Composition for treating retinal or macular disease

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating retinal or macular disease comprising a polypeptide of an amino acid sequence having at least 70% sequence identity to the amino acid sequence of TAFA proteins (e.g., TAFA1 to TAFA4), a nucleic acid encoding the polypeptide, a vector comprising the nucleic acid, a recombinant viral particle comprising the vector and a capsid protein, and a method for preparing the same. Or a cell comprising the nucleic acid or vector. Furthermore, the present invention relates to an adeno-associated viral (AAV) vector comprising a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity to an amino acid sequence of a TAFA protein and a recombinant viral particle comprising the AAV vector and a capsid protein. The AAV vector, the recombinant virus, or the composition comprising the same according to the present invention can be effectively used in the prevention or treatment of retinal or macular diseases by restoring damaged retina.

Description

Technical Field

[0001] The disclosure of the present invention relates to a pharmaceutical composition for preventing or treating retinal or macular diseases, 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 above polypeptide, a vector comprising the above nucleic acid, a recombinant virus particle comprising the above vector and a capsid protein, or a cell comprising the above nucleic acid or vector. Background Art

[0002] The retina is a transparent film located at the innermost part of the eye wall and in contact with the vitreous body. It converts the optical information of objects into electrical signals and transmits the image to the central visual area of ​​the brain through the optic nerve, playing the role of a primary visual information organ. The retina is composed of more than 100 million light-sensitive photoreceptor cells, more than 1 million ganglion cells as 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 and where the image of objects is formed, responsible for central vision. The macula (macula lutea) is composed of a photoreceptor cell layer composed of cone cells and a ganglion cell layer, so the thickness of the retina is relatively thin. In bright light, the electrical signal of the image is converted into a chemical signal and transmitted to the brain through the optic nerve, which is the axon of the ganglion cell. Except for the macula, the retina recognizes the periphery and plays a major role in dark conditions. Therefore, if the retina or macula is abnormal due to aging or external factors, visual impairment accompanied by problems with vision and visual field may lead to blindness. Retinal or macular diseases include diabetic retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal edema, and macular swelling.

[0003] The TAFA proteome is a protein group that is abundantly expressed throughout the brain and consists of five proteins, TAFA1 to 5. These proteins contain structurally conserved cysteine ​​residues, and the C, CC, and CXC motifs between the cysteines are related to chemokines. TAFA1 to 4 have high similarity in terms of the number of cysteines and the space between the cysteines, while TAFA5 has fewer cysteines than them.

[0004] These proteins are highly conserved in vertebrates and have been shown to be very important for the normal function of the central nervous system. It is reported that mice with knockout TAFA1 lose weight, reduce anxiety behavior, and have fear memory impairment (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, it has been reported that when TAFA2 and TAFA3 are knocked out, anxiety behavior increases (Choi JH, Jeong YM, Kim S et al. Proc Natl Acad Sci US A. 2018; 115(5): E1041-E1050. and Kim S, Lee B, Choi JH, Kim JH, Kim CH, Shin HS. Sci Rep. 2017; 7(1): 16503.). It has been reported that TAFA4-null mice exhibit allodynia and hyperalgesia (Delfini MC, Mantileri A, Gaillard S et al. Cell Rep. 2013; 5(2): 378-388.). Behavioral changes such as increased depressive behavior and spatial memory loss have been reported in TAFA5 knockout mice (Huang S, Zheng C, Xie G et al. FAM19A5 / TAFA5, a novel neurokine, plays a crucial role indepressive-like and spatial memory-related behaviors in mice. Mol Psychiatry. 2021; 26(6): 2363-2379.).

[0005] Despite the importance of the roles these proteins play in the CNS, there is very limited knowledge of the functions and pathophysiology of each protein or its function as a therapeutic agent, and limited knowledge of roles outside the CNS.

[0006] The matters described as the background technology above are only for enhancing the understanding of the background technology of the present invention and should not be regarded as an admission that they correspond to the prior art known to those skilled in the art. Summary of the invention

[0007] Problems to be solved by the invention

[0008] The present invention provides a pharmaceutical composition for preventing or treating retinal or macular diseases, comprising: a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75, a nucleic acid encoding the above polypeptide, a vector comprising the above nucleic acid, a recombinant virus particle comprising the above vector and capsid protein, or a cell comprising the above nucleic acid or vector.

[0009] The present invention also provides a therapeutic use (for use in therapy) of a recombinant virus particle comprising the above polypeptide, a nucleic acid encoding the above polypeptide, a vector comprising the above nucleic acid, a recombinant virus particle comprising the above vector and a capsid protein, or a cell comprising the above nucleic acid or vector.

[0010] The present invention also provides a use of the above-mentioned AAV vector or the above-mentioned recombinant virus particle in a method for preventing or treating retinal or macular diseases. The present invention also provides a method for preventing or treating retinal or macular diseases in a subject in need thereof, the method comprising administering the above-mentioned polypeptide, a nucleic acid encoding the above-mentioned polypeptide, a vector comprising the above-mentioned nucleic acid, a recombinant virus particle comprising the above-mentioned vector and a capsid protein, or a cell comprising the above-mentioned nucleic acid or vector to the above-mentioned subject.

[0011] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 above comprises the amino acid sequence of the following general formula 1 (N-terminus to C-terminus):

[0012] <General formula 1>

[0013] X1-X2-X3-GTCEV-X4-A-X5-H-X6-CCN-X7-N-X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X14-VAGTT-X15-X16-X17-PSCV-X18-A-X19-IV -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39

[0014] In the above general formula 1,

[0015] X1 does not exist, or is V, I, or L.

[0016] X2 is K, E, R or Q,

[0017] X3 is G, T, Q, P or A,

[0018] X4 is V or I,

[0019] X5 is A, L, V or I,

[0020] X6 is R or L,

[0021] X7 is K, R or Q,

[0022] X8 is R or K,

[0023] X9 is R or L,

[0024] X10 is V or G,

[0025] X11 is K or N,

[0026] X12 is F or L,

[0027] X13 is P or S,

[0028] X14 is Q or K,

[0029] X15 is R, H or Q,

[0030] X16 is A, N, S or T,

[0031] X17 is A, Q, R, K or T,

[0032] X18 is D or E,

[0033] X19 is S or A,

[0034] X20 is I, E, L, A or V,

[0035] X21 is Q, G or E,

[0036] X22 is K or R,

[0037] X23 is H, Q or E,

[0038] X24 is E, Q, N, D, S or H,

[0039] X25 is L, V or M,

[0040] X26 is E, D, P, L or A,

[0041] X27 is E or D,

[0042] X28 is V, T, A or I,

[0043] X29 is L, N, R, Y, S or Q,

[0044] X30 is S, K or T,

[0045] X31 is S or M,

[0046] X32 is S, A or Y,

[0047] X33 is S, T or R,

[0048] X34 is N or H,

[0049] X35 is V or I,

[0050] X36 is R or K,

[0051] X37 is absent or is V, A, G, M, or N, X38 is absent or is T, I, N, F, or S, and

[0052] X39 does not exist or is R, H, V, K, I, or Q.

[0053] In some embodiments, the above polypeptide further comprises an amino acid sequence of the following general formula 2 (from N-terminus to C-terminus):

[0054] <General formula 2>

[0055] X1-X2-HHKA-X3-H

[0056] In the above general formula 2,

[0057] X1 is A or V,

[0058] X2 is N or I, and

[0059] X3 is H or Q.

[0060] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 2-Formula 1 (N-terminus to C-terminus).

[0061] In some embodiments, the above polypeptide further comprises an amino acid sequence of the following general formula 3 (from N-terminus to C-terminus):

[0062] <General formula 3>

[0063] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0064] In the above general formula 3,

[0065] X1 is A or S,

[0066] X2 is L, T or S,

[0067] X3 is Q, E or H,

[0068] X4 does not exist, or is P, L or H,

[0069] X5 is P or R,

[0070] X6 is T, S or I,

[0071] X7 is A, P, T, S or H,

[0072] X8 is T, A, S or I,

[0073] X9 is V or A, and

[0074] X10 is L or H.

[0075] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 3 to Formula 1 (N-terminus to C-terminus).

[0076] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 4 (from N-terminus to C-terminus):

[0077] <General formula 4>

[0078] X1-X2

[0079] In the above general formula 4,

[0080] X1 is H or Y, and

[0081] X2 is Q, V or L.

[0082] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 4 to Formula 1 (N-terminus to C-terminus).

[0083] In some embodiments, the above polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0084] LHRP (SEQ ID NO. 293);

[0085] LHQSGFTSGHFPHHRKLGE (SEQ ID NO. 294); or

[0086] LAPPGTNIQI (SEQ ID NO. 295).

[0087] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 5 (from N-terminus to C-terminus):

[0088] <General formula 5>

[0089] X1-X2-X3

[0090] In the above general formula 5,

[0091] X1 is P or G,

[0092] X2 is R or H, and

[0093] X3 is T, S or L.

[0094] In some embodiments, the above polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0095] PYTSL (SEQ ID NO. 296); or

[0096] QEDKLK (SEQ ID NO. 297).

[0097] In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1, LHQSGFTSGHFPHHRKLGE-Formula 1, or LAPPGTNIQI-Formula 1. In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of Formula 1-Formula 5, Formula 1-PYTSL, or Formula 1-QEDKLK. In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1-Formula 5, LHQSGFTSGHFPHHRKLGE-Formula 1-Formula 5, LAPPGTNIQI-Formula 1-QEDKLK, or LAPPGTNIQI-Formula 1-PYTSL.

[0098] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NO.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 shown in SEQ ID NO.184 to 238.

[0099] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences shown 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 shown in SEQ ID NOs. 239 to 292.

[0100] In some embodiments, the polypeptide further comprises a signal sequence (signal peptide).

[0101] In some embodiments, the vector is a viral vector. In some embodiments, the virus is an adeno-associated virus (AAV). In some embodiments, the serotype of AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAVrh10.

[0102] In some embodiments, the above-mentioned retinal or macular disease is caused by all or part of the damage of the retina or macula. In some embodiments, the above-mentioned retinal or macular disease is a disease caused by the dysfunction of retinal or macular cells or damage. In certain embodiments, the above-mentioned retinal or macular disease can be a disease caused by the dysfunction of photoreceptor cells (photoreceptor cells) of the retina or macula and / or retinal pigment epithelial (RPE) cells or damage. In some embodiments, the above-mentioned retinal or macular disease includes: 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 its combination. In some embodiments, the above-mentioned macular degeneration includes: age-related macular degeneration, best vitelliform macular dystrophy (BVMD), Sorsby fundus dystrophy, familial dominant drusen (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 above-mentioned 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 above-mentioned retinopathy is retinal dystrophy. In some embodiments, the above-mentioned retinopathy is diabetic retinopathy. In some embodiments, the above-mentioned 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 above-mentioned retinal or macular disease is an inherited retinal disease.In some embodiments, the inherited 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.

[0103] The present invention also provides an adeno-associated virus (AAV) vector, which comprises a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75.

[0104] 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 encoding sequence, a splice donor sequence, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

[0105] In some embodiments, the above-mentioned intron consists of 51 to 117 nucleotides and includes 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 includes: (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).

[0106] In some embodiments, the above-mentioned 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-1αE1 sequence shown in SEQ ID NO.13, or the chicken β-actin E1 sequence shown in SEQ ID NO.14 or 15; (4) a splice donor sequence shown in SEQ ID NO.9 or 10; and / or (5) an EF-1αE2 sequence shown in SEQ ID NO.11.

[0107] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75 above comprises the amino acid sequence of the following general formula 1 (N-terminus to C-terminus):

[0108] <General formula 1>

[0109] X1-X2-X3-GTCEV-X4-A-X5-H-X6-CCN-X7-N-X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X14-VAGTT-X15-X16-X17-PSCV-X18-A-X19-IV -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39

[0110] In the above general formula 1,

[0111] X1 does not exist, or is V, I, or L.

[0112] X2 is K, E, R or Q,

[0113] X3 is G, T, Q, P or A,

[0114] X4 is V or I,

[0115] X5 is A, L, V or I,

[0116] X6 is R or L,

[0117] X7 is K, R or Q,

[0118] X8 is R or K,

[0119] X9 is R or L,

[0120] X10 is V or G,

[0121] X11 is K or N,

[0122] X12 is F or L,

[0123] X13 is P or S,

[0124] X14 is Q or K,

[0125] X15 is R, H or Q,

[0126] X16 is A, N, S or T,

[0127] X17 is A, Q, R, K or T,

[0128] X18 is D or E,

[0129] X19 is S or A,

[0130] X20 is I, E, L, A or V,

[0131] X21 is Q, G or E,

[0132] X22 is K or R,

[0133] X23 is H, Q or E,

[0134] X24 is E, Q, N, D, S or H,

[0135] X25 is L, V or M,

[0136] X26 is E, D, P, L or A,

[0137] X27 is E or D,

[0138] X28 is V, T, A or I,

[0139] X29 is L, N, R, Y, S or Q,

[0140] X30 is S, K or T,

[0141] X31 is S or M,

[0142] X32 is S, A or Y,

[0143] X33 is S, T or R,

[0144] X34 is N or H,

[0145] X35 is V or I,

[0146] X36 is R or K,

[0147] X37 does not exist, or is V, A, G, M, or N,

[0148] X38 is absent, or is T, I, N, F, or S, and

[0149] X39 does not exist or is R, H, V, K, I, or Q.

[0150] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75 above consists of 80 to 130 amino acids, more preferably 90 to 120 amino acids, and most preferably 91 to 119 amino acid sequences.

