Retinal cell-targeting adeno-associated virus variant kap and use thereof

Abstract

Disclosed are an adeno-associated virus (AAV) capsid protein variant having improved infectivity and transduction efficiency into target cells, and a use thereof. Specifically, disclosed are an AAV2 variant capsid protein selected via directed evolution for gene delivery to retinal cells, a recombinant AAV vector comprising same, and a use thereof as a gene delivery vehicle to retinal cells.

Description

Adeno-associated virus variant KAP targeting retinal cells and its uses

[0001] An adeno-associated virus (AAV) capsid protein variant with improved infectivity to target cells and transduction efficiency, and its use are disclosed. Specifically, an AAV2 variant capsid protein selected through induced evolution for gene delivery to retinal photoreceptor cells by intravitreal injection, a recombinant AAV vector containing the same, and its use as a gene delivery vehicle to retinal cells are disclosed.

[0002]

[0003] This research was conducted with support from the Samsung Future Technology Development Project (Project No. SRFC-MA2022-08).

[0004]

[0005] Gene therapy is a method of treating hereditary diseases by introducing normal genes into human cells to correct or compensate for conditions caused by genetic defects and abnormalities, and it has become a key means of treating various hereditary diseases. Adeno-associated viruses (AAVs) are widely used gene delivery vectors in the field of gene therapy because they can infect a wide range of tissues, are non-pathogenic, and have low immunogenicity.

[0006] Luxturna is the first AAV2-based gene therapy approved by the U.S. FDA that targets the retinal pigment epithelium (RPE) via subretinal injection. Since the subretinal space is located between the neural retina and the RPE, subretinal injection is known as a method that can effectively target photoreceptor cells and the RPE. However, subretinal injection requires a complex surgical approach under general or local anesthesia, which carries a risk of serious side effects such as retinal detachment, intraocular hemorrhage, and macular holes. Additionally, there is a limitation in that gene delivery to extensive retinal areas is difficult because gene delivery occurs only around the injection site.

[0007] Accordingly, there is a growing need for intravitreal injection, which enables more non-invasive and extensive gene delivery. Gene therapy via intravitreal injection requires vectors with higher tissue specificity and greater gene delivery and expression efficiency compared to conventional AAV vectors. To achieve this, AAV capsid engineering is being actively researched.

[0008] Directed evolution is a widely used high-throughput screening method for engineering improved biomolecules, a technique that mimics natural selection by repeating the processes of genetic mutation induction / introduction and selection. Directed evolution of AAV capsids is performed by constructing an AAV capsid library with diverse sequences by introducing mutations into the wild-type AAV capsid gene, and then obtaining new capsid variants by screening for those with desired characteristics from this library.

[0009] Through such AAV capsid engineering, recombinant AAV vectors with high tissue specificity and gene delivery efficiency have been developed and utilized (Korean Registered Patent 2234930). In particular, hereditary retinal diseases are often monogenetic diseases and are considered promising indications for gene therapy. Accordingly, AAV variants such as AAV2-7m8, AAV2.GL, and AAV2.NN have been developed (Science Translational Medicine, 5(189), 189ra76 and EMBO Molecular Medicine, 13(4), e13392). However, there is still a need for AAV variants with excellent transduction capabilities that can be delivered with high specificity to desired cells in the retina via intravitreal injection to induce gene expression.

[0010] Accordingly, the inventors completed the present invention by selecting an AAV2 variant with excellent gene transfer ability to photoreceptor cells via intravitreal injection from an AAV library constructed by directed evolution.

[0011]

[0012] The purpose is to provide an AAV2 variant capsid protein capable of gene delivery to photoreceptor cells via intravitreal injection.

[0013] In addition, the invention aims to provide a recombinant AAV2 vector containing an AAV2 variant capsid protein capable of gene transfer to photoreceptor cells via intravitreal injection.

[0014] In addition, the invention aims to provide a therapeutic use for ocular diseases of a recombinant AAV2 vector containing an AAV2 variant capsid protein capable of gene delivery to photoreceptor cells via intravitreal injection.

[0015]

[0016] One aspect of the present invention provides an AAV2 variant capsid protein comprising a peptide KAPPNNP inserted into the GH-loop of a wild-type AAV2 capsid protein.

[0017]

[0018] The AAV2 variant capsid protein of the present invention was obtained by selecting an AAV2 having tropism toward retinal cells, specifically photoreceptor cells, through intravitreal injection via engineering by directed evolution of the wild-type AAV2 capsid protein, and by elucidating its structure and gene delivery activity. The AAV variant capsid protein of the present invention can be used to deliver a target gene to photoreceptor cells through intravitreal injection. In order for the AAV2 variant to target photoreceptor cells through intravitreal injection, it must penetrate the inner limiting membrane (ILM) and the inner retina. Since the target cells, photoreceptor cells, are widely distributed in the outer retina of the entire retina, gene delivery across the entire retina must be possible to obtain a sufficient therapeutic effect.

[0019]

[0020] In one embodiment of the present invention, the peptide KAPPNNP may be inserted at amino acid 587 or a corresponding position of the wild-type AAV2 capsid protein of SEQ ID NO. 1.

[0021] The AAV2 variant capsid protein of the present invention includes the insertion of the peptide KAPPNNP between amino acids N587 and R588 of the wild-type AAV2 capsid protein of SEQ ID NO. 1, and was confirmed to deliver the target gene with high efficiency to retinal cells, including photoreceptor cells, ganglion cells, Müller cells, and bipolar cells by intravitreal injection compared to AAV2.7m8 (which has the insertion of the peptide "LGETTRP" between N587 and R588 of wild-type AAV2: SEQ ID NO. 3) and AAV2.NN (which has the insertion of the peptide "NNPTPSR" between N587 and R588 of wild-type AAV2: SEQ ID NO. 4), which are known to have the ability to deliver genes to wild-type AAV2 and retinal photoreceptor cells, and was named "AAV2.KAP" or "KAP variant".

[0022]

[0023] The wild-type AAV2 capsid protein comprises the amino acid sequence of SEQ ID NO. 1. The KAP variant according to the present invention comprises a GH-loop for the wild-type AAV2 capsid protein and a KAPPNNP inserted between N587 and R588 in the amino acid sequence of SEQ ID NO. 1.

[0024] In one embodiment of the present invention, the AAV2 variant capsid protein may be composed of an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with respect to SEQ ID NO. 2.

