Composition for treating retinal or macular diseases

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating a retinal or macular disease, which comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of a TAFA protein (for example, TAFA1 to TAFA4), a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a recombinant virus particle containing the vector and a capsid protein, or a cell containing the nucleic acid or the vector. The present invention also relates to an adeno-associated virus (AAV) vector containing a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of a TAFA protein, and a recombinant virus particle containing the AAV vector and a capsid protein. The AAV vector, recombinant virus or composition containing the same of the present invention can be usefully used for preventing or treating a retinal or macular disease by restoring the damaged retina.

Description

Detailed Description of the Invention

[0001] [Technical Field] The disclosure of the present invention relates to a pharmaceutical composition for preventing or treating a retinal or macular disease, which comprises a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of a TAFA protein (e.g., TAFA1 to TAFA4), a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a recombinant virus particle containing the vector and a capsid protein, or a cell containing the nucleic acid or the vector.

[0002] [Background Art]

[0003] The retina is a transparent and thin membrane located on the innermost side of the eyeball wall and in contact with the vitreous body. It serves as a primary visual information organ that converts the optical information of things into electrical signals and transmits the images to the central visual region of the brain via the optic nerve. The retina consists of more than 100 million light-sensitive photoreceptor cells, more than 1 million ganglion cells which are visual nerve cells, and nerve cells connecting them. The macula is a nerve tissue located in the center of the retina, where most visual cells gather. It is the place where the image of an object is formed and is responsible for central vision. The macula lutea consists of a photoreceptor cell layer and a ganglion cell layer composed of cone cells. The retina is thin at the macula lutea. While the electrical signal of the image is converted into a chemical signal in a bright light state, it is transmitted to the brain along the optic nerve, which is the axon of the ganglion cell. The retina outside the macula lutea distinguishes the peripheral part and plays a major role in the dark. Therefore, when abnormalities occur in the retina or macula due to aging or external factors, it can lead to visual impairment and even blindness, accompanied by problems in vision and visual field. Retinal or macular diseases include diabetic retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal edema, and the like.

[0004] The TAFA protein group is a group of proteins that are abundantly expressed throughout the brain region and consists of five proteins, namely TAFA1 to 5. These proteins contain structurally conserved cysteine residues, and the C, CC, and CXC motifs between cysteines are related to chemokines. TAFA1 to 4 have a high similarity in the number of cysteines and the space between cysteines, and TAFA5 has fewer cysteines than these.

[0005] These proteins are very well conserved evolutionarily in vertebrates and have been shown to be important for the normal function of the central nervous system. In the case of mice with knockout of TAFA1, weight loss, decreased anxiety behavior, and impairment of memory for fear have been reported (Lei X, Liu L, Terrillion CE, et al. FASEB J. 2019;33(12):14734-14747. and Yong HJ, Ha N, Cho EB, et al. Sci Rep. 2020;10(1):3969.). In contrast, when TAFA2 and TAFA3 were knocked out, an increase in anxiety behavior was reported (Choi JH, Jeong YM, Kim S, et al. Proc Natl Acad Sci USA. 2018;115(5):E1041-E1050. and Kim S, Lee B, Choi JH, Kim JH, Kim CH, Shin HS. Sci Rep. 2017;7(1):16503.). In the case of TAFA4-null mice, allodynia and hyperalgesia have been reported (Delfini MC, Mantilleri A, Gaillard S, et al. Cell Rep. 2013;5(2):378-388.). In mice with knockout of TAFA5, behavioral changes such as increased depressive behavior and loss of spatial memory ability have been reported (Huang S, Zheng C, Xie G, et al. FAM19A5 / TAFA5, a novel neurokine, plays a crucial role in depressive-like and spatial memory-related behaviors in mice. Mol Psychiatry. 2021;26(6):2363-2379.).

[0006] Despite the important roles of these proteins in the central nervous system, very little is known about the function of each protein and its pathophysiological or therapeutic functions, and what has been revealed about its roles outside the central nervous system is also limited.

[0007]

[0008] Each matter described as the above background art is merely for enhancing the understanding of the background of the present invention, and should not be regarded as corresponding to the prior art already known to those having ordinary knowledge in this technical field.

[0009] [Summary of the Invention] [Problems to be Solved by the Invention]

[0010] The present invention provides a pharmaceutical composition for preventing or treating a retinal or macular disease, which comprises a polypeptide having an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a recombinant virus particle containing the vector and a capsid protein, or a cell containing the nucleic acid or the vector.

[0011] The present invention also provides a therapeutic use (for use in therapy) of the polypeptide, the nucleic acid encoding the polypeptide, the vector containing the nucleic acid, the recombinant virus particle containing the vector and a capsid protein, or the cell containing the nucleic acid or the vector.

[0012] The present invention also provides the use of the AAV vector or the recombinant virus particle for use in a method for preventing or treating a retinal or macular disease. The present invention also provides a method for preventing or treating a retinal or macular disease in a subject in need thereof, the method comprising administering to the subject the polypeptide, the nucleic acid encoding the polypeptide, the vector containing the nucleic acid, the recombinant virus particle containing the vector and a capsid protein, or the cell containing the nucleic acid or the vector.

[0013] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 comprises the amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus):

[0014] [General formula 1]

[0015] X1-X2-X3-G-T-C-E-V-X4-A-X5-H-X6-C-C-N-X7-N-X8-I-E-E-X9-S-Q-T-X10-X11-C-S-C-X12-X13-G-X14-V-A-G-T-T-X15-X16-X17-P-S-C-V-X18-A-X19-I-V-X20-X21-X22-W-W-C-X23-M-X24-P-C-X25-X26-G-E-X27-C-K-X28-L-P-D-X29-X30-G-W-X31-C-X32-X33-G-X34-K-X35-K-T-T-X36-X37-X38-X39

[0016] In the general formula 1,

[0017] X1 is absent, or V, I, or L,

[0018] X2 is K, E, R, or Q,

[0019] X3 is G, T, Q, P, or A,

[0020] X4 is V or I,

[0021] X5 is A, L, V, or I,

[0022] X6 is R or L,

[0023] X7 is K, R, or Q,

[0024] X8 is R or K,

[0025] X9 is R or L,

[0026] X10 is V or G,

[0027] X11 is K or N,

[0028] X12 is F or L,

[0029] X13 is P or S,

[0030] X14 is Q or K,

[0031] X15 is R, H or Q,

[0032] X16 is A, N, S or T,

[0033] X17 is A, Q, R, K or T,

[0034] X18 is D or E,

[0035] X19 is S or A,

[0036] X20 is I, E, L, A or V,

[0037] X21 is Q, G or E,

[0038] X22 is K or R,

[0039] X23 is H, Q or E,

[0040] X24 is E, Q, N, D, S or H,

[0041] X25 is L, V or M,

[0042] X26 is E, D, P, L or A,

[0043] X27 is E or D,

[0044] X28 is V, T, A or I,

[0045] X29 is L, N, R, Y, S or Q,

[0046] X30 is S, K or T,

[0047] X31 is S or M,

[0048] X32 is S, A, or Y,

[0049] X33 is S, T, or R,

[0050] X34 is N or H,

[0051] X35 is V or I,

[0052] X36 is R or K,

[0053] X37 is absent, V, A, G, M, or N,

[0054] X38 is absent, T, I, N, F, or S, and

[0055] X39 is absent, R, H, V, K, I, or Q.

[0056] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of the following general formula 2:

[0057] <General formula 2>

[0058] X1-X2-H-H-K-A-X3-H

[0059] In the general formula 2,

[0060] X1 is A or V,

[0061] X2 is N or I, and

[0062] X3 is H or Q.

[0063] In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of general formula 2 - general formula 1.

[0064] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of the following general formula 3:

[0065] <General formula 3>

[0066] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0067] In the above General formula 3,

[0068] X1 is A or S,

[0069] X2 is L, T or S,

[0070] X3 is Q, E or H,

[0071] X4 is absent or is P, L or H,

[0072] X5 is P or R,

[0073] X6 is T, S or I,

[0074] X7 is A, P, T, S or H,

[0075] X8 is T, A, S or I,

[0076] X9 is V or A, and

[0077] X10 is L or H.

[0078] In some embodiments, the polypeptide comprises the amino acid sequence (from the N-terminus to the C-terminus) of General formula 3 - General formula 1.

[0079] In some embodiments, the polypeptide further comprises the amino acid sequence (from the N-terminus to the C-terminus) of the following General formula 4:

[0080] <General formula 4>

[0081] X1-X2

[0082] In the general formula 4,

[0083] X1 is H or Y, and

[0084] X2 is Q, V or L.

[0085] In some embodiments, the polypeptide comprises the amino acid sequence (from the N-terminus to the C-terminus) of general formula 4 - general formula 1.

[0086] In some embodiments, the polypeptide further comprises the following amino acid sequence (from the N-terminus to the C-terminus):

[0087] LHRP (SEQ ID NO: 293);

[0088] LHQSGFTSGHFPHHRKLGE (SEQ ID NO: 294); or

[0089] LAPPGTNIQI (SEQ ID NO: 295).

[0090] In some embodiments, the polypeptide further comprises the following amino acid sequence (from the N-terminus to the C-terminus) of general formula 5.

[0091] <General formula 5>

[0092] X1 - X2 - X3

[0093] In the general formula 5,

[0094] X1 is P or G,

[0095] X2 is R or H, and

[0096] X3 is T, S or L.

[0097] In some embodiments, the polypeptide further comprises the following amino acid sequence (from the N-terminus to the C-terminus).

[0098] PYTSL (SEQ ID NO: 296); or

[0099] QEDKLK (SEQ ID NO: 297).

[0100] In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of LHRP-General Formula 1, LHQSGFTSGHFPHHRKLGE-General Formula 1, or LAPPGTNIQI-General Formula 1. In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 1-General Formula 5, General Formula 1-PYTSL, or General Formula 1-QEDKLK. In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of LHRP-General Formula 1-General Formula 5, LHQSGFTSGHFPHHRKLGE-General Formula 1-General Formula 5, LAPPGTNIQI-General Formula 1-QEDKLK, or LAPPGTNIQI-General Formula 1-PYTSL.

[0101] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 75 to 129. In some embodiments, the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 184 to 238.

[0102] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 130 to 183. In some embodiments, the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 239 to 292.

[0103] In some embodiments, the polypeptide further comprises a signal peptide.

[0104] In some embodiments, the vector is a viral vector. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the AAV has a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10.

[0105] In some embodiments, the retinal or macular disease is due to damage to all or part of the retina or macula. In some embodiments, the retinal or macular disease is a disease that occurs due to abnormal function or damage of retinal or macular cells. In some embodiments, the retinal or macular disease may be a disease that occurs due to abnormal function or damage of photoreceptor cells and / or retinal pigment epithelium (RPE) cells of the retina or macula. In some embodiments, the retinal or macular disease is retinopathy, choroidal neovascularization, macular disease, macular degeneration, retinal degeneration, macular edema, retinal edema, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease, or a combination thereof. In some embodiments, the macular degeneration is age-related macular degeneration, Best macular dystrophy, Sorsby fundus dystrophy, Mallatia Leventinese, Doyne honeycomb retinal dystrophy, Stargardt disease (Stargardt macular dystrophy), myopic macular degeneration, or pigment epithelial detachment-related macular degeneration. In some embodiments, the macular degeneration is age-related macular degeneration. In some embodiments, age-related macular degeneration is wet or dry age-related macular degeneration. In some embodiments, the retinopathy is retinal dystrophy. In some embodiments, the retinopathy is diabetic retinopathy. In some embodiments, the diabetic retinopathy is non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), diabetic maculopathy, diabetic macular edema, or a combination thereof. In some embodiments, the retinal or macular disease is a hereditary retinal disease.In some embodiments, the hereditary retinal disease is retinitis pigmentosa (RP), Leber congenital amaurosis, Stargardt’s disease, Coats retinopathy, cone dystrophy, choroideremia, Usher syndrome, Best’s Disease, X-linked retinoschisis, or unspecified hereditary retinal dystrophy.

[0106] The present invention also provides an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75.

[0107] In some embodiments, the vector further comprises one or more sequences selected from the group consisting of a promoter sequence, an enhancer sequence, an exon sequence, an intron sequence, a signal sequence coding sequence, a splicing donor sequence, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

[0108] In some embodiments, the intron is composed of 51 to 117 nucleotides and contains the nucleotide sequence shown in SEQ ID NO: 57. In some embodiments, the intron has at least 70% sequence identity with (i) nucleotides 871 to 924 of SEQ ID NO: 1 (SEQ ID NO: 58), (ii) nucleotides 861 to 924 of SEQ ID NO: 1 (SEQ ID NO: 59), (iii) nucleotides 852 to 924 of SEQ ID NO: 1 (SEQ ID NO: 3), (iv) nucleotides 851 to 924 of SEQ ID NO: 1 (SEQ ID NO: 61), (v) nucleotides 830 to 924 of SEQ ID NO: 1 (SEQ ID NO: 2), (vi) nucleotides 821 to 924 of SEQ ID NO: 1 (SEQ ID NO: 63), (vii) nucleotides 811 to 924 of SEQ ID NO: 1 (SEQ ID NO: 64), or (viii) nucleotides 808 to 924 of SEQ ID NO: 1 (SEQ ID NO: 65). In some embodiments, the intron comprises (i) nucleotides 871 to 924 of SEQ ID NO: 1 (SEQ ID NO: 58), (ii) nucleotides 861 to 924 of SEQ ID NO: 1 (SEQ ID NO: 59), (iii) nucleotides 852 to 924 of SEQ ID NO: 1 (SEQ ID NO: 3), (iv) nucleotides 851 to 924 of SEQ ID NO: 1 (SEQ ID NO: 61), (v) nucleotides 830 to 924 of SEQ ID NO: 1 (SEQ ID NO: 2), (vi) nucleotides 821 to 924 of SEQ ID NO: 1 (SEQ ID NO: 63), (vii) nucleotides 811 to 924 of SEQ ID NO: 1 (SEQ ID NO: 64), or (viii) nucleotides 808 to 924 of SEQ ID NO: 1 (SEQ ID NO: 65).

[0109] In some embodiments, the vector comprises the following components: (1) the CMV enhancer sequence shown in SEQ ID NO: 4; (2) a promoter sequence selected from the CMV promoter sequence shown in SEQ ID NO: 5 or 6, the EF-1α promoter sequence shown in SEQ ID NO: 7, or the chicken β-actin promoter sequence shown in SEQ ID NO: 8; (3) an exon 1 (E1) sequence selected from the CMV E1 sequence shown in SEQ ID NO: 12, the EF-1α E1 sequence shown in SEQ ID NO: 13, or the chicken β-actin E1 sequence shown in SEQ ID NO: 14 or 15; (4) the splicing donor sequence shown in SEQ ID NO: 9 or 10; and / or (5) the EF-1α E2 sequence shown in SEQ ID NO: 11.

[0110] In some embodiments, an amino acid sequence (from N-terminus to C-terminus) having at least 70% sequence identity with the amino acid sequence presented in SEQ ID NO: 75 comprises the amino acid sequence of General Formula 1 below:

[0111] <General Formula 1>

[0112] X1-X2-X3-G-T-C-E-V-X4-A-X5-H-X6-C-C-N-X7-N-X8-I-E-E-X9-S-Q-T-X10-X11-C-S-C-X12-X13-G-X14-V-A-G-T-T-X15-X16-X17-P-S-C-V-X18-A-X19-I-V-X20-X21-X22-W-W-C-X23-M-X24-P-C-X25-X26-G-E-X27-C-K-X28-L-P-D-X29-X30-G-W-X31-C-X32-X33-G-X34-K-X35-K-T-T-X36-X37-X38-X39

[0113] In the above General Formula 1,

[0114] X1 is absent, V, I or L,

[0115] X2 is K, E, R or Q,

[0116] X3 is G, T, Q, P or A,

[0117] X4 is V or I,

[0118] X5 is A, L, V or I,

[0119] X6 is R or L,

[0120] X7 is K, R or Q,

[0121] X8 is R or K,

[0122] X9 is R or L,

[0123] X10 is V or G,

[0124] X11 is K or N,

[0125] X12 is F or L,

[0126] X13 is P or S,

[0127] X14 is Q or K,

[0128] X15 is R, H or Q,

[0129] X16 is A, N, S or T,

[0130] X17 is A, Q, R, K or T,

[0131] X18 is D or E,

[0132] X19 is S or A,

[0133] X20 is I, E, L, A or V,

[0134] X21 is Q, G or E,

[0135] X22 is K or R,

[0136] X23 is H, Q or E,

[0137] X24 is E, Q, N, D, S or H,

[0138] X25 is L, V or M,

[0139] X26 is E, D, P, L or A,

[0140] X27 is E or D,

[0141] X28 is V, T, A or I,

[0142] X29 is L, N, R, Y, S or Q,

[0143] X30 is S, K or T,

[0144] X31 is S or M,

[0145] X32 is S, A or Y,

[0146] X33 is S, T or R,

[0147] X34 is N or H,

[0148] X35 is V or I,

[0149] X36 is R or K,

[0150] X37 is non - existent or V, A, G, M or N,

[0151] X38 is non - existent or T, I, N, F or S, and

[0152] X39 is non - existent or R, H, V, K, I or Q.

[0153] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 is composed of 80 to 130, more preferably 90 to 120, and most preferably 91 to 119 amino acid sequences.

[0154] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 2 below:

[0155] <General Formula 2>

[0156] X1-X2-H-H-K-A-X3-H

[0157] In the General Formula 2,

[0158] X1 is A or V,

[0159] X2 is N or I, and

[0160] X3 is H or Q.

[0161] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 3 below:

[0162] <General Formula 3>

[0163] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0164] In the General Formula 3,

[0165] X1 is A or S,

[0166] X2 is L, T or S,

[0167] X3 is Q, E or H,

[0168] X4 is absent or P, L or H,

[0169] X5 is P or R,

[0170] X6 is T, S or H,

[0171] X7 is A, P, T, S or I,

[0172] X8 is T, A, S or I,

[0173] X9 is V or A, and

[0174] X10 is L or H.

[0175] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of the following general formula 4:

[0176] <General formula 4>

[0177] X1-X2

[0178] In the general formula 4,

[0179] X1 is H or Y, and

[0180] X2 is Q, V or L.

[0181] In some embodiments, the polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0182] LHRP (SEQ ID NO: 293);

[0183] LHQSGFTSGHFPHHRKLGE (SEQ ID NO: 294); or

[0184] LAPPGTNIQI (SEQ ID NO: 295).

[0185] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of the following general formula 5:

[0186] <General formula 5>

[0187] X1-X2-X3

[0188] In the general formula 5,

[0189] X1 is P or G,

[0190] X2 is R or H, and

[0191] X3 is T, S or L.

[0192] In some embodiments, the polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus).

[0193] PYTSL (SEQ ID NO: 296); or

[0194] QEDKLK (SEQ ID NO: 297).

[0195] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 75 to 129. In some embodiments, the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 184 to 238.

[0196] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 130 to 183. In some embodiments, the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 239 to 292.

[0197] In some embodiments, the AAV vector is for use in gene therapy. In some embodiments, the AAV vector is for use in the expression of a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75.

[0198] The present invention also provides an isolated host cell comprising the AAV vector.

[0199] The present invention also provides an isolated host cell transformed with the AAV vector.

[0200] The present invention also provides a composition comprising the AAV vector, or a host cell comprising the AAV vector, or a host cell transformed with the AAV vector. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is for the prevention or treatment of retinal or macular diseases.

[0201] The present invention also provides a kit comprising the AAV vector, or a host cell comprising the AAV vector, or a host cell transformed with the AAV vector. In some embodiments, the kit further comprises instructions for use.

[0202] The present invention also provides a method for producing recombinant virus particles comprising the steps of: (i) transducing cells with the AAV vector, and (ii) a construct containing the rep and cap genes. In some embodiments, the method further comprises the step of isolating the produced recombinant virus particles.

[0203] The present invention also provides recombinant virus particles produced by the method.

[0204] The present invention also provides recombinant virus particles comprising (a) a capsid protein and (b) the AAV vector.

[0205] Furthermore, the present invention provides a therapeutic use (for use in therapy) of the AAV vector or the recombinant viral particle.

[0206] The present invention also provides the use of the AAV vector or the recombinant viral particle for use in a method of preventing or treating a disease or disorder.

[0207] The present invention also provides a method of preventing or treating a disease or disorder in a subject in need thereof, the method comprising administering the AAV vector or the recombinant viral particle to the subject.

[0208] In some embodiments, the disease or disorder includes a retinal or macular disease. In some embodiments, the retinal or macular disease is caused by damage to all or part of the retina or macula. In some embodiments, the retinal or macular disease is a disease that occurs due to dysfunction or damage of retinal or macular cells. In some embodiments, the retinal or macular disease may be a disease that occurs due to dysfunction or damage of photoreceptor cells and / or retinal pigment epithelium (RPE) cells of the retina or macula. In some embodiments, the retinal or macular disease is retinopathy, choroidal neovascularization, macular disease, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease, or a combination thereof. In some embodiments, the method includes administering an additional therapeutic agent to the subject.

[0209] The present invention also provides a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75, a nucleic acid encoding the polypeptide, a vector comprising the nucleic acid, a recombinant viral particle comprising the vector and a capsid protein, or a cell comprising the nucleic acid or vector, for use in preventing, ameliorating or treating the retinal or macular disease.

[0210] [Means for Solving the Problems]

[0211] The present disclosure provides a polypeptide having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 (e.g., vertebrate TAFA1 to TAFA4 sequences) for use in preventing, ameliorating or treating retinal or macular diseases, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a recombinant virus particle containing the vector and a capsid protein, or a cell containing the nucleic acid or the vector. The present disclosure also provides a pharmaceutical composition for preventing or treating retinal or macular diseases containing the polypeptide, the nucleic acid encoding the polypeptide, the vector containing the nucleic acid, the recombinant virus particle containing the vector and a capsid protein, or the cell containing the nucleic acid or the vector. The present disclosure also provides an adeno-associated virus (AAV) vector containing a nucleic acid encoding the polypeptide; a host cell containing the AAV vector or transformed with the AAV vector; a composition or kit containing the AAV vector or containing or transformed with the AAV vector; a method for producing a recombinant virus particle comprising transducing a cell with the AAV vector and a construct containing the rep and cap genes; a recombinant virus particle produced by the method; a method for preventing or treating a disease or disorder in a subject in need thereof, comprising administering the AAV vector or the recombinant virus particle to the subject.

[0212] As described herein, the applicant has confirmed that the expression of TAFA genes in various species (human TAFA1, human TAFA2, human TAFA3, human TAFA4, mouse TAFA4, zebrafish TAFA4, gecko TAFA4) can recover or protect damaged retinas with excellent ability, and has derived the sequence of the highly conserved human TAFA4 mature protein (SEQ ID NO: 75) that plays a central role in the above effects. As demonstrated herein, in some embodiments, the compositions of the present invention can restore damaged retinas to a level equivalent to normal. Additional aspects of the present disclosure are provided throughout this application.

[0213] To facilitate understanding of the disclosure provided herein, a number of terms and phrases will be defined. Additional definitions are set forth throughout the detailed description.

[0214]

[0215] I. Definitions

[0216] Throughout this disclosure, "a" or "any" entity refers to one or more of that entity; for example, "a polypeptide" is understood to indicate one or more polypeptides. Thus, "a" (or "any"), "one or more", and "at least one" may be used interchangeably herein.

[0217] Also, as used herein, "and / or" must be regarded as a specific disclosure of each of two particular features or components together with, or in place of, the remaining other features or components. Thus, as used herein, the term "and / or" in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following scenarios: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0218] The term "at least" preceding a single number or a series of numbers is understood to include the number following the term "at least" and all subsequent numbers or integers that may logically be included in the context. For example, the number of nucleotides within a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have a given property. When "at least" precedes a series of numbers or a range, it is understood that "at least" modifies each of the numbers in the series or range. Also, "at least" is not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without considering the number of significant figures).

[0219] As used herein, when each embodiment is described with the term "comprising", it is understood that other similar embodiments described in terms of "consisting of" and / or "essentially consisting of" are also provided.

[0220] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the present disclosure. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd ed, 1999, Academic Press; and Oxford Dictionary Of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide many general dictionaries for those of ordinary skill in the art for each of the terms used in the present disclosure.

[0221] Units, prefixes, and symbols are presented in the form approved by these SI (Systeme International de Unites). Numerical ranges include the numbers that limit the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino- to carboxy direction. The headings provided herein are not limitations on the various aspects of the disclosure, and these may refer to the specification as a whole. Accordingly, each term defined immediately below is more fully defined by reference to the entire specification.

[0222] As used herein, the term "about" is used to mean approximately, roughly, on the order of, or within the region of. When the term "about" is used in conjunction with a numerical range, it extends the boundaries above and below the recited numerical values and changes the range. In general, the term "about" can change the numerical values above and below the explicitly stated value by, for example, a variation of up or down (higher or lower) 10%.

[0223] The term "adeno-associated virus" (AAV) as used herein is a single-stranded DNA virus that is a helper-dependent human parvovirus. The genome size is approximately 4.6 kbp. The N-terminal portion of the genome encodes the rep gene involved in viral replication and viral gene expression, and the C-terminal portion encodes the cap gene that encrypts the viral capsid protein. It is composed of inverted terminal repeats (ITRs) with approximately 145 bases inserted at both terminal sites. The 145-bp ITRs (inverted terminal repeats) with a T-shaped structure function as an origin of replication during viral genome replication and act as a primary packaging signal. The ITR is the only cis-acting nucleotide sequence required when making a recombinant AAV construct. It has enhancer activity in the presence of the Rep protein but has very weak activity in the absence of the Rep protein. Therefore, when cloning a foreign gene into a recombinant AAV construct, an expression construct is produced by appropriately configuring an enhancer, a promoter, pA, etc. considering this (RJ Samulski and N Muzyczka, Annu. Rev. Virolo. 2014.1:427-451). Four proteins are translated from the rep gene, which are classified into rep78, rep68, rep52, and rep40 according to their molecular weights and perform important functions in AAV DNA replication. Four proteins are translated from the cap gene. Among them, the VP1, VP2, and VP3 proteins are structural proteins that make up AAV particles, and the assembly-activating protein (AAP) promotes the assembly of AAV particles by the respective structural proteins.For the adeno-associated virus to be efficiently replicated, it requires some proteins and RNAs derived from helper viruses such as adenovirus or herpes simplex virus (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0224] AAV includes, but is not limited to, AAV serotypes and phylogenetic branches disclosed by AAV type 1, AAV type 2, AAV type 3 (including 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, caprine AAV, shrimp AAV, Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and other AAVs currently known or subsequently discovered. For example, reference may be made to VIROLOGY by FIELDS et al., volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV" includes all known derivatives of AAV. In some embodiments, "AAV" includes modified or artificial AAVs.