[0151] In some embodiments, the above polypeptide further comprises an amino acid sequence of the following general formula 2 (from N-terminus to C-terminus):

[0152] <General formula 2>

[0153] X1-X2-HHKA-X3-H

[0154] In the above general formula 2,

[0155] X1 is A or V,

[0156] X2 is N or I, and

[0157] X3 is H or Q.

[0158] In some embodiments, the above polypeptide further comprises an amino acid sequence of the following general formula 3 (from N-terminus to C-terminus):

[0159] <General formula 3>

[0160] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0161] In the above general formula 3,

[0162] X1 is A or S,

[0163] X2 is L, T or S,

[0164] X3 is Q, E or H,

[0165] X4 does not exist, or is P, L or H,

[0166] X5 is P or R,

[0167] X6 is T, S or H,

[0168] X7 is A, P, T, S or I,

[0169] X8 is T, A, S or I,

[0170] X9 is V or A, and

[0171] X10 is L or H.

[0172] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 4 (from N-terminus to C-terminus):

[0173] <General formula 4>

[0174] X1-X2

[0175] In the above general formula 4,

[0176] X1 is H or Y, and

[0177] X2 is Q, V or L.

[0178] In some embodiments, the above polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0179] LHRP (SEQ ID NO. 293);

[0180] LHQSGFTSGHFPHHRKLGE (SEQ ID NO. 294); or

[0181] LAPPGTNIQI (SEQ ID NO. 295).

[0182] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 5 (from N-terminus to C-terminus):

[0183] <General formula 5>

[0184] X1-X2-X3

[0185] In the above general formula 5,

[0186] X1 is P or G,

[0187] X2 is R or H, and

[0188] X3 is T, S or L.

[0189] In some embodiments, the above polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0190] PYTSL (SEQ ID NO. 296); or

[0191] QEDKLK (SEQ ID NO. 297).

[0192] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NO.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 shown in SEQ ID NO.184 to 238.

[0193] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NO. 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 shown in SEQ ID NO. 239 to 292.

[0194] In some embodiments, the AAV vector is used for gene therapy. In some embodiments, the AAV vector is used to express a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75.

[0195] The present invention also provides an isolated host cell comprising the above-mentioned AAV vector.

[0196] The present invention also provides an isolated host cell transformed with the above AAV vector.

[0197] The present invention also provides a composition comprising the above-mentioned AAV vector (AAV vector), or comprising the above-mentioned AAV vector or a host cell transformed with the above-mentioned AAV vector. In some embodiments, the above-mentioned composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the above-mentioned composition is used to prevent or treat retinal or macular diseases.

[0198] The present invention also provides a kit, which comprises the above-mentioned AAV vector, or comprises the above-mentioned AAV vector or a host cell transformed with the above-mentioned AAV vector. In some embodiments, the above-mentioned kit further comprises instructions for use.

[0199] The present invention also provides a method for producing recombinant virus particles, comprising the steps of transducing cells with the above i) AAV vector, and ii) a construct comprising rep and cap genes. In some embodiments, the above method further comprises isolating the produced recombinant virus particles.

[0200] The present invention also provides a recombinant virus particle produced by the method.

[0201] The present invention also provides a recombinant virus particle, which comprises (a) a capsid protein and (b) the above-mentioned AAV vector.

[0202] The present invention also provides a therapeutic use (for use in therapy) of the AAV vector or the recombinant virus particle.

[0203] The present invention also provides a use of the above-mentioned AAV vector or the above-mentioned recombinant virus particle in a method for preventing or treating a disease or condition.

[0204] As a method for preventing or treating a disease or disorder in a subject in need thereof, the present invention also provides a method comprising administering the above-mentioned AAV vector or the above-mentioned recombinant virus particle to the subject.

[0205] In some embodiments, the above-mentioned diseases or conditions include retinal or macular diseases. The above-mentioned retinal or macular diseases are caused by all or part of the damage to the retina or macula. In some embodiments, the above-mentioned retinal or macular diseases are diseases caused by dysfunction or damage of retinal or macular cells. In certain embodiments, the above-mentioned retinal or macular diseases can be diseases caused by dysfunction or damage of photoreceptor cells (photoreceptor cells) of the retina or macula and / or retinal pigment epithelial (RPE) cells. In some embodiments, the above-mentioned retinal or macular diseases include: retinopathy, choroidal neovascularization, maculopathy, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal edema, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal diseases or their combinations. In some embodiments, the above-mentioned method includes applying an additional therapeutic agent to the above-mentioned subject.

[0206] The present invention also provides a polypeptide for preventing or treating retinal or macular diseases, comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75, a nucleic acid encoding the above polypeptide, a vector comprising the above nucleic acid, a recombinant virus particle comprising the above vector and capsid protein, or a cell comprising the above nucleic acid or vector.

[0207] Solutions for solving problems

[0208] The present disclosure provides a polypeptide for preventing, improving or treating retinal or macular diseases, comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 (e.g., TAFA1 to TAFA4 sequences of vertebrates), 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. The present disclosure also provides a pharmaceutical composition for preventing or treating retinal or macular diseases, 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 vector. In addition, the present disclosure also provides an adeno-associated virus (AAV) vector comprising a nucleic acid encoding the above-mentioned polypeptide; a host cell comprising an AAV vector or transformed with the above-mentioned AAV vector; a composition or a kit comprising an AAV vector or a host cell, wherein the above-mentioned host cell comprises the above-mentioned AAV vector or is transformed with the above-mentioned AAV vector; a method for producing recombinant virus particles, comprising transducing cells with the above-mentioned AAV vector and a construct comprising rep and cap genes; a recombinant virus particle produced by the above-mentioned method; a method for preventing or treating a disease or condition in a subject in need thereof, comprising administering the above-mentioned AAV vector or the above-mentioned recombinant virus particle to the subject.

[0209] As described in the present invention, the applicant has confirmed that the expression of TAFA genes of various species (human TAFA1, human TAFA2, human TAFA3, human TAFA4, mouse TAFA4, zebrafish TAFA4, gecko TAFA4) has excellent ability to repair damaged retina or protect retina from damage, and derived the sequence of highly conserved human TAFA4 mature protein (SEQ ID NO.75) that plays a core role in the above-mentioned effects. As demonstrated in this specification, in some embodiments, the composition of the present invention restores damaged retina to a level equivalent to normal. Additional schemes of the present disclosure are provided throughout this application.

[0210] In order to facilitate understanding of the disclosure disclosed in the present invention, a number of terms and phrases are defined. Additional definitions are set forth throughout the detailed description.

[0211] I. Definitions

[0212] Throughout the disclosure, the term "a" or "an" entity refers to one or more of its entities; for example, it should be understood that "a polypeptide" means one or more polypeptides. Therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably in the present invention.

[0213] In addition, the "and / or" used in the present invention should be regarded as a specific disclosure of each of the two specified features or components together with the remaining other features or components or separately. Therefore, the term "and / or" used in statements such as "A and / or B" in the present invention is intended to include "A and B", "A or B", "A" (alone) and "B" (alone). Similarly, the term "and / or" used in statements such as "A, B and / or C" is intended to cover the following aspects: 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).

[0214] The term "at least" before a number or a series of numbers is understood to include the number after the term "at least" and all subsequent numbers or integers that can be clearly and logically included from the context. For example, the number of nucleotides in a nucleic acid must be an integer. For example, "at least 18 nucleotides of a nucleic acid comprising 21 nucleotides" means that 18, 19, 20 or 21 nucleotides have the specified characteristics. If "at least" is located before a series of numbers or ranges, it should be understood that "at least" can modify each number in the above series or range. In addition, "at least" is not limited to integers (for example, "at least 5%" includes 5.0%, 5.1%, 5.18%, regardless of the number of significant figures).

[0215] When the present specification uses the term “comprising” to describe various aspects, it should be understood that other similar aspects described with the terms “consisting of” and / or “essentially consisting of” are also provided.

[0216] Unless otherwise defined, the meaning of all technical and scientific terms used in the present invention is the same as that commonly understood by those of ordinary skill in the art related to the present disclosure. For example, the following references provide a general dictionary of many terms used in the present disclosure for technicians: 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.

[0217] Units, prefixes and symbols are expressed in a form recognized by the International System of Units (Systeme International de Unites, SI). Numerical ranges include numbers that limit the range. Unless otherwise indicated, the writing direction of amino acid sequences is from left to right, from amino to carboxyl. The titles provided in the present invention are not intended to limit the various aspects of the present disclosure, which can be obtained by reference to the entire specification. Therefore, the terms defined below can be more fully defined by reference to the specification as a whole.

[0218] In the present invention, the term "about" is used to mean approximately, roughly, approximately, or within the range thereof. When the term "about" is used with a numerical range, it modifies this range by extending the boundaries above and below the recorded numerical values. In general, the term "about" can change the numerical value above and below the value shown, for example, by 10% (higher or lower) above and below.

[0219] The term "adeno-associated virus" (AAV) used in the present invention is a single-stranded DNA virus, which is a helper-dependent human parvovirus. The genome size is about 4.6 kbp, the N-terminal part of the genome encodes the rep gene, the C-terminal part encodes the cap gene, and is composed of a repeat region (ITR) with about 145 bases inserted at both ends, wherein the rep gene is involved in viral replication and viral gene expression, and the cap gene encodes the viral capsid protein (capsid). The function of the inverted terminal repeat sequence (ITR) with a T-shaped structure and a size of 145 bp plays the role of the replication origin when the viral genome is replicated, and acts as a primary packaging signal. ITR is the only cis-acting nucleotide sequence required for constructing a recombinant AAV construct, and has enhancer activity in the presence of Rep protein, but the activity is very weak in the absence of Rep protein. Therefore, when cloning an exogenous gene into a recombinant AAV construct, this point is taken into account and an expression construct is constructed by appropriately constructing enhancers, promoters, pA, etc. (RJ Samulski and N Muzyczka, Annu. Rev. Virolo. 2014. 1: 427-451). The rep gene translates four proteins, which are divided into rep78, rep68, rep52 and rep40 according to their molecular weight, and play an important role in AAV DNA replication. The cap gene translates four proteins, among which VP1, VP2 and VP3 proteins are structural proteins that constitute AAV particles, and assembly-activating protein (AAP) promotes the assembly of AAV particles through the above structural proteins. In order for the adeno-associated virus to replicate effectively, some partial proteins and RNA derived from helper viruses, such as adenovirus or herpes simplex virus, are required (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0220] AAV includes, but is not limited to, AAV1, AAV2, AAV3 (including 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, sheep AAV, goat AAV, shrimp AAV, Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (J. Virol. 33:375 (2004)) and Morise et al. (J. Virol. 78:6381 (2004)). tal. (Virol. 33: 375 (2004)), and any other AAV currently known or later discovered. For example, see the following references: FIELDS et al., "Virology", volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers) (FIELDS et al. VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers)). In some embodiments, "AAV" includes derivatives of known AAV. In some embodiments, "AAV" includes modified or artificial AAV.

[0221] The terms "administration", "application" and grammatical variants thereof in the present invention refer to the introduction of a composition (e.g., a polynucleotide comprising an exogenous gene and a non-translated nucleic acid sequence described in the present invention) into a subject by a pharmaceutically acceptable route. The composition may be introduced into the subject by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic, intrathecal, periocular, intraocular or topical. Administration includes self-administration and administration to others. The composition or preparation exerts its intended function by a suitable route of administration. For example, if the suitable route is intravenous administration, the composition is administered by introducing the composition or preparation into the subject's vein.

[0222] The term "intraocular" in the present invention refers to the inside or lower part of the eye tissue. The term "intraocular administration" used in the present invention refers to any administration that can deliver the composition to the sub-Tenon, subconjunctival, suprachoroidal, subretinal, vitreous and similar locations of the eye. In some embodiments, intraocular administration includes administration on the choroid, subretinal and vitreous.

[0223] The term "CEE" construct used in the present invention includes a CMV enhancer, an EF-1α promoter, and an EF-1α intron fragment. The "CE" construct contains a CMV enhancer, an EF-1α promoter, but does not contain an intron fragment (e.g., does not contain an EF-1α intron fragment). The "CAE" construct contains a CMV enhancer, a chicken β-actin promoter, and an EF-1α intron fragment. The "CAG" construct used in the present invention includes a CMV enhancer, a chicken β-actin promoter, and a chicken β-actin / rabbit β-globin hybrid intron fragment. The "CA" construct contains 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 Figure 4a , 5a and 5c.

[0224] The term "conservative" used in the present invention refers to that the nucleotides or amino acid residues of each polynucleotide sequence or polypeptide sequence remain unchanged at the same position in two or more sequences to be compared. Relatively conservative nucleotides or amino acids are conserved in more related sequences compared to the nucleotides or amino acids in other parts of the sequence.

[0225] The term "amino acid" used in the present invention 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.) naturally incorporated into peptides, but also D-isomers and modified amino acids. In addition, the above-mentioned peptides may contain non-standard amino acids that have undergone post-translational modification. Post-translational modifications may include phosphorylation, glycosylation, acylation (e.g., acetylation, myristoylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., β-elimination deimidation, deamidation), and structural changes (e.g., formation of disulfide bridges), but are not limited thereto. The above-mentioned peptides may be wild-type peptides identified and isolated from natural sources. On the other hand, the above-mentioned peptides may be artificial variants of amino acid sequences including substitutions, deletions, and / or insertions of more than one amino acid. In addition to artificial variants, amino acid changes in wild-type polypeptides include conservative amino acid substitutions that do not significantly affect the folding and / or activity of the protein. For example, the above conservative replacements may 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 change specific activity are well known in the art.The most common substitutions may 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.