[0025]

[0026] As used herein, the term "AAV" is an abbreviation for adeno-associated virus and refers to the virus itself or its derivatives. Unless otherwise specified, AAV includes all its subtypes and naturally occurring and recombinant forms. AAV is a non-pathogenic parvovirus consisting of a 4.7 kb-long single-stranded DNA genome within a non-encapsulated icosahedral capsid. The genome contains three open translation frames (ORFs) adjacent to an inverted terminal repeat (ITR) that functions as a viral origin for replication and packaging signals: the rep ORF codes for four non-structural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and virion assembly; and the cap ORF codes for three structural proteins (VP1-3) that assemble to form a 60-mer viral capsid; The ORF, which exists as an alternative reading frame within the cap gene, localizes the AAV capsid protein to the nucleolus and codes for the assembly-activating protein (AAP), a viral protein that functions during the capsid assembly process.

[0027] Genome sequences of various serotypes of AAV, sequences of natural terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. These sequences can be found in the literature or in public databases such as GenBank. For example, the genome sequence of AAV2 is registered as GenBank Accession NC_001401.

[0028] The term "tropism" as used herein refers to the preferential targeting of cells of a specific host species or specific cell types within a host species by a virus (e.g., AAV), and is used interchangeably with "tropism." For example, a virus capable of infecting retinal cells with high specificity has high tropism toward retinal cells compared to non-retinal cells. Since the capsid protein of an AAV virus determines infectivity toward target cells or tissues, tropism or tropism is used as a selection pressure to develop AAV variants capable of delivering genes to target cells or tissues with high specificity and efficiency through capsid engineering via directed evolution.

[0029] The term “retinal cell” as used herein means all types of cells present in the retina, including, but not limited to, retinal ganglion cells, bipolar cells, photoreceptor cells, Muller glia cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal blood vessel cells, and retinal pigment epithelium (RPE).

[0030] As used herein, the term "gene delivery capability" refers to the ability or activity of an AAV vector to specifically deliver and express genes in target cells or tissues, and is used interchangeably with "gene delivery efficacy," "infectivity," or "transduction efficacy."

[0031] As used herein, the term "corresponding location" refers to a location that corresponds to a specific location through sequence alignment, or a location determined to perform the same or substantially the same function.

[0032] As used herein, the term "sequence identity" refers to the ratio of the type of amino acid or base at a specific position matching between polypeptides or polynucleotides being compared, and may be used interchangeably with "homology" for sequences. For example, the calculation of the homology (%) of two polypeptide sequences involves optimally aligning the two sequences for comparison purposes and comparing the amino acids at corresponding positions; if an amino acid identical to that at the corresponding position of the second sequence exists at the position of the first sequence, the two sequences are determined to be identical at that position. Sequence homology can be determined using software such as BLASTP, BLASTN, and FASTA.

[0033]

[0034] In one embodiment of the present invention, the AAV2 variant capsid protein can impart increased infectivity to retinal cells compared to the wild-type AAV2 capsid protein.

[0035] In one embodiment of the present invention, the retinal cells may be selected from the group consisting of retinal ganglion cells, bipolar cells, photoreceptor cells, Müller cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal blood vessel cells, and retinal pigment epithelial cells.

[0036] In one embodiment of the present invention, the AAV2 variant capsid protein can impart increased infectivity to photoreceptor cells compared to the wild-type AAV2 capsid protein.

[0037] In one embodiment of the present invention, the AAV2 variant capsid protein can impart increased infectivity to photoreceptor cells compared to the wild-type AAV2 capsid protein by intravitreal injection.

[0038]

[0039] Another aspect of the present invention provides an isolated nucleic acid encoding an AAV variant capsid protein.

[0040] In one embodiment of the present invention, the isolated nucleic acid may comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 2 or a nucleotide sequence having 95% or more sequence homology with respect to it.

[0041]

[0042] Another aspect of the present invention provides a host cell comprising isolated nucleic acid encoding an AAV2 variant capsid protein comprising a peptide KAPPNNP inserted at amino acid 587 or a corresponding position of the wild-type AAV2 capsid protein of SEQ ID NO. 1.

[0043] The above host cell may be used to generate a recombinant AAV virion containing an AAV2 variant capsid protein. In this case, the host cell is referred to as a "packaging cell." The host cell may be selected from various cells including, but not limited to, HEK293 cells, HEK293T cells, HeLa cells, Sf9 cells, 293A cells, 293H cells, etc.

[0044] In one embodiment of the present invention, the isolated nucleic acid is stably or transiently introduced into a host cell using a method including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc.

[0045]

[0046] Another aspect of the present invention is an AAV2 variant capsid protein comprising a peptide KAPPNNP inserted at amino acid 587 or a corresponding position of the wild-type AAV2 capsid protein of SEQ ID NO. 1, and

[0047] A recombinant AAV vector is provided, comprising a heterogeneous nucleic acid containing a sequence encoding a target product.

[0048]

[0049] As used herein, the term "recombinant AAV vector" refers to an AAV vector containing a polynucleotide sequence that is not derived from AAV, that is, a polynucleotide sequence heterogeneous to AAV, and is used interchangeably with "rAAV". Generally, in a recombinant AAV vector, the heterogeneous polynucleotide sequence is a nucleic acid sequence encoding the target product to be delivered to the target cell.

[0050] As used herein, the term "heterogeneity" refers to a source genetically distinct from the AAV2 vector, for example, nucleic acids originating from a different species.

[0051]

[0052] In one embodiment of the present invention, the recombinant AAV vector may exhibit increased transduction into retinal tissue / cells compared to an AAV vector (AAV2 vector) containing wild-type AAV2 capsid protein.

[0053] In one embodiment of the present invention, the recombinant AAV vector may exhibit increased transduction into retinal tissue / cells through intravitreal injection compared to an AAV vector (AAV2 vector) containing a wild-type AAV2 capsid protein.

[0054] A recombinant AAV vector according to one embodiment of the present invention can be used to produce a recombinant AAV virion. When introduced into a suitable host cell, the recombinant AAV vector can provide for the production of the corresponding recombinant AAV virion.

[0055] In one embodiment of the present invention, the target product may be a polypeptide or a nucleic acid. The target product refers to a product capable of producing a therapeutic effect on the target disease by supplementing or enhancing a function that is deficient or lacking in the target, or by reducing or eliminating a defective function. For example, the target product may be a gene product directly or indirectly related to an ocular disease such as a retinal disease, or one that alters the activity thereof, or one that improves the function of retinal cells, and may be, for example, PEDF (pigment epithelium-derived factor) or rhodopsin.