[0225] As used herein, "administer", "administering" and their grammatical variations refer to introducing a composition (e.g., a polynucleotide comprising a foreign gene and untranslated nucleic acid sequence described herein) to a subject via a pharmaceutically acceptable route. The composition is introduced to the subject by any suitable route including intratumoral, oral, intranasal, intrapulmonary, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic, intrathecal, periocular, intraocular or topical administration. Administration includes self-administration and administration by another person. The composition or formulation exerts its intended function via a suitable administration route. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or formulation into a vein of the subject.

[0226] As used herein, the term "intraocular" refers to the interior or lower part of the eye tissue. The term "intraocular administration" as used herein refers to any administration capable of delivering a composition to the sub-Tenon, subconjunctival, suprachoroidal, subretinal, vitreous and similar locations of the eye. In some aspects, intraocular administration includes suprachoroidal, subretinal, and intravitreal administration.

[0227] The "CEE" construct as used herein comprises a CMV enhancer, an EF-1α promoter and an EF-1α intron fragment. The "CE" construct comprises a CMV enhancer and an EF-1α promoter but does not contain an intron fragment (e.g., does not contain an EF-1α intron fragment). The "CAE" construct comprises a CMV enhancer, a chicken β-actin promoter and an EF-1α intron fragment. The "CAG" construct as used herein comprises a CMV enhancer, a chicken β-actin promoter and a chicken β-actin / rabbit β-globin hybrid intron fragment. The "CA" construct comprises a CMV enhancer and a chicken β-actin promoter but does not contain an intron fragment (e.g., does not contain an EF-1α intron fragment). See FIGS. 4a, 5a and 5c.

[0228] As used herein, the term "conserved" refers to each nucleotide or amino acid residue of a polynucleotide sequence or polypeptide sequence that appears unchanged at the same position in two or more arrays being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved among more closely related sequences than the nucleotides or amino acids that appear at other positions within the sequence.

[0229] As used herein, the term "amino acid" includes not only the 22 standard amino acids (arginine (R), lysine (K), histidine (H), glutamic acid (E), aspartic acid (D), glutamine (Q), asparagine (N), leucine (L), isoleucine (I), valine (V), methionine (M), phenylalanine (F), tryptophan (W), tyrosine (Y), glycine (G), alanine (A), serine (S), threonine (T), proline (P), cysteine (C), etc.) that are naturally incorporated into peptides, but also D-isomers and modified amino acids. Further, the peptide can include non-standard amino acids that have been post-translationally modified. Post-translational modifications can include, but are not limited to, phosphorylation, glycosylation, acylation (e.g., acetylation, myristoylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, chemical property changes (e.g., beta-elimination deimidation, deamidation), and structural changes (e.g., formation of disulfide bridges). The peptide can be a wild-type peptide identified and isolated from its natural source as is. On the other hand, the peptide can also be an artificial mutant that includes an amino acid sequence in which one or more amino acids have been substituted, deleted, and / or inserted. Amino acid changes in both artificial mutants and wild-type polypeptides include conservative amino acid substitutions that do not significantly affect protein folding and / or activity.For example, the preservative substitutions can include basic amino acids (arginine (R), lysine (K), and histidine (H)), acidic amino acids (glutamic acid (E) and aspartic acid (D)), polar amino acids (glutamine (Q) and asparagine (N)), hydrophobic amino acids (leucine (L), isoleucine (I), valine (V), and methionine (M)), aromatic amino acids (phenylalanine (F), tryptophan (W), and tyrosine (Y)), and small amino acids (glycine (G), alanine (A), serine (S), and threonine (T)). In general, amino acid substitutions that do not alter specific activity are known in the art. The most frequently occurring exchanges can include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0230] In some embodiments, when two or more sequences are 100% identical to each other, they are said to be "perfectly conserved" or "identical." In some embodiments, when two or more sequences are at least 70%, at least 80%, at least 90%, or at least 95% identical to each other, they are said to be "highly conserved." In some embodiments, when two or more sequences are approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 98%, or approximately 99% identical to each other, they are said to be "highly conserved." In some embodiments, when two or more sequences are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to each other, they are said to be "conserved." In some embodiments, when two or more sequences are approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 98%, or approximately 99% identical to each other, they are said to be "conserved." Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to a portion, region, or feature thereof.

[0231] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each containing a nucleobase sequence) that are related to each other by the Watson-Crick base pairing rules, or between an oligomer and a target gene. For example, the nucleobase sequence T-G-A (5'→ is complementary to the nucleobase sequence A-C-T (3'→. According to the base pairing rules, when the number of all nucleobases of a given nucleobase sequence is less than the number that matches another nucleobase sequence, the complementarity can be "partial". For example, in some embodiments, the complementarity between a given nucleobase sequence and another nucleobase sequence can be about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Thus, in certain embodiments, the term "complementary" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity or complementarity with a target nucleic acid sequence. Or, to continue the illustration, there can be "complete" or "perfect" (100%) complementarity between a given nucleobase sequence and another nucleobase sequence. In some embodiments, the degree of complementarity between nucleobase sequences has a significant impact on the efficiency and strength of hybridization between the sequences.

[0232] As used herein, the expression "deletion of continuous or discontinuous nucleotides" is intended to include continuous or discontinuous nucleotide sequences remaining as a result of the deletion, compared to the wild-type (or original) sequence, but is not intended to include any deletion process. The term can include "deletion of continuous nucleotides", "deletion of non-continuous nucleotides remaining in a non-deleted state between the deleted nucleotides", and "insertion of different types of nucleotides at the positions of the deleted continuous or discontinuous nucleotides". For example, in the case of the nucleotide sequence "A-T-G-C-C-G-T-C", deletion of continuous nucleotides includes deletion of one or more continuous nucleotides such as "A-_-_-_-C-G-T-C", deletion of non-continuous nucleotides means that one or more nucleotides remain in a non-deleted state between the deleted nucleotides such as "A-_-G-_-C-G-T-_", and insertion of different types of nucleotides at the positions of the deleted continuous or discontinuous nucleotides means that one or more nucleotides different from the original nucleotides are inserted at the positions of the deleted nucleotides such as "A-A-G-_-C-G-T-G".

[0233] The term "downstream" refers to a nucleotide sequence located 3' to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence is associated with the sequence after the transcription start point. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0234] As used herein, the term "enhancer" refers to a portion of DNA that contains a sequence capable of providing enhanced transcription and, in some cases, can act regardless of its orientation relative to other regulatory sequences. Enhancers can cooperate with a promoter and / or other enhancer elements or act additionally.

[0235] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance that is added to a pharmaceutical composition to facilitate the administration of a compound, e.g., a polynucleotide containing a foreign gene and untranslated nucleic acid sequences as described herein.

[0236] The term "exon" refers to a nucleic acid sequence that appears in the mature form of an RNA molecule after splicing out a predetermined portion of the nucleic acid encoding a protein or a portion of preprocessed (or precursor) RNA. The mature RNA molecule can be messenger RNA (mRNA) or a functional form of non-coding RNA such as rRNA or tRNA.

[0237] As used herein, the term "expression" refers to the process by which a polynucleotide produces a gene product, e.g., RNA or polypeptide. This includes, but is not limited to, transcribing the polynucleotide into messenger RNA (mRNA) and translating the mRNA into a polypeptide. Expression produces a "gene product". A gene product as used herein can be, for example, a nucleic acid such as RNA produced by transcription of a gene. A gene product as used herein can be a nucleic acid or polypeptide translated from a transcript. Gene products described herein further include post-transcriptional modifications, e.g., nucleic acids by polyadenylation or splicing, or post-translational modifications, e.g., polypeptides by phosphorylation, methylation, glycosylation, lipid addition, association with small subunits of other proteins, or proteolytic cleavage.

[0238] As used herein, the term "identity" refers to overall monomer conservation between each polymer molecule, e.g., polynucleotide molecules. The term "identical" without any additional modifiers, e.g., polynucleotide A is identical to polynucleotide B, means that the polynucleotide sequences are 100% identical (100% sequence identity). Stating that two sequences are, e.g., "70% identical" is the same as stating that they have, e.g., "70% sequence identity".

[0239] For example, the calculation of the identity (percent) between two polypeptide or polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second polypeptide, or polynucleotide sequences, and sequences that are not identical for comparison purposes can be ignored). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. Thereafter, the amino acids at corresponding amino acid positions are compared, or in the case of polynucleotides, the bases are compared.

[0240] If a particular position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the respective molecules are identical at that position. The identity (percent) between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each of those gaps. Sequence comparison and determination of the identity (percent) between two sequences can be performed using a mathematical algorithm.

[0241] Suitable software programs that can be used to align mutually different sequences (e.g., polynucleotide sequences) are available from many suppliers. One suitable program for determining sequence identity (percent) is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. bl2seq performs a comparison between two sequences using the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the bioinformatics program EMBOSS suite and are available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0242] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X, or Clustal Omega), MUSCLE, and the like.

[0243] Mutually different regions within a single polynucleotide or polypeptide target sequence aligned with a polynucleotide or polypeptide reference sequence can each have their own sequence identity (percent). Note that the sequence identity (percent) value is rounded / truncated to the nearest first decimal place. For example, 80.11, 80.12, 80.13, and 80.14 are truncated to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded to 80.2. Also note that the length value can always be an integer.

[0244] In certain embodiments, the identity (percent, %ID) of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100×(Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in an alignment of the first and second sequences (when aligned by visual inspection or by a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the identity (percent) of the first sequence to the second sequence can be higher than the identity (percent) of the second sequence to the first sequence.

[0245] One of ordinary skill in the art will understand that the generation of a sequence alignment for calculating sequence identity (percent) is not limited to a binary sequence-sequence comparison that is entirely processed by primary sequence data. It will also be understood that sequence alignments can be performed by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., positions of mutations), or phylogenetic data. Suitable programs for integrating heterogeneous data and making multiple sequence alignments are available at www.tcoffee.org, such as, among others, T-Coffee which is also available from, for example, EBI. It will also be understood that the final alignment used to calculate sequence identity (percent) can be systematized either automatically or manually.

[0246] As used herein, the term "intron" refers to the portion of DNA within a gene (intervening sequence) that is spliced out of the mRNA transcribed from the gene before the mRNA is sent out of the cell nucleus without coding for a portion of the protein produced by the gene. An "intron sequence" refers to the nucleic acid sequence of an intron. Such sequences are also referred to herein as "untranslated nucleic acid sequences." Thus, an intron is each region of a DNA sequence that is transcribed along with the coding sequences (exons) but is removed during the formation of mature mRNA.

[0247] As used herein, the term "intron fragment" refers to a fragment derived from the full-length EF-1α intron A sequence (i.e., for example, the first intron of EF-1α as shown in the first sequence of the sequence listing). The fragment is meant to exclude the full-length EF-1α intron. In some embodiments, the "intron fragment" includes the minimum number of nucleotides or composition necessary to achieve an expression level that exceeds the expression level achieved with the corresponding construct in which all nucleotides of the EF-1α intron A are missing. Thus, the intron fragments of the present disclosure (also referred to herein as "untranslated nucleic acid sequences") include fragments of the EF-1α intron and are not particularly limited as long as they can increase the expression of a foreign gene. As demonstrated herein, in some embodiments, the intron fragment (i.e., the untranslated nucleic acid sequence) can increase the transcription of a foreign gene and thereby increase the expression of the foreign gene. Thus, in some embodiments, the intron fragments described herein can be untranslated regulatory elements.

[0248] As used herein, the terms "isolated," "purified," "extracted," and grammatical variations thereof are used interchangeably and refer to the state of a desired composition of the present disclosure, such as a formulation of a polynucleotide containing a foreign gene and an untranslated nucleic acid sequence, that has undergone one or more purification processes. In some embodiments, isolation or purification as used herein is the process of removing or partially removing (e.g., fractionating) a composition of the present disclosure, such as a polynucleotide described herein, from a sample containing contaminants.

[0249] In some embodiments, the isolated composition has no detectable undesirable activity, or alternatively, the level or amount of undesirable activity is below an acceptable level or amount. In other embodiments, the isolated composition has an amount and / or concentration of the desired composition of the present disclosure that is equal to or greater than an acceptable amount and / or concentration and / or activity. In other embodiments, the isolated composition is concentrated relative to the starting material from which the composition is obtained. Such concentration can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999% greater than the starting material, or can be greater than 99.9999%.

[0250] In some embodiments, the isolated formulation is substantially free of residual biological products. In some embodiments, the isolated formulation is free of any biological contaminants by 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, or at least about 90%. Residual biological products can include abiotic substances (including chemicals), or undesired nucleic acids, proteins, lipids, or metabolites.

[0251] As used herein, the term "linked" refers to a first amino acid sequence or polynucleotide sequence that is covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence can be directly linked or juxtaposed to the second amino acid or polynucleotide sequence, but, in contrast, intervening sequences can covalently link the first sequence to the second sequence. The term "linked" means not only fusing a first polynucleotide sequence to a second polynucleotide sequence at the 5'-end or 3'-end, but also includes inserting the entire sequence of the first polynucleotide (or the second polynucleotide sequence) into any two nucleotides within the second polynucleotide sequence (or the first polynucleotide sequence), respectively. The first polynucleotide sequence can be linked to the second polynucleotide sequence by a phosphodiester bond or a linker. The linker can be, for example, a polynucleotide.

[0252] As used herein, the term "retinal disease" or "macular disease" refers to a disease, disorder or condition that affects or is associated with a part or region of the retina or macula of the eye. The retinal or macular disease can be a disease, disorder or condition resulting from damage to the whole or a part of the retina or macula. The retinal or macular disease can occur due to abnormal function or damage of retinal or macular cells. Also, the retinal or macular disease can occur due to abnormal function or damage of photoreceptor cells and / or retinal pigment epithelium (RPE) cells of the retina or macula.

[0253] Sodium iodate (NaIO3) has been reported to induce retinal damage and / or degeneration over a part or the whole of the outer and inner parts of the retina, and to induce damage to the retinal pigment epithelium (RPE) and photoreceptors (A.E.-H. Koh, et al. Journal of Photochemistry & Photobiology, B: Biology 196 (2019) 111514). Therefore, using the NaIO3 model, it is possible to confirm the preventive or therapeutic effect of a disease caused by damage to the retina or macula (e.g., dysfunction or damage of photoreceptor cells and / or retinal pigment epithelial cells). Examples of diseases caused by dysfunction or damage of the photoreceptor cells and / or retinal pigment epithelial cells include retinopathy, choroidal neovascularization, macular disease, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal diseases, or combinations thereof.

[0254] As used herein, the term "retinal pigment epithelium" (RPE) refers to a single layer of epithelial cells located on the posterior side of the vertebrate eye between the choroidal blood supply (choroidal capillary layer) and the neural retina. The RPE acts as one of the components of the blood-retinal barrier, and RPE cells play important roles in maintaining the visual cycle, phagocytosis of the outer segments of photoreceptors, and the transport of nutrients, metabolic wastes, ions, and body fluids between the distal retina and the choroidal capillary layer.

[0255] As used herein, the term "retinopathy" refers to a disease or damage of the retina (i.e., the tissue covering the inner surface on the posterior side of the eye that captures the image passing through the cornea and the lens) or retinal cells.

[0256] As used herein, the term "diabetic retinopathy" (DR) refers to retinopathy induced by complications associated with diabetes. Depending on the severity of the disease, DR can be asymptomatic, cause minor vision problems, or lead to blindness. DR is the result of microvascular retinal changes. Hyperglycemia-induced death of intramural perivascular cells and thickening of the basement membrane can lead to vascular wall failure. These damages change the formation of the blood-retinal barrier and make retinal blood vessels more permeable. The death of perivascular cells can be induced when hyperglycemia continuously activates PKC-δ and p38 mitogen-activated protein kinase (MAPK) encoded by protein kinase C-δ and increases the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase that is a previously unknown target of PKC-δ signaling. This signaling cascade leads to PDGF receptor dephosphorylation and decreased downstream signaling from this receptor, resulting in possible "perivascular cell death". Small blood vessels in the eye, for example, can be particularly vulnerable to poor regulation of blood sugar. Excessive accumulation of glucose and / or fructose can damage small blood vessels in the retina.

[0257] DR can be classified into two distinct stages (Wu L., et al., World J Diabetes 4(6):290-294(2013)). The first stage, non-proliferative diabetic retinopathy (NPDR), is associated with early diabetic retinopathy. NPDR is generally asymptomatic or associated with mild vision distortion caused by vessels leaking fluid into the surrounding tissue. The only way to detect NPDR is by fundus photography, which reveals microaneurysms (tiny bulges filled with blood in the artery wall). If left untreated, DR patients can progress to the second stage, proliferative diabetic retinopathy (PDR). PDR is characterized by abnormal new blood vessel formation (i.e., angiogenesis) that can cause rupture and bleeding, leading to blurred vision. Other symptoms of PDR include spots or dark lines floating in the field of vision ("floaters"), changes in vision, color vision impairment, dark or empty areas in the field of vision, pain, uneven vision, and complete vision loss.

[0258] The term "diabetic retinopathy" includes, but is not limited to, non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), diabetic maculopathy, and diabetic macular edema, and includes all types of diabetic retinopathy.

[0259] In some aspects, PDR occurs after the onset of NPDR (e.g., after initially being diagnosed with NPDR, the disease progresses to PDR). In other aspects, PDR occurs independently of NPDR. Also, as used herein, the term "diabetic retinopathy" includes all types of diabetic retinopathy and any and all symptoms of diabetic retinopathy, regardless of cause. Non-limiting examples of risk factors for diabetic retinopathy include diabetes duration, genetics, excessive alcohol consumption, smoking, high blood pressure, obesity, dyslipidemia, high cholesterol, kidney disease, pregnancy, and kidney damage.

[0260] As used herein, the term "maculopathy" means any pathological condition of the macula, which is the central area of the retina associated with very sensitive and accurate vision. In some aspects, the terms "maculopathy" and "retinopathy" may be used interchangeably (i.e., when only the macula is affected). In some aspects, the maculopathy is diabetic maculopathy.

[0261] "Diabetic maculopathy" occurs when the macula is affected by retinal changes caused by diabetes. The term includes two distinct eye diseases, diabetic macular edema and diabetic ischemic maculopathy. These two types of maculopathy are often comorbid. That is, people with macular edema often have ischemic maculopathy. Ischemic maculopathy occurs with macular edema and can also occur when macular edema is mild. In some aspects, the retinal changes associated with diabetic maculopathy include a decrease in retinal potential within the subject's retina, loss of perivascular cells, acellular capillary formation, vascular congestion, vascular dysfunction, vascular leakage, vascular occlusion, tissue swelling (edema), tissue ischemia, or any combination thereof.

[0262] As used herein, the term "acellular capillary" means a vessel tube having the size of a capillary that has no nucleus anywhere along its length.

[0263] As used herein, the term "vascular congestion" refers to a type of vascular injury and is an important factor in the etiology of various eye diseases disclosed herein (e.g., diabetic macular edema). Vascular congestion is associated with the accumulation of body fluids (e.g., blood within blood vessels) within vascular tissue. In some aspects, vascular congestion can be caused by hyperglycemia (i.e., high blood sugar).

[0264] As used herein, the term "retinal dystrophy" (RD) refers to retinopathy (degenerative disorders of the retina) caused by genetic or other causes.

[0265] As used herein, the term "macular degeneration" refers to any number of disorders and pathologies in which the central portion of the retina (i.e., the macula) degenerates or loses functional activity. The degeneration or loss of functional activity can occur, for example, as a result of cell death, decreased cell proliferation, loss of normal biological function, or combinations thereof. Macular degeneration can lead to and / or manifest as changes in the structural integrity of cells and / or the extracellular matrix of the macula, changes in the normal cell and / or extracellular matrix composition, and / or loss of function of macular cells. The cells can be any cell type that normally exists within or near the macula, including RPE cells, photoreceptors, and / or capillary endothelial cells. Age-related macular degeneration is the most frequently seen form of macular degeneration, but the term "macular degeneration" does not necessarily exclude macular degeneration in non-elderly patients. Non-limiting examples of macular degeneration include: age-related macular degeneration (wet or dry); Best macular dystrophy, Sorsby fundus dystrophy, Malattia Leventinese, Doyne honeycomb retinal dystrophy, Stargardt disease (also referred to as Stargardt macular dystrophy, juvenile macular degeneration, or fundus flavimaculatus), and pigment epithelial detachment-related macular degeneration.

[0266] As used herein, the term "age-related macular degeneration" (AMD) generally refers to a retinopathy that affects primarily the elderly and is associated with loss of central vision due to damage to the central portion of the retina (i.e., the macula). AMD is generally characterized by the progressive accumulation or aggregation of drusen, yellowish insoluble extracellular deposits consisting of extracellular proteins and lipids such as amyloid β, within the macula (primarily between the retinal pigment epithelium (RPE) and the underlying choroid). The accumulation or aggregation of these deposits within the macula can gradually deteriorate the macula and result in damage to central vision. As used herein, the term "macula" refers to the central portion of the retina responsible for central high-resolution color vision.

[0267] Several theories, including oxidative stress, mitochondrial dysfunction, and inflammatory processes, have been proposed, but the etiology of age-related macular degeneration is not well understood. An imbalance between the production and degradation of damaged cellular components leads to the accumulation of harmful products such as intracellular lipofuscin and extracellular drusen. Early atrophy is characterized by areas of thinning or depigmentation of the retinal pigment epithelium (RPE) that precede geographic atrophy in the early stages of AMD. Atrophic changes in the RPE (geographic atrophy) and / or the development of new blood vessels (neovascularization) in the advanced stages of AMD result in photoreceptor death and loss of central vision. In dry (non-exudative) AMD, drusen, cellular debris, accumulate between the retina and choroid, causing atrophy and scarring in the retina. In the more severe wet (exudative) AMD, blood vessels grow in the choroid behind the retina (neovascularization), and exudates and fluid leak out, sometimes inducing bleeding.

[0268] Depending on the extent of drusen present, AMD can be classified into three major stages: (i) early, (ii) intermediate, and (iii) advanced or late. Early AMD is characterized by the presence of numerous small (e.g., less than about 63 microns in diameter) drusen, or a few intermediate-sized (e.g., about 63 to 124 microns in diameter) drusen. At the early stage, patients do not experience vision loss and have no obvious symptoms. The intermediate stage is characterized by the presence of many intermediate-sized drusen or one or more large (e.g., greater than about 125 microns in diameter) drusen. At this stage, some patients may experience blurred spots in the center of their visual field. Advanced or late AMD is characterized by extensive damage to retinal tissue, resulting in a central scotoma and ultimately loss of central vision. Based on the type of damage (e.g., presence or absence of neovascularization), advanced or late AMD can be further divided into two subtypes: (i) geographic atrophy (also called atrophic AMD) and (ii) wet AMD (also called neovascular or exudative AMD).

[0269] AMD has two main forms: (i) dry AMD and (ii) wet AMD. Unless otherwise specified, the term "age-related macular degeneration" includes both dry AMD and wet AMD. Also, as used herein, the term "age-related macular degeneration" includes all types of age-related macular degeneration, and any and all symptoms of age-related macular degeneration, regardless of cause. Non-limiting examples of symptoms associated with macular degeneration (e.g., age-related macular degeneration) include: loss of central vision, distortion, decreased contrast sensitivity, blurring of the visual field, difficulty adapting to low light levels, sudden onset and rapid worsening of symptoms, and decreased color vision. In some embodiments, macular degeneration (e.g., age-related macular degeneration) can cause macular edema (i.e., swelling of the macula due to the accumulation of fluid and protein deposits above or below the macula).

[0270] As used herein, the term "dry AMD" (also referred to as atrophic age-related macular degeneration or non-exudative AMD) refers to all forms of AMD that are not wet (neovascular) AMD. This includes not only the early and intermediate forms of AMD, but also the advanced form of dry AMD known as geographic atrophy. Patients with dry AMD tend to have a faster onset and minimal symptoms initially; loss of visual function occurs more frequently when the symptoms progress to geographic atrophy.

[0271] As used herein, the term "wet AMD" (also referred to as neovascular age-related macular degeneration or exudative AMD) refers to retinal symptoms characterized by the presence of retinal neovascularization and is the most advanced form of AMD. In wet AMD, blood vessels grow from the choroidal capillaries through defects in Bruch's membrane and, in some cases, from the underlying retinal pigment epithelium (choroidal neovascularization or neovascularization). Organization of the serous or hemorrhagic exudates leaking from these blood vessels can cause fibrovascular scarring in the macular area, along with associated degeneration of the neurosensory retina, detachment and rupture of the retinal pigment epithelium, vitreous hemorrhage, and permanent damage to central vision.