[0226] In some embodiments, two or more sequences are said to be "fully conserved" or "identical" if they are 100% identical to one another. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be "highly conserved" if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, as well as to portions, regions, or features thereof.

[0227] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each including a nucleotide sequence) or oligomers and target genes that are related to each other by Watson-Crick (Watson-Crick) base pairing rules. For example, the nucleotide sequence "TGA (5'→)" is complementary to the nucleotide sequence "ACT (3'→)". Complementarity can be "partial", wherein less than all the core bases of a given nucleotide sequence match another nucleotide sequence according to the base pairing rules. For example, in some embodiments, the complementarity between a given nucleotide sequence and another nucleotide sequence can be about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. Therefore, in some aspects, the term "complementary" refers to a match or complementarity with a target nucleic acid sequence of 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%. Alternatively, there may be "complete" or "perfect" (100%) complementarity between a given nucleotide sequence and another nucleotide sequence to continue the example. In some embodiments, the degree of complementarity between nucleotide sequences has a significant effect on the efficiency and strength of hybridization between the sequences.

[0228] The expression "continuous or non-continuous nucleotide deletion" used in the present invention is intended to include a continuous or non-continuous nucleotide sequence remaining due to deletion compared to the wild-type (or original) sequence, but does not include any deletion process. The above terms may include "deletion of continuous nucleotides", "deletion of non-continuous nucleotides remaining between the deleted nucleotides" and "insertion of different types of nucleotides into the position of the continuous or non-continuous nucleotides of the deletion". For example, in the case of the nucleotide sequence "ATGCCGTC", the deletion of continuous nucleotides includes the deletion of one or more continuous nucleotides (such as "A-_-_-_-CGTC"), non-continuous nucleotide deletion means that one or more nucleotides remain non-deleted between the deleted nucleotides (such as "A-_-G-_-CGT-_"), and insertion of different types of nucleotides into the position of the continuous or non-continuous nucleotides of the deletion means that one or more nucleotides different from the original nucleotides are inserted at the position of the deleted nucleotides (such as "AAG-_-CGTG").

[0229] The term "downstream" refers to a nucleotide sequence located at the 3' position of a reference nucleotide sequence. In some aspects, the downstream nucleotide sequence is related to the sequence after the transcription start point. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0230] The term "enhancer" as used in the present invention refers to a DNA fragment containing a sequence that can provide enhanced transcription and in some cases can function independently of its orientation relative to another control sequence. An enhancer can function synergistically or additively with a promoter and / or other enhancer elements.

[0231] The terms "excipient" and "carrier" are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound (eg, a polynucleotide comprising an exogenous gene and an untranslated nucleic acid sequence as described herein).

[0232] The term "exon" refers to a defined segment in a nucleic acid that encodes a protein or refers to a nucleic acid sequence that is represented in the mature form of an RNA molecule after removing any part of a preprocessed (or precursor) RNA by splicing. The mature RNA molecule may be a messenger RNA (mRNA) or a functional form of a non-coding RNA (such as rRNA or tRNA).

[0233] The term "expression" used in the present invention refers to the process by which a polynucleotide produces a gene product such as RNA or a polypeptide. It includes but is not limited to transcribing a polynucleotide into messenger RNA (mRNA), and translating mRNA into a polypeptide. Expression produces a "gene product". The gene product of the present invention may be a substance such as a nucleic acid, such as RNA produced by gene transcription. The gene product of the present invention may be a nucleic acid or a polypeptide translated from a transcript. The gene product of the present invention further includes a nucleic acid that has been post-transcriptionally modified (e.g., polyadenylated or spliced), or a polypeptide that has been post-translationally modified (e.g., phosphorylation, methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage).

[0234] The term "identity" as used in the present invention refers to the overall monomer conservation between polymer molecules (e.g., between polynucleotide molecules). The term "identical" without any additional qualifiers (e.g., polynucleotide A is identical to polynucleotide B) implies that the polynucleotide sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity".

[0235] Calculating the identity (percentage) of two polypeptide or polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., for optimal comparison purposes, gaps can be introduced in one or both of the first polypeptide or polynucleotide sequence and for comparison purposes, non-identical sequences can be ignored). In some aspects, the length of the sequence aligned 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. The amino acids at corresponding amino acid positions are then compared or, in the case of polynucleotides, the bases are compared.

[0236] When a specific position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at said position. The identity (percentage) between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of spaces and the length of each space, and it is necessary to introduce said function to perform the best comparison of the two sequences. The comparison of the sequence and the determination of the identity (percentage) between the two sequences can be completed using a mathematical algorithm.

[0237] Suitable software programs that can be used for comparing different sequences (e.g., polynucleotide sequences) can be obtained from various sources. Bl2seq is a suitable program for determining sequence identity (percentage), which is a part of the BLAST program suite that can be obtained through the National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to perform the comparison between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs include Needle, Stretcher, Water or Matcher, etc., as part of the EMBOSS bioinformatics program suite, available through the European Bioinformatics Institute (EBI) website (www.ebi.ac.uk / Tools / psa).

[0238] When performing sequence alignment, methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc., can be used.

[0239] Different regions within a single polynucleotide or polypeptide target sequence aligned to a polynucleotide or polypeptide reference sequence can each have their own sequence identity (percentage). Note that the sequence identity (percentage) is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Also note that length values ​​will always be integers.

[0240] In certain aspects, the identity (percentage, %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), wherein Y is the number of amino acid residues (or nucleobases) scored as identical matches when aligning the first and second sequences (such as by visual inspection or alignment by a specific 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 (percentage) of the first sequence to the second sequence will be higher than the identity (percentage) of the second sequence to the first sequence.

[0241] It will be appreciated by those skilled in the art that the generation of sequence alignments for calculating sequence identity (percentage) is not limited to binary (binary) sequence-sequence comparisons driven only by primary sequence data. It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources (such as structural data (e.g., crystallographic protein structure), functional data (e.g., position of mutation) or phylogenetic data). An appropriate program for integrating heterogeneous data to produce multiple sequence alignments is T-Coffee, which can be obtained at www.tcoffee.org, and alternatively can be obtained from, for example, EBI. It will also be appreciated that the final alignment for calculating sequence identity (percentage) can be automatically or manually planned.

[0242] The term "intron" used in the present invention refers to a DNA segment (intervening sequence) within a gene that does not encode the protein portion of the gene production and is sheared out from the mRNA transcribed from the gene before being exported from the nucleus. "Intron sequence" refers to the nucleic acid sequence of an intron. Such sequences are also referred to as "non-translated nucleic acid sequences" herein. Therefore, introns are those regions in a DNA sequence that are transcribed together with the coding sequence (exon) but are removed during the formation of mature mRNA.

[0243] The term "intron fragment" used in the present invention refers to a fragment derived from the full-length EF-1α intron A sequence (i.e., the first intron of EF-1α, such as the intron shown in SEQ ID NO.1). The above-mentioned fragment is intended to exclude the full-length EF-1α intron. In some embodiments, the "intron fragment" includes the minimum number of nucleotides or elements required to achieve an expression level exceeding the expression level achieved by the corresponding construct in which all nucleotides of EF-1α intron A are deleted. Therefore, the intron fragments disclosed in the present invention (also referred to as "non-translated nucleic acid sequences" herein) are not particularly limited, as long as they include fragments of EF-1α introns and can increase the expression of exogenous genes. As verified herein, in some embodiments, intron fragments (i.e., non-translated nucleic acid sequences) can increase the transcription of exogenous genes, thereby resulting in enhanced expression of exogenous genes. Therefore, in some embodiments, the intron fragments described in the present invention can be non-translated regulatory elements.

[0244] The terms "separation", "purification", "extraction" and grammatical variants thereof used in the present invention are used interchangeably and refer to the preparation state of the desired composition of the present disclosure (e.g., polynucleotides including exogenous genes and non-translated nucleic acid sequences) that has undergone one or more purification processes. In some embodiments, separation or purification as used in the present invention is a process of removing, partially removing (e.g., a fraction) the composition of the present disclosure (e.g., polynucleotides described in the present invention) from a sample containing contaminants.

[0245] In some embodiments, the composition of separation does not have detectable undesirable activity, or alternatively, the level or amount of undesirable activity is at or below an acceptable level or amount. In other aspects, the composition of separation has the amount and / or concentration of the desired composition of the present disclosure at or above an acceptable amount and / or concentration and / or activity. In other aspects, compared with the starting material from which the composition is obtained, the concentrated composition of separation. Compared with the starting material, this 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% or greater than 99.9999%.

[0246] In some embodiments, the preparation of separation is substantially free of residual biological products. In some embodiments, the preparation of separation is 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free or at least about 90% free of any contaminating biological substances. Residual biological products can include non-biological materials (including chemicals) or unwanted nucleic acids, proteins, lipids or metabolites.

[0247] The term "linked" used in the present invention refers to a first amino acid sequence or polynucleotide sequence that is covalently or non-covalently bound 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, or alternatively, an intervening sequence can covalently link the first sequence to the second sequence. The term "linked" not only means that the first polynucleotide sequence is fused to the second polynucleotide sequence at the 5' end or the 3' end, but also includes inserting the entire first polynucleotide sequence (or the second polynucleotide sequence) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence), respectively. The first polynucleotide sequence can be connected to the second polynucleotide sequence by a phosphodiester bond or a joint. The joint can be, for example, a polynucleotide.

[0248] The term "retinal disease" or "macular disease" used in the present invention refers to a part or region of the retina or macula that affects the eyeball, or a disease, condition or symptom associated therewith. The above-mentioned retinal or macular disease can be a disease, disease or symptom caused by all or part of damage to the retina or macula. The above-mentioned retinal or macular disease may be caused by dysfunction or damage of retinal or macular cells. In addition, the above-mentioned retinal or macular disease can be caused by dysfunction or damage of photoreceptor cells (photoreceptor cells) of the retina or macula and / or retinal pigment epithelial (RPE) cells of the retina or macula.

[0249] It is reported that sodium iodate (NaIO 3 ) induces retinal damage and / or degeneration in the outer and inner part of the retina, and induces damage to the retinal pigment epithelium (RPE) and photoreceptors (AE-H. Koh, et al. Journal of Photochemistry & Photobiology, B: Biology 196 (2019) 111514). Therefore, the use of NaIO 3The model can confirm the effect of preventing or treating diseases caused by retinal or macula damage (e.g., dysfunction or damage of photoreceptor cells and / or retinal pigment epithelial cells). Examples of diseases caused by dysfunction or damage of the above-mentioned photoreceptor cells and / or retinal pigment epithelial cells include: retinopathy, choroidal neovascularization, maculopathy, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal edema, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease or a combination thereof.

[0250] The term "retinal pigment epithelium" (RPE) used in the present invention refers to the single-layer epithelial cell between the choroid blood supply (choroid capillaries) and the neural retina at the back of the vertebrate eye. RPE is one of the components of the blood-retina barrier, and RPE cells play an important role in the transportation of nutrients, metabolic waste, ions and liquids for maintaining the visual cycle, the phagocytosis of the photoreceptor outer segment and between the distal retina and the choroid capillaries.

[0251] As used herein, the term "retinal disease" refers to a disease or damage to the retina (ie, the tissue covering the inner surface of the back of the eye that captures images passing through the cornea and lens) or retinal cells.

[0252] The term "diabetic retinopathy" (DR) as used in the present invention refers to a retinal disease caused by complications associated with diabetes. Depending on the severity of the above-mentioned disease, DR may cause asymptomatic or mild vision problems or blindness. DR is the result of changes in retinal microvessels. Hyperglycemia-induced intramural perivascular cell death and basement membrane thickening can lead to vascular wall failure. These injuries change the formation of the blood-retinal barrier and allow better penetration of retinal blood vessels. When hyperglycemia continues to activate PKC-δ and p38 mitogen-activated protein kinase (MAPK) encoded by protein kinase C-δ, as a previously unknown target of PKC-δ signal transduction, increasing the expression of Src homology-2 domain-containing phosphatase 1 (SHP-1) as a protein tyrosine phosphatase, perivascular cell death may be induced. This signal cascade can lead to platelet-derived growth factor (PDGF) receptor-dephosphorylation and a decrease in downstream signaling of the receptor, resulting in "perivascular cell death". Small blood vessels, such as those in the eye, are particularly susceptible to poor glycemic control. Excessive accumulation of glucose and / or fructose may damage the small blood vessels in the retina.

[0253] DR can be divided into two separate stages (Wu L. et al., World J Diabetes 4(6):290-294(2013)). The first stage is called non-proliferative diabetic retinopathy (NPDR), which is associated with early diabetic retinopathy. NPDR usually has no obvious symptoms or is associated with mild vision distortion caused by the flow of fluid to the blood vessels of the surrounding tissue. The only way to detect NPDR is through fundus imaging, which can see microaneurysms (bulges filled with blood on the arterial wall). If left untreated, DR patients may progress to a more advanced second stage called proliferative diabetic retinopathy (PDR). PDR is characterized by the formation of abnormal new blood vessels (i.e., neovascularization), which can rupture and bleed, leading to blurred vision. Other symptoms of PDR include floating points or dark lines in the visual field ("floaters"), changes in vision, impaired color vision, dark or blank areas in the visual field, pain, patches of vision, and complete loss of vision.

[0254] The term "diabetic retinopathy" refers to all types of diabetic retinal diseases including, but not limited to, non-proliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), diabetic maculopathy, and diabetic macular edema.