[0056] In one embodiment of the present invention, the target product may be a therapeutic nucleic acid and may target any gene associated with an ocular disease such as color blindness, age-related macular degeneration (such as wet AMD or dry AMD), cataract, panchoroidal atrophy, glaucoma, optic neuropathy, Marfan syndrome, myopia, nodular choroidal vasculopathy, retinitis pigmentosa, Stargardt disease, Usher syndrome, Leber congenital glaucoma, Leber hereditary optic neuropathy, uveal melanoma or X-linked retinal detachment, or any hereditary disease affecting ocular tissue. Target genes are, for example, VEGFA, VEGFB, ANGPT2, FLT1, KDR, PDGFB, CFH, ARMS2, HTRA1, TIMP3, C3, C2, CFB, SERPINF1, ANGPT1, CFI, CD59, ABCA1, ABCA4, RHO, RPGR, PRPF3, PRPF31, PRPF8, PDE6A, PDE6B, PDE6G, CNGB1, CNGA1, AIPL1, CRX, CRB1, CEP290, RS1, USH2A, MYO7A, USH1C, CLRN1, BBS1, BBS10, BEST1, PROM1, ELOVL4, PRPH2, RDH12, RPE65, CDH3, PCDH15, GUCY2D, GUCA1A, TULP1, LRAT, GNAT2, CNGB3, CNGA3, PDE6C, PDE6H, ATF6, GRM6, GPR179, TRPM1, NYX, CABP4, NR2E3, NRL, IMPDH1, IMPDH2, SNRNP200, HK1, GNAT1, KCNV2, RAX2, CHM, MERTK, EYS, NMNAT1, RP1, RP2, LCA5, RD3, RLBP1, MT-ND4, MT-ND1, MT-ND6, NDP, LRP5, TSPAN12, FZD4, CAPN5, TEK, CYP1B1, CYP4V2, MYOC, OPTN, OPA1, IL10, IL1RN, TNFRSF1A, HGF, PEDF, sFLT1, sTNFR, GNAQ, GNA11,BAP1, SF3B1, EIF1AX, TP53, TNFSF10, DDEF1, CDKN2A, p14ARF, IFNB1, HSV-TK, BDNF, GDNF, CNTF, NTF3, NGF, WDR87, APE1, MIP, GJA3, GJA8, CRYAA, CRYBB2, PRX, POLR3B, XRCC1, ZNF350, EPHA2, REP1, CALM2, MPP7, LOX1, CAV1, CAV2, PITX2, FOXC1, PAX6, LTBP2, FBN1, TGFBR2, MTHFR, MTR, MTRR, MET, UMODL1, It may be a gene encoding a protein selected from the group consisting of MMP1, MMP2, CBS, IGF1, UHRF1BP1L, PTPRR, PPFIA2, P4HA2, SERPING1, FGD6, ABCG1, LOC387715, CETP, PTEN, and HERC2 / OCA2, but is not limited thereto.

[0057] In one embodiment of the present invention, the target product may be a therapeutic protein or polypeptide, for example, VEGFA, VEGFB, ANGPT2, FLT1, KDR, PDGFB, CFH, ARMS2, HTRA1, TIMP3, C3, C2, CFB, SERPINF1, ANGPT1, CFI, CD59, ABCA1, ABCA4, RHO, RPGR, PRPF3, PRPF31, PRPF8, PDE6A, PDE6B, PDE6G, CNGB1, CNGA1, AIPL1, CRX, CRB1, CEP290, RS1, USH2A, MYO7A, USH1C, CLRN1, BBS1, BBS10, BEST1, PROM1, ELOVL4, PRPH2, RDH12, RPE65, CDH3, PCDH15, GUCY2D, GUCA1A, TULP1, LRAT, GNAT2, CNGB3, CNGA3, PDE6C, PDE6H, ATF6, GRM6, GPR179, TRPM1, NYX, CABP4, NR2E3, NRL, IMPDH1, IMPDH2, SNRNP200, HK1, GNAT1, KCNV2, RAX2, CHM, MERTK, EYS, NMNAT1, RP1, RP2, LCA5, RD3, RLBP1, MT-ND4, MT-ND1, MT-ND6, NDP, LRP5, TSPAN12, FZD4, CAPN5, TEK, CYP1B1, CYP4V2, MYOC, OPTN, OPA1, IL10, IL1RN, TNFRSF1A, HGF, PEDF, sFLT1, sTNFR, GNAQ, GNA11, BAP1, SF3B1, EIF1AX, TP53, TNFSF10, DDEF1, CDKN2A, p14ARF, IFNB1, HSV-TK, BDNF, GDNF, CNTF, NTF3, NGF, TAFIA, WDR87, APE1, MIP, GJA3, GJA8, CRYAA, CRYBB2, PRX, POLR3B, XRCC1, ZNF350, EPHA2,It may be selected from the group consisting of REP1, CALM2, MPP7, LOX1, CAV1, CAV2, PITX2, FOXC1, PAX6, LTBP2, FBN1, TGFBR2, MTHFR, MTR, MTRR, MET, UMODL1, MMP1, MMP2, CBS, IGF1, UHRF1BP1L, PTPRR, PPFIA2, P4HA2, SERPING1, FGD6, ABCG1, LOC387715, CETP, PTEN, and HERC2 / OCA2, but is not limited thereto.

[0058]

[0059] Another aspect of the present invention provides the use of the aforementioned recombinant AAV vector in the treatment of ocular diseases.

[0060] In one embodiment of the present invention, a pharmaceutical composition for treating an eye disease is provided, comprising the aforementioned recombinant AAV vector.

[0061] In one embodiment of the present invention, a method for treating an eye disease is provided, comprising the step of administering a therapeutically effective amount of the aforementioned recombinant AAV vector to a subject who requires treatment for an eye disease.

[0062] As used here, the term "ocular disease" refers to a disease related to the eye or a part or specific area of ​​the eye, such as the retina or choroid.

[0063] In one embodiment of the present invention, the eye disease may be a retinal disease, a choroidal disease, or a retinal / choroidal disease.

[0064] In one embodiment of the present invention, the ocular disease may be selected from retinal vascular disease, optic neuropathy, hereditary retinal disease, hereditary choroidal disease, and intraocular tumor.

[0065] In one embodiment of the present invention, the ocular disease may be selected from retinal-vitreous-choroidal related disease, retinal vascular disease, macular disease, hereditary retinal disease, hereditary vitreous disease, hereditary choroidal disease, intraocular inflammatory disease, intraocular tumor, glaucoma, and optic neuropathy.

[0066] In one embodiment of the present invention, ocular diseases include age-related macular degeneration, diabetic retinopathy, retinal vascular occlusion such as retinal artery / vein occlusion, and macular telangiectasia; optic neuropathy including glaucoma; Hereditary retinal / choroidal diseases including uveitis, Leber congenital amaurosis, retinitis pigmentosa, Usher syndrome, Stargardt's Disease, Best's Disease, X-linked retinoschisis, congenital stationary night blindness, choroideremia, achromatopsia, cone dystrophy, gyrate atrophy, and Bardet-Biedl syndrome; intraocular tumors such as uveal melanoma, intraocular lymphoma, and retinoblastoma, but are not limited thereto.