[0272] As used herein, the term "angiogenesis" refers to the growth of new abnormal blood vessels in different parts of the eye that can induce bleeding and cause vision loss. As used herein, the term "choroidal angiogenesis" refers to the abnormal growth of new blood vessels in the choroid (i.e., the vascular layer of the eye that contains connective tissue and is located between the retina and the sclera). In wet AMD, the new blood vessels can grow into the retina through the retinal pigment epithelium (RPE) and the choroid, and can damage visual function by blood and lipid leakage. As used herein, the term "retinal angiogenesis" refers to the abnormal development, proliferation, and / or growth of blood vessels in the upper or inner part of the retina, such as on the retinal surface. Retinal angiogenesis can occur in many retinopathies associated with retinal ischemia, such as diabetic retinopathy, sickle cell retinopathy, Eales disease, ocular ischemic syndrome, carotid cavernous fistula, familial exudative vitreoretinopathy, hyperviscosity syndrome, radiation retinopathy, retinal vein occlusion, retinal artery occlusion, retinal embolism, birdshot retinochoroidopathy, choroidal melanoma, chronic retinal detachment, anterior ischemic optic neuropathy (AION), non-arteritic anterior ischemic optic neuropathy (NAION), and incontinentia pigmenti. Methods for detecting angiogenesis are known in the art and include, but are not limited to, measuring the expression of CD31 (platelet endothelial cell adhesion molecule, also known as PECAM-1) and vascular endothelial growth factor (VEGF) in tissues. For example, reference may be made to Schluter A., et al., BMC Cancer l8(1):272(2018).

[0273] As used herein, the term "hereditary retinal disease" refers to a retinal disease in which the structure and function of retinal cells are abnormal due to gene defects or abnormalities. Hereditary retinal diseases vary in onset time and symptoms depending on the causative gene. Non-limiting examples of hereditary retinal diseases include retinitis pigmentosa (RP), Leber congenital amaurosis, Stargardt's disease, Coats retinopathy, cone dystrophy, choroideremia, Usher syndrome, Best's Disease, X-linked retinoschisis, and unspecified hereditary retinal dystrophy.

[0274] As used herein, the term "retinitis pigmentosa" (RP) refers to a retinal disease in which the optic cells and retinal pigment epithelial cells of the retina are damaged (degenerated). Night blindness initially appears due to damage to the optic cells, and the visual field gradually narrows, eventually leading to blindness. Defects in genes involved in the mechanism of converting light into electrical signals within the optic cells are the main cause, and gene abnormalities may also be found in some retinal pigment epithelium, and these gene abnormalities cause extensive retinal damage.

[0275] The most prominent symptom of retinitis pigmentosa is the gradual destruction of rod cells and cone cells, with rod cells being damaged earlier than cone cells. When the destruction of the optic cells progresses to the cone cells, the central visual field can no longer be seen, resulting in complete blindness.

[0276] As used herein, the term "Leber congenital Amaurosis" refers to a hereditary retinal disease that can cause congenital blindness at birth or shortly after birth. In children with Leber congenital amaurosis, since there are no rod cells and cone cells in the retina with normal function, both the cone cell response and the rod cell response disappear in electroretinogram examination. Leber congenital amaurosis is a disease caused by gene abnormalities, and gene mutations are found in 40% to 50% of patients. Among the 12 known gene abnormalities, 11 are autosomal recessive mutations (GUCY2D, RPE65, SPATA7, AIPL1, LCA5, RPGRIP1, CRB1, CEP290, IMPDH1, RD3, RDH12), and rarely there is also an autosomal dominant case (CRX).

[0277] As used herein, the term "Stargardt’s disease" refers to a type of autosomal recessive retinal dystrophy. Stargardt’s disease appears between the ages of 8 and 15, and gradually causes a decrease in central vision due to macular degeneration in both eyes. Currently, it is considered that a gene mutation called ABCA4 causes Stargardt’s disease. The ABCA4 gene mutation causes the accumulation of a substance similar to lipofuscin in the retinal pigment epithelium layer, leading to RPE cell death and photoreceptor disappearance. The ABCA4 mutation is also associated with dysplasia of cone cells and rod cells and severe retinal dystrophy.

[0278] As used herein, the term "Coats retinopathy" refers to a retinal vascular disease that causes dilation of retinal capillaries and hemangiomas, accumulates exudates in the retina and subretinal space, and causes exudative retinal detachment. It is known to be caused by a somatic mutation in the NDP gene on the X chromosome, resulting in a lack of norrin protein necessary for retinal development.

[0279] As used herein, the term "Cone dystrophy" refers to a disease in which cone cells responsible for color vision and central vision among the cells of the retina degenerate due to genetic abnormalities and central vision is lost. Cone dystrophy can be broadly classified into Pure cone dystrophy and Cone-rod dystrophy. The modes of inheritance are diverse, such as autosomal dominant, autosomal recessive, and X-chromosome related, and it can also occur due to its own mutations without showing a specific mode of inheritance.

[0280] As used herein, the term "Choroideremia" refers to a rarely occurring X-chromosome related progressive degeneration of the choroid, retinal pigment epithelium, and photoreceptors. Patients lack REP1 (Rab escort protein 1) due to CHM gene mutations and gradually die while losing the functions of each photoreceptor cell in the retina. In most cases, the symptoms of the disease appear in males, typically starting with night blindness in childhood, and as the disease progresses, peripheral vision decreases, and it is known that central vision is preserved to a certain extent in the early stage. In the case of an ordinary male patient in his forties, the vision is good, but the visual field is very narrow, and vision is also lost when he reaches the age of 50 to 70. In some patients, a decrease in color perception ability may also appear.

[0281] As used herein, the term "Usher syndrome" refers to a genetic disease in which visual impairment progresses along with hearing impairment. The hearing impairment in Usher syndrome is caused by abnormalities in the inner ear, and the visual impairment is related to retinitis pigmentosa (RP).

[0282] As used herein, the term "Best Disease" refers to a hereditary retinal disease caused by mutations in the BEST1 (VMD2) gene. This disease progresses gradually and can cause a decline in central vision. Mutations in the BEST1 (VMD2) gene lead to abnormal function of Bestrophin, a calcium-chloride channel protein in the basolateral plasma membrane of the retinal pigment epithelium, resulting in impaired water movement through the retinal pigment epithelium, which can lead to serous retinal detachment or retinal pigment epithelial detachment.

[0283] As used herein, the term "X-linked Retinoschisis" refers to a disease in which separation occurs between the inner retinal layers due to mutations in the retinoschisis gene (RS1). Retinoschisis causes visual impairment due to abnormal separation of the nerve fiber layer among the ten layers that make up the retina. Juvenile retinoschisis is an X-linked recessive genetic disease that occurs rarely with a worldwide prevalence of 1:120,000.

[0284] The terms "miRNA", "miR", and "microRNA" are used interchangeably and refer to microRNA molecules found in eukaryotes that are involved in RNA-based gene regulation. These terms can be used to refer to single-stranded RNA molecules processed from precursors. In some embodiments, the term "antisense oligomer" may also be used to describe the microRNA molecules of the present disclosure. The names of the miRNAs and their sequences associated with the present disclosure are provided herein. MicroRNAs recognize target mRNAs through imperfect base pairing, bind to the target mRNAs, and downregulate target gene expression by leading to destabilization or translational inhibition of the target mRNAs. Conversely, miRNA targeting through molecules containing miRNA binding sites (generally molecules containing sequences complementary to the seed region of the miRNA) can reduce or inhibit miRNA-induced translational inhibition and can result in upregulation of the target gene.

[0285] The terms "nucleic acid", "nucleic acid molecule", "nucleotide sequence", "polynucleotide" and their grammatical variants are used interchangeably and refer to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented herein in the 5' to 3' direction. The polynucleotides of the present disclosure can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are presented herein in single letter codes such as adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).

[0286] As used herein, the terms "operably linked" or "operatively linked" mean that each DNA sequence being linked is positioned adjacent to each other so as to perform the desired function. For example, when a particular promoter facilitates the initiation of transcription of a coding sequence (e.g., a foreign gene), such a promoter can be operably linked to the coding region. The promoter and the coding region do not necessarily have to be positioned adjacent to each other as long as such functional relationships are maintained.

[0287] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient" and their grammatical variants include all carriers or diluents that are approved by the regulatory agencies of the United States federal government or listed in the United States Pharmacopeia for use in animals including humans, and that do not cause the occurrence of physiologically undesirable effects to the extent that they make the administration of the composition to the subject impossible, and that prevent the elimination of the biological activity and properties of the administered complex. They include excipients and carriers that are useful in the manufacture of pharmaceutical compositions, generally safe and non-toxic, and preferred.

[0288] As used herein, the term "pharmaceutical composition" refers to one or more of the compositions described herein (e.g., polynucleotides, vectors, cells and / or recombinant viruses) that are mixed with or suspended in one or more other chemical components such as pharmaceutically acceptable carriers and excipients.

[0289] As used herein, the terms "promoter" and "promoter sequence" are interchangeable and refer to a DNA sequence capable of regulating the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' to the promoter sequence. A promoter can be entirely derived from a native gene, can be composed of different elements each derived from different naturally-occurring promoters, or can also contain synthetic DNA portions. One of ordinary skill in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. A promoter that expresses most genes in most host cell types is usually referred to as a "constitutive promoter". A promoter that expresses a gene in a specific cell type is usually referred to as a "cell-specific promoter" or "tissue-specific promoter". A promoter that expresses a gene at a specific developmental or cell differentiation stage is usually referred to as a "development-specific promoter" or "cell differentiation-specific promoter". A promoter that is induced to express a gene after exposing or treating cells with an agent, biological molecule, chemical, ligand, light, etc. that induces the promoter is usually referred to as an "inducible promoter" or "regulatory promoter". Also, in most cases, since the exact boundaries of regulatory sequences have not been fully defined, each DNA fragment of different lengths is recognized as capable of having the same promoter activity.

[0290] The promoter sequence typically extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a detectable level of background aberrations, bordering the transcription start site at its 3' end. Within the promoter sequence, not only protein-binding domains (consensus sequences) responsible for the binding of RNA polymerase but also transcription start sites (e.g., conveniently defined by mapping with nuclease S1) can be found. In some embodiments, promoters that can be used in the present disclosure include tissue-specific promoters.

[0291] As used herein, the term "gene regulatory region" or "regulatory region" refers to a nucleotide sequence located within the upstream (5' untranslated sequence) or downstream (3' untranslated sequence) of a coding region and affecting the transcription, RNA processing, stability or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites or stem-loop structures. When the coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences can typically be located 3' to the coding sequence.

[0292] In some embodiments, the polynucleotides described herein (e.g., including foreign genes and untranslated nucleic acid sequences) can include a promoter and / or other expression (e.g., transcription) regulatory elements operably associated with one or more coding regions. In an operable association, the coding region for a gene product is associated with one or more regulatory regions in such a way that the expression of the gene product is placed under the influence or control of the regulatory region. For example, a coding region and a promoter are "operably associated" when induction of the promoter function causes transcription of an mRNA encoding the gene product encoded by the coding region and the linkage characteristics between the promoter and the coding region do not interfere with the ability of the promoter to direct the expression of the gene product or the ability of the DNA template to be transcribed. Also, other expression regulatory elements other than promoters, such as enhancers, operators, repressors and transcription termination signals, can be operably associated with a coding region that directs gene product expression.

[0293] As used herein, the terms "subject", "patient", "individual", "host" and variants thereof are interchangeable and refer to any mammalian subject to which any of the compositions described herein (e.g., polynucleotides, recombinant expression constructs, vectors, cells, pharmaceutical compositions or recombinant viruses) are administered. Non-limiting examples include humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.) and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.) in need of diagnosis, treatment or therapy, particularly humans. The methods described herein are applicable to all human prophylactic or therapeutic and veterinary uses.

[0294] As used herein, the phrase "subject in need" includes subjects such as mammalian subjects that would benefit from administration of the compositions described herein.

[0295] As used herein, the term "therapeutically effective amount" is an amount of a reagent or pharmaceutical complex, including a composition of the present disclosure (e.g., a polynucleotide comprising a foreign gene and untranslated nucleic acid sequences), sufficient to obtain a desired therapeutic, pharmacological and / or physiological effect in a subject in need. A therapeutically effective amount can be a "prophylactically effective amount" since prophylaxis can be considered treatment.

[0296] As used herein, the term "foreign gene" refers to at least one polynucleotide, polynucleotide region, expression product of said polynucleotide or polynucleotide region, polypeptide or multiple polypeptides encoded by a recombinant expression construct, or a nucleic acid that promotes or regulates. In some embodiments, the foreign gene can be heterologous to the cell into which it is inserted (or transduced) (i.e., not naturally expressed in the cell).

[0297] As used herein, the terms "treatment" or "treating" refer to, for example, reducing the severity of a disease or condition, reducing the duration of the disease course, improving or eliminating one or more symptoms associated with the disease or condition, and providing a beneficial effect to a subject having the disease or condition without necessarily curing the disease or condition. The terms also include preventing or precluding a disease, condition or its symptoms.

[0298] The term "upstream" means a nucleotide sequence located 5' to a reference nucleotide sequence.

[0299] As used herein, the terms "vector" or "construct" refer to any vehicle into which a nucleic acid or gene can be inserted, e.g., a delivery vehicle that can be introduced into a cell and in which a nucleic acid sequence can be inserted and replicated. The nucleic acid sequence that can be inserted into the vector can be exogenous or heterologous. The nucleic acid sequence can be a foreign gene. Examples of constructs include, but are not limited to, plasmids, cosmids, and viruses (e.g., AAV). Those skilled in the art can produce the vector or construct by standard recombinant techniques (such as Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.). As used herein, the terms "expression vector" or "expression construct" mean a vector or construct that contains a nucleotide sequence encoding at least a portion of the gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression constructs can contain various regulatory elements. Along with regulatory sequences that regulate transcription and translation, vectors and expression vectors can also contain nucleotide sequences that provide other functions. Viruses that can be utilized in the present invention include, but are not limited to, retroviruses, herpes simplex viruses, lentiviruses, poxviruses, vaccinia viruses, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, adeno-associated viruses (AAV), etc.

[0300] The vector can be engineered to encode a selectable marker or reporter provided for the selection or identification of cells transfected with the vector. Expression of the selectable marker or reporter enables the identification and / or selection of host cells that integrate and express other coding regions contained in the vector. Examples of selectable marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamides, etc.; and genes used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentenyl transferase genes, and the like. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. A selectable marker can also be regarded as a reporter.

[0301] Generally, recombinant adeno-associated virus (AAV) is produced by triple transfection of host cells (e.g., HEK293 cells). It is 1) an AAV production plasmid having a gene expression cassette flanked by ITRs (inverted terminal repeats), 2) a "Rep-Cap plasmid" that provides the Rep proteins required for replication of the adeno-associated virus genome and the capsid proteins that make up the viral particles, and finally, 3) a "helper plasmid" that provides the adenovirus proteins (E2a, E4) and RNA (VA RNA) that facilitate the life cycle of AAV. AAV is produced when these three types of plasmids are transfected into HEK293 cells, etc., that provide the adenovirus E1 and E3 genes.

[0302] As used herein, the term "dual helper plasmid" means a plasmid that can provide two or more of the requirements for generating AAV in cells. As is apparent from the present disclosure, in some aspects, the dual helper plasmids described herein provide the above-described requirements 2) "plasmid" and 3) "helper plasmid". For example, in some aspects, the dual helper plasmid contains the rep gene, the cap gene, the E2a gene, the E4 gene, and the VA RNA gene.

[0303] The dual helper plasmids described herein not only contain the above-described genes, but the genes are arranged in a specific configuration within the plasmid. For example, in some aspects, the E2a gene, the E4 gene, and the VA RNA gene are sequentially linked within the dual helper plasmid, and the rep gene and the cap gene (collectively referred to herein as the "rep-cap gene") are sequentially linked clockwise (from 5' to 3') between the 5'-end of the E2a gene and the 3'-end of the VA RNA gene. More specifically, in some aspects, the 5'-end of the rep-cap gene is linked to the 5'-end of the E2a gene, where the 3'-end of the rep-cap gene is linked to the 3'-end of the VA RNA gene. In some aspects, the E2a gene, the E4 gene, and the VA RNA gene are sequentially linked, and the rep-cap gene is counterclockwise (from 3' to 5') between the 5'-end of the E2a gene and the 3'-end of the VA RNA gene. More specifically, in some aspects, the 3'-end of the rep-cap gene is linked to the 5'-end of the E2a gene, where the 5'-end of the rep-cap gene is linked to the 3'-end of the VA RNA gene.

[0304] As used herein, the term "cell" includes eukaryotic and prokaryotic cells and means any transformable cell capable of replicating the vector or expressing the gene encoded by the vector. A cell can be transfected, transduced or transformed by the vector, which means the process by which an exogenous polynucleotide (nucleic acid molecule) is transmitted or introduced into a host cell. As used herein, the term "transformation" is used to mean including the transfection and transduction.

[0305] The (host) cells of the present invention include, but are not limited to, preferably insect cells or mammalian cells, more preferably Sf9 in the case of insect cells, and HEK293 cells, HeLa cells, ARPE-19 cells, RPE-1 cells, HepG2 cells, Hep3B cells, Huh-7 cells, C8D1a cells, Neuro2A cells, CHO cells, MES13 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, MCF-7 cells, HC70 cells, HCC1428 cells, BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, MIA PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells, PC-12 cells, NIH3T3 cells, etc. can be used. In some embodiments, the host cell is an isolated host cell.

[0306] As used herein, the terms "family having sequence similarity 19", "FAM19", or "TAFA" mean proteins belonging to the TAFA family of 5 proteins (also known as the FAM19 family) and mainly expressed in the brain and spinal cord. FAM19A1 is also known as TAFA1, FAM19A2 is also known as TAFA2, FAM19A3 is also known as TAFA3, FAM19A4 is also known as TAFA4, and FAM19A5 is also known as TAFA5.

[0307] The human TAFA1 gene encodes a 133-amino acid sequence, and there are different views on the lengths of the signal peptide and the mature protein. However, in the case of the human TAFA1 protein (SEQ ID NO: 302), it is predicted to be composed of a 35-amino acid signal peptide and a 98-amino acid mature protein. TAFA1 shows high expression in the prefrontal cortex, temporal cortex, occipital cortex, and posterior medial cortex, and low expression in the basal ganglia, lateral ventricle, and cerebellum. Through cell experiments, it has been revealed that TAFA1 affects the differentiation fate determination of neural stem cells, inhibits the differentiation of neural stem cells into astrocytes, and promotes the differentiation into neurons. Through TAFA1 Knock-Out (KO) mouse experiments, it is known that TAFA1 can regulate motor activity, behavior towards anxiety, learning and memory, and somatosensory function.

[0308] The human TAFA2 gene encodes a 131-amino acid sequence, and there are different views on the lengths of the signal peptide and the mature protein. However, in the case of the human TAFA2 protein (SEQ ID NO: 303), it is predicted to be composed of a 30-amino acid signal peptide and a 101-amino acid mature protein. TAFA2 is abundantly expressed in the occipital and prefrontal cortex and the medulla oblongata within the central nervous system. When the TAFA2 recombinant protein is injected into the third ventricle of mice, it is known to increase the food intake and meal frequency of mice, increase energy consumption, and increase the respiratory exchange ratio. Therefore, it is suggested that TAFA2 may play a role in food intake and energy metabolism regulation. Also, it is known that when TAFA2 is suppressed in zebrafish and mice, anxiety-related behaviors increase.

[0309] The human TAFA3 gene encodes a 133-amino acid sequence, and there are differences in the lengths of the signal peptide and the mature protein. However, in the case of the human TAFA3 protein (SEQ ID NO: 304), it is predicted to be composed of a 30-amino acid signal peptide and a 103-amino acid mature protein. In a transient focal cerebral ischemia mouse model, the expression of TAFA3 increases in microglia, and it is known that microglia treated with TAFA3 polarize into anti-inflammatory microglia. Also, in a mouse model, when TAFA3 is knocked out, three major behavioral deficits observed in the autism spectrum, such as reduced response to social novelty, social communication disorder, and increased repetitive behavior, appear, suggesting that TAFA3 is involved in the normal function of forming social relationships.

[0310] The human TAFA4 gene encodes a 140-amino acid sequence, and there are differences in the lengths of the signal peptide and the mature protein. However, in the case of the human TAFA4 protein (SEQ ID NO: 299), it is predicted to be composed of a 45-amino acid signal peptide and a 95-amino acid mature protein. TAFA4 is mainly expressed in sensory neurons of the peripheral nervous system. The TAFA4 protein is specifically expressed in particular low-threshold mechanoreceptors (C-low-threshold mechanoreceptors), and is considered to regulate the activity of interneurons, particularly GABAergic neurons, and reduce pain.

[0311] The human TAFA5 gene encodes a 132-amino acid sequence, and although there are different views on the lengths of the signal peptide and mature protein, in the case of the human TAFA5 protein, it is expected to be composed of a 43-amino acid signal sequence and an 89-amino acid mature protein. TAFA5 shows high expression in the basal ganglia region and the cerebellum. It is known that the expression of TAFA5 increases in the hypothalamus of mice by inflammatory stimuli such as TNF-α, and when TAFA5 is KO, the food intake, weight loss, and increase in inflammatory cytokines decreased by TNF-α are partially restored.

[0312] The five TAFA families are expected to have different functions in vivo. However, in the case of TAFA1 to TAFA4 mature proteins, they show high sequence identity (for example, human TAFA4 has a 73.7% sequence identity with TAFA1, an 85.3% sequence identity with TAFA2, and an 81.1% sequence identity with TAFA3). On the other hand, TAFA5 shows low sequence identity with TAFA1 to 4 (for example, human TAFA5 has a 48.9% sequence identity with TAFA1, a 51.1% sequence identity with TAFA2, a 47.7% sequence identity with TAFA3, and a 50.0% sequence identity with TAFA4), and it can be expected that they have completely different functions from each other (see Figure 21).

[0313] Human TAFA4 (TAFA Chemokine Like Family Member 4) protein (SEQ ID NO: 299) is one of the TAFA proteins (TAFA1-5) that are abundantly expressed throughout the brain regions. The TAFA4, which is composed of 140 amino acids, has 95 amino acids (SEQ ID NO: 171) excluding the first 45 amino acids at the front, and these 95 amino acids are very well conserved evolutionarily within vertebrates. Also, among the TAFA proteins, it is confirmed that TAFA1 to 4 have very well conserved sequence identity among species within vertebrates. i) The mature human TAFA4 amino acid sequence shows a sequence identity of 90% or more compared to TAFA4 of mammals and amphibians, 95% or more compared to TAFA4 of birds, and 85% or more compared to TAFA4 of reptiles and fish (see Figure 19). ii) The human TAFA1 amino acid sequence shows a sequence identity of 90% or more compared to TAFA1 of mammals and amphibians, 95% or more compared to TAFA1 of birds, and 85% or more compared to TAFA1 of reptiles and fish (see Figure 16). iii) The human TAFA2 amino acid sequence shows a sequence identity of 90% or more compared to TAFA2 of mammals and amphibians, 95% or more compared to TAFA2 of birds, and 80% or more compared to TAFA2 of reptiles and fish (see Figure 17). iv) The human TAFA3 amino acid sequence shows a sequence identity of 75% or more compared to TAFA3 of mammals and amphibians, 80% or more compared to TAFA3 of birds, and 75% or more compared to TAFA3 of reptiles and fish (see Figure 18). Thus, it can be confirmed that regardless of species, they show very high sequence identity.

[0314]

[0315] II. Polynucleotide

[0316] II.A. Un-translated Nucleic Acid Sequence

[0317] The present disclosure relates to a polynucleotide comprising an un-translated nucleic acid sequence, wherein the un-translated nucleic acid sequence can increase the expression of a foreign gene during translation. Specifically, the present specification provides an elongation factor-1 alpha (EF-1α) intron sequence shorter than the full-length EF-1α intron.

[0318] Elongation factor 1 alpha (EF-1α) is a gene located on chromosome 6 (nucleotides 73,489,308 to 73,525,587 of GenBank accession number NC_000006.12; minus strand direction). The EF-1α gene contains 8 exons and 7 introns and encodes the eukaryotic elongation factor 1A (also known as eEF1A1 and eEF1A) protein, which plays an important role in mRNA translation (for example, transporting aminoacyl-tRNA as a ternary complex eEF1A1-GTP-aa-tRNA to the A site of the ribosome). See Scaggiante et al., Atlas Genet Cytogenet Oncol Haematol 19(4):256-265 (March 2015). The nucleotide sequence of the full-length EF-1α intron is set forth in SEQ ID NO: 1 (924 nucleotides in length). As described herein, the untranslated nucleic acid sequences of the present disclosure (i.e., EF-1α intron fragment sequences) provide distinct advantages over the full-length EF-1α intron (or other introns known in the art). For example, in some embodiments, the untranslated nucleic acid sequences described herein can further increase the expression of foreign genes more greatly than the full-length EF-1α intron. Also, since the untranslated nucleic acid sequences of the present disclosure are shorter than their full-length counterparts, in some embodiments, they can be used in combination with larger foreign genes. For example, an AAV capsid (the same as those described herein) can accommodate nucleic acids up to approximately 4.7 kb. Thus, in some embodiments, larger foreign genes and / or additional cis-elements can be integrated with the EF-1α intron fragments (i.e., untranslated nucleic acid sequences) described herein to further increase gene expression.

[0319] In some embodiments, the untranslated nucleic acid sequence described herein (i.e., the EF-1α intron fragment) includes nucleotides at positions 874 to 924 of the sequence shown in SEQ ID NO: 1, but does not include SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence consists essentially of nucleotides 874 to 924 of SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence consists of nucleotides 874 to 924 of SEQ ID NO: 1. Such an EF-1α intron fragment is also referred to herein as the "T3.2 fragment" and is shown in SEQ ID NO: 57. In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 874 to 924 of SEQ ID NO: 1, and the untranslated nucleic acid sequence does not include SEQ ID NO: 1.