[0255] In some embodiments, PDR occurs after the onset of NPDR (e.g., NPDR is initially diagnosed and the disease progresses to PDR). In other aspects, the occurrence of PDR is unrelated to NPDR. The term "diabetic retinopathy" used in the present invention also includes any and all symptoms of all types of diabetic retinopathy and diabetic retinopathy, regardless of the cause. Non-limiting examples of risk factors for diabetic retinopathy include diabetes course, heredity, excessive drinking, smoking, hypertension, obesity, dyslipidemia, high cholesterol, nephropathy, pregnancy and renal injury.

[0256] As used herein, the term "maculopathy" refers to any pathological condition of the macula, which is the central area of ​​the retina associated with highly sensitive and accurate vision. In some embodiments, the terms "maculopathy" and "retinopathy" can be used interchangeably (i.e., when only the macula is affected). In some embodiments, the above-mentioned maculopathy is diabetic maculopathy.

[0257] "Diabetic maculopathy" occurs when the macula is affected by retinal changes caused by diabetes. The above term covers two different eye diseases: diabetic macular edema and diabetic ischemic maculopathy. The above two types of maculopathy are often comorbid, that is, people with macular edema usually also have ischemic maculopathy. Ischemic maculopathy occurs at the same time as macular edema, even if the macular edema is mild. In some embodiments, retinal changes associated with diabetic maculopathy include decreased retinal potential within the subject's retina, loss of pericytes, formation of acellular capillaries, vascular congestion, vascular dysfunction, vascular leakage, vascular occlusion, tissue swelling (edema), tissue ischemia, or any combination thereof.

[0258] As used herein, the term "acellular capillaries" refers to capillary-sized vessel tubes that do not have cell nuclei anywhere along their length.

[0259] As used herein, the term "vascular congestion" refers to a type of vascular damage and is an important factor in the pathogenesis of various eye diseases (e.g., diabetic macular edema) disclosed herein. Vascular congestion is associated with the accumulation of fluid (e.g., blood in blood vessels) within vascular tissue. In some embodiments, vascular congestion may be caused by hyperglycemia (i.e., high blood sugar).

[0260] The term "retinal dystrophy" (RD) used in the present invention refers to a retinal disease (retinal degenerative disease) caused by hereditary or other causes.

[0261] The term "macular degeneration" used in the present invention refers to any number of diseases and morbidities of degeneration or loss of functional activity in the central part of the retina (i.e., macula). The loss of the above-mentioned degeneration or functional activity may be caused by, for example, apoptosis, cell proliferation reduction, loss of normal biological function or the result of its combination. Macular degeneration can cause and / or show changes in the structural integrity of the extracellular matrix of cells and / or macula, changes in the composition of normal cells and / or extracellular matrix, and / or the loss of macular cell function. The above-mentioned cells can be any cell type normally present in or near the macula, including RPE cells, photoreceptors and / or capillary endothelial cells. Age-related macular degeneration is the most common in 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, familial dominant drusen (Mallatia Leventinese), Doyne honeycomb retinal dystrophy, Stargardt disease (Stargardt macular dystrophy), juvenile macular degeneration or fundus flavimaculatus, and macular degeneration associated with pigment epithelial detachment.

[0262] The term "age-related macular degeneration" (AMD) used in the present invention refers to a retinopathy that is usually affected in the elderly and is associated with the central vision loss caused by damage to the central part of the retina (i.e., macula). AMD is usually characterized by the gradual accumulation or coagulation of yellow insoluble extracellular deposits called drusen (the accumulation of extracellular proteins and lipids, such as beta amyloid) in the macula (mainly located between the retinal pigment epithelium (RPE) and the choroid below it). These deposits accumulate or coagulate in the macula, causing the macula to gradually deteriorate, thereby causing central vision to be impaired. The term "macula" used in the present invention refers to the central part of the retina responsible for central high-resolution color vision.

[0263] The pathogenesis of age-related macular degeneration is unclear, although several theories have been proposed, including oxidative stress, mitochondrial dysfunction, and inflammatory processes. 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 bounded by areas of thinning or depigmentation of the retinal pigment epithelium (RPE), which precede the appearance of geographic atrophy in the early stages of AMD. In the later stages of AMD, RPE atrophy (geographic atrophy) and / or new blood vessel development (neovascularization) lead to photoreceptor death and central vision loss. In dry (non-exudative) AMD, cellular debris called drusen accumulate between the retina and choroid, causing retinal atrophy and scarring. In the more severe wet (exudative) AMD, blood vessels grow in the choroid behind the retina (neovascularization), exudate and fluid leak, and may cause bleeding.

[0264] According to the degree of drusen present, AMD can be classified into three main stages: (i) early stage; (ii) mid-stage: and (iii) late stage or terminal stage. The feature of early stage AMD is the presence of several small (e.g., diameter less than about 63 microns) drusen or several medium-sized (e.g., diameter between about 63 to 124 microns) drusen. During the early stage, the patient has no obvious symptoms and no vision loss. The feature of mid-stage is the presence of many medium-sized drusen or one or more large (e.g., diameter greater than about 125 microns) drusen. During this period, some patients may begin to experience blurred spots in the center of their vision. The feature of late stage or terminal stage AMD is that retinal tissue is damaged over a large area, thereby causing central blind spots and eventually losing central vision. Based on the type of damage (e.g., the presence or absence of new blood vessels), late stage or terminal stage AMD can be further divided into two subtypes: (i) geographic atrophy (also referred to as atrophic AMD) and (ii) wet AMD (also referred to as neovascular or exudative AMD).

[0265] 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. As used in the present invention, the term "age-related macular degeneration" also includes all types of age-related macular degeneration, regardless of the cause of age-related macular degeneration and any and all symptoms. The non-limiting example of the symptom associated with macular degeneration (for example, age-related macular degeneration) includes: central vision loss, distortion, contrast sensitivity decrease, blurred vision, difficulty in adapting to low light levels, sudden onset of symptoms and rapid deterioration and color vision decrease. In some embodiments, macular degeneration (for example, age-related macular degeneration) may cause macular edema (that is, macula swelling caused by fluid and protein deposits converging on or under the macula).

[0266] The term "dry AMD" (also referred to as atrophic age-related macular degeneration or non-exudative AMD) used in the present invention refers to all AMD forms that do not belong to wet (neovascular) AMD. This includes early and mid-stage forms of AMD and late-stage forms of dry AMD (known as geographic atrophy). Dry AMD patients often have mild symptoms in the early stages; if the condition develops to geographic atrophy, loss of visual function occurs more often.

[0267] The term "wet AMD" (also referred to as neovascular age-related macular degeneration or exudative AMD) used in the present invention refers to a retinal condition characterized by the presence of retinal neovascularization and is the most advanced form of AMD. In wet AMD, blood vessels grow from choriocapillaris due to defects in Bruch's membrane, and in some cases, grow from the underlying retinal pigment epithelium (choroidal neovascularization or neovascularization). Tissue serum or hemorrhagic exudates escape from these blood vessels and may cause fibrovascular scarring in the macula region, with permanent loss of neuroretinal degeneration, retinal pigment epithelium detachment and tearing, vitreous hemorrhage, and central vision.

[0268] The term "neovascularization" used in the present invention refers to the growth of new abnormal blood vessels in different parts of the eye, which can cause bleeding and lead to vision loss. The term "choroidal neovascularization" used in the present invention refers to the abnormal growth of new blood vessels in the choroid (i.e., the vascular layer of the eye comprising connective tissue and between the retina and the sclera). In wet AMD, new blood vessels can grow into the retina through the retinal pigment epithelium (RPE) and the choroid, and may damage visual function due to blood and lipid leakage. The term "retinal neovascularization" used in the present invention refers to the abnormal development, proliferation and / or growth of blood vessels on the upper or inner part of the retina, such as the surface of the retina. Retinal neovascularization can occur in many retinal diseases, 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, etc., retinal diseases associated with retinal ischemia. Methods for detecting neovascularization are known in the art and include measuring the expression of CD31 (also known as platelet endothelial cell adhesion molecule, PECAM-1) and vascular endothelial growth factor (VEGF) in tissues, but are not limited thereto. For example, see Schluter A., ​​et al., BMC Cancer 18(1):272 (2018).

[0269] The term "inherited retinal disease" used in the present invention refers to a retinal disease in which the structure and function of retinal cells are abnormal due to genetic defects or abnormalities. The onset and symptoms of inherited retinal diseases vary depending on the pathogenic gene. Non-limiting examples of inherited 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 caused by unspecified genetic abnormalities.

[0270] The term "retinitis pigmentosa" (RP) used in the present invention refers to a retinal disease in which the optic cells and retinal pigment epithelial cells of the retina are damaged (degenerated). Due to damage to the optic cells, night blindness will occur in the early stage, the visual field will gradually narrow, and eventually lead to blindness. The main reason is the genetic defects in the optic cells involved in the mechanism of converting light into electrical signals, and genetic abnormalities have also been found in some retinal pigment epithelial cells, which lead to extensive retinal damage.

[0271] The first obvious symptom of retinitis pigmentosa is the gradual destruction of rod cells and cone cells, with rod cells being damaged before cone cells. If the destruction of visual cells progresses to cone cells, the central field of vision cannot be seen, leading to complete blindness.

[0272] The term "Leber congenital amaurosis" used in the present invention refers to a hereditary retinal disease that can cause congenital blindness at birth or shortly after birth. In children with Leber congenital amaurosis, there are no rods and cones with normal function in the retina, so both cone and cone responses are lost in the electroretinogram. Leber congenital amaurosis is a disease caused by genetic abnormalities, and 40% to 50% of patients have gene mutations. Among the 12 known genetic abnormalities, there are 11 mutations that are inherited in an autosomal recessive manner (GUCY2D, RPE65, SPATA7, AIPL1, LCA5, RPGRIP1, CRB1, CEP290, IMPDH1, RD3, RDH12), and in rare cases, there are also cases where it is inherited in an autosomal dominant manner (CRX).

[0273] The term "Stargardt's disease" as used in the present invention refers to a type of retinal dystrophy that is inherited in an autosomal recessive manner. Stargardt's disease appears between the ages of 8 and 15, and central vision gradually deteriorates due to macular degeneration in both eyes. It is currently believed that the cause of Stargardt's disease is a mutation in the ABCA4 gene. ABCA4 gene mutations cause the accumulation of lipofuscin-like substances in the retinal pigment epithelium, leading to RPE cell death and loss of photoreceptors. ABCA4 mutations are associated with malformations of cones and rods, as well as severe retinal dystrophy.

[0274] The term "Coats retinopathy" used in this specification refers to a retinal vascular disease that causes exudative retinal detachment by causing retinal capillary dilation and hemangiomas, leading to accumulation of exudates in the inner and subretinal spaces of the retina. It is known that due to somatic mutation of the NDP gene on the X chromosome, the norrin protein required for retinal development is deficient.

[0275] The term "Cone dystrophy" used in this specification refers to a disease in which the cone cells in the retina responsible for color vision and central vision degenerate due to genetic abnormalities, resulting in loss of central vision. Cone dystrophy is mainly divided into pure cone dystrophy and cone-rod dystrophy. There are various forms of inheritance, including autosomal dominant inheritance, recessive inheritance, sex chromosome linkage, etc., and it can also not be manifested as an inherited form, but caused by the patient's own mutation.

[0276] The term "Choroideremia" as used in this specification refers to a rare X-linked progressive degeneration of the choroid, retinal pigment epithelium, and photoreceptors. In patients, due to the loss of Rabescort protein 1 (REP1) protein caused by mutations in the CHM gene, the photoreceptor cells in the retina lose their function and gradually die. It is reported that the symptoms of the disease generally appear in men, and usually the first symptom is night blindness in childhood. As the disease progresses, peripheral vision decreases, while in the early stages, central vision is preserved to a certain extent. Typically, male patients in their 40s have good vision, but a narrow field of vision, and vision will be lost by the age of 50 to 70. Some patients may also experience a decrease in color perception.

[0277] The term "Usher syndrome" used in this specification refers to a genetic disease in which visual impairment progresses along with hearing impairment. The hearing impairment in Usher syndrome is caused by inner ear abnormalities, while the visual impairment is related to retinitis pigmentosa (RP).

[0278] The term "Best's Disease" used in this specification refers to an inherited retinal disease caused by a mutation in the BEST1 (VMD2) gene. It progresses slowly and may lead to deterioration of central vision. The BEST1 (VMD2) gene mutation causes dysfunction of the Bestropin (Bestropin), a calcium-activated chloride channel protein in the basal plasma membrane of the retinal pigment epithelium, causing obstruction of water movement through the retinal pigment epithelium, which may cause serous retinal detachment or retinal pigment epithelial detachment, etc.

[0279] The term "X-linked retinoschisis" used in this specification refers to a disease in which the inner retinal layers separate due to a mutation in the retinoschisis gene (RS1). Retinoschisis is a visual impairment caused by abnormal separation of the nerve fiber layer among the 10 layers that make up the retina. Juvenile retinoschisis is a rare X-chromosome recessive genetic disease with a global prevalence of 1:120,000.

[0280] The terms "miRNA", "miR" and "microRNA" are used interchangeably and refer to microRNA molecules found in eukaryotic organisms involved in RNA-based gene regulation. The above terms will be used to refer to single-stranded RNA molecules processed from precursors. In some embodiments, "antisense oligomers" can also be used to describe microRNA molecules of the present disclosure. The names of miRNAs and their sequences related to the present disclosure are provided herein. MicroRNA recognizes and binds to target mRNA through incomplete base pairing (causing instability or translation inhibition of the target mRNA) and thereby downregulates target gene expression. In contrast, targeting miRNA by molecules comprising miRNA binding sites (usually molecules comprising sequences complementary to the miRNA seed region) can reduce or inhibit miRNA-induced translation inhibition, thereby leading to upregulation of target genes.