[0067] In one embodiment of the present invention, the ocular disease may be selected from macular degeneration, diabetic retinopathy, retinal vascular occlusion, macular telangiectasia, retinopathy of prematurity, myopic degeneration, retinitis pigmentosa, Leber congenital amaurosis, Best's disease, Stargardt's disease, congenital non-progressive night blindness, X-linked retinal detachment, Vietti's crystal retinopathy, total color blindness, cone cell dystrophy, cone-rod cell dystrophy, macular dystrophy, Usher syndrome, Bardett-Beadle syndrome and other syndromic retinitis pigmentosa, panchoroidal atrophy, centrochoroidal dystrophy, cerebral choroidal atrophy, glaucoma, other optic neuropathy, uveitis, uveal melanoma, intraocular lymphoma, retinoblastoma, retinal detachment, and other retinal damage.

[0068]

[0069] In one embodiment of the present invention, the recombinant AAV vector may be administered via any suitable route of administration for delivery to the retina, such as intravitreal injection, subretinal injection, ocular injection by choroidal injection, or periocular, intravenous, arterial, or nasal administration, but not limited thereto.

[0070] In one embodiment of the present invention, the recombinant AAV vector may be administered by intravitreal injection. When the recombinant AAV vector of the present invention is administered via intravitreal injection, it can move through the vitreous humor, pass through the ILM, and move through the retinal layer more efficiently than an AAV vector containing wild-type AAV2 capsid protein.

[0071] In one embodiment of the present invention, the recombinant AAV vector may be administered by intraocular injection.

[0072] The recombinant AAV vector according to the present invention has an increased ability to deliver and express heterogeneous nucleic acids across the ILM to retinal cells compared to an AAV2 vector containing a wild-type capsid protein when administered via intravitreal injection.

[0073] The recombinant AAV vector according to the present invention can cross the ILM and also reach photoreceptor cells by traversing a cell layer including Müller cells, bipolar cells, etc. For example, when administered via intravitreal injection, the recombinant AAV vector according to the present invention can cross the ILM and also reach photoreceptor cells by traversing a cell layer including Müller cells, bipolar cells, etc.

[0074] In some embodiments, the recombinant AAV vector specifically infects retinal cells, and in particular, can specifically infect photoreceptor cells.

[0075]

[0076] As used herein, the term "therapeutic effective dose" refers to an amount sufficient to alleviate (e.g., relieve, reduce, or mitigate) at least one of the symptoms associated with a diseased state. For example, the therapeutic effective dose for in vivo injection, i.e., direct injection into the retina, is approximately 10 6 to about 10 15 , for example, about 10 8 to 10 12 It may be within the range of GC (genome copy). For in vitro transduction, the effective amount of rAAV virion delivered to cells is approximately 10 8 to about 10 13 The therapeutic effective dose of the recombinant AAV vector according to the present invention can be easily determined by a person skilled in the art by taking into account the target disease, age, gender, height, and severity of the disease to be treated.

[0077] In one embodiment of the present invention, a recombinant viral vector is administered in an amount sufficient to induce infection (or transduction) and expression of a heterogeneous nucleic acid sequence in the retinal cells of the subject.

[0078] In one embodiment of the present invention, the retinal cells may be selected from the group consisting of photoreceptor cells, retinal ganglion cells, bipolar cells, Müller cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal blood vessel cells, and retinal pigment epithelial (RPE) cells.

[0079] In one embodiment of the present invention, the retinal cells are photoreceptor cells, e.g., cone cells and / or rod cells.

[0080] In one embodiment of the present invention, the retinal cell is a retinal ganglion cell (RGC).

[0081] In one embodiment of the present invention, the retinal cell is an RPE cell.

[0082] In one embodiment of the present invention, the pharmaceutical composition may be administered one or more times over periods of various intervals, for example, daily, weekly, monthly, or annually, to achieve a desired level of gene expression.

[0083] In one embodiment of the present invention, the pharmaceutical composition may include a pharmaceutically acceptable carrier, excipient, or additive.

[0084]

[0085] Another aspect of the present invention provides an in vitro method for delivering a heterogeneous nucleic acid encoding a target product to a retinal cell, comprising the step of contacting the aforementioned recombinant AAV vector with the retinal cell.

[0086] According to the present invention, delivering heterogeneous nucleic acids to retinal cells can be used for the treatment of retinal diseases.

[0087] In a specific embodiment of the present invention, the target product and retinal cells are as described above.

[0088]

[0089] The KAP variant according to the present invention has been confirmed to have excellent gene delivery efficiency by passing through the ILM via intravitreal injection, delivering target genes to retinal cells including photoreceptor cells, inducing strong gene expression, providing increased gene expression and lateral spreading compared to wild-type AAV2, and exhibiting increased photoreceptor-specific gene delivery and expression compared to wild-type AAV2 in human retinal organoids. Therefore, the KAP variant according to the present invention can be usefully employed in gene therapy for the treatment of ocular diseases.

[0090]

[0091] Figure 1 shows a schematic diagram of the process of selecting an AAV2 variant capsid protein according to one embodiment of the present invention by directed evolution and the NGS results. A heptmeric peptide insertion AAV library (primary library) was constructed by inserting a heptmeric peptide of a random sequence at position 587 of a wild-type AAV2 capsid protein (SEQ No. 1), and AAV variants having tropism toward photoreceptor cells were selected by screening human retinal organoids (CRX-tdTomato retinal organoid) and mouse (NRL-EGFP) retinas. After that, a cap gene was obtained from a variant selected from human retinal organoids (O) and mouse photoreceptor cells (M), and a primary screening was performed by NGS analysis and selection. This was then repackaged to construct a photoreceptor-directed AAV library (secondary library). The secondary libraries (O and M) selected from human retinal organoids and mouse retinas, respectively, were subjected to secondary screening of such transduction, NGS analysis, and selection in human retinal organoids and mouse retinas, respectively, to select AAV2 variant capsid proteins that showed improved transduction into photoreceptor cells compared to AAVs with wild-type AAV2 capsid.

[0092] Figure 2 shows an image of the vp3 structure of AAV2.KAP containing a KAP variant according to one embodiment of the present invention, predicted by ColabFold. The part indicated by the dotted circle represents the inserted heptameric peptide.