[0320] In some embodiments, the untranslated nucleic acid sequence described herein (i.e., the EF-1α intron fragment) includes nucleotides at positions 852 to 924 of SEQ ID NO: 1, but does not include SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence of the present disclosure consists essentially of nucleotides 852 to 924 of SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence consists of nucleotides 852 to 924 of SEQ ID NO: 1. Such an EF-1α intron fragment is also referred to herein as the "T3.1.2 fragment" and is described in SEQ ID NO: 3. In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 852 to 924 of SEQ ID NO: 1, and the untranslated nucleic acid sequence does not include SEQ ID NO: 1.

[0321] In some embodiments, the untranslated nucleic acid sequence (i.e., the EF-1α intron fragment) described herein includes nucleotides 830 to 924 of SEQ ID NO: 1, but does not include SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence of the present disclosure is essentially composed of nucleotides 830 to 924 of SEQ ID NO: 1. In some embodiments, the untranslated nucleic acid sequence consists of nucleotides 830 to 924 of SEQ ID NO: 1. Such an EF-1α intron fragment is also referred to herein as the "T3.1.1 fragment" and is set forth in SEQ ID NO: 2. In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 830 to 924 of SEQ ID NO: 1, and the untranslated nucleic acid sequence does not include SEQ ID NO: 1.

[0322] In some embodiments, the untranslated nucleic acid sequence (i.e., the EF-1α intron fragment) described herein is composed of 29 to 174 nucleotides, and the untranslated nucleic acid sequence includes a nucleotide sequence having at least about 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 57. In some embodiments, the untranslated nucleic acid sequence is composed of 51 to 117 nucleotides, and the untranslated nucleic acid sequence includes a nucleotide sequence having at least about 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 57. In some embodiments, the untranslated nucleic acid sequence is composed of 51 to 117 nucleotides, and the untranslated nucleic acid sequence includes the nucleotide sequence shown in SEQ ID NO: 57.

[0323] In some embodiments, the untranslated nucleic acid sequences described herein have at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with (i) nucleotides 871 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 58), (ii) nucleotides 861 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 59), (iii) nucleotides 852 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 60), (iv) nucleotides 851 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 61), (v) nucleotides 830 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2), (vi) nucleotides 821 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 63), (vii) nucleotides 811 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 64), (viii) nucleotides 808 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 65), (ix) nucleotides 801 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 66), (x) nucleotides 751 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 67), (xi) nucleotides 721 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 68), (xii) nucleotides 701 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 69), (xiii) nucleotides 651 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 70), (xiv) nucleotides 601 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 71), (xv) nucleotides 570 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 72), (xvi) nucleotides 551 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 73), or (xvii) nucleotides 501 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 74).

[0324] In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with the nucleotides from position 871 to position 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 58). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with the nucleotides from position 861 to position 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 59). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with the nucleotides from position 852 to position 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 60). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with the nucleotides from position 851 to position 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 61). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with the nucleotides from position 830 to position 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 2). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with the nucleotides from position 821 to position 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 63).In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 811 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 64). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 808 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 65). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 801 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 66). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 751 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 67). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 721 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 68).In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 701 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 69). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 651 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 70). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 601 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 71). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 570 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 72). In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 551 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 73).In some embodiments, the untranslated nucleic acid sequence has at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or about 100% sequence identity with nucleotides 501 to 924 of SEQ ID NO: 1 (i.e., SEQ ID NO: 74).

[0325] As is apparent from the disclosure, in some embodiments, a polynucleotide comprising the untranslated nucleic acid sequence described herein further comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at the 5' end ("5' region") of the untranslated nucleic acid sequence. In some embodiments, a polynucleotide described herein further comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at the 3' end ("3' region") of the untranslated nucleic acid sequence. In some embodiments, a polynucleotide comprising the untranslated nucleic acid sequence further comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 nucleotides at both the 5' end ("5' region") and the 3' end ("3' region") of the untranslated nucleic acid sequence.

[0326] In some embodiments, the polynucleotide comprises one or more contiguous or non - contiguous nucleotides corresponding to positions 1 to 873 in SEQ ID NO: 1 in the 5' region of the untranslated nucleic acid sequence.

[0327]

[0328] II.B. Foreign gene

[0329] In some embodiments, a polynucleotide comprising the untranslated nucleic acid sequence described herein further comprises a foreign gene. In some embodiments, the foreign gene is a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the nucleic acid has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 184.

[0330]

[0331] II.C. Regulatory element

[0332] In some embodiments, the polynucleotide described herein further comprises a regulatory element. Thus, in some embodiments, the polynucleotide comprises (1) a regulatory element, (2) the untranslated nucleic acid sequence described herein, and (3) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184).

[0333] As used herein, the term "regulatory element" refers to a nucleic acid sequence that regulates (e.g., increases or decreases) the expression of an operably linked nucleic acid. Regulatory elements useful in the present disclosure include enhancers (e.g., CMV enhancer), promoters (e.g., CMV promoter, EF-1α promoter or β-actin promoter), exons (e.g., exon 1 or exon 2), splicing donor sequences, acceptor sequences or combinations thereof. In some embodiments, the regulatory element can include a sequence for transcriptional termination (e.g., poly A), a sequence for stable expression of a foreign gene (e.g., WPRE sequence), a sequence for reducing the occurrence of foreign gene-specific immunity (e.g., miRNA target sequence) or combinations thereof.

[0334]

[0335] II.C.1. Enhancer

[0336] In some embodiments, the regulatory element is an enhancer. Thus, in some embodiments, the polynucleotide described herein comprises (1) an enhancer, (2) an untranslated nucleic acid sequence, and (3) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184), in any order. In some embodiments, the polynucleotide described herein comprises, in the 5' to 3' direction: (1) an enhancer, (2) an untranslated nucleic acid sequence and (3) a foreign gene.

[0337] Any suitable enhancer known in the art can be used in the present disclosure. Non-limiting examples of suitable enhancers include: cytomegalovirus (CMV) enhancer, SV40 early enhancer, adenovirus 5 E1A enhancer, HBV enhancer-1 regulatory region (Eh-1), HPV-16 or -18 E6 / 7 long regulatory region (LCR), HIV-1 long terminal repeat (LTR), or any combination thereof. In some embodiments, the enhancer is a cytomegalovirus (CMV) enhancer. In some embodiments, the CMV enhancer comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 4. In some embodiments, the cytomegalovirus (CMV) enhancer comprises the nucleotide sequence shown in SEQ ID NO: 4.

[0338]

[0339] II.C.2. Promoter

[0340] In some embodiments, the regulatory element is a promoter. Thus, in some embodiments, the polynucleotides described herein include, without a particular order, (1) a promoter, (2) an untranslated nucleic acid sequence, and (3) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184). In some embodiments, the polynucleotide includes, in the 5' to 3' direction: (1) a promoter, (2) an untranslated nucleic acid sequence, and (3) a foreign gene. In some embodiments, the regulatory element includes both an enhancer and a promoter. In such embodiments, the polynucleotide can include, without a particular order, (1) an enhancer, (2) a promoter, (3) an untranslated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the polynucleotide includes, in the 5' to 3' direction: (1) an enhancer, (2) a promoter, (3) an untranslated nucleic acid sequence, and (4) a foreign gene. Any suitable promoter known in the art can be used in the present disclosure.

[0341] In some embodiments, the promoter includes the cytomegalovirus (CMV) promoter, EF-1α promoter, β-actin promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, 70-kDa heat shock protein (HSP70) promoter, 78-kDa glucose-regulated protein (GRP78) promoter, eukaryotic initiation factor-4A (eIF4a) promoter, alpha-1-antitrypsin (AAT) promoter, transthyretin (TTR) promoter, glial fibrillary acidic protein (GFAP) promoter, early promoter of simian virus 40 (SV40), synapsin I (SYN1) promoter, G protein-coupled receptor kinase (GRK) promoter, rhodopsin (Rho) promoter, or a combination thereof.

[0342] In some embodiments, a promoter useful in the present disclosure is the CMV promoter. Thus, in some embodiments, the polynucleotide comprises (1) an enhancer (e.g., the CMV enhancer), (2) the CMV promoter, (3) an untranslated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the CMV promoter comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 5 or 6. In some embodiments, the CMV promoter comprises the nucleotide sequence shown in SEQ ID NO: 5 or 6.

[0343] In some embodiments, a promoter that can be used in the present disclosure is the EF-1α promoter. In some embodiments, the polynucleotide comprises (1) an enhancer (e.g., the CMV enhancer), (2) the EF-1α promoter, (3) an untranslated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the EF-1α promoter comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 7. In some embodiments, the EF-1α promoter comprises the nucleotide sequence shown in SEQ ID NO: 7.

[0344] In some embodiments, the promoter is a β-actin promoter. Thus, in some embodiments, the polynucleotide described herein comprises (1) an enhancer (e.g., CMV enhancer), (2) a β-actin promoter, (3) an untranslated nucleic acid sequence, and (4) a foreign gene. In some embodiments, the β-actin promoter comprises a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 8. In some embodiments, the β-actin promoter is the same chicken β-actin promoter as shown in SEQ ID NO: 8.

[0345] As demonstrated herein (see Figure 4a), in some embodiments, the polynucleotides described herein (e.g., those comprising a foreign gene and an untranslated nucleic acid sequence) can comprise multiple promoters. For example, in some embodiments, the polynucleotide comprises a combination of a CMV promoter, an EF-1α promoter, and / or a β-actin promoter. In some embodiments, the polynucleotide comprises both two of the CMV promoter and the EF-1α promoter. In such embodiments, the CMV promoter can be a part of the full-length CMV promoter, such as the sequence shown in SEQ ID NO: 5 (i.e., the first 31 nucleotides from the 5'-end of SEQ ID NO: 6).

[0346]

[0347] II.C.3. Splicing donor sequence

[0348] In some embodiments, the polynucleotides described herein (i.e., including untranslated nucleic acid sequences) include splicing donor sequences. As used herein, the term "splicing donor sequence" or "splicing donor site" refers to a guanine-thymine (GT)-rich domain present at the 5'-end of an intron (e.g., the EF-1α intron), which implies the boundary between the intron and the exon. As demonstrated herein, such sequences can be targeted to generate the untranslated nucleic acid sequences of the present disclosure. In some embodiments, the splicing donor sequence is ligated upstream of an EF-1α intron fragment (i.e., an untranslated nucleic acid sequence). For example, in some embodiments, the polynucleotides described herein include, in no particular order: (1) an enhancer (e.g., the CMV enhancer), (2) a promoter (e.g., the CMV promoter, the EF-1α promoter, and / or the β-actin promoter), (3) a splicing donor sequence, (4) an untranslated nucleic acid sequence, and (5) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184). In some embodiments, the polynucleotide comprises, in the 5' to 3' direction: (1) an enhancer (e.g., the CMV enhancer), (2) a promoter (e.g., the CMV promoter, the EF-1α promoter, and / or the β-actin promoter), (3) a splicing donor sequence, (4) an untranslated nucleic acid sequence, and (5) a foreign gene.

[0349] In some embodiments, the splicing donor sequences useful in the present disclosure have at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequences shown in SEQ ID NO: 9 or 10. In some embodiments, the splicing donor sequence comprises the nucleotide sequence shown in SEQ ID NO: 9 or 10.

[0350]

[0351] II.C.4. Exon Sequences

[0352] As demonstrated in this specification, in some embodiments, the polynucleotides of the present disclosure further include one or more exon sequences. For example, in some embodiments, the polynucleotides described herein include the EF-1α exon 2 (E2) sequence. Thus, in some embodiments, the polynucleotides described herein include the following features (in no particular order): (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter and / or β-actin promoter), (3) a splicing donor sequence, (4) an untranslated nucleic acid sequence, (5) the EF-1α E2 sequence, and (6) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184). In some embodiments, such polynucleotides include, in the 5' to 3' direction: (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter and / or β-actin promoter), (3) a splicing donor sequence, (4) an untranslated nucleic acid sequence, (5) the EF-1α E2 sequence, and (6) a foreign gene. In some embodiments, the EF-1α E2 sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 11. In some embodiments, the EF-1α E2 sequence includes the nucleotide sequence shown in SEQ ID NO: 11.

[0353] In some embodiments, one or more exon sequences that may be included in the polynucleotides described herein include cytomegalovirus (CMV), EF-1α or beta-actin exon 1 (E1) sequences. In some embodiments, the polynucleotides described herein can include both of the two E1 and E2 sequences. For example, in some embodiments, the polynucleotide includes (in no particular order): (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter and / or beta-actin promoter), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence and / or beta-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, and (7) a foreign gene. In some embodiments, the polynucleotide includes (in the 5' to 3' direction): (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter and / or beta-actin), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence and / or beta-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, and (7) a foreign gene.

[0354] In some embodiments, the E1 sequence that can be used in the present disclosure is the CMV E1 sequence. In some embodiments, the CMV E1 sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 12. In some embodiments, the CMV E1 sequence includes the nucleotide sequence shown in SEQ ID NO: 12.

[0355] In some embodiments, the E1 sequence is an EF-1α E1 sequence. In some embodiments, the EF-1α E1 sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 13. In some embodiments, the EF-1α E1 sequence comprises the nucleotide sequence shown in SEQ ID NO: 13. In some embodiments, the E1 sequence is a β-actin E1 sequence. In some embodiments, the β-actin E1 sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 14 or 15. In some embodiments, the β-actin E1 sequence comprises the nucleotide sequence shown in SEQ ID NO: 14 or 15.

[0356]

[0357] II.C.5. miRNA Target Sequence

[0358] As described herein, in some embodiments, the regulatory elements of the polynucleotides described herein include one or more target sequences (“miRNA target sequences”) for microRNAs (miRNAs) specific to immune cells. Thus, in some embodiments, the polynucleotides described herein include the following features (in no particular order): (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter and / or β-actin promoter), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence and / or β-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184), and (8) one or more miRNA target sequences. In some embodiments, the polynucleotide comprises, in the 5' to 3' direction: (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter and / or β-actin promoter), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence and / or β-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) a foreign gene, and (8) one or more miRNA target sequences.

[0359] In some embodiments, the polynucleotides described herein include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more miRNA target sequences. In some embodiments, the number of said target sequences for miRNAs that can be included in the polynucleotides described herein is from about 2 to about 6 (e.g., 2 to 6). In some embodiments, said multiple miRNA target sequences are identical. In some embodiments, one or more of said multiple miRNA target sequences are different from each other.

[0360] As is apparent from the present disclosure, the inclusion of one or more miRNA target sequences can improve the specificity of the polynucleotides described herein. For example, when inhibition of the foreign gene is preferred in a specific cell type (e.g., immune cells), the expression of the foreign gene can be suppressed in immune cells using a target sequence for an miRNA specific to immune cells, and as a result, the generation of foreign gene-specific immunity by immune cells can be blocked. Therefore, by using different miRNA target sequences, the expression of foreign genes can be regulated in different cells / tissues.

[0361] Any suitable miRNA target sequence known in the art can be used in the present disclosure. In some embodiments, the miRNA target sequence is specific for miR142-3p or miR142-5p. In some embodiments, the target sequence for the miRNA is an antisense oligonucleotide, anti-miR, short hairpin RNA (shRNA) molecule, short interfering RNA (siRNA) molecule, ribozyme, peptide nucleic acid (PNA) oligonucleotide, locked nucleic acid (LNA) oligonucleotide, or a combination thereof having a sequence complementary to the full-length or partial sequence of miR142-3p or miR142-5p.

[0362] In some embodiments, the miRNA target sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 16. In some embodiments, the miRNA target sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 17. In some embodiments, the miRNA target sequence comprises the nucleotide sequence shown in SEQ ID NO: 16 or 17.

[0363]

[0364] II.C.6. WPRE Sequence

[0365] In some embodiments, the polynucleotides described herein (i.e., including untranslated nucleic acid sequences) further comprise a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. Thus, in some embodiments, the polynucleotides described herein comprise, in no particular order: (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter, and / or β-actin promoter), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence, and / or β-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184), (8) one or more miRNA target sequences, and (9) a WPRE sequence. In some embodiments, the polynucleotides described herein comprise, in the 5' to 3' direction: (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter, and / or β-actin promoter), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence, and / or β-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) a foreign gene, (8) one or more miRNA target sequences, and (9) a WPRE sequence.

[0366] In some embodiments, the WPRE sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 18. In some embodiments, the WPRE sequence comprises the nucleotide sequence set forth in SEQ ID NO: 18.

[0367]

[0368] II.C.7. Polyadenylation sequence

[0369] In some embodiments, the polynucleotides described herein (i.e., including untranslated nucleic acid sequences) further include one or more polyadenylation (pA) sequences. Thus, in some embodiments, the polynucleotide includes, in no particular order: (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter, and / or β-actin promoter), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence, and / or β-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184), (8) one or more miRNA target sequences, (9) a WPRE sequence, and (10) one or more pA sequences. In some embodiments, the polynucleotide includes, in the 5' to 3' direction: (1) an enhancer (e.g., CMV enhancer), (2) a promoter (e.g., CMV promoter, EF-1α promoter, and / or β-actin), (3) an E1 sequence (e.g., CMV E1 sequence, EF-1α E1 sequence, and / or β-actin E1 sequence), (4) a splicing donor sequence, (5) an untranslated nucleic acid sequence, (6) an EF-1α E2 sequence, (7) a foreign gene, (8) one or more miRNA target sequences, (9) a WPRE sequence, and (10) one or more pA sequences.

[0370] Any suitable pA sequence known in the art can be used in the present disclosure. In some embodiments, examples of polyadenylation sequences include, but are not limited to, the human growth hormone (hGH) pA sequence, the bovine growth hormone (bGH) pA sequence, the simian virus 40 (SV40) early pA sequence, and the SV40 late pA sequence.

[0371] In some embodiments, the pA sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 19. In some embodiments, the pA sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the pA sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 21. In some embodiments, the pA sequence has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 22. In some embodiments, the polyadenylation sequence is selected from the group consisting of each nucleotide sequence shown in SEQ ID NOs: 19 to 22.

[0372] As described above, in some embodiments, the polynucleotides described herein comprise (i) a foreign gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO: 184), and (ii) a regulatory element operably linked to the foreign gene, the regulatory element comprising, in the 5' to 3' direction: (1) the CMV enhancer sequence shown in SEQ ID NO: 4, (2) the CMV promoter sequence shown in SEQ ID NO: 5 or 6, the EF-1α promoter sequence shown in SEQ ID NO: 7, or the chicken β-actin promoter sequence shown in SEQ ID NO: 8, (3) the CMV E1 sequence shown in SEQ ID NO: 12, the EF-1α E1 sequence shown in SEQ ID NO: 13, or the chicken β-actin E1 sequence shown in SEQ ID NO: 14 or 15, (4) the splicing donor sequence shown in SEQ ID NO: 9 or 10, (5) the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 57, or an EF-1α intron fragment sequence (i.e., an untranslated nucleic acid sequence) essentially consisting of or consisting of the nucleotide sequence, and (6) the EF-1α E2 sequence shown in SEQ ID NO: 11.

[0373]

[0374] III.TAFA

[0375] The present disclosure provides a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of a TAFA protein (e.g., TAFA1 to TAFA4), a nucleic acid encoding the polypeptide, a vector comprising the nucleic acid, a recombinant virus particle comprising the vector and a capsid protein, or a cell comprising the nucleic acid or the vector.

[0376]

[0377] III.A. Polypeptide

[0378] The present disclosure provides a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 comprises the amino acid sequence of General Formula 1 below (from N-terminus to C-terminus):

[0379] <General Formula 1>

[0380] X1-X2-X3-G-T-C-E-V-X4-A-X5-H-X6-C-C-N-X7-N-X8-I-E-E-X9-S-Q-T-X10-X11-C-S-C-X12-X13-G-X14-V-A-G-T-T-X15-X16-X17-P-S-C-V-X18-A-X19-I-V-X20-X21-X22-W-W-C-X23-M-X24-P-C-X25-X26-G-E-X27-C-K-X28-L-P-D-X29-X30-G-W-X31-C-X32-X33-G-X34-K-X35-K-T-T-X36-X37-X38-X39

[0381] In General Formula 1 above,

[0382] X1 is absent, V, I, or L,

[0383] X2 is K, E, R, or Q,

[0384] X3 is G, T, Q, P, or A,

[0385] X4 is V or I,

[0386] X5 is A, L, V, or I,

[0387] X6 is R or L,

[0388] X7 is K, R, or Q,

[0389] X8 is R or K,

[0390] X9 is either R or L,

[0391] X10 is either V or G,

[0392] X11 is either K or N,

[0393] X12 is either F or L,

[0394] X13 is either P or S,

[0395] X14 is either Q or K,

[0396] X15 is either R, H or Q,

[0397] X16 is either A, N, S or T,

[0398] X17 is either A, Q, R, K or T,

[0399] X18 is either D or E,

[0400] X19 is either S or A,

[0401] X20 is either I, E, L, A or V,

[0402] X21 is either Q, G or E,

[0403] X22 is either K or R,

[0404] X23 is either H, Q or E,

[0405] X24 is either E, Q, N, D, S or H,

[0406] X25 is either L, V or M,

[0407] X26 is either E, D, P, L or A,

[0408] X27 is either E or D,

[0409] X28 is V, T, A or I,

[0410] X29 is L, N, R, Y, S or Q,

[0411] X30 is S, K or T,

[0412] X31 is S or M,

[0413] X32 is S, A or Y,

[0414] X33 is S, T or R,

[0415] X34 is N or H,

[0416] X35 is V or I,

[0417] X36 is R or K,

[0418] X37 is absent, V, A, G, M or N,

[0419] X38 is absent, T, I, N, F or S, and

[0420] X39 is absent, R, H, V, K, I or Q.

[0421] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 is composed of an amino acid sequence of 80 to 130, more preferably 90 to 120, and most preferably 91 to 119 amino acids.

[0422] In some embodiments, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75.

[0423] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 2 below:

[0424] <General Formula 2>

[0425] X1-X2-H-H-K-A-X3-H

[0426] In General Formula 2,

[0427] X1 is A or V,

[0428] X2 is N or I, and

[0429] X3 is H or Q.

[0430] In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 2 - General Formula 1.

[0431] In some embodiments, the polypeptide further comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 3 below:

[0432] <General Formula 3>

[0433] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0434] In the general formula 3,

[0435] X1 is A or S,

[0436] X2 is L, T or S,

[0437] X3 is Q, E or H,

[0438] X4 is absent or P, L or H,

[0439] X5 is P or R,

[0440] X6 is T, S or I,

[0441] X7 is A, P, T, S or H,

[0442] X8 is T, A, S or I,

[0443] X9 is V or A, and

[0444] X10 is L or H.

[0445] In some embodiments, the polypeptide comprises the amino acid sequence (from the N-terminus to the C-terminus) of general formula 3 - general formula 1.

[0446] In some embodiments, the polypeptide further comprises the amino acid sequence (from the N-terminus to the C-terminus) of the following general formula 4:

[0447] <General formula 4>

[0448] X1 - X2

[0449] In the general formula 4,

[0450] X1 is H or Y, and

[0451] X2 is Q, V or L.

[0452] In some embodiments, the polypeptide comprises the amino acid sequence of Formula 4 to Formula 1 (from the N-terminus to the C-terminus).

[0453] In some embodiments, the polypeptide further comprises the following amino acid sequence (from the N-terminus to the C-terminus):

[0454] LHRP (SEQ ID NO: 293);

[0455] LHQSGFTSGHFPHHRKLGE (SEQ ID NO: 294); or

[0456] LAPPGTNIQI (SEQ ID NO: 295).

[0457] In some embodiments, the polypeptide further comprises the following amino acid sequence of Formula 5 (from the N-terminus to the C-terminus):

[0458] <Formula 5>

[0459] X1-X2-X3

[0460] In Formula 5,

[0461] X1 is P or G,

[0462] X2 is R or H, and

[0463] X3 is T, S or L.

[0464] In some embodiments, the polypeptide further comprises the following amino acid sequence (from the N-terminus to the C-terminus).

[0465] PYTSL (SEQ ID NO: 296); or

[0466] QEDKLK (SEQ ID NO: 297).

[0467] In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of LHRP-General Formula 1, LHQSGFTSGHFPHHRKLGE-General Formula 1, or LAPPGTNIQI-General Formula 1. In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 1-General Formula 5, General Formula 1-PYTSL, or General Formula 1-QEDKLK. In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of LHRP-General Formula 1-General Formula 5LHQSGFTSGHFPHHRKLGE-General Formula 1-General Formula 5, LAPPGTNIQI-General Formula 1-QEDKLK, or LAPPGTNIQI-General Formula 1-PYTSL.

[0468] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 75 to 129. In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 130 to 183. In some embodiments, the polypeptide further comprises a signal sequence. In some embodiments, it comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 298 to 304.

[0469]

[0470] III.B. Nucleic Acids Encoding Polypeptides

[0471] The present disclosure provides a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75.

[0472] The nucleic acids useful in the present disclosure are not particularly limited as long as they can be translated into a polypeptide when the nucleic acid is transfected into a cell. In some embodiments, the nucleic acid encodes the polypeptide (or any variant thereof) or a fusion protein.

[0473] In some embodiments, the nucleic acid encodes a protein useful for preventing or treating the same disease or disorder as described herein. In some embodiments, the nucleic acid encodes a peptide for preventing or treating a specific disease, for the purpose of sustained expression in the body of a subject or patient. In some embodiments, the nucleic acid has at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 184.