[0281] "Nucleic acid", "nucleic acid", "nucleotide sequence", "polynucleotide" and grammatical variants thereof are used interchangeably and refer to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are presented in the present invention in a 5' to 3' orientation. The polynucleotides described in the present disclosure can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. In the present invention, the nucleotide bases are represented by a single letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I) and uracil (U).

[0282] As used herein, the term "operably linked" or "operably connected" means that the DNA sequences to be linked are adjacent to each other to perform the intended function. For example, when a promoter helps initiate transcription of a coding sequence (e.g., an exogenous gene), the promoter is operably linked to the coding region. As long as this functional relationship remains unchanged, the promoter does not need to be connected to the coding region.

[0283] The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient" and their grammatical variations encompass any pharmaceutical agent approved by the U.S. federal government regulatory agency or listed in the United States Pharmacopeia for use in animals (including humans), as well as any carrier or diluent that does not produce adverse physiological effects that prohibit administration of the composition to a subject; and does not abrogate the biological activity and properties of the administered compound. Excipients and carriers that are useful in preparing pharmaceutical compositions and are generally safe and non-toxic are included.

[0284] The term "pharmaceutical composition" used in the present invention refers to one or more compositions described in the present invention (such as polynucleotides, vectors, cells and / or recombinant viruses) mixed or intermingled or suspended with one or more other chemical components such as pharmaceutically acceptable carriers and excipients.

[0285] The terms "promoter" and "promoter sequence" used in the present invention are interchangeable and refer to a DNA sequence that can control the expression of a coding sequence or functional RNA. The coding sequence is generally located at the 3' end of the promoter sequence. The promoter can be completely derived from a natural gene, or composed of different elements derived from different promoters in nature, or even contain synthetic DNA fragments. It should be understood by those skilled in the art that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or under different environmental or physiological conditions. Promoters that cause gene expression in most periods and most cell types are generally referred to as "constitutive promoters". Promoters that cause gene expression in specific cell types are generally referred to as "cell-specific promoters" or "tissue-specific promoters". Promoters that cause gene expression in specific developmental or cell differentiation stages are generally referred to as "developmental-specific promoters" or "cell differentiation-specific promoters". Promoters that are induced and cause gene expression after contacting or treating cells with substances that induce promoters such as agents, biomolecules, chemicals, ligands, light, etc. are generally referred to as "inducible promoters" or "regulatory promoters". It will further be appreciated that, since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0286] The above promoter sequence is usually bounded at its 3' end by the transcription start site, extending upstream (5' direction), and includes the minimum number of bases or elements required to initiate transcription at a detectable level above the background. The above promoter sequence contains a transcription start site (e.g., which can be easily defined by mapping with nuclease S1), as well as a protein binding domain (consensus sequence) responsible for RNA polymerase binding. In some embodiments, a promoter that can be used in conjunction with the present disclosure includes a tissue-specific promoter.

[0287] The term "gene regulatory region" or "regulatory region" as used in the present invention refers to a nucleotide sequence located upstream (5' non-coding sequence), internal or downstream (3' non-coding sequence) of a coding region, which affects the transcription, RNA processing, stability or translation of the associated coding region. The regulatory region may include a promoter, a translation leader sequence, an intron, a polyadenylation recognition sequence, an RNA processing site, an effector binding site or a stem-loop structure. If the coding region is used for expression in eukaryotic cells, a polyadenylation signal and a transcription termination sequence are usually located at the 3' end of the coding sequence.

[0288] In some embodiments, polynucleotides of the present invention (e.g., including foreign genes and non-translated nucleic acid sequences) may include promoters and / or other expression (e.g., transcription) control elements operably associated with one or more coding regions. In operable association, the coding region of 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, if the inducing promoter function results in transcription of an mRNA encoding the gene product encoded by the coding region, and if the nature of the connection between the promoter and the coding region does 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, the coding region and the promoter are "operably associated". In addition to the promoter, other expression control elements (e.g., enhancers, operators, repressors, and transcription termination signals) may also be operably associated with the coding region to direct the expression of the gene product.

[0289] The terms "subject", "patient", "individual" and "host" and their variants used in the present invention are interchangeable and refer to any mammalian subject to which any composition (e.g., polynucleotide, recombinant expression construct, cell, pharmaceutical composition or recombinant virus) described herein is administered. Non-limiting examples include humans, domestic animals (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.) and experimental animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), particularly humans, in need of diagnosis, treatment or therapy. The methods described herein are applicable to both human therapy and veterinary applications.

[0290] As used herein, the phrase "a subject in need thereof" includes subjects (eg, mammalian subjects) who would benefit from the administration of the compositions described herein.

[0291] The term "therapeutically effective amount" used in the present invention is an amount of an agent or pharmaceutical compound including a composition of the present disclosure (e.g., a polynucleotide including an exogenous gene and a non-translated nucleic acid sequence) sufficient to produce a desired therapeutic effect, pharmacological effect and / or physiological effect on a subject in need thereof. Prevention can be considered as therapy, so a therapeutically effective amount can be a "preventatively effective amount".

[0292] The term "exogenous gene" as used in the present invention refers to at least one polynucleotide or polynucleotide region encoded in a recombinant expression construct, or the expression product of the above polynucleotide or polynucleotide region, a polynucleotide encoding a polypeptide or multi-polypeptide, or a modulating or regulating nucleic acid. In some embodiments, the exogenous gene may be heterologous to the cell into which it is inserted (or transduced) (i.e., not naturally expressed in the cell).

[0293] The terms "treat", "treatment" or "treating" used in the present invention refer to, for example, reducing the severity of a disease or condition; shortening the duration of the disease course; improving or eliminating one or more symptoms associated with a disease or condition; providing a beneficial effect to a subject suffering from a disease or condition, but not necessarily curing the disease or condition. The above terms also include preventing or avoiding a disease or condition or its symptoms.

[0294] The term "upstream" refers to a nucleotide sequence located 5' to a reference nucleotide sequence.

[0295] The term "vector" or "construct" used in the present invention refers to any vehicle into which a nucleic acid or gene can be inserted, such as a delivery vehicle into which a nucleic acid sequence can be inserted to be introduced into a cell into which it can replicate. The nucleic acid sequence that can be inserted into the vector can be exogenous or heterologous. The above-mentioned nucleic acid sequence can be an exogenous gene. Examples of constructs include, but are not limited to, plasmids, cosmids, and viruses (e.g., AAV). Those skilled in the art can construct vectors or constructs by standard recombinant techniques (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (NY), 1988; and Ausubel et al., Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, New York, 1994, etc.). The term "expression vector" or "expression construct" used in the present invention refers to a vector or construct comprising a nucleotide sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide or peptide. The expression construct may include various control elements. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also include nucleotide sequences that supply other functions. Viruses that can be used in the present invention include retroviruses, herpes simplex viruses, lentiviruses, poxviruses, vaccinia viruses, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, and adeno-associated viruses (Adeno-associated virus: AAV), etc., but are not limited thereto.

[0296] The vector can be engineered to encode a selectable marker or reporter gene that provides selection and identification of cells that have been incorporated into the above-mentioned vector. The expression of the selectable marker or reporter gene allows identification and / or selection of host cells that have been incorporated and expressed in other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes that provide resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, etc.; and genes used as phenotypic markers, i.e. anthocyanidin regulatory genes, isopentanol transferase genes, etc. Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selectable markers can also be considered as reporter genes.

[0297] Recombinant adeno-associated virus (AAV) is usually produced by triple transfection of host cells (such as HEK293 cells). They are: 1) AAV construction plasmid, which has a gene expression cassette flanked by inverted terminal repeats (ITRs); 2) "Rep-Cap plasmid", which provides the Rep protein required for replication of the adeno-associated virus genome and the capsid protein that constitutes the virus particle; and finally 3) "helper plasmid", which provides adenoviral proteins (E2a, E4) and RNA (VARNA) for assisting the life cycle of AAV. When these three plasmids are transfected into HEK293 cells, etc., which provide the E1 and E3 genes of the adenovirus, AAV is produced.

[0298] The term "double helper plasmid" as used in the present invention refers to a plasmid that can provide two or more conditions required for producing AAV in a cell. It is apparent from the present disclosure that in some embodiments, the double helper plasmid of the present invention satisfies the above conditions 2) "plasmid" and 3) "helper plasmid". For example, in some embodiments, the double helper plasmid includes a rep gene, a cap gene, an E2a gene, an E4 gene, and a VA RNA gene.

[0299] The double helper plasmid of the present invention not only contains the above-mentioned genes, but also these genes are arranged in a specific configuration in the plasmid. For example, in some embodiments, the E2a gene, the E4 gene and the VA RNA gene are sequentially connected in the double helper plasmid, and the rep gene and the cap gene (collectively referred to as "rep-cap gene" in the present invention) are sequentially connected in a clockwise direction (from 5' to 3' direction) between the 5' end of the E2a gene and the 3' end of the VA RNA gene. More specifically, in some embodiments, the 5' end of the rep-cap gene is connected to the 5' end of the E2a gene, wherein the 3' end of the rep-cap gene is connected to the 3' end of the VARNA gene. In some embodiments, the E2a gene, the E4 gene and the VARNA gene are sequentially connected, and the rep-cap gene is located in a counterclockwise direction (from 3' to 5') between the 5' end of the E2a gene and the 3' end of the VARNA gene. More specifically, in some embodiments, the 3' end of the rep-cap gene is connected to the 5' end of the E2a gene, wherein the 5' end of the rep-cap gene is connected to the 3' end of the VARNA gene.

[0300] The term "cell" used in the present invention includes eukaryotic cells and prokaryotic cells, and refers to any transformable cell capable of replicating the above-mentioned vector or expressing a gene encoded by the vector. The cell can be transfected, transduced or transformed by the above-mentioned vector, which refers to the process of transferring or introducing an exogenous polynucleotide (nucleic acid) into a host cell. The term "transformation" used in the present invention is used to include the above-mentioned transfection and transduction.

[0301] The (host) cells of the present invention are not limited, but preferably insect cells or mammalian cells, more preferably Sf9 cells can be used 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, HC7 cells can be used in the case of mammalian cells. 0 cells, HCC1428 cells, BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, MIAPaCa-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. In some embodiments, the above host cells are isolated host cells.

[0302] The term "family with sequence similarity 19" or "FAM19", or "TAFA" as used in the present invention refers to a protein belonging to the TAFA family (also called the FAM19 family) of five proteins and mainly expressed in the brain and spinal cord. It is reported that FAM19A1 is also called TAFA1, FAM19A2 is also called TAFA2, FAM19A3 is also called TAFA3, FAM19A4 is also called TAFA4, and FAM19A5 is also called TAFA5.

[0303] The human TAFA1 gene encodes a 133 amino acid sequence. There are different opinions on the length of the signal sequence (signal peptide) and the mature protein (mature protein), but for the human TAFA1 protein (SEQ ID NO.302), it is predicted to be composed of a signal sequence of 35 amino acids and a mature protein of 98 amino acids. TAFA1 shows high expression in the frontal, temporal, occipital and parietal cortices, and low expression in the basal ganglia, lateral canal and cerebellum. Cell experiments have found that TAFA1 affects the differentiation fate of neural stem cells, inhibits the differentiation of neural stem cells into astrocytes, and promotes their differentiation into neurons. Through TAFA1 knockout (KO) mouse experiments, it is known that TAFA1 can regulate motor activity, anxiety-related behaviors, learning and memory, and somatosensory functions.

[0304] The human TAFA2 gene encodes a 131 amino acid sequence. There are differences of opinion on the length of the signal sequence and the mature protein, but for the human TAFA2 protein (SEQ ID NO.303), it is predicted to consist of a 30 amino acid signal sequence and a 101 amino acid mature protein. TAFA2 is expressed in large quantities in the occipital lobe, frontal cortex, and medulla in the central nervous system. It is reported that when TAFA2 recombinant protein is injected into the third ventricle of mice, the food intake and meal frequency of the mice increase, energy consumption increases, and the respiratory exchange ratio increases. Therefore, this suggests that TAFA2 may play a role in regulating food intake and energy metabolism. In addition, it is well known that when TAFA2 is inhibited in zebrafish and mice, anxiety-related behaviors increase.

[0305] The human TAFA3 gene encodes a 133 amino acid sequence, and there are differences of opinion on the length of the signal sequence and the mature protein, but for the human TAFA3 protein (SEQ ID NO.304), it is predicted to consist of a 30 amino acid signal sequence and a 103 amino acid mature protein. It is known that in a mouse model of transient focal cerebral ischemia, the expression of TAFA3 in microglia is increased, and microglia treated with TAFA3 are polarized into anti-inflammatory microglia. In addition, when TAFA3 is knocked out in a mouse model, three major behavioral defects observed in the autism spectrum, such as reduced responsiveness to social novelties, social communication impairment, and increased repetitive behaviors, are shown, indicating that TAFA3 is involved in the normal function of social relationship formation.

[0306] The human TAFA4 gene encodes a 140 amino acid sequence. There are different opinions on the length of the signal sequence and the mature protein, but for the human TAFA4 protein (SEQ ID NO.299), it is predicted to consist of a 45 amino acid signal sequence and a 95 amino acid mature protein. TAFA4 is mainly expressed in sensory neurons of the peripheral nervous system. TAFA4 protein is specifically expressed in low-threshold mechanoreceptors (C-low-threshold mechanoreceptors), showing that it relieves pain by regulating the activity of interneurons, especially GABAergic neurons.