[0093] Figures 3a and 3b show the gene delivery ability of AAV2.KAP containing a KAP variant according to one embodiment of the present invention verified in human retinal organoids. When recombinant AAV2.KAP (SEQ No. 2), wild-type AAV2 (SEQ No. 2), improved AAV2.7m8 (SEQ No. 3), and AAV2.NN (SEQ No. 4), loaded with the GFP (green fluorescent protein) gene as the target gene, were transduced into human retinal organoids (CRX-tdTomato), it was confirmed that AAV2.KAP exhibited superior gene expression efficiency throughout the retinal organoid compared to wild-type AAV2 and improved AAV2.7m8 and AAV2.NN. Figure 3a is a low-magnification image, and Figure 4b is a high-magnification image.

[0094] Figures 4a and 4b show the results of a quantitative analysis of the gene transfer efficiency of AAV2.KAP according to one embodiment of the present invention confirmed in human retinal organoids. tdTomato fluorescence labels photoreceptor cells, and GFP fluorescence labels the cells to which the gene was transferred. In Figure 4b, the gene transfer efficiency for all cells (Total) was analyzed as the ratio of GFP-positive cells to all cells, the gene transfer efficiency to photoreceptor cells was analyzed as the ratio of GFP-positive cells to tdTomato-positive cells, and the gene transfer efficiency to non-photoreceptor cells was analyzed as the ratio of GFP-positive cells to tdTomato-negative cells. AAV2.KAP showed significantly higher gene transfer efficiency than wild-type AAV2 and improved types AAV2.7m8 and AAV2.NN in the retinal organoid total cells, photoreceptor cells, and non-photoreceptor cells, respectively, and this was statistically significant.

[0095] Figure 5 shows the results of immunofluorescence staining indicating the gene transfer efficiency of recombinant AAV2 vectors loaded with a gene encoding GFP as a target gene on AAV2.KAP, wild-type AAV2, improved AAV2.7m8, and AAV2.NN according to one embodiment of the present invention. Antibodies labeling photoreceptor cells and Müller cells (CRX and Recoverin antibodies: photoreceptor cells, CARLBP and SOX9: Müller cells) and antibodies against GFP fluorescence were used.

[0096] Figure 6 shows the results of verifying the in vivo gene delivery efficiency of AAV2.KAP according to one embodiment of the present invention. It shows the expression patterns of the target gene observed after intravitreal administration of AAV2.KAP loaded with the tdTomato gene as the target gene, wild-type AAV2, improved AAV2.7m8, and AAV2.NN as controls to reporter mice (Nrl-EGFP) labeled with EGFP fluorescence in rod cells. In the figure, RGC refers to retinal ganglion cells, INL refers to the inner nuclear layer, ONL refers to the outer nuclear layer, and OS refers to the outer segment. While wild-type AAV2 was confirmed to infect RGC and some INL, it was confirmed that the tdTomato gene delivered by AAV2.KAP was significantly high in expression not only in RGC and INL but also in the ONL and OS regions, which are the nuclear layers of photoreceptor cells.

[0097] Figure 7 shows the results of a quantitative analysis of the in vivo gene transfer efficiency of AAV2.KAP according to one embodiment of the present invention. It shows the expression patterns of the target gene confirmed by FACS analysis after intravitreal administration of AAV2.KAP loaded with the tdTomato gene as the target gene, and wild-type AAV2, improved AAV2.7m8, and AAV2.NN as controls, respectively, into mice (Nrl-EGFP). EGFP fluorescence labels rod cells, and tdTomato fluorescence labels the cells to which the gene was transferred. The gene transfer efficiency to all retinal cells (Total) was measured as the ratio of tdTomato-positive cells to the total cells, the gene transfer efficiency to rod cells was measured as the ratio of tdTomato-positive cells to EGFP-positive cells, and the gene transfer efficiency to retinal cells other than rods was measured as the ratio of tdTomato-positive cells to EGFP-negative cells. AAV2.KAP demonstrated significantly higher gene delivery efficiency than wild-type AAV2 and improved types AAV2.7m8 and AAV2.NN in whole cells, rod cells, and non-rod cells, respectively, and this was statistically significant.

[0098] FIG. 8 shows the gene transfer efficiency of AAV2 variants to retinal cells confirmed by immunofluorescence staining after intravitreal injection of a recombinant AAV2 vector loaded with GFP as the target gene into a wild-type mouse (C57BL / 6J) according to one embodiment of the present invention, a wild-type AAV2, a control, an improved AAV2.7m8, and an AAV2.NN. Immunofluorescence staining performed using antibodies labeling photoreceptor cells (rod and cone cells), ganglion cells, Müller cells, and bipolar cells, and antibodies against GFP fluorescence delivered by the recombinant AAV2 vector, shows that AAV2.KAP exhibits high gene transfer efficiency to photoreceptor cells, ganglion cells, Müller cells, and bipolar cells compared to wild-type AAV2, and has higher gene transfer efficiency to photoreceptor cells than the improved AAV2.7m8 and AAV2.NN.

[0099]

[0100] The present disclosure will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present disclosure is not limited to the embodiments and may be implemented in various forms. The embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined by the claims.

[0101]

[0102] Example 1. Selection of KAP variants

[0103] Wild-type AAV2 is difficult to deliver genes to photoreceptor cells via intravitreal injection. Therefore, AAV2 capsid engineering was performed by directed evolution to select AAV2 capsid variants capable of delivering and expressing target genes in photoreceptor cells via intravitreal injection. A schematic diagram of the directed evolution method is shown in Figure 1.

[0104]

[0105] In vitro and in vivo directed evolution techniques are being utilized to develop AAV variants that are superior to existing AAV-based gene delivery vectors. Directed evolution techniques are known in the art, for example, as described in PCT Publication WO 2014 / 194132. Directed evolution is a capsid engineering method that mimics natural evolution through repetitive genetic diversification and selection processes, enabling the accumulation of beneficial mutations that progressively improve the function of biomolecules such as AAV-based virions. In this approach, various variants are introduced using error-prone PCR to introduce random point mutations into wild-type AAVcap genes, DNA shuffling to generate random chimeras, random peptide insertion, transposon-mediated mutagenesis, etc., and AAV particles are packaged to create a library of AAV variants, and selective pressure is applied to isolate unique variants with superior phenotypes capable of overcoming gene delivery barriers.

[0106]

[0107] In this embodiment, screening was performed on retinas (in vitro human-derived retinal organoids and in vivo mouse retinas) using a heptmeric peptide insertion library in which a random heptmeric peptide was inserted at position 587 of the wild-type AAV2 capsid sequence (SEQ No. 1). The insertion site of the heptmeric peptide is known to be involved in the interaction with the external receptor of AAV, and thus may be involved in cell specificity and gene delivery efficiency.