[0474]

[0475] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence (from N-terminus to C-terminus) of General Formula 1 below:

[0476]

[0477] <General Formula 1> X1-X2-X3-G-T-C-E-V-X4-A-X5-H-X6-C-C-N-X7-N-X8-I-E-E-X9-S-Q-T-X10-X11-C-S-C-X12-X13-G-X14-V-A-G-T-T-X15-X16-X17-P-S-C-V-X18-A-X19-I-V-X20-X21-X22-W-W-C-X23-M-X24-P-C-X25-X26-G-E-X27-C-K-X28-L-P-D-X29-X30-G-W-X31-C-X32-X33-G-X34-K-X35-K-T-T-X36-X37-X38-X39

[0478] In General Formula 1,

[0479] X1 is absent, V, I or L,

[0480] X2 is K, E, R or Q,

[0481] X3 is G, T, Q, P or A,

[0482] X4 is V or I,

[0483] X5 is A, L, V or I,

[0484] X6 is R or L,

[0485] X7 is K, R or Q,

[0486] X8 is R or K,

[0487] X9 is R or L,

[0488] X10 is V or G,

[0489] X11 is K or N,

[0490] X12 is F or L,

[0491] X13 is P or S,

[0492] X14 is Q or K,

[0493] X15 is R, H or Q,

[0494] X16 is A, N, S or T,

[0495] X17 is A, Q, R, K or T,

[0496] X18 is D or E,

[0497] X19 is S or A,

[0498] X20 is I, E, L, A or V,

[0499] X21 is Q, G or E,

[0500] X22 is K or R,

[0501] X23 is H, Q or E,

[0502] X24 is E, Q, N, D, S or H,

[0503] X25 is L, V or M,

[0504] X26 is E, D, P, L or A,

[0505] X27 is E or D,

[0506] X28 is V, T, A or I,

[0507] X29 is L, N, R, Y, S or Q,

[0508] X30 is S, K or T,

[0509] X31 is S or M,

[0510] X32 is S, A or Y,

[0511] X33 is S, T or R,

[0512] X34 is N or H,

[0513] X35 is V or I,

[0514] X36 is R or K,

[0515] X37 is absent or is V, A, G, M or N,

[0516] X38 is absent or is T, I, N, F or S, and

[0517] X39 is absent or is R, H, V, K, I or Q.

[0518] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of the following general formula 2 (from N-terminus to C-terminus) and the amino acid sequence of the general formula 1:

[0519] <General formula 2>

[0520] X1-X2-H-H-K-A-X3-H

[0521] In the above general formula 2,

[0522] X1 is A or V,

[0523] X2 is N or I, and

[0524] X3 is H or Q.

[0525] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of general formula 2 - general formula 1 (from N-terminus to C-terminus).

[0526] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of the following general formula 3 (from N-terminus to C-terminus) and the amino acid sequence of general formula 1:

[0527] <General formula 3>

[0528] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0529] In the above general formula 3,

[0530] X1 is A or S,

[0531] X2 is L, T or S,

[0532] X3 is Q, E or H,

[0533] X4 is absent, P, L or H,

[0534] X5 is P or R,

[0535] X6 is T, S or I,

[0536] X7 is A, P, T, S or H,

[0537] X8 is T, A, S or I,

[0538] X9 is V or A, and

[0539] X10 is L or H.

[0540] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of Formula 3 - Formula 1 (from N - terminus to C - terminus).

[0541] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of the following Formula 4 (from N - terminus to C - terminus) and the amino acid sequence of Formula 1:

[0542] <General Formula 4>

[0543] X1 - X2

[0544] In the general formula 4,

[0545] X1 is H or Y, and

[0546] X2 is Q, V or L.

[0547] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of Formula 4 - Formula 1 (from N - terminus to C - terminus).

[0548] In some embodiments, in addition to the amino acid sequence of Formula 1, the nucleic acid encodes a polypeptide comprising the following amino acid sequence (from N - terminus to C - terminus):

[0549] LHRP (SEQ ID NO: 293);

[0550] LHQSGFTSGHFPHHRKLGE (SEQ ID NO: 294); or

[0551] LAPPGTNIQI (SEQ ID NO: 295).

[0552] In some embodiments, the nucleic acid encodes a polypeptide comprising, in addition to the amino acid sequence of the general formula 1, the amino acid sequence of the following general formula 5 (from N-terminus to C-terminus):

[0553] <General formula 5>

[0554] X1-X2-X3

[0555] In the general formula 5,

[0556] X1 is P or G,

[0557] X2 is R or H, and

[0558] X3 is T, S or L.

[0559] In some embodiments, the polypeptide encodes a polypeptide comprising the following amino acid sequence (from N-terminus to C-terminus).

[0560] PYTSL (SEQ ID NO: 296); or

[0561] QEDKLK (SEQ ID NO: 297).

[0562] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence (from N-terminus to C-terminus) of LHRP - General Formula 1, LHQSGFTSGHFPHHRKLGE - General Formula 1, or LAPPGTNIQI - General Formula 1. In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence (from N-terminus to C-terminus) of General Formula 1 - General Formula 5, General Formula 1 - PYTSL, or General Formula 1 - QEDKLK. In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence (from N-terminus to C-terminus) of LHRP - General Formula 1 - General Formula 5, LHQSGFTSGHFPHHRKLGE - General Formula 1 - General Formula 5, LAPPGTNIQI - General Formula 1 - QEDKLK, or LAPPGTNIQI - General Formula 1 - PYTSL.

[0563] In some embodiments, the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 184 to 238. In some embodiments, the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 239 to 292. In some embodiments, the nucleic acid encoding the polypeptide further comprises a sequence encoding a signal sequence. In some embodiments, the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 305 to 311.

[0564]

[0565] IV. Vectors Containing Nucleic Acids Encoding Peptides

[0566] The present disclosure provides a vector comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75.

[0567] The nucleic acid is as described in III.B.

[0568] As described herein, such vectors are useful for recombinant expression in host cells and cells targeted for therapeutic intervention. In some embodiments, vectors useful for delivery of the polynucleotides described herein (e.g., nucleic acids encoding polypeptides having an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 75, or nucleic acids having at least 70% sequence identity to SEQ ID NO: 184) and non-translated nucleic acid sequences) include viral vectors. Examples of viruses that can be used as vectors in the present disclosure include, but are not limited to, retroviruses, herpes simplex viruses, lentiviruses, poxviruses, vaccinia viruses, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, adeno-associated viruses (AAV), baculoviruses, and combinations thereof. In some embodiments, vectors that can be used in the present disclosure include non-viral vectors. Non-limiting examples of such vectors include plasmids, cosmids, yeast artificial chromosomes (YACs), bacteriophages, and combinations thereof.

[0569] In some embodiments, a vector comprising a nucleic acid encoding a polypeptide having an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 75 is an adeno-associated virus (AAV) vector.

[0570] In some embodiments, the vector further comprises one or more sequences selected from the group consisting of a promoter sequence, an enhancer sequence, an exon sequence, an intron sequence, a signal sequence coding sequence, a splicing donor sequence, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

[0571] In some embodiments, the intron does not include the nucleotide sequence shown in SEQ ID NO: 1.

[0572] In some embodiments, the intron is composed of 29 to 174 nucleotides. In some embodiments, the intron is composed of 51 to 117 nucleotides.

[0573] In some embodiments, the intron comprises the nucleotide sequence shown in SEQ ID NO: 57. In some embodiments, the intron comprises the nucleotide sequence shown in SEQ ID NO: 58. In some embodiments, the intron comprises the nucleotide sequence shown in SEQ ID NO: 59. In some embodiments, the intron comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0574] In some embodiments, the intron has at least 70% sequence identity with (i) nucleotides 871 to 924 of SEQ ID NO: 1 (SEQ ID NO: 58), (ii) nucleotides 861 to 924 of SEQ ID NO: 1 (SEQ ID NO: 59), (iii) nucleotides 852 to 924 of SEQ ID NO: 1 (SEQ ID NO: 3), (iv) nucleotides 851 to 924 of SEQ ID NO: 1 (SEQ ID NO: 61), (v) nucleotides 830 to 924 of SEQ ID NO: 1 (SEQ ID NO: 2), (vi) nucleotides 821 to 924 of SEQ ID NO: 1 (SEQ ID NO: 63), (vii) nucleotides 811 to 924 of SEQ ID NO: 1 (SEQ ID NO: 64), or (viii) nucleotides 808 to 924 of SEQ ID NO: 1 (SEQ ID NO: 65).

[0575] In some embodiments, the intron comprises (i) nucleotides 871 to 924 of SEQ ID NO: 1 (SEQ ID NO: 58), (ii) nucleotides 861 to 924 of SEQ ID NO: 1 (SEQ ID NO: 59), (iii) nucleotides 852 to 924 of SEQ ID NO: 1 (SEQ ID NO: 3), (iv) nucleotides 851 to 924 of SEQ ID NO: 1 (SEQ ID NO: 61), (v) nucleotides 830 to 924 of SEQ ID NO: 1 (SEQ ID NO: 2), (vi) nucleotides 821 to 924 of SEQ ID NO: 1 (SEQ ID NO: 63), (vii) nucleotides 811 to 924 of SEQ ID NO: 1 (SEQ ID NO: 64), or (viii) nucleotides 808 to 924 of SEQ ID NO: 1 (SEQ ID NO: 65).

[0576] In some embodiments, the vector comprises the following configuration:

[0577] (1) The CMV enhancer sequence shown in SEQ ID NO: 4;

[0578] (2) A promoter sequence selected from the CMV promoter sequence shown in SEQ ID NO: 5 or 6, the EF-1α promoter sequence shown in SEQ ID NO: 7, or the chicken β-actin promoter sequence shown in SEQ ID NO: 8;

[0579] (3) An exon 1 (E1) sequence selected from the CMV E1 sequence shown in SEQ ID NO: 12, the EF-1α E1 sequence shown in SEQ ID NO: 13, or the chicken β-actin E1 sequence shown in SEQ ID NO: 14 or 15;

[0580] (4) A splicing donor sequence shown in SEQ ID NO: 9 or 10; and / or

[0581] (5) The EF-1α E2 sequence shown in SEQ ID NO: 11.

[0582] In some embodiments, the AAV vector is for use in gene therapy. In some embodiments, the AAV vector is for use in the expression of a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75.

[0583]

[0584] V.AAV

[0585] In some embodiments, the polynucleotides described herein (e.g., including foreign genes and untranslated nucleic acid sequences) are delivered to cells, for example, using AAV. Adeno-associated virus (AAV), as a single-stranded DNA virus, is a helper-dependent human parvovirus. The AAV genome has a size of about 4.7 kbp and consists of an N-terminus encoding the rep gene involved in viral replication and expression of viral genes, a C-terminus encoding the cap gene encoding the capsid protein of the virus, and inverted terminal repeats (ITRs) with about 145 bases inserted at each end. The 145 bp inverted terminal repeat (ITR) has a T-shaped structure, functions as an origin of replication during replication of the viral genome, and acts as a primary packaging signal. The ITR is the only cis-acting nucleotide sequence required when making a recombinant AAV (rAAV) construct. The ITR has enhancer activity in the presence of the Rep protein but very weak activity in the absence of the Rep protein. When cloning a foreign gene into a recombinant AAV construct, considering these characteristics, an expression construct is produced by appropriately configuring enhancers, promoters, pA, etc. (RJ Samulski and N Muzyczka, Annu. Rev. Virol. 2014.1:427-451). Four proteins are translated from the rep gene. These proteins are classified as rep78, rep68, rep52, and rep40 according to their molecular weights and perform important functions in AAV DNA replication. Four proteins are translated from the cap gene. Among them, VP1, VP2, and VP3 proteins are each structural protein constituting the AAV particle, and the assembly-activating protein (AAP) promotes the assembly of AAV particles by each of the structural proteins. For efficient replication of adeno-associated virus, some proteins and RNAs derived from helper viruses such as adenovirus or herpes simplex virus are required (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0586] AAV is a vector for delivering foreign DNA to cells and has unique features that make it attractive. In culture, AAV infection of cells is generally non-cytopathic, and natural infection in humans and other animals is asymptomatic while being symptom-free. Furthermore, AAV can infect many different types of mammalian cells and target many different tissues in the body. Also, AAV promotes a weak immune response compared to other forms of gene delivery and includes those that persistently express based on non-integrating episomal vector DNA in all of dividing and quiescent cells, having additional advantages that make it a particularly attractive viral system for gene delivery. Also, since AAV can withstand the conditions (56°C to 65°C for several hours) used to inactivate adenoviruses, cryopreservation of rAAV-based vaccines becomes less important.

[0587] Adeno-associated virus types or serotypes that can be used in the present disclosure include AAVrh.10 (AAVrh10), AAV-DJ (AAVDJ), AAV-DJ8 (AAVDJ8), AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R variant, AAVrh8R R533A variant, AAAV, BAAV, Caprine AAV, Bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, B P61 AAV, B P62 AAV, B P63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10 and Japanese AAV 10 serotypes, including but not limited to these.

[0588] In some embodiments, the serotype of the adeno-associated virus is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10. In some embodiments, the serotype of the AAV is AAV2. In some embodiments, the serotype of the AAV is AAV5. In some embodiments, the serotype of the AAV is AAV8. In some embodiments, the serotype of the AAV is AAV9.

[0589]

[0590] VI. Cells

[0591] In some embodiments, the present specification provides cells comprising any polynucleotide of each polynucleotide described herein (e.g., including untranslated nucleic acid sequences). For example, in some embodiments, the cells described herein are transduced, transfected, or transformed with a recombinant expression construct comprising a foreign gene (e.g., a nucleic acid encoding a polypeptide having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 or a nucleic acid having at least 70% sequence identity with SEQ ID NO: 184) and an untranslated nucleic acid sequence for foreign gene expression.

[0592] Although not bound by any one theory, in some embodiments, the cells described herein (e.g., transfected with a polynucleotide comprising an untranslated nucleic acid sequence) are useful for producing proteins such as those encoded by the foreign genes described herein (e.g., a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 75). As described herein, in some embodiments, the untranslated nucleic acid sequences described herein (i.e., EF-1α intron fragments) can enhance the expression of the protein encoded by the foreign gene (the "encoded protein") in cells. Thus, in some embodiments, the cells described herein (e.g., transfected with a polynucleotide comprising a foreign gene and an untranslated nucleic acid sequence of the present disclosure) exhibit greater expression of the encoded protein compared to a reference cell. In some embodiments, the reference cell is transfected with the polynucleotide but lacks the untranslated nucleic acid sequence.

[0593] In some embodiments, the cells described herein are capable of producing the protein encoded by the foreign gene in vitro. In certain embodiments, the cells described herein are capable of producing the encoded protein in vivo (e.g., in a subject administered the polynucleotide described herein). In some embodiments, the cells described herein are capable of producing the encoded protein both in vitro and in vivo.

[0594] In some embodiments, cells that can be used to produce a protein encoded by a foreign gene (e.g., in vitro) include host cells. As used herein, the term "host cell" is intended to include cells of any organism that are transfected with the expression construct (e.g., an AAV vector) and can replicate the expression construct or express the gene encoded by the expression construct. Such cells include eukaryotic and prokaryotic cells. As used herein, the term "transfection" is intended to include transduction and transformation. The host cell can be transfected, transduced or transformed by the expression construct. This process means that an exogenous nucleic acid molecule is transmitted or introduced into the host cell. In some embodiments, the host cell is an isolated host cell comprising the AAV vector. In some embodiments, the host cell is an isolated host cell transformed with the AAV vector.

[0595] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from the group consisting of mammalian cells, insect cells, yeast cells, transgenic mammalian cells, and plant cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell.

[0596] In some embodiments, the host cell is an insect cell. In some embodiments, the insect cell is Sf9. In some embodiments, the host cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used in the present disclosure include HEK293, HeLa, ARPE-19, RPE-1, HepG2, Hep3B, Huh-7, C8D1a, Neuro2A, CHO, MES13, BHK-21, COS7, COP5, A549, MCF-7, HC70, HCC1428, BT-549, PC3, LNCaP, Capan-1, Panc-1, MIA PaCa-2, SW480, HCT166, LoVo, A172, MKN-45, MKN-74, Kato-III, NCI-N87, HT-144, SK-MEL-2, SH-SY5Y, C6, HT-22, PC-12, NIH3T3 cells and combinations thereof.

[0597] In some embodiments, cells that can be used to produce a protein encoded by a foreign gene described herein (e.g., in vivo) include human cells. In some embodiments, the human cells are cells of a subject to whom the nucleic acid molecule described herein has been administered. In certain embodiments, the human cells are derived from a donor (e.g., a healthy human subject).

[0598] In some embodiments, the present disclosure provides a composition comprising an AAV vector, or a host cell comprising or transformed with the AAV vector.

[0599]

[0600] VII. Compositions

[0601] The present specification provides a composition comprising the polypeptide, a nucleic acid encoding the polypeptide, a vector comprising the nucleic acid, a recombinant virus particle comprising the vector and a capsid protein, or a cell comprising the nucleic acid or the vector.

[0602] In some embodiments, the composition is a pharmaceutical composition.

[0603] In some embodiments, this specification discloses a pharmaceutical composition comprising (a) any polynucleotide of each polynucleotide described herein (e.g., including foreign genes and untranslated nucleic acid sequences), and (b) one or more pharmaceutically acceptable carriers. In some embodiments, this specification discloses a pharmaceutical composition comprising (a) a vector described herein (e.g., rAAV), and (b) one or more pharmaceutically acceptable carriers. In some embodiments, this specification discloses a pharmaceutical composition comprising (a) a cell described herein, and (b) one or more pharmaceutically acceptable carriers.

[0604] In some embodiments, the pharmaceutical composition described herein comprises a polypeptide having an amino acid sequence with at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 75 (e.g., vertebrate TAFA1 to TAFA4 sequences), a nucleic acid encoding the polypeptide, a vector comprising the nucleic acid, a recombinant virus particle comprising the vector and a capsid protein, or a cell comprising the nucleic acid or vector.

[0605] In some embodiments, the amino acid sequence having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 75 comprises the amino acid sequence of the following general formula 1 (from N-terminus to C-terminus):

[0606] <General formula 1>

[0607] X1-X2-X3-G-T-C-E-V-X4-A-X5-H-X6-C-C-N-X7-N-X8-I-E-E-X9-S-Q-T-X10-X11-C-S-C-X12-X13-G-X14-V-A-G-T-T-X15-X16-X17-P-S-C-V-X18-A-X19-I-V-X20-X21-X22-W-W-C-X23-M-X24-P-C-X25-X26-G-E-X27-C-K-X28-L-P-D-X29-X30-G-W-X31-C-X32-X33-G-X34-K-X35-K-T-T-X36-X37-X38-X39

[0608] In the general formula 1,

[0609] X1 is absent, or is V, I or L,

[0610] X2 is K, E, R or Q,

[0611] X3 is G, T, Q, P or A,

[0612] X4 is V or I,

[0613] X5 is A, L, V or I,

[0614] X6 is R or L,

[0615] X7 is K, R or Q,

[0616] X8 is R or K,

[0617] X9 is R or L,

[0618] X10 is V or G,

[0619] X11 is K or N,

[0620] X12 is F or L,

[0621] X13 is P or S,

[0622] X14 is Q or K,

[0623] X15 is R, H or Q,

[0624] X16 is A, N, S or T,

[0625] X17 is A, Q, R, K or T,

[0626] X18 is D or E,

[0627] X19 is S or A,

[0628] X20 is I, E, L, A or V,

[0629] X21 is Q, G or E,

[0630] X22 is K or R,

[0631] X23 is H, Q or E,

[0632] X24 is E, Q, N, D, S or H,

[0633] X25 is L, V or M,

[0634] X26 is E, D, P, L or A,

[0635] X27 is E or D,

[0636] X28 is V, T, A or I,

[0637] X29 is L, N, R, Y, S or Q,

[0638] X30 is S, K or T,

[0639] X31 is S or M,

[0640] X32 is S, A or Y,

[0641] X33 is S, T or R,

[0642] X34 is N or H,

[0643] X35 is V or I,

[0644] X36 is R or K,

[0645] X37 is non - existent or V, A, G, M or N,

[0646] X38 is absent, or is T, I, N, F, or S, and

[0647] X39 is absent, or is R, H, V, K, I, or Q.

[0648] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 is composed of an amino acid sequence of 80 to 130, more preferably 90 to 120, and most preferably 91 to 119 amino acids.

[0649] In some embodiments, the polypeptide has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75.

[0650] In some embodiments, the polypeptide further comprises the amino acid sequence of the following general formula 2 (from the N-terminus to the C-terminus):

[0651] <General formula 2>

[0652] X1-X2-H-H-K-A-X3-H

[0653] In the general formula 2,

[0654] X1 is A or V,

[0655] X2 is N or I, and

[0656] X3 is H or Q.

[0657] In some embodiments, the polypeptide comprises the amino acid sequence of formula 2 - formula 1 (from the N-terminus to the C-terminus).

[0658] In some embodiments, the polypeptide further comprises the amino acid sequence of the following general formula 3 (from the N-terminus to the C-terminus):

[0659] <General formula 3>

[0660] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0661] In the general formula 3,

[0662] X1 is A or S,

[0663] X2 is L, T or S,

[0664] X3 is Q, E or H,

[0665] X4 is absent or P, L or H,

[0666] X5 is P or R,

[0667] X6 is T, S or I,

[0668] X7 is A, P, T, S or H,

[0669] X8 is T, A, S or I,

[0670] X9 is V or A, and

[0671] X10 is L or H.

[0672] In some embodiments, the polypeptide comprises the amino acid sequence of formula 3 - formula 1 (from the N-terminus to the C-terminus).

[0673] In some embodiments, the polypeptide further comprises the amino acid sequence (from the N-terminus to the C-terminus) of General Formula 4 below:

[0674] <General Formula 4>

[0675] X1-X2

[0676] In General Formula 4,

[0677] X1 is H or Y, and

[0678] X2 is Q, V or L.

[0679] In some embodiments, the polypeptide comprises the amino acid sequence (from the N-terminus to the C-terminus) of General Formula 4 - General Formula 1.

[0680] In some embodiments, the polypeptide further comprises the following amino acid sequence (from the N-terminus to the C-terminus):

[0681] LHRP (SEQ ID NO: 293);

[0682] LHQSGFTSGHFPHHRKLGE (SEQ ID NO: 294); or

[0683] LAPPGTNIQI (SEQ ID NO: 295).

[0684] In some embodiments, the polypeptide further comprises the amino acid sequence (from the N-terminus to the C-terminus) of General Formula 5 below:

[0685] <General Formula 5>

[0686] X1-X2-X3

[0687] In General Formula 5,

[0688] X1 is P or G,

[0689] X2 is R or H, and

[0690] X3 is T, S, or L.

[0691] In some embodiments, the polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus).

[0692] PYTSL (SEQ ID NO: 296); or

[0693] QEDKLK (SEQ ID NO: 297).

[0694] In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of LHRP-General Formula 1, LHQSGFTSGHFPHHRKLGE-General Formula 1, or LAPPGTNIQI-General Formula 1. In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of General Formula 1-General Formula 5, General Formula 1-PYTSL, or General Formula 1-QEDKLK. In some embodiments, the polypeptide comprises the amino acid sequence (from N-terminus to C-terminus) of LHRP-General Formula 1-General Formula 5, LHQSGFTSGHFPHHRKLGE-General Formula 1-General Formula 5, LAPPGTNIQI-General Formula 1-QEDKLK, or LAPPGTNIQI-General Formula 1-PYTSL.

[0695] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 75 to 129. In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 130 to 183. In some embodiments, the polypeptide further comprises a signal sequence. The polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 298 to 304.

[0696] In some embodiments, the pharmaceutical composition described herein comprises a recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the recombinant adeno-associated virus comprises: (a) an AAV8 capsid protein; and (b) a polynucleotide comprising: (i) a foreign gene having at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO: 184; and (ii) regulatory elements operably linked to the foreign gene, the regulatory elements comprising, in the 5' to 3' direction: (1) a CMV enhancer sequence shown in SEQ ID NO: 4; (2) a chicken β-actin promoter sequence shown in SEQ ID NO: 8; (3) a chicken β-actin exon 1 (E1) sequence shown in SEQ ID NO: 15; (4) a splicing donor sequence of the chicken β-actin intron shown in SEQ ID NO: 10; (5) a non-translated nucleic acid sequence comprising, consisting essentially of, or consisting of the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 57; and (6) an EF-1α exon 2 (E2) sequence shown in SEQ ID NO: 11.

[0697] In some embodiments, the pharmaceutical composition is a pharmaceutical composition for preventing, ameliorating, or treating a retinal or macular disease.

[0698] In some embodiments, the retinal or macular disease is caused by damage to all or part of the retina or macula. In some embodiments, the retinal or macular disease is a disease that occurs due to abnormal function or damage of retinal or macular cells. In some embodiments, the retinal or macular disease may be a disease that occurs due to abnormal function or damage of photoreceptor cells and / or retinal pigment epithelium (RPE) cells of the retina or macula. In some embodiments, the retinal or macular disease is retinopathy, choroidal neovascularization, macular disease, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease, or a combination thereof. In some embodiments, the macular degeneration is age-related macular degeneration, Best macular dystrophy, Sorsby fundus dystrophy, Mallatia Leventinese, Doyne honeycomb retinal dystrophy, Stargardt disease (Stargardt macular dystrophy), myopic macular degeneration, or pigment epithelial detachment-related macular degeneration. In some embodiments, the macular degeneration is age-related macular degeneration. In some embodiments, age-related macular degeneration is wet or dry age-related macular degeneration. In some embodiments, the retinopathy is retinal dystrophy. In some embodiments, the retinopathy is diabetic retinopathy. In some embodiments, the diabetic retinopathy is non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), diabetic maculopathy, diabetic macular edema, or a combination thereof. In some embodiments, the retinal or macular disease is a hereditary retinal disease.In some embodiments, the hereditary retinal disease is retinitis pigmentosa (RP), Leber congenital amaurosis, Stargardt’s disease, Coats retinopathy, cone dystrophy, choroideremia, Usher syndrome, Best’s Disease, X-linked retinoschisis, or unspecified hereditary retinal dystrophy.

[0699] The pharmaceutically acceptable carriers that can be used in the present disclosure are those commonly used in formulation. Examples of the pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical compositions of the present disclosure can further include one or more additives selected from the group consisting of lubricants, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents, and preservatives. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington’s Pharmaceutical Sciences (19th ed., 1995).