[0307] The human TAFA5 gene encodes a 132 amino acid sequence. There are different opinions on the length of the signal sequence and the mature protein, but for the human TAFA5 protein, it is predicted to consist of a 43 amino acid signal sequence and an 89 amino acid mature protein. TAFA5 shows high expression in the basal ganglia and cerebellum. It is reported that under inflammatory stimuli such as TNF-α, the expression of TAFA5 in the mouse hypothalamus increases. When TAFA5 is knocked out (KO), the reduced food intake, weight loss, and increased inflammatory cytokines caused by TNF-α are partially restored.

[0308] The five TAFA families described above are predicted to have different functions in vivo, but TAFA1 to TAFA4 mature proteins show high sequence identity (e.g., human TAFA4 has 73.7% sequence identity with TAFA1, 85.3% sequence identity with TAFA2, and 81.1% sequence identity with TAFA3). On the other hand, TAFA5 shows low sequence identity with TAFA1 to 4 (e.g., human TAFA5 has 48.9% sequence identity with TAFA1, 51.1% sequence identity with TAFA2, 47.7% sequence identity with TAFA3, and 50.0% sequence identity with TAFA4), so they are expected to have completely different functions (see Fig.21 ).

[0309] Human TAFA4 (TAFA Chemokine Like Family Member 4) protein (SEQ ID NO.299) is one of the TAFA protein family (TAFA1-5) and is expressed in large quantities throughout the brain area. The above-mentioned TAFA4 consists of 140 amino acids, and except for the first 45 amino acids, the remaining 95 amino acids (SEQ ID NO.171) are highly conserved in evolution among vertebrates. In addition, it has been confirmed that in the TAFA protein group, the sequence identity of the above-mentioned TAFA1 to TAFA4 is highly conserved between species within vertebrates. i) The amino acid sequence of human mature TAFA4 has more than 90% sequence identity compared with TAFA4 of mammals and amphibians, more than 95% sequence identity compared with TAFA4 of birds, and more than 85% sequence identity compared with TAFA4 of reptiles and fish (see Fig.19 ), ii) the amino acid sequence of human TAFA1 shows more than 90% sequence identity with TAFA1 of mammals and amphibians, more than 95% sequence identity with TAFA1 of birds, and more than 85% sequence identity with TAFA1 of reptiles and fish (see Fig.16), iii) the amino acid sequence of human TAFA2 shows more than 90% sequence identity with TAFA2 of mammals and amphibians, more than 95% sequence identity with TAFA2 of birds, and more than 80% sequence identity with TAFA2 of reptiles and fish (see Fig.17 ), iv) the amino acid sequence of human TAFA3 shows more than 75% sequence identity with TAFA3 of mammals and amphibians, more than 80% sequence identity with TAFA3 of birds, and more than 75% sequence identity with TAFA3 of reptiles and fish (see Fig.18 ), it can be confirmed that, regardless of species, they all show very high sequence identity.

[0310] II. Polynucleotides

[0311] II.A. Non-translated nucleic acid sequences

[0312] The present disclosure relates to polynucleotides including non-translated nucleic acid sequences, wherein the non-translated nucleic acid sequences can increase the expression of exogenous genes when translated. Specifically, provided herein is an elongation factor-1α (EF-1α) intron sequence that is shorter than the full-length EF-1α intron.

[0313] Elongation factor-1α (EF-1α) is a gene located on chromosome 6 (GenBank accession number is NC_000006.12, nucleotides 73489308 to 73525587; negative strand orientation). The EF-1α gene includes 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 (e.g., carrying aminoacyl-tRNA to the A site of the ribosome as a ternary complex eEF1A1-GTP-aatRNA). Scaggiante et al., Genetics and Cytogenetics in Oncology and Hematology (Atlas Genet Cytogenet Oncol Haematol) 19 (4): 256-265 (March 2015). The nucleotide sequence of the above-mentioned full-length EF-1α intron is shown in SEQ ID NO.1 (924 nucleotides in length). As described herein, the non-translated nucleic acid sequences of the present disclosure (i.e., EF-1α intron fragment sequences) provide significant advantages over the full-length EF-1α intron (or any other intron known in the art). For example, in some embodiments, the non-translated nucleic acid sequences of the present invention can increase the expression of exogenous genes to a greater extent than the full-length EF-1α intron. Moreover, because the non-translated nucleic acid sequences of the present disclosure are shorter than their full-length counterparts, in some embodiments, they can be used in conjunction with larger exogenous genes. For example, AAV capsids (such as those described herein) can accommodate up to about 4.7 kb of nucleic acid. Therefore, in some embodiments, larger exogenous genes and / or additional cis-elements can be incorporated into the EF-1α intron fragments (i.e., non-translated nucleic acid sequences) of the present invention to greatly enhance gene expression.

[0314] In some embodiments, the non-translated nucleic acid sequence of the present invention (i.e., the EF-1α intron fragment) includes nucleotides 874 to 924 of the sequence shown in SEQ ID NO.1, but does not include SEQ ID NO.1. In some embodiments, the non-translated nucleic acid sequence is necessarily composed of nucleotides 874 to 924 of SEQ ID NO.1. In some embodiments, the non-translated nucleic acid sequence is composed of nucleotides 874 to 924 of SEQ ID NO.1. Such EF-1α intron fragments are also referred to herein as "T3.2 fragments" and are shown in SEQ ID NO.57. In some embodiments, the non-translated 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, wherein the above-mentioned non-translated nucleic acid sequence does not include SEQ ID NO.1.

[0315] In some embodiments, the non-translated nucleic acid sequence of the present invention (i.e., the EF-1α intron fragment) includes nucleotides 852-924 of SEQ ID NO.1, but does not include SEQ ID NO.1. In some embodiments, the non-translated nucleic acid sequence of the present disclosure is necessarily composed of nucleotides 852-924 of SEQ ID NO.1. In some embodiments, the non-translated nucleic acid sequence consists of nucleotides 852-924 of SEQ ID NO.1. Such EF-1α intron fragment is also referred to herein as "T3.1.2 fragment" and is shown in SEQ ID NO.3. In some embodiments, the non-translated 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 852-924 of SEQ ID NO.1, wherein the non-translated nucleic acid sequence does not include SEQ ID NO.1.

[0316] In some embodiments, the non-translated nucleic acid sequence of the present invention (i.e., the EF-1α intron fragment) includes nucleotides 830-924 of SEQ ID NO.1, but does not include SEQ ID NO.1. In some embodiments, the non-translated nucleic acid sequence of the present disclosure is necessarily composed of nucleotides 830-924 of SEQ ID NO.1. In some embodiments, the non-translated nucleic acid sequence consists of nucleotides 830-924 of SEQ ID NO.1. Such EF-1α intron fragment is also referred to herein as "T3.1.1 fragment" and is shown in SEQ ID NO.2. In some embodiments, the non-translated 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 830-924 of SEQ ID NO.1, wherein the above-mentioned non-translated nucleic acid sequence does not include SEQ ID NO.1.

[0317] In some embodiments, the non-translated nucleic acid sequence of the present invention (i.e., the EF-1α intron fragment) consists of 29 to 174 nucleotides, and the non-translated nucleic acid sequence comprises a nucleotide sequence having at least about 70% sequence identity with the nucleotide sequence shown in SEQ ID NO. 57. In some embodiments, the non-translated nucleic acid sequence consists of 51 to 117 nucleotides, and the non-translated nucleic acid sequence comprises a nucleotide sequence having at least about 70% sequence identity with the nucleotide sequence shown in SEQ ID NO. 57. In some embodiments, the non-translated nucleic acid sequence consists of 51 to 117 nucleotides, and the non-translated nucleic acid sequence comprises the nucleotide sequence shown in SEQ ID NO. 57.

[0318] In some embodiments, the non-translated nucleic acid sequence of the present invention 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: (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).

[0319] In some embodiments, the non-translated 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 871 to 924 of SEQ ID NO.1 (i.e., SEQ ID NO.58). In some embodiments, the non-translated 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 861 to 924 of SEQ ID NO.1 (i.e., SEQ ID NO.59). In some embodiments, the non-translated 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 (i.e., SEQ ID NO.60). In some embodiments, the non-translated 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 851 to 924 of SEQ ID NO.1 (i.e., SEQ ID NO.61). In some embodiments, the non-translated 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 (i.e., SEQ ID NO.2). In some embodiments, the non-translated 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 821 to 924 of SEQ ID NO.1 (i.e., SEQ ID NO.63). In some embodiments, the non-translated 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 non-translated 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 non-translated 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 non-translated 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 non-translated 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 non-translated 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 non-translated 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 non-translated 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 non-translated 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 non-translated 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 non-translated 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).

[0320] As is apparent from the above disclosure, in some embodiments, the polynucleotide comprising a non-translated nucleic acid sequence of the present invention 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 non-translated nucleic acid sequence. In some embodiments, the polynucleotides of the present invention further include 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 non-translated nucleic acid sequence. In some embodiments, the polynucleotide comprising the above-mentioned non-translated nucleic acid sequence further includes at least about 1, at least about 2, at least 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 above-mentioned non-translated nucleic acid sequence.

[0321] In some embodiments, the polynucleotide includes one or more consecutive or non-consecutive nucleotides corresponding to positions 1 to 873 in SEQ ID NO. 1 at the 5' region of the non-translated nucleic acid sequence.

[0322] II.B. Foreign genes

[0323] In some embodiments, the polynucleotide comprising the non-translated nucleic acid sequence of the present invention further comprises an exogenous gene. In some embodiments, the exogenous gene is a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75. In some embodiments, the exogenous gene comprises a nucleotide sequence having 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 SEQ ID NO.184.

[0324] II.C. Control elements

[0325] In some embodiments, the polynucleotides of the present invention further include control elements. Thus, in some embodiments, the polynucleotides include (1) control elements; (2) the non-translated nucleic acid sequence of the present invention; and (3) an exogenous gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO. 184).

[0326] As used herein, the term "control element" refers to a nucleic acid sequence that regulates (e.g., increases or decreases) the expression of an operably linked nucleic acid. Control elements that can be used in the present disclosure include enhancers (e.g., CMV enhancers), promoters (e.g., CMV promoters, EF-1α promoters or β-actin promoters), exons (e.g., exon 1 or exon 2), splicing donor sequences, acceptor sequences, or combinations thereof. In some embodiments, control elements may include sequences for transcription termination (e.g., poly A), sequences for stable exogenous gene expression (e.g., WPRE sequences), sequences for reducing exogenous gene-specific immunity (e.g., miRNA target sequences), or combinations thereof.

[0327] II.C.1. Enhancers

[0328] In some embodiments, the control element is an enhancer. Thus, in some embodiments, the polynucleotide of the present invention includes (in no particular order) (1) an enhancer; (2) a non-translated nucleic acid sequence; and (3) an exogenous gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO. 184). In some embodiments, the polynucleotide of the present invention includes (from 5' to 3' direction): (1) an enhancer; (2) a non-translated nucleic acid sequence; and (3) an exogenous gene.

[0329] Any suitable enhancer known in the art can be used with the present disclosure. Non-limiting examples of enhancers include: cytomegalovirus (CMV) enhancer, SV40 early enhancer, adenovirus 5E1A enhancer, HBV enhancer-1 regulatory region (Eh-1), HPV-16 or HPV-18E6 / 7 long control 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 includes a sequence having 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 SEQ ID NO.4. In some embodiments, the cytomegalovirus (CMV) enhancer includes the nucleotide sequence shown in SEQ ID NO.4.

[0330] II.C.2. Promoter

[0331] In some embodiments, the control element is a promoter. Thus, in some embodiments, the polynucleotides of the present invention include (in no particular order) (1) a promoter; (2) a non-translated nucleic acid sequence; and (3) an exogenous gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO. 184). In some embodiments, the polynucleotides include (from 5' to 3'): (1) a promoter; (2) a non-translated nucleic acid sequence; and (3) an exogenous gene. In some embodiments, the control element includes both an enhancer and a promoter. In such aspects, the polynucleotides may include (in no particular order) (1) an enhancer; (2) a promoter; (3) a non-translated nucleic acid sequence; and (4) an exogenous gene. In some embodiments, the polynucleotides include (from 5' to 3'): (1) an enhancer; (2) a promoter; (3) a non-translated nucleic acid sequence; and (4) an exogenous gene. Any suitable promoter known in the art may be used with the present disclosure.

[0332] In some embodiments, the above-mentioned promoter includes a cytomegalovirus (CMV) promoter, an EF-1α promoter, a β-actin promoter, a 3-phosphoglyceraldehyde dehydrogenase (GAPDH) promoter, a 70-kDa heat shock protein (HSP70) promoter, a 78-kDa glucose-regulated protein (GRP78) promoter, a eukaryotic initiation factor-4A (eIF4a) promoter, an α-1-antitrypsin (AAT) promoter, a transthyretin (TTR) promoter, a glial fibrillary acidic protein (GFAP) promoter, an early promoter of the simian vacuolating virus 40 (SV40) promoter, a synapsin I (SYN1) promoter, a G protein-coupled receptor kinase (GRK) promoter, an opsin gene (Rho) promoter, or a combination thereof.

[0333] In some embodiments, the promoter useful in the present disclosure is a CMV promoter. Thus, in some embodiments, the polynucleotide comprises (1) an enhancer (e.g., a CMV enhancer); (2) a CMV promoter; (3) a non-translated nucleic acid sequence; and (4) an exogenous gene. In some embodiments, the CMV promoter comprises a sequence having 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 to SEQ ID NO. 5 or 6. In some embodiments, the CMV promoter comprises the nucleotide sequence shown in SEQ ID NO. 5 or 6.