[0108]

[0109] 1.1. Preparation of Human Retinal Organoids In Vitro

[0110] Human retinal organoids to be used to evaluate the gene delivery efficacy of AAV2 variants to photoreceptor cells were prepared according to the description in Wahlin, KJ et al. Photoreceptor Outer Segment-like Structures in Long-Term 3D Retinas from Human Pluripotent Stem Cells. Sci Rep 7,766 (2017). Specifically, human retinal organoids were prepared using human embryonic stem cells that specifically express tdTomato fluorescent protein in photoreceptor cells. Human embryonic stem cells expressing tdTomato fluorescent protein specifically in photoreceptor cells were cultured in mTesR1 basal medium (Stem Cell Technologies, Vancouver, Canada) on plates coated with Matrigel (Corning, NY, USA). Before inducing differentiation into retinal organoids, the cultured stem cells were cultured for 4-5 days. After dissociating the cultured stem cells using Accutase (BD Biosciences, CA, USA), they were aggregated in low-cell adhesion 96-well plates (Corning) with U-shaped bottoms at a density of 3,000 cells per well. Subsequently, human retinal organoids were obtained by differentiating and culturing for more than 30 weeks. All cultures for the preparation of human retinal organoids were maintained at 37°C in a humidified environment with 5% carbon dioxide.

[0111]

[0112] 1.2. In Vitro and In Vivo Screening

[0113] 5x10 heptmeric peptide insertion libraries into human retinal organoids derived from reporter embryonic stem cells labeled with tdTomato fluorescence in photoreceptor cells prepared in 1.1 10After transduction with GC (Genome Copy) and 3 weeks later, variants that are preferentially delivered to photoreceptor cells were separated by FACS using tdTomato fluorescence and screened in vitro.

[0114] In addition, 2x10 heptmeric peptide insertion libraries were introduced into EGFP-fluorescence-labeled mice (B6.Cg-Tg(Nrl-EGFP)1Asw / J) in rod cells. 10 In vivo screening was performed to identify variants capable of infecting photoreceptor cells in the mouse retina via intravitreal injection by FACS isolation with EGFP fluorescence three weeks after intravitreal injection with GC.

[0115]

[0116] For Fluorescence-activated cell sorting (FACS), human retinal organoids and mouse retinas were dissociated into single cells using the Papain Dissociation System (Worthington Biochemical Corporation, NJ, USA). FACS was performed using the BD FACSymphony S6 Cell Sorter (BD Biosciences, CA, USA). For human retinal organoids, tdTomato-positive cells and tdTomato-negative cells were separated, and for mouse retinas, EGFP-positive cells and EGFP-negative cells were separated.

[0117]

[0118] After recovering the isolated cells by centrifugation, DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, 51306). PCR was performed to amplify the 229 bp containing the inserted heptameric peptide from the extracted DNA, and the target DNA was obtained as a PCR product using the QIAquick Gel Extraction Kit (QIAGEN, 51306). Next Generation Sequencing (NGS) analysis of the PCR product was performed using the MiSeq Reagent Kits v2 (ILLUMINA, MS-102-2003) according to the method provided by the manufacturer.

[0119]

[0120] A secondary library (organoid-secondary library (O) / mouse-secondary library (M)) was constructed using a pool of variants selected from human retinal organoids / mouse photoreceptor cells through primary screening, and secondary screening was performed. The organoid-secondary library and the mouse-secondary library were transduced into photoreceptor-fluorescent human retinal organoids (O), respectively, and injected intravitreally into rod-fluorescent mice (M). After FACS isolation using tdTomato / EGFP, the samples were analyzed via NGS. The 'KAPPNNP' variant (hereinafter AAV2.KAP), which was detected in common across a total of four samples (OO, OM, MM, and MO) and identified as having the highest copy number within each sample, was selected. This variant was named the "KAP variant."

[0121]

[0122] The amino acid sequence of the capsid protein of AAV2.KAP (SEQ No. 2), in which the KAP variant (indicated in red) is inserted at amino acid 587 of the AAV2 wild-type capsid protein (SEQ No. 1), is as follows:

[0123]

[0124]

[0125] In addition, the vp3 structure image of AAV2.KAP containing the KAP variant was confirmed using ColabFold (Nature Methods, 19(6), 679-682). Figure 2 shows the vp3 structure image of AAV2.KAP containing the ADN variant according to one embodiment of the present invention, predicted by ColabFold.

[0126]

[0127] Example 2. In vitro verification of AAV2.KAP variants: Human retinal organoids

[0128] To verify the ability of the AAV2.KAP variant selected in Example 1 to be delivered and expressed into photoreceptor cells, the expression and delivery of the target gene were confirmed in human retinal organoids.

[0129]

[0130] 2.1. Confirmation of Gene Transfer and Expression by Fluorescence Microscopy

[0131] Recombinant vectors AAV2.KAP-EGFP, wild-type AAV2-EGFP, positive controls AAV2.NN-EGFP, and AAV2.7m8-EGFP (see Science Translational Medicine, 5(189), 189ra76 and EMBO Molecular Medicine, 13(4), e13392) loaded with the gene encoding EGFP were applied to photoreceptor reporter (CRX-tdTomato) retinal organoids that specifically express tdTomato fluorescence as described in Example 1, 5x10 10 Gene transfer was confirmed after transduction with GC.

[0132]

[0133] In human retinal organoids, photoreceptor cells, which are the target cells of AAV2.KAP, are located at the outermost layer. When a gene expressing EGFP was delivered, it was confirmed that, as is known, wild-type AAV2 could not penetrate the retina layer and thus could not infect the interior. Furthermore, in human retinal organoids treated with AAV2.KAP, the region appearing yellow due to the merging of photoreceptor-specific tdTomato fluorescent protein and EGFP fluorescent protein was wider than in human retinal organoids treated with AAV2, AAV2.7m8, and AAV2.NN. Therefore, AAV2.KAP demonstrated significantly higher EGFP gene delivery efficiency throughout the human retinal organoid compared to wild-type AAV2 and the improved versions AAV2.7m8 and AAV2.NN. The results are illustrated in Figures 3a and 3b.

[0134] The AAV2.KAP variant was confirmed to have superior gene delivery to human photoreceptor cells compared to wild-type AAV2, AAV2.7m8, and AAV2.NN.

[0135]

[0136] 2.2. Quantitative Analysis of Gene Transfer Efficiency by FACS

[0137] The gene delivery and expression efficiency of AAV2.KAP in human retinal organoids were quantitatively analyzed.