[0700] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration. Examples of suitable parenteral routes of administration include intravenous infusion, transdermal administration, subcutaneous injection, intramuscular injection, intraocular (e.g., sub-Tenon, subconjunctival, suprachoroidal, suprachoroidal space, subretinal, intravitreal, and any administration that can be delivered to similar locations) injection, eye drop administration, intracerebroventricular injection, intrathecal injection, intra-amniotic injection, intra-arterial injection, intra-articular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracerebral cistern injection, intracoronary injection, intracranial injection, intradural injection, epidural injection, intrahippocampal injection, intranasal injection, intramedullary injection, intraperitoneal injection, intrathoracic injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injection, intratumoral injection, local injection, intraperitoneal injection, and combinations thereof. In some aspects, intraocular administration includes suprachoroidal, subretinal, and intravitreal administration.

[0701] In some embodiments, the pharmaceutical composition is administered at a daily dosage of from 0.0001 mg / kg to 100 mg / kg.

[0702] The pharmaceutical composition of the present disclosure can be formulated with one or more pharmaceutically acceptable carriers and / or excipients. The pharmaceutical composition can be provided in unit dosage form or dispensed into multi-dose containers. The dosage form can be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or can be in the form of an extract, powder, granule, tablet, or capsule. The dosage form can further include a dispersing agent or a stabilizing agent.

[0703]

[0704] VIII. Kit

[0705] In addition, this specification discloses a kit comprising one or more polypeptides, one or more polynucleotides (e.g., including foreign genes and untranslated nucleic acid sequences) disclosed herein, one or more vectors (e.g., AAV vectors) disclosed herein, one or more cells (e.g., host cells containing or transformed with the AAV vector) disclosed herein, any composition disclosed herein, or any combination thereof. In some embodiments, the kit includes instructions for use.

[0706] As used herein, the terms "kit" and "system" are, in some embodiments, intended to refer to at least one or more polynucleotides disclosed herein, one or more vectors (e.g., AAV vectors) disclosed herein, one or more host cells disclosed herein, any pharmaceutical composition disclosed herein, or any combination thereof, combined with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packages such as commercial packaging, instructions for use, etc.).

[0707]

[0708] IX. Uses and Methods

[0709] IX.A. Production Methods

[0710] In addition, this specification discloses a method for producing a polypeptide encoded by a foreign gene (for example, a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75, or a nucleic acid having at least 70% sequence identity with SEQ ID NO: 184). In some embodiments, such a method comprises culturing a cell described herein (for example, transfected with a polynucleotide comprising a foreign gene and an untranslated nucleic acid molecule) under appropriate conditions and recovering the encoded protein. In certain embodiments, a method for producing a polypeptide encoded by a foreign gene comprises administering to a subject in need of a polynucleotide of the present disclosure (for example, comprising a foreign gene and an untranslated nucleic acid molecule) and producing the polypeptide encoded in said subject. Additional disclosure regarding such in vivo methods of producing polypeptides is provided in other parts of the present disclosure (see, for example, therapeutic uses).

[0711] In some embodiments, the present disclosure provides a method for producing recombinant adeno-associated virus particles comprising a polynucleotide described herein (for example, comprising a foreign gene and an untranslated nucleic acid sequence). In some embodiments, such a method for producing recombinant AAV comprises culturing a cell transduced with an AAV vector described herein under conditions for producing recombinant AAV. In some embodiments, the method further comprises separating the produced recombinant virus particles.

[0712] In some embodiments, the present disclosure provides recombinant virus particles produced by the method.

[0713] In some embodiments, the recombinant viral particles can be produced by transducing cells with (i) an AAV vector containing the foreign gene (see, e.g., FIG. 6b), and (ii) a construct containing the rep and cap genes. Additionally, (iii) it can be produced using a helper construct for transducing the foreign gene into the host cell. In such an embodiment, the helper construct can contain the E2A gene that promotes AAV genome replication and gene transcription, the E4 gene that allows AAV mRNA to move from the nucleus to the cytoplasm, and the VA region that generates two VA RNAs that play a role in regulating translation.

[0714] In some embodiments, the three constructs described above can be replaced by two constructs for transduction into the host cell. In such an embodiment, the AAV construct contains the foreign gene and the untranslated nucleic acid sequence, and a separate construct contains the rep and cap genes, the E2A gene, the E4 gene, and the VA region. Additional methods for producing the AAV particles described herein are generally known in the art. See, for example, Clement et al., Mol Ther Methods Clin Dev 3:16002 (March 2016); Clark, Kidney Int. 61:S9-15 (January 2002); and Xiao et al., J Virol 72(3):2224-32 (March 1998), each of which is incorporated herein by reference in its entirety.

[0715] Also disclosed herein are recombinant viral particles comprising (a) a capsid protein, and (b) the AAV vector.

[0716]

[0717] IX.B. Therapeutic Uses

[0718] The polypeptides, nucleic acids (e.g., including foreign genes and untranslated nucleic acid sequences), vectors and recombinant viruses (e.g., rAAV) carrying such nucleic acids, cells containing said nucleic acids or vectors, and methods described herein have many in vitro and in vivo utilities. For example, the polypeptides, polynucleotides, e.g., vectors, e.g., AAV vectors, described herein can be used to prevent or treat diseases by administration to cells in culture, in vitro or ex vivo, or to humans, e.g., in vivo. Thus, in some aspects, the present disclosure provides for the therapeutic use of the polypeptides, any polynucleotide of each polynucleotide (e.g., including foreign genes and untranslated nucleic acid sequences) described herein, the recombinant expression constructs or vectors described herein, the cells described herein, the pharmaceutical compositions described herein, or the recombinant viruses described herein. In some aspects, the present disclosure describes a method of expressing a foreign gene in a subject in need thereof, comprising administering to the subject a polynucleotide (e.g., including foreign genes and untranslated nucleic acid sequences) disclosed herein, a vector disclosed herein, a recombinant virus (e.g., rAAV) disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein, wherein after said administration, the expression of the foreign gene in the subject is increased.

[0719] As described herein, the untranslated nucleic acid sequences of the present disclosure can increase the expression of the foreign gene when the foreign gene is translated. Thus, in some embodiments, the present disclosure relates to a method of increasing the expression of a foreign gene in a cell, the method comprising contacting the cell with any polynucleotide, vector or recombinant virus (e.g., rAAV) of each polynucleotide, each vector or each recombinant virus disclosed herein. The contacting can occur in vitro or in vivo. When the contacting occurs in vivo, the method can further comprise administering to the subject any polynucleotide, vector or recombinant virus of each polynucleotide, each vector or each recombinant virus prior to the contacting.

[0720] In some embodiments, the expression of the foreign gene after the contacting is increased by at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold or at least about 10-fold or more compared to the reference expression. In some embodiments, the reference expression is the expression of the foreign gene in the cell before the contacting. In some embodiments, the reference expression is the expression of the foreign gene in the cell that does not contact the polypeptide, polynucleotide, vector or recombinant virus (e.g., lacking the untranslated nucleic acid sequence or comprising the nucleotide sequence of SEQ ID NO: 1) described herein.

[0721] Still other aspects of the present disclosure are methods for preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of any polypeptide, polynucleotide, vector, cell, recombinant virus or pharmaceutical composition of each of the polypeptides, polynucleotides, vectors, cells, recombinant viruses or pharmaceutical compositions. As is apparent from the present disclosure, the compositions described herein (e.g., polypeptides, polynucleotides, recombinant expression constructs, cells, pharmaceutical compositions or recombinant viruses) can be used, for example, to prevent or treat any subject disease by modifying foreign genes.

[0722] In some aspects, the method can further comprise administering to the subject an additional therapeutic agent (e.g., an inhibitor of vascular endothelial growth factor (“VEGF”)). In some aspects, the additional therapeutic agent can be administered to the subject simultaneously with, prior to, or after administration of the polypeptide, polynucleotide, vector, cell, recombinant virus or pharmaceutical composition.

[0723] The diseases that can be prevented, ameliorated or treated by the present disclosure are not limited and include all diseases for which it is necessary to reduce the number of drug administrations. Non-limiting examples of such diseases include retinal or macular diseases. In some aspects, the retinal or macular disease is selected from retinopathy, choroidal neovascularization, macular disease, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal diseases and combinations thereof.

[0724] In some embodiments, retinal or macular diseases that can be prevented or treated by the present disclosure include macular degeneration. In some embodiments, the macular degeneration includes age-related macular degeneration (AMD). Age-related macular degeneration can be divided into dry (atrophic) macular degeneration and wet (neovascular or exudative) macular degeneration. Age-related macular degeneration can also be divided into early AMD, intermediate AMD, and late or advanced AMD (geographic atrophy). In some embodiments, retinal or macular diseases that can be prevented or treated by the present disclosure include diabetic retinopathy. In some embodiments, the diabetic retinopathy is non-proliferative diabetic retinopathy (NPDR). In some embodiments, the diabetic retinopathy is proliferative diabetic retinopathy (PDR). In some embodiments, the diabetic retinopathy is diabetic maculopathy. In some embodiments, the diabetic retinopathy is diabetic macular edema. In some embodiments, the diabetic retinopathy is any retinopathy associated with ischemic damage in the retina. Unless otherwise specified, the present disclosure can be used to prevent or treat all forms of AMD and / or diabetic retinopathy.

[0725] Still other aspects of the present disclosure provide gene therapy agents or methods for preventing or treating diseases that can achieve sustained foreign gene expression.

[0726] Using the viral delivery system described herein, administration of the compositions described herein can be at intervals of about one week, about two weeks, about three weeks, about one month, about two months, about three months, about four months, about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months, about one year, about two years, about three years, about four years, about five years or about ten years or more. In some embodiments, the interval is about two to about three months. In some embodiments, the interval is about six months. In some embodiments, the interval is about one year. In some embodiments, the interval is at least about one year. In some embodiments, the interval is at least about two years. In some embodiments, the interval is at least about three years. In some embodiments, the interval is at least about four years. In some embodiments, the interval is at least about five years. In some embodiments, the interval is at least about ten years. That is, using the viral delivery system described herein, the number of administrations of the composition can be epochally reduced, and the inconvenience caused by repeated administration of the composition to a doctor, patient or subject can be avoided. Depending on the symptoms or needs of the patient, the composition can initially be administered at least two to three times at intervals of one to two weeks, and then once every two to three months, every six months, every year or more, or every two to ten years or more.

[0727] [Advantages of the Invention]

[0728] The features and advantages of the present invention are summarized as follows:

[0729] (i) The present invention provides a pharmaceutical composition for preventing or treating a retinal or macular disease, comprising a polypeptide having an amino acid sequence having at least 70% sequence identity with the amino acid sequence of a TAFA protein (e.g., TAFA1 to TAFA4), a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a recombinant viral particle containing the vector and a capsid protein, or a cell containing the nucleic acid or vector.

[0730] (ii) Further, the present invention provides an adeno-associated virus (AAV) vector a comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of the TAFA protein, and a recombinant virus particle comprising the AAV vector and a capsid protein.

[0731] (iii) The AAV vector, recombinant virus or composition comprising the same of the present invention can be usefully used for the prevention or treatment of retinal or macular diseases by restoring damaged retina.

[0732] [Brief Description of Drawings]

[0733] These and / or other aspects and advantages of the present disclosure will be apparent and readily understood from the description hereinafter of each aspect with reference to the accompanying drawings:

[0734] [FIG. 1] Cleavage map of the pAAV-eGFP expression construct described herein.

[0735] [FIGS. 2a and 2b] Show increased expression of eGFP in cells transduced with an expression construct containing the full-length EF-1α intron together with the EF-1α promoter (“CEE-FL”). Control group cells were transduced with the expression construct lacking the EF-1α intron (“CE”). In FIG. 2a, an ITR-lacking animal cell expression construct was used. In FIG. 2b, an ITR-containing pAAV expression construct was used. In all of FIGS. 2a and 2b, eGFP expression in the following transduced cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7 and Hep3B) is shown.

[0736] [Figs. 3a, 3b, and 3c] Expression of eGFP in cells transfected with an expression construct containing the full-length EF-1α intron along with various promoters (the "CCE-FL") is shown. Control cells were transfected with the expression construct lacking the EF-1α intron. In Fig. 3a, a combination of the CMV enhancer and CMV promoter was inserted into an animal cell expression construct (i.e., lacking ITR) and used to transfect the cells. In Fig. 3b, a combination of the CMV enhancer and CMV promoter was inserted into a pAAV expression construct and used to transfect the cells. In Fig. 3c, the cells were transfected with an animal cell expression construct containing a combination of the CMV enhancer and chicken-β-actin promoter. In each of Figs. 3a, 3b, and 3c, eGFP expression in the following transfected cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) is shown.

[0737] [Figs. 4a and 4b] show the increasing or decreasing effects of various EF-1α intron fragment sequences (i.e., the untranslated nucleic acid sequences described herein) on gene expression. Fig. 4a schematically shows a series of CEE constructs produced by sequentially deleting the EF-1α intron sequence. Each of the illustrated constructs includes the following: (1) CMV enhancer (380 base pairs); (2) a promoter containing a part of the CMV promoter (the first 31 base pairs from the 5' end) and the EF-1α promoter (201 base pairs); and (3) EF-1α exon 1 (E1) sequence (29 base pairs). The control "CE" construct contained the EF-1 alpha intron sequence and no additional components. The other constructs further included the following additional components: (4) a splice donor sequence consisting of the first 19 base pairs from the 5'-end of the full-length EF-1 alpha intron sequence (i.e., the first sequence of SEQ ID NO); (5) the EF-1α intron sequence; and (6) EF-1α exon 2 (E2) sequence (9 base pairs). The EF-1α intron sequence was either the full-length sequence (924 base pairs) ("CEE-FL") or; (ii) nucleotides 570 to 924 of the first sequence of SEQ ID NO (355 base pairs) ("CEE-T2"); (iii) nucleotides 721 to 924 of the first sequence of SEQ ID NO (204 base pairs) ("CEE-T3"); (iv) nucleotides 808 to 924 of the first sequence of SEQ ID NO (117 base pairs) ("CEE-T3.1"); (v) nucleotides 830 to 924 of the first sequence of SEQ ID NO (95 base pairs) ("CEE-T3.1.1"); (vi) nucleotides 852 to 924 of the first sequence of SEQ ID NO (73 base pairs) ("CEE-T3.1.2"); (vii) nucleotides 874 to 924 of the first sequence of SEQ ID NO (51 base pairs) ("CEE-T3.2"); and (viii) nucleotides 896 to 924 of the first sequence of SEQ ID NO (29 base pairs) ("CEE-T4"). The full length of each of the constructs is provided on the right side.Figure 4b shows the effects of different EF-1α intron sequences (full-length or deletion) on eGFP (i.e., foreign gene) expression in five different cell lines (HeLa, Hep3B, Huh-7, ARPE-19, and RPE-1). ARPE-19 and RPE-1 cells were derived from the retina. Huh-7 and Hep3B cells were derived from the liver. HeLa cells were derived from the cervix. eGFP expression was shown as the expression rate (%) observed from cells transfected with the foreign gene using the CEE-FL construct. "ns" = no significance. "**" = p < 0.01 and "***" = p < 0.001.

[0738] [Figs. 5a, 5b, 5c, and 5d] The EF-1α intron fragments T3.1.1 (i.e., nucleotides 830 to 924 (95 base pairs) of the first sequence of SEQ ID NO.) and T3.1.2 (i.e., nucleotides 852 to 924 (73 base pairs) of the first sequence of SEQ ID NO.) that increase foreign gene expression are shown. Fig. 5a schematically shows a series of CAE constructs containing the EF-1α intron fragments T3.1.1 and T3.1.2 along with the following additional components: (1) CMV enhancer (380 base pairs); (2) chicken β-actin promoter (279 base pairs); (3) chicken β-actin exon 1 (E1) sequence (32 base pairs); (4) EF-1α exon 1 (E1) sequence (29 base pairs); (5) a splicing donor sequence consisting of the first 19 base pairs from the 5'-end of the full-length EF-1α intron sequence (i.e., the first sequence of SEQ ID NO.); and (6) EF-1α exon 2 (E2) sequence (9 base pairs). The control "CA" construct contains only the CMV enhancer, chicken β-actin promoter, and chicken β-actin E1 sequence. The control "CAE-FL" construct contains the full-length EF-1α intron sequence. The full lengths of the said constructs are provided on the left side. Fig. 5b shows eGFP (i.e., foreign gene) expression in HeLa (left graph) and ARPE-19 (right graph) cells transfected with the CAE-T3.1.1 and CAE-T3.1.2 constructs. Gene expression was shown as the expression rate (%) in the said cells transfected with the CA construct (i.e., lacking the EF-1α intron sequence). Fig. 5c schematically shows a series of hybrid intron CA constructs (i.e., CA-T3.1.1 and CA-T3.1.2 constructs) that contain all of the EF-1α intron fragment T3.1.1 or T3.1.2 and chicken β-actin intron fragments. The said two constructs further contain: (1) CMV enhancer; (2) chicken β-actin promoter; (3) chicken β-actin E1 sequence; and (4) EF-1α E2 sequence.The control group "CAG-FL" construct includes the following: (1) CMV enhancer (380 base pairs); (2) chicken β-actin promoter (279 base pairs); (3) chicken β-actin E1 sequence (93 base pairs); (4) chimeric intron (including the introns of chicken β-actin and rabbit β-globin) (924 base pairs); and (5) rabbit β-globin exon 3 (E3) sequence (48 base pairs). The "CA" construct is the same as that described in Fig. 5a. Fig. 5d shows eGFP expression in HeLa (left graph) and Hep3B (right graph) cells transfected with constructs CA-T3.1.1 and CA-T3.1.2. Gene expression was shown as the expression rate (%) in the cells transfected with the CA construct (i.e., lacking the EF-1α intron sequence).

[0739] [Fig. 6] Cleavage map of the AAV construct for producing AAV8. Fig. 6a is a pAAV construct without a transgene, and Fig. 6b is a pAAV-mTAFA4 construct containing the mouse TAFA4 gene as a transgene.

[0740] [Fig. 7] Results of analyzing the improvement efficacy of retinal damage by fluorescence angiography (FA) when the control group (control AAV8) or the experimental group (AAV8.mouse TAFA4) was subretinally administered to the NaIO3-induced retinal damage model. OD (oculus dexter) means the right eye, and OS (oculus sinister) means the left eye.

[0741] [Fig. 8] Results of measuring the change range of the amplitudes of the A wave and B wave of the electroretinogram (ERG) between the control group (control AAV8) and the experimental group (AAV8.mouse TAFA4).

[0742] [Fig. 9] Results of relatively comparing the TAFA4 expression levels before and after NaIO3 administration and the TAFA4 mRNA expression level after administration in the experimental group.

[0743] [Figure 10] Results of comparing the interspecies sequence identity of the full-length TAFA4 protein.

[0744] [Figure 11] Results of analyzing the improvement efficacy of retinal damage by fluorescence angiography (FA) when the control group (control AAV8) or experimental groups (AAV8.human TAFA4, AAV8.gecko TAFA4, AAV8.fish TAFA4) were subretinally administered to the NaIO3-induced retinal damage model. OD (oculus dexter) means the right eye, and OS (oculus sinister) means the left eye.

[0745] [Figure 12] Results of measuring the change ranges of the amplitudes of the A and B waves of the electroretinogram (ERG) between the control group (control AAV8) and the experimental groups (AAV8.human TAFA4, AAV8.gecko TAFA4, AAV8.fish TAFA4).

[0746] [Figure 13] Results of comparing the mRNA and protein expressions of TAFA4 by the signal sequence.

[0747] [Figure 14] Results of analyzing the improvement efficacy of retinal damage by fluorescence angiography (FA) when the control group (control AAV8) or experimental groups (AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, AAV8.TAFA4) were subretinally administered to the NaIO3-induced retinal damage model. OD (oculus dexter) means the right eye, and OS (oculus sinister) means the left eye.

[0748] [Figure 15] Results of measuring the change ranges of the amplitudes of the A and B waves of the electroretinogram (ERG) between the control group (control AAV8) and the experimental groups (AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, AAV8.TAFA4).

[0749] [Fig. 16] Results of comparing the interspecies sequence identity of the mature TAFA1 protein.

[0750] [Fig. 17] Results of comparing the interspecies sequence identity of the mature TAFA2 protein.

[0751] [Fig. 18] Results of comparing the interspecies sequence identity of the mature TAFA3 protein.

[0752] [Fig. 19] Results of comparing the interspecies sequence identity of the mature TAFA4 protein.

[0753] [Fig. 20] Results of comparing the interspecies amino acid sequence identity of full-length TAFA1, TAFA2, TAFA3, and TAFA4. Fig. 20a shows the comparison of the amino acid sequences from position 1 to 70, Fig. 20b shows the comparison of the amino acid sequences from position 71 to 140, Fig. 20c shows the comparison of the amino acid sequences from position 141 to 210, and Fig. 20d shows the comparison of the amino acid sequences from position 211 to 244.

[0754] [Fig. 21] Results of comparing the sequence identity of the mature proteins of human TAFA1, TAFA2, TAFA3, and TAFA4.

[0755] [Mode for Carrying Out the Invention]

[0756] Hereinafter, the present invention will be described in more detail through examples. These examples are merely for more specifically explaining the present invention, and it will be apparent to those having ordinary knowledge in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0757]

[0758] [Examples]

[0759] Materials and Methods

[0760] Example 1. Production of pAAV-eGFP construct without enhancer-promoter-intron array

[0761] Example 1-1. Insertion of bGH polyadenylation signal sequence

[0762] Using the pcDNA5 / FRT / TO construct (Invitrogen, USA, Cat No. V6520-20) as a template, polymerase chain reaction (PCR) was performed using oligo #001 and #002 to obtain a bovine growth hormone (bGH) polyadenylation signal A sequence (poly A) fragment. Next, human growth hormone (hGH) poly A was removed using the BglII / BstEII site of the pAAV-MCS-promoterless plasmid (Cellbiolabs, USA, Cat No. VPK-411), and bGH poly A was inserted.

[0763]

[0764] Example 1-2. Insertion of eGFP

[0765] A human codon-optimized eGFP gene derived from the pUCIDT-KAN-eGFP construct (GeneArt, Germany) was obtained and cloned into the BamHI / HindIII site of the pAAV-bGH construct prepared in Example 1-1.

[0766]

[0767] Example 1-3. Insertion of WPRE sequence

[0768] The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence was obtained with the pUC57-WPRE construct (GenScript, USA) and cloned into the HindIII / BglII site of the pAAV-eGFP-bGH construct prepared in Example 1-2.

[0769]

[0770] Example 1-4. Insertion of 4 copies of miRNA142-3p target sequence

[0771] Oligo #003 and #004, and #005 and #006 were annealed respectively to prepare two DNA fragments each containing two copies of the miRNA142-3p target sequence. The two short DNA fragments were cloned into the HindIII / SalI site of the pAAV-eGFP-WPRE-bGH construct prepared in Examples 1-3 such that a total of four copies of the miRNA142-3p target sequence were inserted. Through this, a pAAV-eGFP construct without an enhancer-promoter-intron sequence was prepared (Figure 1).

[0772]

[0773] Example 2. Preparation of each construct containing various types of enhancer-promoter-intron sequences

[0774] Example 2-1. Preparation of each construct containing the CEE series (CEE-FL, CEE-T2, -T3, -T3, -T3.1, -T3.1.1, -T3.1.2, -T3.2, -T4) and the CE sequence

[0775] Example 2-1-1. Preparation of a construct containing the CEE-FL (full length) nucleotide sequence

[0776] A DNA fragment of CEE-FL (CMV enhancer (SEQ ID NO: 4) - 31 bp of CMV promoter (SEQ ID NO: 5) - EF-1α promoter (SEQ ID NO: 7) - 29 bp of EF-1α exon 1 (SEQ ID NO: 13) - EF-1α intron (SEQ ID NO: 1) - 9 bp of EF-1α exon 2 (SEQ ID NO: 11)) was secured from the pMK-RQ3_PEM construct (GeneArt, Germany) and cloned into the EcoRI / BamHI site of the construct prepared in Example 1.

[0777]

[0778] Example 2-1-2. Preparation of a construct containing the CEE-T2 nucleotide sequence

[0779] Using the oligo #007 / 008 combination and the #009 / 010 combination, PCR was performed with the construct produced in Example 2-1-1 as a template, and two DNA fragments were ligated through Gibson Assembly (registered trademark) (NEB, USA, Cat No. E2611) to produce a construct having the CEE-T2 sequence.

[0780]

[0781] Example 2-1-3. Production of a construct containing the CEE-T3 base sequence

[0782] Using the oligo #011 / 008 combination and the #012 / 010 combination, PCR was performed with the construct produced in Example 2-1-1 as a template, and two DNA fragments were ligated through Gibson Assembly (registered trademark) to produce a construct having the CEE-T3 sequence.

[0783]

[0784] Example 2-1-4. Production of a construct containing the CEE-T3.1 base sequence

[0785] Using the oligo #013 / 008 and #014 / 010 combinations, PCR was performed with the construct produced in Example 2-1-1 as a template, and two DNA fragments were ligated through Gibson Assembly (registered trademark) to produce a construct having the CEE-T3.1 sequence.

[0786]

[0787] Example 2-1-5. Production of a construct containing the CEE-T3.2 base sequence

[0788] Using the oligo #015 / 008 and #016 / 010 combinations, PCR was performed with the construct produced in Example 2-1-1 as a template, and two DNA fragments were ligated through Gibson Assembly (registered trademark) to produce a construct having the CEE-T3.2 sequence.

[0789]

[0790] Example 2-1-6. Production of a construct containing the CEE-T4 nucleotide sequence

[0791] Using the Oligo #017 / 008, #018 / 010 combination, PCR was performed with the construct produced in Example 2-1-1 as a template, and two DNA fragments were ligated through Gibson Assembly (registered trademark) to produce a construct having the CEE-T4 sequence.