[0334] In some embodiments, the promoter that can be used with the present disclosure is the EF-1α promoter. In some embodiments, the polynucleotide includes (1) an enhancer (e.g., a CMV enhancer); (2) an EF-1α promoter; (3) a non-translated nucleic acid sequence; and (4) an exogenous gene. In some embodiments, the EF-1α promoter includes a sequence having 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 SEQ ID NO.7. In some embodiments, the EF-1α promoter includes the nucleotide sequence shown in SEQ ID NO.7.

[0335] In some embodiments, the above-mentioned promoter is a β-actin promoter. Therefore, in some embodiments, the polynucleotides described in the present invention include (1) an enhancer (e.g., a CMV enhancer); (2) a β-actin promoter; (3) a non-translated nucleic acid sequence; and (4) an exogenous gene. In some embodiments, the β-actin promoter includes a sequence having 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 SEQ ID NO. 8. In some embodiments, the β-actin promoter is a chicken β-actin promoter as shown in SEQ ID NO. 8.

[0336] As shown in this manual (see Figure 4a ), in some embodiments, the polynucleotides of the present invention (e.g., including exogenous genes and non-translated nucleic acid sequences) may include multiple promoters. For example, in some embodiments, the polynucleotides include a combination of a CMV promoter, an EF-1α promoter, and / or a β-actin promoter. In some embodiments, the polynucleotides include both a CMV promoter and an EF-1α promoter. In such aspects, the CMV promoter may be a portion of a 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).

[0337] II.C.3. Splice donor sequence

[0338] In some embodiments, the polynucleotides described herein (i.e., including non-translated nucleic acid sequences) include a splice donor sequence. As used herein, the term "splice donor sequence" or "splice donor site" refers to a guanine-thymine (GT)-rich domain (indicating the boundary between an intron and an exon) present at the 5' end of an intron (e.g., an EF-1α intron). As demonstrated herein, such sequences can be targeted to produce non-translated nucleic acid sequences of the present disclosure. In some embodiments, the splice donor sequence is connected upstream of the EF-1α intron fragment (i.e., non-translated nucleic acid sequence). For example, in some embodiments, the polynucleotides described herein include (in no particular order) (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter); (3) a splice donor sequence; (4) a non-translated nucleic acid sequence; and (5) an exogenous gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO.184). In some embodiments, the polynucleotide includes (from 5' to 3' direction): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter and / or a β-actin promoter); (3) a splice donor sequence; (4) a non-translated nucleic acid sequence; and (5) an exogenous gene.

[0339] In some embodiments, the splice donor sequence useful in the present disclosure 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 to the nucleotide sequence shown in SEQ ID NO. 9 or SEQ ID NO. 10. In some embodiments, the splice donor sequence includes the nucleotide sequence shown in SEQ ID NO. 9 or 10.

[0340] II.C.4. Exon sequences

[0341] As demonstrated herein, in some embodiments, the polynucleotides of the present disclosure further include one or more exon sequences. For example, in some embodiments, the polynucleotides of the present disclosure include an EF-1α exon 2 (E2) sequence. Thus, in some embodiments, the polynucleotides of the present disclosure include the following features (in no particular order): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter); (3) a splice donor sequence; (4) a non-translated nucleic acid sequence; (5) an EF-1αE2 sequence; and (6) an exogenous gene (e.g., a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO. 184). In some embodiments, such polynucleotides include (from 5' to 3' direction): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter); (3) a splice donor sequence; (4) an untranslated nucleic acid sequence; (5) an EF-1αE2 sequence; and (6) an exogenous gene. In some embodiments, the EF-1αE2 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 SEQ ID NO. 11. In some embodiments, the EF-1αE2 sequence includes the nucleotide sequence shown in SEQ ID NO. 11.

[0342] In some embodiments, the one or more exon sequences that can be included in the polynucleotides of the present invention include cytomegalovirus (CMV), EF-1α or β-actin exon 1 (E1) sequences. In some embodiments, the polynucleotides of the present invention can include both E1 sequences and E2 sequences. For example, in some embodiments, the polynucleotides include (in no particular order): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) a non-translated nucleic acid sequence; (6) an EF-1αE2 sequence; and (7) an exogenous gene. In some embodiments, the polynucleotide includes (from 5' to 3' direction): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) an untranslated nucleic acid sequence; (6) an EF-1αE2 sequence; and (7) an exogenous gene.

[0343] In some embodiments, the E1 sequence that can be used with the present disclosure is a CMVE1 sequence. In some embodiments, the CMVE1 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 to SEQ ID NO. 12. In some embodiments, the CMVE1 sequence includes the nucleotide sequence shown in SEQ ID NO. 12.

[0344] In some embodiments, the E1 sequence is an EF-1αE1 sequence. In some embodiments, the EF-1αE1 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 SEQ ID NO.13. In some embodiments, the EF-1αE1 sequence includes 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 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 SEQ ID NO.14 or 15. In some embodiments, the β-actin E1 sequence includes the nucleotide sequence shown in SEQ ID NO. 14 or 15.

[0345] II.C.5. miRNA target sequences

[0346] As described herein, in some embodiments, the control elements of the polynucleotides described herein include one or more target sequences for microRNA (miRNA) specific to immune cells ("miRNA target sequences"). Thus, in some embodiments, the polynucleotides described herein include the following features (in no particular order): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence, and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) an untranslated nucleic acid sequence; (6) an EF-1αE2 sequence; (7) an exogenous 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 includes (from 5' to 3' direction): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) an untranslated nucleic acid sequence; (6) an EF-1αE2 sequence; (7) an exogenous gene; and (8) one or more miRNA target sequences.

[0347] In some embodiments, the polynucleotides of the present invention include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more miRNA target sequences. In some embodiments, the number of target sequences for miRNA that can be included in the polynucleotides of the present invention is about two to about six (e.g., 2 to 6). In some embodiments, the multiple miRNA target sequences are the same. In some embodiments, one or more of the multiple miRNA target sequences are different.

[0348] As is apparent from the present disclosure, including one or more miRNA target sequences can enhance the specificity of the polynucleotides of the present invention. For example, in certain cell types (e.g., immune cells) where it is desired to inhibit exogenous genes, miRNA target sequences specific to immune cells can be used to inhibit the expression of exogenous genes in immune cells, with the result that immune cells can be prevented from producing exogenous gene-specific immunity. Therefore, by using different miRNA target sequences, the expression of exogenous genes in different cells / tissues can be regulated.

[0349] Any suitable miRNA target sequence known in the art can be used with the present disclosure. In some embodiments, the above-mentioned miRNA target sequence is specific to miR142-3p or miR142-5p. In some embodiments, the above-mentioned miRNA target sequence is selected from antisense oligonucleotides, antagomirs, small hairpin RNA (shRNA) molecules, small interfering RNA (siRNA) molecules, ribozymes, peptide nucleic acid (PNA) oligonucleotides, locked nucleic acid (LNA) oligonucleotides or combinations thereof having a sequence complementary to the full-length sequence or partial sequence of miR142-3p or miR142-5p.

[0350] In some embodiments, the miRNA target 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 SEQ ID NO. 16. In some embodiments, the miRNA target 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 SEQ ID NO. 17. In some embodiments, the miRNA target sequence includes the nucleotide sequence shown in SEQ ID NO. 16 or SEQ ID NO. 17.

[0351] II.C.6. WPRE sequence

[0352] In some embodiments, the polynucleotides of the present invention (i.e., including non-translated nucleic acid sequences) further include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence. Thus, in some embodiments, the polynucleotides of the present invention include (in no particular order): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence, and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) a non-translated nucleic acid sequence; (6) an EF-1αE2 sequence; (7) an exogenous 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 in the present invention include (from 5' to 3' direction): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) an untranslated nucleic acid sequence; (6) an EF-1αE2 sequence; (7) an exogenous gene; (8) one or more miRNA target sequences; and (9) a WPRE sequence.

[0353] In some embodiments, the WPRE 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 SEQ ID NO. 18. In some embodiments, the WPRE sequence includes the nucleotide sequence shown in SEQ ID NO. 18.

[0354] II.C.7. Polyadenylation sequence

[0355] In some embodiments, the polynucleotides described herein (i.e., including non-translated nucleic acid sequences) further include one or more polyadenylation (pA) sequences. Thus, in some embodiments, the polynucleotides include (in no particular order): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter, and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence, and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) a non-translated nucleic acid sequence; (6) an EF-1αE2 sequence; (7) an exogenous 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 (from 5' to 3' direction): (1) an enhancer (e.g., a CMV enhancer); (2) a promoter (e.g., a CMV promoter, an EF-1α promoter and / or a β-actin promoter); (3) an E1 sequence (e.g., a CMVE1 sequence, an EF-1αE1 sequence and / or a β-actin E1 sequence); (4) a splice donor sequence; (5) an untranslated nucleic acid sequence; (6) an EF-1αE2 sequence; (7) an exogenous gene; (8) one or more miRNA target sequences; (9) a WPRE sequence; and (10) one or more pA sequences.

[0356] Any suitable pA sequence known in the art can be used with the present disclosure. In some embodiments, examples of polyadenylation sequences include, but are not limited to, human growth hormone (hGH) pA sequence, bovine growth hormone (bGH) pA sequence, simian vacuolating virus 40 (SV40) early pA sequence, and SV40 late pA sequence.

[0357] In some embodiments, the pA 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 SEQ ID NO. 19. In some embodiments, the pA 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 SEQ ID NO. 20. In some embodiments, the pA 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 SEQ ID NO. 21. In some embodiments, the pA 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 to SEQ ID NO. 22. In some embodiments, the polyadenylation sequence is selected from the group consisting of the nucleotide sequences shown in SEQ ID NOs. 19 to 22.

[0358] As described above, in some embodiments, the polynucleotide of the present invention comprises (i) an exogenous gene (e.g., a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO.184) and (ii) a control element operably linked to the exogenous gene, wherein the control element comprises (from 5' to 3' direction): (1) a CMV enhancer sequence as shown in SEQ ID NO.4; (2) a CMV promoter sequence as shown in SEQ ID NO.5 or 6, an EF-1α promoter sequence as shown in SEQ ID NO.7, or a chicken β-actin promoter sequence as shown in SEQ ID NO.8; (3) a CMVE1 sequence as shown in SEQ ID NO.12, an EF-1αE1 sequence as shown in SEQ ID NO.13, or a chicken β-actin E1 sequence as shown in SEQ ID NO.14 or 15; (4) a splice donor sequence as shown in SEQ ID NO.9 or 10; (5) an EF-1α intron fragment sequence (i.e., a non-translated nucleic acid sequence), wherein the EF-1α intron fragment sequence comprises SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4; The nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO.57, essentially consists of or consists of the nucleotide sequence shown in SEQ ID NO.11; and (6) the EF-1αE2 sequence shown in SEQ ID NO.11.

[0359] III.TAFA

[0360] 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 above polypeptide, a vector comprising the above nucleic acid, a recombinant virus particle comprising the above vector and a capsid protein, or a cell comprising the above nucleic acid or vector.

[0361] III.A. Peptides

[0362] The present disclosure provides a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75. In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75 comprises the following amino acid sequence of Formula 1 (N-terminus to C-terminus):

[0363] <General formula 1>

[0364] X1-X2-X3-GTCEV-X4-A-X5-H-X6-CCN-X7-N-X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X14-VAGTT-X15-X16-X17-PSCV-X18-A-X19-IV -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39

[0365] In the above general formula 1,

[0366] X1 does not exist, or is V, I, or L.

[0367] X2 is K, E, R or Q,

[0368] X3 is G, T, Q, P or A,

[0369] X4 is V or I,

[0370] X5 is A, L, V or I,

[0371] X6 is R or L,

[0372] X7 is K, R or Q,

[0373] X8 is R or K,

[0374] X9 is R or L,

[0375] X10 is V or G,

[0376] X11 is K or N,

[0377] X12 is F or L,

[0378] X13 is P or S,

[0379] X14 is Q or K,

[0380] X15 is R, H or Q,

[0381] X16 is A, N, S or T,

[0382] X17 is A, Q, R, K or T,

[0383] X18 is D or E,

[0384] X19 is S or A,

[0385] X20 is I, E, L, A or V,

[0386] X21 is Q, G or E,

[0387] X22 is K or R,

[0388] X23 is H, Q or E,

[0389] X24 is E, Q, N, D, S or H,

[0390] X25 is L, V or M,

[0391] X26 is E, D, P, L or A,

[0392] X27 is E or D,

[0393] X28 is V, T, A or I,

[0394] X29 is L, N, R, Y, S or Q,

[0395] X30 is S, K or T,

[0396] X31 is S or M,

[0397] X32 is S, A or Y,

[0398] X33 is S, T or R,

[0399] X34 is N or H,

[0400] X35 is V or I,

[0401] X36 is R or K,

[0402] X37 does not exist, or is V, A, G, M, or N,

[0403] X38 is absent, or is T, I, N, F, or S, and

[0404] X39 does not exist or is R, H, V, K, I, or Q.

[0405] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75 above consists of 80 to 130 amino acids, more preferably 90 to 120 amino acids, and most preferably 91 to 119 amino acid sequences.

[0406] 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 shown in SEQ ID NO.75 above.

[0407] In some embodiments, the above polypeptide further comprises an amino acid sequence of the following general formula 2 (from N-terminus to C-terminus):

[0408] <General formula 2>

[0409] X1-X2-HHKA-X3-H

[0410] In the above general formula 2,

[0411] X1 is A or V,

[0412] X2 is N or I, and

[0413] X3 is H or Q.

