said inserted peptide having the formula I: spacer 1-X1-X2-X3-X4-X5-X6-X7-spacer 2 (I) wherein: 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; X7 is selected from K, P and N; The spacers 1 and 2 each independently comprise one or more amino acids selected from A, L, and G. A recombinant adeno-associated virus (rAAV).

13. The inserted peptide has the formula II: Y1-Y2-Y3-X1-X2-X3-X4-X5-X6-X7-Y4-Y5 (II) wherein: Spacer 1 consists of the amino acids Y1, Y2 and Y3, spacer 2 consists of the 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; X7 is selected from K, P and N; The rAAV of claim 12, comprising:

14. The rAAV of claim 12 or 13, wherein 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).

15. 13. The rAAV of claim 12, wherein 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).

16. 16. The rAAV of any one of claims 12-15, 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 over the full length, amino acids 138-735, or amino acids 203-735.

17. The rAAV of any one of claims 12 to 16, wherein the peptide is inserted between positions corresponding to positions 587 and 588 of SEQ ID NO:

1.

18. 18. The rAAV of any one of claims 12-17, 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 NO: 2, 3, 4, or 5, or amino acids 203 to 747 of SEQ ID NO: 2, 3, 4, or 5.

19. A pharmaceutical composition comprising the rAAV of any one of claims 12 to 18.

20. A method for treating a retinal disease comprising administering an rAAV according to any one of claims 12 to 18 or a pharmaceutical composition according to claim 19 to the eye of a subject in need of such treatment.

21. 20. An rAAV according to any one of claims 12 to 18 or a pharmaceutical composition according to claim 19 for use in treating a retinal disease.

22. Use of an rAAV according to any one of claims 12 to 18 or a pharmaceutical composition according to claim 19 in the preparation of a medicament for treating a retinal disease.

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