[0792]

[0793] Example 2-1-7. Production of a construct containing the CEE-T3.1.1 nucleotide sequence

[0794] The CEE-T3.1.1 nucleotide sequence was obtained from the pUC57-T3.1.1 construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct produced in Example 2-1-1.

[0795]

[0796] Example 2-1-8. Production of a construct containing the CEE-T3.1.2 nucleotide sequence

[0797] The CEE-T3.1.2 nucleotide sequence was obtained from the pUC57-T3.1.2 construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct produced in Example 2-1-1.

[0798]

[0799] Example 2-1-9. Production of a construct containing the CE nucleotide sequence

[0800] Using Oligo #019 and #020, a CE fragment was obtained from the construct produced in Example 2-1-1 through PCR and cloned into the EcoRI / BamHI site of the construct produced in Example 2-1-1.

[0801]

[0802] Example 2-2. Production of each construct containing CCE-FL and CC sequences

[0803] Example 2-2-1. Production of a construct containing the CCE-FL sequence

[0804] A DNA fragment of the CCE-FL (CMV enhancer (SEQ ID NO: 4) - CMV promoter (SEQ ID NO: 6) - 30 bp of CMV exon 1 (SEQ ID NO: 12) - 29 bp of EF-1α exon 1 (SEQ ID NO: 13) - EF-1α intron (SEQ ID NO: 1) - 9 bp of EF-1α exon 2 (SEQ ID NO: 11) sequence) was secured from the pMK-RQ4_PME construct (GeneArt, Germany) and cloned into the EcoRI / BamHI site of the pAAV-eGFP construct secured in Example 1.

[0805]

[0806] Example 2-2-2. Production of a construct containing the CC sequence

[0807] Using Oligo #019 and #021, a CC fragment was secured from the construct produced in Example 2-2-1 through PCR and cloned into the EcoRI / BamHI site of the construct produced in Example 2-2-1.

[0808]

[0809] Example 2-3. Production of each construct containing CAG-FL, CA-T3.1.1, CA-T3.1.2, and CA sequences

[0810] Example 2-3-1. Production of a construct containing the CAG-FL sequence

[0811] A CAG fragment was secured from the pCAG-Neo construct (Wako Pure Chemical Industries, Ltd., Japan, Cat No. 163-25601) and cloned into the SnaBI / BamHI site of the construct secured in Example 2-1.

[0812]

[0813] Example 2-3-2. Production of a construct containing the CA array

[0814] The CA fragment was secured from the pUC57-CA construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct produced in Example 2-3-1.

[0815]

[0816] Example 2-3-3. Production of a construct containing the CA-T3.1.1 array

[0817] Using oligos #022 and #023, the T3.1.1 fragment was secured from the construct obtained in Example 2-1-7 through PCR and cloned into the AfeI / BamHI site of the construct produced in Example 2-3-1.

[0818]

[0819] Example 2-3-4. Production of a construct containing the CA-T3.1.2 array

[0820] The T3.1.2 fragment was secured by annealing oligos #024 and #025 and cloned into the AfeI / BamHI site of the construct produced in Example 2-3-1.

[0821]

[0822] Example 2-4. Production of each construct containing the CAE-FL, CAE-T3.1.1, and CAE-T3.1.2 arrays

[0823] Example 2-4-1. Production of a construct containing the CAE-FL array

[0824] A CAE (CMV enhancer (SEQ ID NO: 4) - chicken β-actin promoter (SEQ ID NO: 8) - 32 bp of chicken β-actin exon 1 (SEQ ID NO: 14) - 29 bp of EF-1α exon 1 (SEQ ID NO: 13) - 924 bp of EF-1α intron (SEQ ID NO: 1) - 9 bp of EF-1α exon 2 (SEQ ID NO: 11)) fragment was secured from the pUC57-CAE construct (GenScript, USA), and this was cloned into the EcoRI / BamHI site of the construct produced in Example 1.

[0825]

[0826] Example 2-4-2. Production of a construct containing the CAE-T3.1.1 sequence

[0827] Using Oligo #026 and #027, a CAE-T3.1.1 fragment was secured from the construct produced in Example 2-1-7, and this was cloned into the KpnI / BamHI site of the construct produced in Example 2-1-7.

[0828]

[0829] Example 2-4-3. Production of a construct containing the CAE-T3.1.2 sequence

[0830] Using Oligo #026 and #027, a CAE-T3.1.2 fragment was secured from the construct produced in Example 2-1-8, and this was cloned into the KpnI / BamHI site of the construct produced in Example 2-1-8.

[0831]

[0832] Example 2-5. Production of a pAAV construct containing the aflibercept gene

[0833] Using Oligo#028 and #029, the aflibercept gene (SEQ ID NO: 23) DNA fragment secured by PCR from the pcDNA3.1(+)-hIgG-aflibercept construct (GenScript, USA) was cloned into the BamHI / HindIII sites of the construct produced in Example 2-3-3, respectively.

[0834]

[0835] Example 2-6. Production of constructs containing TAFA 1, 2, 3, and 4 sequences

[0836] Example 2-6-1. Production of a construct containing the mouse TAFA4 sequence

[0837] The gene DNA fragment of mouse TAFA4 (mTAFA4) obtained from the pMA-RQ-mouse TAFA4 construct (Geneart, Thermo Fisher Scientific, USA) with restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (containing the CA-T3.1.1 fragment) produced in Example 2-5. The mouse TAFA4 gene was used with codon optimization of SEQ ID NO: 305 for mice.

[0838]

[0839] Example 2-6-2. Production of a construct containing the human TAFA4 sequence

[0840] The gene DNA fragment of human TAFA4 (hTAFA4) obtained from the pMA-RQ-human TAFA4 construct (Geneart, Thermo Fisher Scientific, USA) with restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (containing the CA-T3.1.1 fragment) produced in Example 2-5. The human TAFA4 gene was used with codon optimization of SEQ ID NO: 306 for humans.

[0841]

[0842] Example 2-6-3. Production of a construct containing the gecko TAFA4 sequence

[0843] The gene DNA fragment of gecko TAFA4 (gTAFA4) obtained from the pMK-RQ-gecko TAFA4 construct (Geneart, Thermo Fisher Scientific, USA) with restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (including the CA-T3.1.1 fragment) prepared in Example 2-6-2. The gecko TAFA4 gene was used with codon optimization for mouse against SEQ ID NO: 307.

[0844]

[0845] Example 2-6-4. Preparation of a construct containing the fish TAFA4 sequence

[0846] The gene DNA fragment of fish TAFA4 obtained from the pMK-RQ-fish TAFA4 construct (Geneart, Thermo Fisher Scientific, USA) with restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (including the CA-T3.1.1 fragment) prepared in Example 2-6-2. The fish TAFA4 gene was used with codon optimization for mouse against SEQ ID NO: 308.

[0847]

[0848] Example 2-6-5. Preparation of a construct containing the human TAFA1 sequence

[0849] The gene DNA fragment of human TAFA1 obtained from the pMK-RQ-human TAFA1 construct (GeneArt, Germany) with restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (including the CA-T3.1.1 fragment) prepared in Example 2-6-2. The human TAFA1 gene was used with codon optimization for human against SEQ ID NO: 309.

[0850]

[0851] Example 2-6-6. Preparation of a construct containing the human TAFA2 sequence

[0852] The gene DNA fragment of human TAFA2 obtained from the pMK-RQ-human TAFA2 construct (GeneArt, Germany) with restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (including the CA-T3.1.1 fragment) prepared in Example 2-6-2. The human TAFA2 gene was used with codon optimization of SEQ ID NO: 310 for humans.

[0853]

[0854] Example 2-6-7. Production of a construct containing the human TAFA3 sequence

[0855] The gene DNA fragment of human TAFA3 obtained from the pMK-RQ-human TAFA3 construct (GeneArt, Germany) with BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (including the CA-T3.1.1 fragment) prepared in Example 2-6-2. The human TAFA3 gene was used with codon optimization of SEQ ID NO: 311 for humans.

[0856]

[0857] Example 2-7. Confirmation of the base sequence

[0858] For all constructs obtained using cloning, the base sequence was confirmed through base sequence analysis (DNA sequencing) (Macrogen, Korea, or Bionics, Korea).

[0859]

[0860] Example 3. Cell culture

[0861] The HEK293 and HeLa cell lines were cultured under humidified conditions at 5% CO2 and 37 °C using MEM medium (Gibco, USA, Cat No. 42360-032), the ARPE-19 cell line was cultured using DMERM / F12 medium (Gibco, USA, Cat No. 11330-032), and the RPE-1 and Hep3B cell lines were cultured using DMEM medium (Gibco, USA, Cat. No. 10569-010). 10% fetal bovine serum (FBS, Gibco, USA, Cat No. 16000-044) and 1% penicillin-streptomycin (Gioco, USA, Cat No. 15140-163) were added to all media. The Expi293 cell line was cultured under humidified conditions at 8% CO2 and 37 °C with shaking at 250 rpm using Expi293 medium (Gibco, USA, Cat.No. A14351-01) supplemented with 1% penicillin-streptomycin.

[0862]

[0863] Example 4. Transduction

[0864] Example 4-1. Transduction of adherent cells

[0865] For transduction, each cell line was washed twice with DPBS (Gibco, USA, Cat No. 14190-250), then treated with trypsin-EDTA (Gibco, USA, Cat No. 25200-114), detached from the culture dish and harvested, and then seeded in a 12-well plate at 80% confluence. After culturing for 24 hours, transduction was performed using Lipofectamine 3000 (Thermo Fisher Scientific, USA, Cat No. L300075) with each plasmid DNA.

[0866]

[0867] Example 4-2. Transduction of suspension cells for AAV production

[0868] For AAV production, 6×10 8 cells were inoculated into 220 ml of expi293 medium in a 1 L Erlenmeyer culture flask. For stabilization, after culturing for about 3 to 4 hours, 3.73 pmole each of pHelper plasmid DNA, pUC-RC2 plasmid DNA, pUC-RC8 plasmid DNA, and AAV construct plasmid DNA containing the foreign gene were dissolved in 10 ml of Opti-MEM (Gibco, USA, Cat No. 51985-034). Then, polyethyleneimine (PEI, Polyscience, USA, Cat No. 23966-1) corresponding to twice the total amount of DNA was diluted in 10 ml of Opti-MEM, and immediately after that, the two solutions were mixed to prepare the transduction solution. After reacting at room temperature for 30 minutes, a total of 20 ml of the transduction solution was added to the culture flask.

[0869]

[0870] Example 5. Purification of AAV

[0871] Example 5-1. Purification of AAV2

[0872] At 72 hours after transduction in Example 4-2, the cell culture supernatant was removed, and cells were obtained by removing the medium by centrifugation. The cell precipitate was washed with DPBS and the cells were resuspended in 16 ml of DPBS. Each cell was lysed by performing a freezing / thawing cycle three times, and the supernatant containing AAV2 was obtained by centrifuging this. The supernatant and AAVanced TM Concentration Reagent (System Bioscience, USA, Cat No. AAV110A-1) were mixed at a ratio of 4:1, mixed at 4°C for 16 hours, and then the supernatant was removed by centrifugation to obtain a precipitate containing AAV2. The precipitate was washed with 500 μl of Opti-MEM, all the supernatant was removed, and finally, AAV was obtained by resuspending in 400 μl of ice-cooled DPBS.

[0873]

[0874] Example 5-2. AAV8 Purification

[0875] At 72 hours after transduction in Example 4-2, the cell culture medium was removed. Cell debris was removed with a 0.45 μm filter, and AAV was obtained using anion exchange and affinity chromatography methods.

[0876]

[0877] Example 6. Titration of AAV

[0878] To determine the titer of AAV2 purified in Example 5, qPCR (Bio-Rad, USA, CFX96) was performed. AAV was treated with DNase I at 37°C for 1 hour with DNase I reaction buffer (New England Biolab, USA, M0303S). Then, proteinase K (Invitrogen, USA, Cat No. AM2548) was treated with the DNase I-treated sample at 55°C for 30 minutes and inactivated by reacting at 95°C for 15 minutes. Each prepared sample was used as a template for qPCR, and an AAV construct was used to obtain a standard curve (7.4×10 8 -7.4×10 4, 10-fold dilution), Recombinant Adeno-associated Virus 2 Reference Standard Stock (rAAV2-RSS, ATCC, USA, Cat No. VR-1616) or Recombinant Adeno-associated Virus 8 Reference Standard Stock (rAAV8-RSS, ATCC, USA, Cat No. VR-1816) was used as the positive control group. For qPCR for titer determination, 2×SsoAdvanced Universal Probe Supermix (Bio-Rad, USA, Cat No. 172-5282) and AAV2-ITR specific primers (#030, #031) and probe (#032, FAM-CACT CCCTCTCTGCGCGCTCG-BHQ1) were used. Each qPCR cycle consisting of denaturation at 95°C for 10 minutes, reaction at 95°C for 30 seconds, and reaction at 60°C for 1 minute was repeated 40 times. The standard curve and quantification were analyzed using Bio-Rad CFX Maestro 1.1 software (Bio-Rad, USA).

[0879]

[0880] Example 7. Measurement of GFP gene expression intensity through flow cytometry

[0881] The expression of eGFP was measured through flow cytometry (Beckman Coulter, USA, CytoFlex). The measured value of eGFP was calculated by correcting the transduction efficiency with the measurement of red fluorescence by the co-transduced pCMV-dsRed (Clontech, Japan, Cat No. 632416) construct. At 72 hours after transduction, the cells were washed with DPBS and detached with trypsin. The cells were secured by centrifugation at 1500 rpm for 5 minutes, 500 μl of DPBS supplemented with 2% FBS was added, and the cells were resuspended. In flow cytometry, the single-cell region was delineated through the FSC vs SSC plot, and among them, FL1-A (green) and FL2-A (red) were measured. After complementation was carried out using each sample transfected with a single fluorescent vector respectively (pEGFP-C1, Clontech, Japan, Cat No. PT3286-1 and pCMV-DsRed-Expression2), this was reflected in the measurement results. All flow cytometry results were analyzed using FlowJo software 10.5.3 (Becton Dickinson & Company, USA).

[0882]

[0883]

Table 1

[0884] Oligonucleotide sequences used in the present disclosure

[0885]

[0886] Experimental results

[0887] 1. Increase in the expression of the eGFP gene by the EF-1α intron in combination with the EF-1α promoter

[0888] In the eGFP gene expression regulated by the combination of the cytomegalovirus (CMV) enhancer and the human elongation factor-1 alpha (EF-1α) promoter, the effect of the EF-1α intron was tested. First, as a result of comparison with animal cell expression constructs without ITR, in all cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B), the expression of eGFP was increased by the EF-1α intron (459.6%, 276.3%, 181.8%, 163.4%, 471.1%, 494.3%) (Figure 2a). Similarly, in the pAAV construct containing ITR, the expression of eGFP was also increased by the EF-1α intron (224.0%, 167.7%, 218.7%, 229.5%, 202.5%, 260.5%) (Figure 2b). This result suggests that the EF-1α intron combined with the EF-1α promoter can increase gene expression in many forms of exogenous gene expression constructs.

[0889]

[0890] 2. Increase in the expression of the eGFP gene by the EF-1α intron in combination with various promoters

[0891] In addition to the EF-1α promoter, it was tested whether an increase in gene expression by the EF-1α intron was observed through combination with other promoters. First, in the expression of the eGFP gene induced by the combination of the cytomegalovirus (CMV) enhancer and the CMV promoter, the effect of the EF-1α intron was tested. First, as a result of comparison with animal cell expression constructs without ITR, it was confirmed that the expression of eGFP increased by the EF-1α intron (284.3%, 464.0%, 217.5%, 180.4%, 405.7%, 370.6%) in all cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) (Figure 3a).

[0892] Similarly, in pAAV constructs containing ITR, the EF-1α intron was also confirmed to increase the expression of eGFP regulated by the combination of the cytomegalovirus (CMV) enhancer and the CMV promoter (241.4%, 494.8%, 266.8%, 185.5%, 415.5%, 367.8%) (Figure 3b).

[0893] Next, the effect of the EF-1α intron on the expression of the eGFP gene regulated by the combination of the CMV enhancer and the chicken β-actin promoter was confirmed. As a result of comparison in animal cell expression constructs, it was confirmed that the EF-1α intron increased the expression of eGFP in all cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) (415.2%, 396.7%, 233.9%, 217.9%, 353.7%, 297.7%) (Figure 3c). Such results suggest that gene expression is increased by the EF-1α intron even when combined with various promoters other than the EF-1α promoter.

[0894]

[0895] 3. Determination of the minimum length of the EF-1α intron fragment showing the gene expression increasing effect

[0896] 3-A. Fabrication of a series of CEE constructs with sequential deletions of the EF-1α intron sequence

[0897] The CEE-FL construct was designed to contain a part of the CMV enhancer and CMV promoter (31 bp at the 5'-end), the EF-1α promoter, 29 bp of EF-1α exon 1, 924 bp of EF-1α intron, and 9 bp of EF-1α exon 2. In the core sequence related to the splicing function of the intron, the 3'-end splicing acceptor containing the 5'-end splicing donor and the branch point site (BPS) was located. To search for the minimum-length EF-1α intron sequence capable of increasing gene expression, the splicing donor sequence at the 5'-end of the intron was conserved, and the subsequent intron sequences were sequentially deleted. That is, the CEE constructs were prepared as in Example 2-1, which commonly included the 19 bp at the 5'-end of the EF-1α intron, which was presumed to be the splicing donor consensus sequence of the EF-1α intron, and the subsequent 659 bp (T2), 720 bp (T3), 807 bp (T3.1), 829 bp (T3.1.1), 851 bp (T3.1.2), 873 bp (T3.2), and 895 bp (T4) were deleted (Figure 4a).

[0898]

[0899] 3-B. Effects of sequential deletion of the EF-1α intron sequence on gene expression increase or decrease

[0900] The eGFP expression from a total of eight constructs, including those with the full-length EF-1α intron sequence (CEE-FL) and those with sequential deletions, was compared among five types of animal cell lines. First, in the HeLa, Hep3B, and Huh-7 cell lines, it was confirmed that eGFP gene expression was maintained from CEE-T2 to CEE-T3.1.2 when compared with the full-length EF-1α intron A. However, in the cases of CEE-T3.2 and CEE-T4, gene expression decreased rapidly. Next, in the ARPE-19 and RPE-1 cell lines, eGFP expression decreased to approximately 50% level in the case of CEE-T2 compared with CEE-FL, but showed eGFP expression similar to or higher than that of CEE-FL from CEE-T3 to CEE-T3.1.2. However, as in the case of each previous cell line, expression decreased rapidly in CEE-T3.2 and CEE-T4. Each of the above results indicates that the intron A fragment possessing the gene expression increasing function in the EF-1α intron A is from T2 to T3.2, preferably a fragment with a length of T3 (117 nucleotides) or less, and a fragment with a length of T3.2 (51 nucleotides) or more is presented as a gene expression increasing fragment (Figure 4b).

[0901]

[0902] 4. Gene expression increase by T3.1.1 and T3.1.2, which are EF-1α intron fragments

[0903] 4-A. Production of EF-1α intron fragments T3.1.1 and T3.1.2 combined with the chicken β-actin promoter

[0904] The CAE-FL construct was designed to contain the CMV enhancer, chicken β-actin promoter, 32 bp of chicken β-actin exon 1, 29 bp of EF-1α exon 1, 924 bp of EF-1α intron A, and 9 bp of EF-1α exon 2. CA is a construct in which the entire EF-1α intron is deleted. The CAE-T3.1.1 construct contains 19 bp at the 5'-end and the T3.1.1 sequence at the 3'-end of the EF-1α intron, and is identical to CAE-FL except that 829 bp in between is deleted. The CAE-T3.1.2 construct contains 19 bp at the 5'-end of EF-1α intron A and the T3.1.2 sequence at the 3'-end, and is identical to CAE-FL except that 851 bp in between is deleted (Figure 5a).

[0905]

[0906] 4-B. Increased gene expression by the EF-1α intron fragments T3.1.1 or T3.1.2 combined with the chicken β-actin promoter

[0907] Using three constructs, CA in which all nucleotides of the EF-1α intron were completely deleted, and CAE-T3.1.1 and CAE-T3.1.2 containing partially deleted EF-1α introns, the eGFP expression was compared in two animal cell lines (HeLa, ARPE-19). In the CAE-T3.1.1 and CAE-T3.1.2 groups, when compared with the CA construct group, eGFP expression levels of 330.5% and 243.9% in HeLa, and 170.8% and 165.9% in ARPE-19 were observed (Figure 5b). Such results suggest that T3.1.1 and T3.1.2, which are fragments of the EF-1α intron, can increase gene expression even in combination with the chicken β-actin promoter.

[0908]

[0909] 4-C. Construction of constructs in which the EF-1α intron fragments T3.1.1 and T3.1.2 are combined with the splicing donor of chicken β-actin

[0910] CA-T3.1.1 and CA-T3.1.2 constructs with a hybrid intron structure containing a 95-bp (T3.1.1) or 73-bp (T3.1.2) 3'-terminal fragment of the EF-1α intron and 9 bp of the EF-1α exon 2 were constructed while maintaining the CMV enhancer, chicken β-actin promoter, 93 bp of chicken β-actin exon 1, and 43 bp of the intron. A part of the chicken β-actin exon 1 and the chicken β-actin intron was expected to function as a splicing donor, and the EF-1α intron A fragment was expected to function as a splicing acceptor. As a control, CA constructs without intron sequences were both tested (Figure 5c).

[0911]

[0912] 4-D. Increased gene expression by an intron hybridized with a splicing donor fragment of chicken β-actin and 3'-terminal fragments of T3.1.1 and T3.1.2 of the EF-1α intron A

[0913] After transducing HeLa and Hep3B cell lines with CA, CA-T3.1.1, and CA-T3.1.2 constructs respectively and comparing the levels of gene expression, it was confirmed that gene expression increased in CA-T3.1.1 and CA-T3.1.2 compared to CA without introns (HeLa: 215.7%, 211.0%, Hep3B: 155.0%, 167.5%). Such results suggest that T3.1.1 and T3.1.2, which are EF-1α intron fragments, can also increase gene expression when combined with splicing donors of other genes (Figure 5d).

[0914]

[0915] 5. Delivery of the TAFA4 gene using AAV

[0916] 5-A. Evaluation of the efficacy of TAFA4 in a NaIO3 (sodium iodate)-induced retinal injury model

[0917] To evaluate the efficacy of TAFA4 in improving retinal damage, NaIO3 was administered, and changes in fluorescence angiography (FA) and electroretinogram (ERG) were evaluated by administering AAV8.mouse TAFA4 to a mouse model with induced retinal damage.

[0918] Control AAV8 or AAV8.mouse TAFA4 was subretinally injected (SRI) into C57BL / 6 mice (Orient Bio). Control AAV8 was produced using the pAAV construct in Figure 6a, and AAV8.mouse TAFA4 was produced using the pAAV-mTAFA4 construct (Example 2-6-1) in Figure 6b.

[0919] Mouse TAFA4 can be sufficiently expressed. After subretinally injecting (SRI) 5×10 8 vg of AAV8 into both eyes of the mouse and waiting for 56 days, NaIO3 was administered via the tail vein at a dose of 20 mg / kg to induce an AMD model. On the 10th day after model induction (66 days after SRI administration), a fluorescent contrast agent was injected through the tail vein. Then, after aligning the image with the fundus using a Micron-IV imaging camera (Phoenix), a fluorescence angiography (FA) image was taken. On the 11th day after model induction (67 days after SRI administration), a scotopic ERG evaluation was performed. The mouse was placed on the ERG stage, and after the ERG probe was brought into contact with the tail, head, and cornea respectively, the amplitudes of the A and B waves were measured. The analysis of ERG was performed using the "LabScribe ERG (iWorx Data Acquisition Software)" program.

[0920] On the FA images taken on the 10th day after model induction, compared with the healthy (AMD not induced) animal (naive, control AAV8-administered) group, leakage of the fluorescent contrast agent due to damage to the outer retina was confirmed in the retina of the NaIO3-administered group (control group, control-AAV8-administered). On the other hand, in the group administered with AAV8.mouse TAFA4 (experimental group), similar to the naive group, almost normal retinal findings were shown (Figure 7). In the scotopic ERG results evaluated on the 11th day after model induction, similar to FA, abnormal findings in the retina were shown by NaIO3, and the amplitudes and potential inductions of both A and B waves were all significantly decreased. On the other hand, in the group administered with AAV8.mouse TAFA4 (experimental group), the amplitudes of both A and B waves were all restored, and in particular, the amplitude of the B wave was restored to a level almost close to normal (Figure 8).

[0921]

[0922] 5-B. mRNA expression analysis of TAFA4 in the NaIO3 (sodium iodate)-induced retinal damage model

[0923] On the 56th day after subretinal administration of control AAV8 or AAV8.mouse TAFA4 to C57BL / 6 mice, a retinal damage model was induced by intravenous administration of NaIO3 at a dose of 20 mg / kg via the tail vein. On the 14th day after model induction (70 days after SRI administration), the eyeballs were enucleated. RNA was extracted from the enucleated eyeballs using the RNeasy Mini kit (Qiagen). After synthesizing cDNA from the extracted RNA using the Primescript 1st strand cDNA synthesis kit (Takara), qPCR was performed using the cDNA as a template to confirm the mRNA level of TAFA4. The TAFA4 mRNA level was the relative amount to GAPDH mRNA and was shown using the ΔΔCq value.

[0924] Compared with the naive group, in the group treated with NaIO3 (control group, control-AAV8), the mouse TAFA4 mRNA level hardly changed separately from retinal damage. However, in the group administered with AAV8.mouse TAFA4 (experimental group), it was confirmed that the mouse TAFA4 mRNA level was significantly higher than that in the non-administered group, and it was found that the retinal damage was improved by the thus-expressed mouse TAFA4 (Figure 9).