[0138]

[0139] Specifically, AAV2.KAP loaded with a gene encoding EGFP as the target gene and control wild-type AAV2, improved AAV2.7m8, and AAV2.NN were loaded into human retinal organoids that specifically express tdTomato fluorescence in photoreceptor cells, prepared as described in 1.1., 5x10 10Human retinal organoids were transfected with GC, and FACS analysis was performed after 3 weeks. Human retinal organoids were dissociated using the Papain Dissociation System (Worthington Biochemical Corporation, NJ, USA). FACS was performed using BD FACS Aria II (BD Biosciences, CA, USA), and analysis was performed using FlowJo software (Tree Star, OR, USA).

[0140]

[0141] tdTomato fluorescence labels photoreceptor cells, and GFP fluorescence labels cells to which the target gene has been delivered. The gene delivery efficiency to all retinal cells was analyzed as the ratio of GFP-positive cells to total cells, the gene delivery efficiency to photoreceptor cells was analyzed as the ratio of GFP-positive cells to tdTomato-positive cells, and the gene delivery efficiency to retinal cells other than photoreceptor cells was analyzed as the ratio of GFP-positive cells to tdTomato-negative cells.

[0142] According to FACS analysis, the proportion of GFP-positive cells among total human retinal organoid cells was 79.22 ± 1.11% for AAV2.KAP, which was significantly higher and statistically significant than that of the wild-type AAV2 (10.19 ± 1.09%), improved AAV2.7m8 (39.08 ± 4.26%), and AAV2.NN (63.76 ± 3.05%) used as controls. Additionally, the proportion of GFP-positive cells among photoreceptor cells (tdTomato+) was 77.69 ± 4.67% for AAV2.KAP, which was significantly higher and statistically significant than that of AAV2 (14.31 ± 1.84%) and AAV2.7m8 (46.01 ± 2.37%). Figures 4a and 4b show representative result images and quantitative analysis results of the FACS analysis, respectively.

[0143]

[0144] 2.3. Detection of Gene Expression by Immunofluorescence Staining

[0145] To confirm gene transfer to photoreceptor cells and Muller glia cells, immunofluorescence staining was performed using antibodies labeling these cells and antibodies against GFP delivered to AAV2.

[0146]

[0147] Human retinal organoids transfected with AAV2.KAP-GFP and the control wild-type AAV2-GFP, improved AAV2.NN-GFP, and AAV2.7m8-GFP, respectively, were fixed in a 5% sucrose solution containing 4% paraformaldehyde for 25 minutes. After fixation, they were sequentially treated in 10%, 20%, and 30% sucrose solutions for 1 hour each. For sectioning, human retinal organoids were placed using an OCT (Optical Coherence Tomography) compound and rapidly frozen using liquid nitrogen vapor. Subsequently, 7 μm thick sections were prepared using a cryostat (Leica Microsystems, Wetzlar, Germany) to confirm differences in GFP gene expression in the human retinal organoids. The results are shown in Figure 5. The expression of the GFP gene delivered to AAV2 variants and wild-type AAV2 merged with the fluorescence of photoreceptor cell labeling antibodies (CRX and Recoverin antibodies) and Müller glial cell labeling antibodies (CRALBP and SOX9 antibodies), respectively. The GFP gene expression induced by the AAV2.KAP variant had a wider merged region in photoreceptor cells and Müller glial cells than that of wild-type AAV2 and improved AAV2.NN and AAV2.7m8. Therefore, it was confirmed that the selected AAV2.KAP had a higher gene delivery efficiency to photoreceptor cells and Müller glial cells than that of wild-type AAV2 and improved AAV2.7m8 and AAV2.NN.

[0148]

[0149] Example 3. Phosphoric In vivo verification of AAV2.KAP variants: Mouse

[0150] The gene transfer ability of the AAV2.KAP variant selected in Example 1 into photoreceptor cells was confirmed in reporter mice (B6.Cg-Tg(Nrl-EGFP)1Asw / J) in which rod cells were labeled with EGFP fluorescence.

[0151]

[0152] 3.1. Confirmation of Gene Transfer and Expression by Fluorescence Microscopy

[0153] To verify the gene delivery efficiency of AAV2.KAP in mice, 8-week-old mice (B6.Cg-Tg(Nrl-EGFP)1Asw / J) expressing EGFP fluorescence in rod cells were treated with AAV2.KAP loaded with the gene encoding tdTomato and AAV2 / AAV2.7m8 / AAV2.NN as controls, each at a rate of 1 x 10⁶ 10 Intravitreal injection was performed via GC, and after 3 weeks, the mice were sacrificed and the eyes were enucleated. The enucleated eyes were fixed in 1% paraformaldehyde for 1 hour, and then sequentially treated in 10%, 20%, and 30% sucrose solutions for 1 hour each. For section preparation, the eyes were positioned using an OCT (Optical Coherence Tomography) compound to ensure horizontal alignment of the optic nerve and the central corneal junction, and then rapidly frozen using liquid nitrogen vapor. Subsequently, 7 µm thick sections were prepared using a cryostat (Leica Microsystems, Wetzlar, Germany). After treatment with DAPI, a cell nucleus label, the expression of EGFP and tdTomato was imaged using a fluorescence microscope.

[0154]

[0155] It was confirmed that wild-type AAV2 infected retinal ganglion cells (RGCs) and some inner nuclear layers (INL). In contrast, it was confirmed that AAV2.KAP delivered the tdTomato gene to significantly high expression not only in RGCs and INLs but also in the outer nuclear layer (ONL) and outer segment (OS) regions of photoreceptor cells. The results are shown in Figure 6.

[0156]

[0157] 3.2. Quantitative Analysis of Gene Transfer Efficiency by FACS

[0158] To quantify the gene delivery efficiency of AAV2.KAP in mice, 8-week-old mice (B6.Cg-Tg(Nrl-EGFP)1Asw / J) with EGFP fluorescence-labeled rod cells were treated with AAV2.KAP loaded with the gene encoding tdTomato and controls AAV2, AAV2.7m8, and AAV2.NN, each at a rate of 1 x 10⁶ 10 Intravitreal injection was performed via GC, and after 3 weeks, mice were sacrificed and eyes were enucleated. The cells were dissociated into single cells using the Papain Dissociation System (Worthington Biochemical Corporation, NJ, USA), and FACS was performed using the BD FACSymphony S6 Cell Sorter (BD Biosciences, CA, USA). FACS analysis was performed using FlowJo software (Tree Star, OR, USA).

[0159]

[0160] EGFP fluorescence labels rod cells, and tdTomato fluorescence labels cells to which the gene has been delivered. The gene delivery efficiency to total retinal cells was measured as the ratio of tdTomato-positive cells to total cells, the gene delivery efficiency to rod cells was measured as the ratio of tdTomato-positive cells to EGFP-positive cells, and the gene delivery efficiency to retinal cells excluding rod cells was measured as the ratio of tdTomato-positive cells to EGFP-negative cells.