[0925]

[0926] 5-C. Comparison of sequence identity of TAFA4 among species

[0927] The sequence identities of the full-length TAFA4 proteins of mammals (Human, Monkey, Pig, Rabbit, Rat, Mouse), birds (Chicken), reptiles (Komodo dragon, Wall lizard, Fence lizard, Gecko), amphibians (Frog), and fish (Fish) were compared, and the consensus sequence among species was confirmed (Figure 10). The amino acid sequence of the mature human TAFA4 protein (95 a.a.) showed a sequence identity of more than 90% compared with mammals and amphibians, more than 95% compared with birds, and more than 85% compared with reptiles and fish, and it was confirmed that it showed a very high sequence identity regardless of species (Figure 19).

[0928]

[0929] 5-D. Retinal damage protection effect by TAFA4 sequence identity in the NaIO3 (sodium iodate)-induced retinal damage model

[0930] Since the protective effect of AAV8.mouse TAFA4 on retinal damage was confirmed in a NaIO3 (sodium iodate)-induced retinal damage model, experiments were conducted to determine whether other types of TAFA4 also have a protective effect on retinal damage. Experiments were carried out to confirm the efficacy of human TAFA4 and TAFA4 from reptiles (Gecko TAFA4; sequence identity 87.4%) and fish (fish TAFA4; sequence identity 86.3%), which have the lowest sequence identity with human TAFA4. C57BL / 6 mice (Orient Bio) were subretinally injected (SRI) with control AAV8 or various AAV8.TAFA4. The AAV8 and various AAV8.TAFA4 used in the experiment were produced by the same method as described in 5-A above. AAV8.human TAFA4 was produced using the pAAV-human TAFA4 construct (Example 2-6-2), AAV8.gecko TAFA4 was produced using the pAAV-gecko TAFA4 construct (Example 2-6-3), and AAV8.fish TAFA4 was produced using the pAAV-fish TAFA4 construct (Example 2-6-4). To ensure sufficient expression of TAFA4, 1×10

[0931] C57BL / 6 mice (Orient Bio) were subretinally injected (SRI) with control AAV8 or various AAV8.TAFA4. The AAV8 and various AAV8.TAFA4 used in the experiment were produced by the same method as described in 5-A above.

[0932] AAV8.human TAFA4 was produced using the pAAV-human TAFA4 construct (Example 2-6-2), AAV8.gecko TAFA4 was produced using the pAAV-gecko TAFA4 construct (Example 2-6-3), and AAV8.fish TAFA4 was produced using the pAAV-fish TAFA4 construct (Example 2-6-4).

[0933] TAFA4 could be sufficiently expressed, so 1×10 9After subretinal injection (SRI) of vg AAV8 into both eyes of mice and waiting for 42 days, an AMD model was induced by intravenous injection of NaIO3 at a dose of 20 mg / kg into the tail vein. On the 9th day after model induction (51 days after SRI administration), a fluorescent contrast agent was injected through the tail vein. Subsequently, after aligning the image with the fundus using a Micron-IV imaging camera (Phoenix), a fluorescence angiography (FA) image was taken. On the 7th day after model induction (49 days after SRI administration), scotopic ERG evaluation was performed. The mice were placed on the ERG stage, and after the ERG probes were brought into contact with the tail, head, and cornea respectively, the amplitudes of the A and B waves were measured. The analysis of ERG was carried out using the "LabScribe ERG (iWorx Data Acquisition Software)" program.

[0934] On the FA images taken on the 9th day after model induction, leakage of the fluorescent contrast agent due to damage to the outer retina was confirmed in the retina of the NaIO3-administered group (control group, control-AAV8 administration) compared to the healthy animal (naive, control AAV8 administration) group. On the other hand, in the groups administered with AAV8.human TAFA4, AAV8.gecko TAFA4, and AAV8.fish TAFA4 (experimental groups), almost normal retinal findings were shown, similar to the naive group (Figure 11). In the scotopic ERG results evaluated on the 11th day after model induction, abnormal findings of the retina were also confirmed, where the amplitude of the A and B waves and the amplitude of the induced potential were all significantly decreased by NaIO3. On the other hand, in the groups administered with AAV8.human TAFA4, AAV8.gecko TAFA4, and AAV8.fish TAFA4 (experimental groups), the induced potential changes in the amplitudes of the A and B waves caused by NaIO3 were restored (Figure 12).

[0935]

[0936] Comparison of TAFA4 expression by 5-F.signal peptide difference

[0937] To confirm the presence or absence of normal expression of the 95 - amino acid sequence predicted to be the mature protein, the signal sequence was replaced and the expression of human TAFA4 protein was confirmed. For this purpose, a full - length TAFA4 construct, a construct in which the 45 - amino acid sequence at the N - terminus was replaced with the signal sequence of the murine IgG heavy - chain variable region 3 (MGWSCIILFLVATATGVHS, SEQ ID NO: 312), and a construct in which the 45 - amino acid sequence at the C - terminus was replaced with the signal sequence of human Interleukin 2 (IL2) (MYRMQLLSCIALSLALVTNS, SEQ ID NO: 313) were prepared in the pcDNA3.1( - ) vector. HEK293 cells were attached to a 12 - well plate at 6×10 5 cells / well and cultured for 18 hours. Then, the constructs were transfected using Lipofectamin 3000 (Invitrogen) together with pCMV DsRED - Express2 (TAKARA). After culturing for 72 hours, the cells were harvested and RNA was extracted using the RNeasy Mini kit (Qiagen). The extracted RNA was synthesized into cDNA using the Primescript 1st strand cDNA synthesis kit (Takara). Then, qPCR was performed using the cDNA as a template to confirm the mRNA level of TAFA4. The TAFA4 mRNA level was the relative amount to GAPDH mRNA, calculated using the ΔΔCq value, and then divided by the ratio of cells expressing DsRed for comparison. To confirm the amount of TAFA4 protein, the culture medium was removed, and the supernatant was separated by centrifugation at 12000 rpm and analyzed by the method provided by the manufacturer using a human TAFA4 ELISA kit (Abbexa). The calculated values were divided by the ratio of cells expressing DsRed for comparison.

[0938] As a result of the confirmation, no significant change in expression due to the signal sequence was shown in either mRNA expression or protein expression (p>0.05) (Figure 13).

[0939]

[0940] 6. Delivery of TAFA1-3 genes using AAV

[0941] 6-A. Protective effect against retinal damage due to sequence identity of TAFA4 paralogs in a NaIO3 (sodium iodate)-induced retinal damage model

[0942] It is known that there is high sequence identity between TAFA4 and the TAFA protein group. Therefore, it was confirmed whether human TAFA1, TAFA2, and TAFA3 in a mouse model also exhibit a retinal protective effect against NaIO3-induced retinal damage. NaIO3 was administered, and changes in fluorescence angiography (FA) and electroretinogram (ERG) were evaluated by administering AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, and AAV8.TAFA4 to a mouse model in which AMD was induced.

[0943] Control AAV8, AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, or AAV8.TAFA4 was subretinally injected (SRI) into C57BL / 6 mice (Orient Bio). The AAV8, AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, and AAV8.TAFA4 used in the experiment were produced by the same method as described in 5-A above.

[0944] AAV8.human TAFA1 was produced using the pAAV-human TAFA1 construct (Example 2-6-5), AAV8.human TAFA2 was produced using the pAAV-human TAFA2 construct (Example 2-6-6), AAV8.human TAFA3 was produced using the human TAFA1 construct (Example 2-6-7), and AAV8.human TAFA4 was produced using the human TAFA1 construct (Example 2-6-2).

[0945] To confirm the retinal protective effects of TAFA1, TAFA3, and TAFA4, 1×10 9After waiting for 42 days following subretinal injection (SRI) of vg of AAV8 into both eyes of mice, an AMD model was induced by intravenous injection of NaIO3 at a dose of 20 mg / kg via the tail vein. On the 9th day after model induction (51 days after SRI administration), a fluorescent contrast agent was injected through the tail vein. Subsequently, after aligning the image with the fundus using a Micron-IV imaging camera (Phoenix), a fluorescence angiography (FA) image was taken. On the 7th day after model induction (49 days after SRI administration), a scotopic ERG evaluation was performed. The mice were placed on the ERG stage, and after the ERG probe was brought into contact with the tail, head, and cornea respectively, the amplitudes of the A and B waves were measured. The analysis of ERG was carried out using the "LabScribe ERG (iWorx Data Acquisition Software)" program.

[0946] To confirm the retinal protective effect of TAFA2, 1×10 9 After waiting for 56 days following subretinal injection (SRI) of vg of AAV8 into both eyes of mice, an AMD model was induced by intravenous injection of NaIO3 at a dose of 20 mg / kg via the tail vein. On the 9th day after model induction (65 days after SRI administration), a fluorescent contrast agent was injected through the tail vein. Subsequently, after aligning the image with the fundus using a Micron-IV imaging camera (Phoenix), a fluorescence angiography (FA) image was taken. On the 7th day after model induction (63 days after SRI administration), a scotopic ERG evaluation was performed. The mice were placed on the ERG stage, and after the ERG probe was brought into contact with the tail, head, and cornea respectively, the amplitudes of the A and B waves were measured. The analysis of ERG was carried out using the "LabScribe ERG (iWorx Data Acquisition Software)" program.

[0947] In the FA images, leakage of the fluorescent contrast agent due to damage to the outer retina was confirmed in the retina of the NaIO3-administered group (control group, control-AAV8 administration) compared to the healthy animal (naive, control AAV8 administration) group. On the other hand, in the groups administered with AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, or AAV8.TAFA4 (experimental groups), almost normal retinal findings were shown as in the naive group (Figure 14). In the scotopic ERG results, abnormal findings of the retina in which the amplitude potential induction widths of both the A-wave and B-wave were significantly decreased by NaIO3 were confirmed. On the other hand, in the groups administered with AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, or AAV8.TAFA4 (experimental groups), the potential induction changes in the amplitudes of the A-wave and B-wave caused by NaIO3 were restored (Figure 15).

[0948]

[0949] Sequence identity comparison between 6-B.human TAFA4 and TAFA paralogs of various species

[0950] In the retinal damage model induced by NaIO3, the retinal protective effects of not only Human TAFA1, human TAFA2, human TAFA3, human TAFA4, but also gecko TAFA4 and fish TAFA4 were confirmed. Therefore, considering that the common sequences of various TAFA proteins have retinal protective effects, in order to confirm the common sequences, the sequence identities of full-length TAFA1, full-length TAFA2, full-length TAFA3, and full-length TAFA4 proteins of mammals (Human, Monkey, Pig, Rabbit, Rat, Mouse), birds (Chicken), reptiles (Komodo dragon, Wall lizard, Fence lizard, Gecko), amphibians (Frog), and fish (Fish) were compared. The amino acid sequences of full-length TAFA1 to TAFA4 in each vertebrate are shown in Figures 20a to 20d. Figure 16 shows the comparison of interspecies sequence identities of mature TAFA1 protein, Figure 17 shows the comparison of interspecies sequence identities of mature TAFA2 protein, Figure 18 shows the comparison of interspecies sequence identities of mature TAFA3 protein, and Figure 19 shows the comparison results of interspecies sequence identities of mature TAFA4 protein.

[0951] The 93 sequences of human TAFA4 (IKQGTCEVVAVHRCCNKNRIEERSQTVKCSCFPGQVAGTTRAQPSCVEASIVIQKWWCHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR, SEQ ID NO: 75) were confirmed to have very high sequence identities with TAFA4 sequences of other species and the common sequences of TAFA1 to TAFA3 from human to fish. The sequence identities within the common sequences are as shown in Table 2.

[0952]

[0953]

Table 2

[0954] The sequence identities were confirmed using Clustal Omega.

[0955] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes as if each individual publication, patent, patent application, or other document were individually incorporated by reference for all purposes.

[0956] Having described the present disclosure with reference to the foregoing aspects, those of ordinary skill in the art will understand that various modifications and variations are possible by adding, changing, deleting, or inserting components, without departing from the spirit of the present disclosure as set forth in the claims. It will be understood that such modifications and variations are within the scope of the present disclosure.

Brief Description of the Drawings

[0957]

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Claims

1. A pharmaceutical composition for preventing or treating a retinal or macular disease, comprising a polypeptide having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75, a nucleic acid encoding the polypeptide, a vector containing the nucleic acid, a recombinant virus particle containing the vector and a capsid protein, or a cell containing the nucleic acid or the vector.

2. The pharmaceutical composition according to claim 1, wherein the amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 comprises the amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus): <General formula 1> X1 - X2 - X3 - G - T - C - E - V - X4 - A - X5 - H - X6 - C - C - N - X7 - N - X8 - I - E - E - X9 - S - Q - T - X10 - X11 - C - S - C - X12 - X13 - G - X14 - V - A - G - T - T - X15 - X16 - X17 - P - S - C - V - X18 - A - X19 - I - V - X20 - X21 - X22 - W - W - C - X23 - M - X24 - P - C - X25 - X26 - G - E - X27 - C - K - X28 - L - P - D - X29 - X30 - G - W - X31 - C - X32 - X33 - G - X34 - K - X35 - K - T - T - X36 - X37 - X38 - X39 In the general formula 1, X1 is absent, V, I or L, X2 is K, E, R or Q, X3 is G, T, Q, P or A, X4 is V or I, X5 is A, L, V or I, X6 is R or L, X7 is K, R or Q, X8 is R or K, X9 is R or L, X10 is V or G, X11 is K or N, X12 is F or L, X13 is P or S, X14 is Q or K, X15 is R, H or Q, X16 is A, N, S or T, X17 is A, Q, R, K or T, X18 is D or E, X19 is S or A, X20 is I, E, L, A or V, X21 is Q, G or E, X22 is K or R, X23 is H, Q or E, X24 is E, Q, N, D, S or H, X25 is L, V or M, X26 is E, D, P, L or A, X27 is E or D, X28 is V, T, A or I, X29 is L, N, R, Y, S or Q, X30 is S, K or T, X31 is S or M, X32 is S, A or Y, X33 is S, T or R, X34 is N or H, X35 is V or I, X36 is R or K, X37 is absent, V, A, G, M or N, X38 is absent, T, I, N, F or S, and X39 is absent, R, H, V, K, I or Q.

3. The pharmaceutical composition according to claim 1, wherein the polypeptide further comprises an amino acid sequence (from the N-terminus to the C-terminus) of the following general formula 2: <General formula 2> X1-X2-H-H-K-A-X3-H In the general formula 2, X1 is A or V, X2 is N or I, and X3 is H or Q.

4. The pharmaceutical composition according to claim 1, wherein the polypeptide further comprises an amino acid sequence (from the N-terminus to the C-terminus) of the following general formula 3: <General formula 3> X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 In the general formula 3, X1 is A or S, X2 is L, T or S, X3 is Q, E or H, X4 is absent, P, L or H, X5 is P or R, X6 is T, S or I, X7 is A, P, T, S or H, X8 is T, A, S or I, X9 is V or A, and X10 is L or H.

5. The pharmaceutical composition according to claim 1, wherein the polypeptide further comprises an amino acid sequence (from the N-terminus to the C-terminus) of the following general formula 4: <General formula 4> X1-X2 In the general formula 4, X1 is H or Y, and X2 is Q, V or L.

6. The pharmaceutical composition according to claim 1, wherein the polypeptide further comprises an amino acid sequence (from the N-terminus to the C-terminus) of: LHRP (SEQ ID NO: 293); LHQSGFTSGHFPHHRKLGE (SEQ ID NO: 294); or LAPPGTNIQI (SEQ ID NO: 295)

7. The pharmaceutical composition according to claim 1, wherein the polypeptide further comprises an amino acid sequence (from the N-terminus to the C-terminus) of the following general formula 5: <General formula 5> X1-X2-X3 In the general formula 5, X1 is P or G, X2 is R or H, and X3 is T, S or L.

8. The pharmaceutical composition according to claim 1, wherein the polypeptide further comprises an amino acid sequence (from the N-terminus to the C-terminus) of: PYTSL (SEQ ID NO: 296); or QEDKKL (SEQ ID NO: 297).

9. The pharmaceutical composition according to claim 1, wherein the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 75 to 129.

10. The pharmaceutical composition according to claim 1, wherein the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 184 to 238.

11. The pharmaceutical composition according to claim 1, wherein the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 130 to 183.

12. The pharmaceutical composition according to claim 1, wherein the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 239 to 292.

13. The pharmaceutical composition according to claim 1, wherein the vector is a viral vector.

14. The pharmaceutical composition according to claim 1 or 13, wherein the virus is an adeno-associated virus (AAV).

15. The pharmaceutical composition according to claim 14, wherein the AAV has a serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAVrh10.

16. The pharmaceutical composition according to claim 1, wherein the retinal or macular disease comprises diabetic retinopathy, choroidal neovascularization, macular disease, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease or a combination thereof.

17. The pharmaceutical composition according to claim 16, wherein the macular degeneration is age-related macular degeneration.

18. The pharmaceutical composition according to claim 17, wherein the age-related macular degeneration is wet or dry.

19. An adeno-associated virus (AAV) vector comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO:

75.

20. The AAV vector according to claim 19, further comprising at least one sequence selected from the group consisting of a promoter sequence, an enhancer sequence, an exon sequence, an intron sequence, a signal sequence coding sequence, a splicing donor sequence, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

21. The AAV vector according to claim 20, wherein the intron is composed of 51 to 117 nucleotides and contains the nucleotide sequence shown in SEQ ID NO:

57.

22. The AAV vector according to claim 20, wherein the intron has at least 70% sequence identity with (i) nucleotides 871 to 924 of SEQ ID NO: 1 (SEQ ID NO: 58), (ii) nucleotides 861 to 924 of SEQ ID NO: 1 (SEQ ID NO: 59), (iii) nucleotides 852 to 924 of SEQ ID NO: 1 (SEQ ID NO: 3), (iv) nucleotides 851 to 924 of SEQ ID NO: 1 (SEQ ID NO: 61), (v) nucleotides 830 to 924 of SEQ ID NO: 1 (SEQ ID NO: 2), (vi) nucleotides 821 to 924 of SEQ ID NO: 1 (SEQ ID NO: 63), (vii) nucleotides 811 to 924 of SEQ ID NO: 1 (SEQ ID NO: 64), or (viii) nucleotides 808 to 924 of SEQ ID NO: 1 (SEQ ID NO: 65).

23. The AAV vector according to claim 22, wherein the intron contains (i) nucleotides 871 to 924 of SEQ ID NO: 1 (SEQ ID NO: 58), (ii) nucleotides 861 to 924 of SEQ ID NO: 1 (SEQ ID NO: 59), (iii) nucleotides 852 to 924 of SEQ ID NO: 1 (SEQ ID NO: 3), (iv) nucleotides 851 to 924 of SEQ ID NO: 1 (SEQ ID NO: 61), (v) nucleotides 830 to 924 of SEQ ID NO: 1 (SEQ ID NO: 2), (vi) nucleotides 821 to 924 of SEQ ID NO: 1 (SEQ ID NO: 63), (vii) nucleotides 811 to 924 of SEQ ID NO: 1 (SEQ ID NO: 64), or (viii) nucleotides 808 to 924 of SEQ ID NO: 1 (SEQ ID NO: 65).

24. The AAV vector according to claim 20, wherein the vector comprises the following configuration: (1) The CMV enhancer sequence shown in SEQ ID NO: 4; (2) A promoter sequence selected from the CMV promoter sequence shown in SEQ ID NO: 5 or 6, the EF-1α promoter sequence shown in SEQ ID NO: 7, or the chicken β-actin promoter sequence shown in SEQ ID NO: 8; (3) An exon 1 (E1) sequence selected from the CMV E1 sequence shown in SEQ ID NO: 12, the EF-1α E1 sequence shown in SEQ ID NO: 13, or the chicken β-actin E1 sequence shown in SEQ ID NO: 14 or 15; (4) A splicing donor sequence shown in SEQ ID NO: 9 or 10; and / or (5) The EF-1α E2 sequence shown in SEQ ID NO:

11. [

25. ] The amino acid sequence having at least 70% sequence identity with the amino acid sequence presented in SEQ ID NO: 75 comprises the amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus), and is characterized in that the AAV vector according to claim 19: <General formula 1> X1-X2-X3-G-T-C-E-V-X4-A-X5-H-X6-C-C-N-X7-N-X8-I-E-E-X9-S-Q-T-X10-X11-C-S-C-X12-X13-G-X14-V-A-G-T-T-X15-X16-X17-P-S-C-V-X18-A-X19-I-V-X20-X21-X22-W-W-C-X23-M-X24-P-C-X25-X26-G-E-X27-C-K-X28-L-P-D-X29-X30-G-W-X31-C-X32-X33-G-X34-K-X35-K-T-T-X36-X37-X38-X39 In the above general formula 1, X1 is absent, V, I or L, X2 is K, E, R or Q, X3 is G, T, Q, P or A, X4 is V or I, X5 is A, L, V or I, X6 is R or L, X7 is K, R or Q, X8 is R or K, X9 is R or L, X10 is V or G, X11 is K or N, X12 is F or L, X13 is P or S, X14 is Q or K, X15 is R, H or Q, X16 is A, N, S or T, X17 is A, Q, R, K or T, X18 is D or E, X19 is S or A, X20 is I, E, L, A or V, X21 is Q, G or E, X22 is K or R, X23 is H, Q or E, X24 is E, Q, N, D, S or H, X25 is L, V or M, X26 is E, D, P, L or A, X27 is E or D, X28 is V, T, A or I, X29 is L, N, R, Y, S or Q, X30 is S, K or T, X31 is S or M, X32 is S, A or Y, X33 is S, T or R, X34 is N or H, X35 is V or I, X36 is R or K, X37 is absent, V, A, G, M or N, X38 is absent, T, I, N, F or S, and X39 is absent, R, H, V, K, I or Q.

26. The polypeptide further comprises an amino acid sequence of the following general formula 2 (from the N-terminus to the C-terminus), and the AAV vector according to claim 19 is characterized in that: <General formula 2> X1-X2-H-H-K-A-X3-H In the general formula 2, X1 is A or V, X2 is N or I, and X3 is H or Q.

27. The polypeptide further comprises an amino acid sequence of the following general formula 3 (from the N-terminus to the C-terminus), and the AAV vector according to claim 19 is characterized in that: <General formula 3> X1-X2-X3-X4-X5-X6-X7-X8-X9-X10 In the general formula 3, X1 is A or S, X2 is L, T or S, X3 is Q, E or H, X4 is absent, P, L or H, X5 is P or R, X6 is T, S or H, X7 is A, P, T, S or I, X8 is T, A, S or I, X9 is V or A, and X10 is L or H.

28. The polypeptide further comprises an amino acid sequence of the following general formula 4 (from the N-terminus to the C-terminus), and the AAV vector according to claim 19 is characterized in that: <General formula 4> X1-X2 In the general formula 4, X1 is H or Y, and X2 is Q, V or L.

29. The polypeptide further comprises an amino acid sequence of the following (from the N-terminus to the C-terminus), and the AAV vector according to claim 19 is characterized in that: LHRP (SEQ ID NO: 293); LHQSGFTSGHFPHHRKLGE (SEQ ID NO: 294); or LAPPGTNIQI (SEQ ID NO: 295).

30. The polypeptide further comprises an amino acid sequence of the following general formula 5 (from the N-terminus to the C-terminus), and the AAV vector according to claim 19 is characterized in that: <General formula 5> X1-X2-X3 In the general formula 5, X1 is P or G, X2 is R or H, and X3 is T, S or L.

31. The AAV vector according to claim 19, wherein the polypeptide further comprises the following amino acid sequence (from the N-terminus to the C-terminus): PYTSL (SEQ ID NO: 296); or QEDKLK (SEQ ID NO: 297).

32. The AAV vector according to claim 19, wherein the amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO: 75 is composed of 91 to 119 amino acid sequences.

33. The AAV vector according to claim 19, wherein the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 75 to 129.

34. The AAV vector according to claim 19, wherein the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 184 to 238.

35. The AAV vector according to claim 19, wherein the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 130 to 183.

36. The AAV vector according to claim 19, wherein the nucleic acid encoding the polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 239 to 292.

37. The AAV vector according to claim 19, which is for use in gene therapy.

38. The AAV vector according to claim 19, which is for use in the expression of a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence set forth in SEQ ID NO:

75.

39. An isolated host cell comprising the AAV vector of claim 19.

40. An isolated host cell transformed with the AAV vector of claim 19.

41. A composition comprising the AAV vector of claim 19 or a host cell comprising the AAV vector or transformed with the AAV vector.

42. The composition according to claim 41, further comprising a pharmaceutically acceptable carrier.

43. The composition according to claim 42, characterized in that it is for the prevention or treatment of retinal or macular diseases.

44. A kit comprising the AAV vector of claim 19, or a host cell comprising or transformed with said AAV vector and an instruction manual.

45. A method for producing recombinant virus particles comprising the step of transducing cells with a construct containing the AAV vector of claim 19 and the rep and cap genes.

46. The method according to claim 45, characterized in that the method further comprises the step of separating the produced recombinant virus particles.

47. Recombinant virus particles produced by the method of claim 45.

48. Recombinant virus particles comprising (a) a capsid protein and (b) the AAV vector of claim 19.

49. A method for preventing or treating a disease or disorder in a subject in need thereof, comprising administering to the subject the AAV vector according to claim 19 or the recombinant virus particles according to claim 48.

50. The method according to claim 49, characterized in that the disease or disorder comprises a retinal or macular disease.

51. The retinal or macular disease according to claim 50, characterized in that it comprises retinopathy, choroidal neovascularization, maculopathy, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease or a combination thereof.

52. The method according to claim 51, characterized in that the macular degeneration comprises age-related macular degeneration (AMD).

53. The pharmaceutical composition according to claim 52, characterized in that the age-related macular degeneration is wet or dry.

54. The pharmaceutical composition according to claim 51, characterized in that the retinopathy is diabetic retinopathy.

55. The method according to claim 51, comprising administering an additional therapeutic agent to the subject.

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