[0161] FACS analysis showed that the proportion of tdTomato-positive cells among total mouse retinal cells was 47.48 ± 2.90% for AAV2.KAP, which is significantly higher and statistically significant than that of wild-type AAV2 (6.18 ± 1.09%), improved-type AAV2.7m8 (21.30 ± 7.18%), and AAV2.NN (25.84 ± 2.24%) used as controls. Additionally, the proportion of tdTomato-positive cells among rod cells (EGFP+) was 40.14 ± 4.17% for AAV2.KAP, which is significantly higher and statistically significant than that of AAV2 (6.52 ± 2.00%), AAV2.7m8 (15.95 ± 3.42%), and AAV2.NN (15.78 ± 3.09%). The results are shown in Figure 7.

[0162]

[0163] 3.3. Detection of Gene Expression by Immunofluorescence Staining

[0164] To verify the gene delivery efficiency of AAV2.KAP, AAV2.KAP, wild-type AAV2 as a control, and the improved AAV2.7m8 and AAV2.NN loaded with the EGFP expression gene were each injected intravitreally into C57BL / 6J wild-type mice.

[0165] Specifically, 8-week-old C58BL / 6 mice were given 2x10 AAV2.KAP loaded with the EGFP-coding gene and AAV2 / AAV2.7m8 / AAV2.NN as a control group. 10Intravitreal injection was performed via GC, and after 3 weeks, the mice were sacrificed and the eyes were enucleated. The enucleated eyes were fixed in 1% paraformaldehyde for 1 hour. After fixation, they were sequentially treated in 10%, 20%, and 30% sucrose solutions for 1 hour each. For section preparation, the eyes were positioned using an OCT (Optical Coherence Tomography) compound so that the optic nerve and the central corneal junction were horizontally aligned, and then rapidly frozen using liquid nitrogen vapor. Subsequently, 7 µm thick sections were prepared using a cryostat (Leica Microsystems, Wetzlar, Germany). Immunofluorescence staining was performed on the prepared sections using antibodies labeling retinal cells (Recoverin antibody: photoreceptor cells, Rhodopsin antibody: rod cells, L / M opsin antibody: L / M cone cells, HuC / D antibody: ganglion cells, SOX9 antibody: Müller glial cells, PKCα antibody: bipolar cells) and antibodies against EGFP fluorescence induced by AAV2 delivery. After treatment with DAPI, which labels cell nuclei, EGFP expression was captured using a fluorescence microscope. The results are shown in Figure 8.

[0166] In the case of AAV2.KAP, it was confirmed that it spreads and infects the entire retina in low-magnification fluorescent images. AAV2.KAP was found to have a higher gene delivery efficiency to photoreceptor cells, ganglion cells, Müller cells, and bipolar cells compared to wild-type AAV2, and to have a higher gene delivery efficiency to photoreceptor cells than the improved types AAV2.7m8 and AAV2.NN.

[0167]

[0168] Through these results, it was confirmed that the KAP variant selected in Example 1, compared to wild-type AAV2 and improved AAV2.7m8 and AAV2.NN, delivers the target gene to photoreceptor cells by passing through the ILM from the injection site at a high rate through intravitreal injection and expresses the target gene at a high rate.

Claims

1. An AAV2 variant capsid protein comprising the peptide KAPPNNP inserted into the GH-loop of a wild-type AAV2 capsid protein.

2. The AAV2 variant capsid protein of Claim 1, wherein the peptide is inserted at amino acid 587 or a corresponding position of the wild-type AAV2 capsid protein of SEQ ID NO.

1.

3. The AAV2 variant capsid protein of claim 1, wherein the AAV2 variant capsid protein confers increased infectivity to retinal cells through intravitreal injection compared to the wild-type AAV2 capsid protein.

4. The AAV2 variant capsid protein of claim 3, wherein the retinal cells are selected from the group consisting of photoreceptor cells, retinal ganglion cells, bipolar cells, Müller cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal blood vessel cells, and retinal pigment epithelial cells.

5. Isolated nucleic acid encoding the AAV2 variant capsid protein of Claim 1.

6. A host cell comprising isolated nucleic acid encoding the AAV2 variant capsid protein of Claim 1.

7. AAV2 variant capsid protein of Claim 1, and A recombinant AAV vector comprising a heterogeneous nucleic acid containing a sequence encoding a target product.

8. The recombinant AAV vector of claim 7, wherein the recombinant AAV vector results in increased transduction into retinal cells via intravitreal injection compared to an AAV vector containing wild-type AAV2 capsid protein.

9. The recombinant AAV vector of claim 8, wherein the retinal cells are selected from the group consisting of photoreceptor cells, retinal ganglion cells, bipolar cells, Müller cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal vascular cells, and retinal pigment epithelial cells.

10. A recombinant AAV vector according to claim 7, wherein the object product is a polypeptide or nucleic acid.

11. A pharmaceutical composition for treating ocular diseases comprising the recombinant AAV vector of claim 7.

12. A pharmaceutical composition according to claim 11, wherein the ocular disease is selected from retinal-vitreous-choroidal related disease, retinal vascular disease, macular disease, hereditary retinal disease, hereditary vitreous disease, hereditary choroidal disease, intraocular inflammatory disease, intraocular tumor, glaucoma, and optic neuropathy.

13. A pharmaceutical composition according to claim 11, wherein the eye disease is selected from macular degeneration, diabetic retinopathy, retinal vascular occlusion, macular telangiectasia, retinopathy of prematurity, myopic degeneration, retinitis pigmentosa, Leber congenital amaurosis, Best's disease, Stargardt's disease, congenital non-progressive night blindness, X-linked retinal detachment, Vietti's crystal retinopathy, total color blindness, cone cell dystrophy, cone-rod cell dystrophy, macular dystrophy, Usher syndrome, Bardett-Beadle syndrome, syndromic retinitis pigmentosa, panchoroidal atrophy, centrochoroidal dystrophy, cerebral choroidal atrophy, glaucoma, optic neuropathy, uveitis, uveal melanoma, intraocular lymphoma, retinoblastoma, retinal detachment, and retinal injury.

14. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is administered by intravitreal injection, subretinal injection, or macula injection.

15. An in vitro method for delivering heterogeneous nucleic acids to retinal cells, comprising the step of contacting the recombinant AAV vector of claim 7 with retinal cells.

16. The method of claim 15, wherein the retinal cells are selected from the group consisting of photoreceptor cells, retinal ganglion cells, bipolar cells, Müller cells, horizontal cells, amacrine cells, astrocytes, microglia, retinal blood vessel cells, and retinal pigment epithelial cells.

Images