[0414] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 2-Formula 1 (N-terminus to C-terminus).

[0415] In some embodiments, the above polypeptide further comprises an amino acid sequence of the following general formula 3 (from N-terminus to C-terminus):

[0416] <General formula 3>

[0417] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0418] In the above general formula 3,

[0419] X1 is A or S,

[0420] X2 is L, T or S,

[0421] X3 is Q, E or H,

[0422] X4 does not exist, or is P, L or H,

[0423] X5 is P or R,

[0424] X6 is T, S or I,

[0425] X7 is A, P, T, S or H,

[0426] X8 is T, A, S or I,

[0427] X9 is V or A, and

[0428] X10 is L or H.

[0429] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 3 to Formula 1 (N-terminus to C-terminus).

[0430] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 4 (from N-terminus to C-terminus):

[0431] <General formula 4>

[0432] X1-X2

[0433] In the above general formula 4,

[0434] X1 is H or Y, and

[0435] X2 is Q, V or L.

[0436] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 4 to Formula 1 (N-terminus to C-terminus).

[0437] In some embodiments, the above polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0438] LHRP (SEQ ID NO. 293);

[0439] LHQSGFTSGHFPHHRKLGE (SEQ ID NO. 294); or

[0440] LAPPGTNIQI (SEQ ID NO. 295).

[0441] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 5 (from N-terminus to C-terminus):

[0442] <General formula 5>

[0443] X1-X2-X3

[0444] In the above general formula 5,

[0445] X1 is P or G,

[0446] X2 is R or H, and

[0447] X3 is T, S or L.

[0448] In some embodiments, the above polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0449] PYTSL (SEQ ID NO. 296); or

[0450] QEDKLK (SEQ ID NO. 297).

[0451] In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1, LHQSGFTSGHFPHHRKLGE-Formula 1, or LAPPGTNIQI-Formula 1. In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of Formula 1-Formula 5, Formula 1-PYTSL, or Formula 1-QEDKLK. In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1-Formula 5, LHQSGFTSGHFPHHRKLGE-Formula 1-Formula 5, LAPPGTNIQI-Formula 1-QEDKLK, or LAPPGTNIQI-Formula 1-PYTSL.

[0452] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NO.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 shown in SEQ ID NO.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 shown in SEQ ID NO.298 to 304.

[0453] III.B. Nucleic Acids Encoding Polypeptides

[0454] 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 shown in SEQ ID NO.75.

[0455] The nucleic acid that can be used in the present invention is not particularly limited, as long as the nucleic acid can be translated into a polypeptide when transduced into a cell. In some embodiments, the nucleic acid encodes a polypeptide (or any variant thereof) or a fusion protein.

[0456] In some embodiments, the nucleic acid encodes a protein as described herein that can be used to prevent or treat a disease or condition. In some embodiments, the nucleic acid encodes a peptide for preventing or treating a specific disease, the purpose of which is to be continuously expressed in 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 at least 100% sequence identity with SEQ ID NO.184.

[0457] In some embodiments, the above nucleic acid encodes a polypeptide comprising the amino acid sequence (N-terminus to C-terminus) of the following formula 1:

[0458] <General formula 1>

[0459] X1-X2-X3-GTCEV-X4-A-X5-H-X6-CCN-X7-N-X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X14-VAGTT-X15-X16-X17-PSCV-X18-A-X19-IV -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39

[0460] In the above general formula 1,

[0461] X1 does not exist, or is V, I, or L.

[0462] X2 is K, E, R or Q,

[0463] X3 is G, T, Q, P or A,

[0464] X4 is V or I,

[0465] X5 is A, L, V or I,

[0466] X6 is R or L,

[0467] X7 is K, R or Q,

[0468] X8 is R or K,

[0469] X9 is R or L,

[0470] X10 is V or G,

[0471] X11 is K or N,

[0472] X12 is F or L,

[0473] X13 is P or S,

[0474] X14 is Q or K,

[0475] X15 is R, H or Q,

[0476] X16 is A, N, S or T,

[0477] X17 is A, Q, R, K or T,

[0478] X18 is D or E,

[0479] X19 is S or A,

[0480] X20 is I, E, L, A or V,

[0481] X21 is Q, G or E,

[0482] X22 is K or R,

[0483] X23 is H, Q or E,

[0484] X24 is E, Q, N, D, S or H,

[0485] X25 is L, V or M,

[0486] X26 is E, D, P, L or A,

[0487] X27 is E or D,

[0488] X28 is V, T, A or I,

[0489] X29 is L, N, R, Y, S or Q,

[0490] X30 is S, K or T,

[0491] X31 is S or M,

[0492] X32 is S, A or Y,

[0493] X33 is S, T or R,

[0494] X34 is N or H,

[0495] X35 is V or I,

[0496] X36 is R or K,

[0497] X37 does not exist, or is V, A, G, M, or N,

[0498] X38 is absent, or is T, I, N, F, or S, and

[0499] X39 does not exist or is R, H, V, K, I, or Q.

[0500] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of the following formula 2 (N-terminus to C-terminus) and the amino acid sequence of the above formula 1:

[0501] <General formula 2>

[0502] X1-X2-HHKA-X3-H

[0503] In the above general formula 2,

[0504] X1 is A or V,

[0505] X2 is N or I, and

[0506] X3 is H or Q.

[0507] In some embodiments, the above nucleic acid encodes a polypeptide comprising an amino acid sequence of Formula 2-Formula 1 (N-terminus to C-terminus).

[0508] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of the following formula 3 (N-terminus to C-terminus) and the amino acid sequence of the above formula 1:

[0509] <General formula 3>

[0510] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0511] In the above general formula 3,

[0512] X1 is A or S,

[0513] X2 is L, T or S,

[0514] X3 is Q, E or H,

[0515] X4 does not exist, or is P, L or H,

[0516] X5 is P or R,

[0517] X6 is T, S or I,

[0518] X7 is A, P, T, S or H,

[0519] X8 is T, A, S or I,

[0520] X9 is V or A, and

[0521] X10 is L or H.

[0522] In some embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence (N-terminus to C-terminus) of Formula 3-Formula 1.

[0523] In some embodiments, the nucleic acid encodes a polypeptide comprising the amino acid sequence of the following general formula 4 (N-terminus to C-terminus) and the amino acid sequence of the above general formula 1:

[0524] <General formula 4>

[0525] X1-X2

[0526] In the above general formula 4,

[0527] X1 is H or Y, and

[0528] X2 is Q, V or L.

[0529] In some embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence (N-terminus to C-terminus) of Formula 4 to Formula 1.

[0530] In some embodiments, the nucleic acid encodes a polypeptide comprising the following amino acid sequence (N-terminus to C-terminus) in addition to the amino acid sequence of the general formula 1:

[0531] LHRP (SEQ ID NO. 293);

[0532] LHQSGFTSGHFPHHRKLGE (SEQ ID NO. 294); or

[0533] LAPPGTNIQI (SEQ ID NO. 295).

[0534] In some embodiments, the above nucleic acid encodes a polypeptide comprising an amino acid sequence (N-terminus to C-terminus) of the following formula 5 in addition to the amino acid sequence of the above formula 1:

[0535] <General formula 5>

[0536] X1-X2-X3

[0537] In the above general formula 5,

[0538] X1 is P or G,

[0539] X2 is R or H, and

[0540] X3 is T, S or L.

[0541] In some embodiments, the above polypeptide encodes a polypeptide comprising the following amino acid sequence (N-terminus to C-terminus).

[0542] PYTSL (SEQ ID NO. 296); or

[0543] QEDKLK (SEQ ID NO. 297).

[0544] In some embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1, LHQSGFTSGHFPHHRKLGE-Formula 1, or LAPPGTNIQI-Formula 1. In some embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence (N-terminus to C-terminus) of Formula 1-Formula 5, Formula 1-PYTSL, or Formula 1-QEDKLK. In some embodiments, the nucleic acid encodes a polypeptide comprising an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1-Formula 5, LHQSGFTSGHFPHHRKLGE-Formula 1-Formula 5, LAPPGTNIQI-Formula 1-QEDKLK, or LAPPGTNIQI-Formula 1-PYTSL.

[0545] In some embodiments, the nucleic acid encoding the above polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences shown in SEQ ID NO.184 to 238. In some embodiments, the nucleic acid encoding the above polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences shown in SEQ ID NO.239 to 292. In some embodiments, the nucleic acid encoding the above polypeptide further comprises a sequence encoding a signal sequence. In some embodiments, the nucleic acid encoding the above polypeptide is one or more nucleotide sequences selected from the group consisting of the nucleotide sequences shown in SEQ ID NO.305 to 311.

[0546] IV. Vectors containing nucleic acids encoding polypeptides

[0547] The present disclosure provides a vector comprising a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75.

[0548] The above nucleic acids are as described in III.B.

[0549] As described herein, such vectors can be used for recombinant expression in host cells and cells targeted for therapeutic intervention. In some embodiments, vectors (e.g., including exogenous genes (encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 or a nucleic acid having at least 70% sequence identity with SEQ ID NO.184) and non-translated gene sequences) that can be used to deliver the polynucleotides described in the present invention include viral vectors. Examples of viruses that can be used as vectors in the present disclosure include retroviruses, herpes simplex viruses, lentiviruses, poxviruses, vaccinia viruses, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, adeno-associated viruses (AAV), baculoviruses, and combinations thereof, but are not limited thereto. In some embodiments, vectors that can be used in the present invention include non-viral vectors. Non-limiting examples of such vectors include plasmids, cosmids, yeast artificial chromosomes (YACs), bacteriophages, and combinations thereof.

[0550] In some embodiments, the vector comprising a nucleic acid encoding a polypeptide is an adeno-associated virus (AAV) vector, and the polypeptide comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75.

[0551] 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 encoding sequence, a splice donor sequence, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

[0552] In some embodiments, the above-mentioned intron does not include the nucleotide sequence shown in SEQ ID NO.1.

[0553] In some embodiments, the intron consists of 29 to 174 nucleotides. In some embodiments, the intron consists of 51 to 117 nucleotides.

[0554] 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.

[0555] 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).

[0556] In some embodiments, the above-mentioned intron includes: (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).

[0557] In some embodiments, the above-mentioned carrier comprises the following components:

[0558] (1) the CMV enhancer sequence shown in SEQ ID NO.4;

[0559] (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;

[0560] (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;

[0561] (4) the splice donor sequence shown in SEQ ID NO. 9 or 10; and / or

[0562] (5) The EF-1αE2 sequence shown in SEQ ID NO.11.

[0563] In some embodiments, the AAV vector is used for gene therapy. In some embodiments, the AAV vector is used to express a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75.

[0564] V.AAV

[0565] In some embodiments, the polynucleotides of the present invention (e.g., including exogenous genes and non-translated nucleic acid sequences) are delivered to, for example, cells using AAV. Adeno-associated virus (AAV) is a single-stranded DNA virus belonging to a helper-dependent human parvovirus. The size of the above-mentioned AAV genome is about 4.7 kbp, and is composed of the N-terminus of the rep gene involved in encoding viral replication and viral gene expression, the C-terminus of the cap gene encoding the capsid protein of the above-mentioned virus, and the reverse terminal repeats (ITR) with about 145 bases inserted at each end. The above-mentioned 145bp reverse terminal repeats (ITR) have a T-shaped structure, which is used as the origin of replication during viral genome replication and serves as a major packaging signal. ITR is only the cis-acting nucleotide sequence required for preparing a recombinant AAV (rAAV) construct. ITR has enhancer activity in the presence of Rep protein, but its activity is very weak in the absence of Rep protein. In view of these characteristics, when the foreign gene is cloned into the recombinant AAV construct, enhancers, promoters, pA, etc. are appropriately assembled to prepare the expression construct (RJ Samulski and N Muzyczka, "Annual Review of Virology (Annu. Rev. Virolo.)" 2014.1: 427-451). There are four proteins translated from the rep gene. These proteins are divided into rep78, rep68, rep52 and rep40 according to their molecular weight, and perform important functions in AAV DNA replication. There are four proteins translated from the cap gene. Among them, VP1, VP2 and VP3 proteins are structural proteins that constitute AAV particles, and the assembly activation protein (AAP) promotes the formation (assembly) of AAV particles through structural proteins. Some proteins and RNA derived from helper viruses such as adenovirus or herpes simplex virus are required for efficient replication of adeno-associated virus (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0566] AAV has unique characteristics that make it attractive as a vector system for delivering foreign DNA into cells. AAV infection of cells in culture is usually non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. In addition, AAV infects many different types of mammalian cells, making it possible to target many different tissues in vivo. AAV also has additional advantages that make it a particularly attractive viral system for gene delivery, including promoting a relatively milder immune response compared to other forms of gene delivery and based on non-integrating episomal vector DNA (non-integrating, episomal vector DNA) in both dividing cells and resting cells. In addition, AAV withstands conditions for inactivating adenovirus (56°C to 65°C for several hours), making cold storage of rAAV-based vaccines less important.

[0567] Types of viruses or serotypes that can be used together with this publication include: AAVrh.10(AAVrh10), AAV-DJ(AAVDJ), AAV-DJ8(AAVDJ8), AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AA V4-4、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 AV9.84、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、AAV4211、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.6 2、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 / rh.54、AAV4-9 / rh.54、AAV4-1 9 / 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、AA Vrh.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, A AVcy.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. hu.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. u.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, AAVrh8RA586R variant, AAVrh8RR533A variant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, 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, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAVLK06, 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-precursor-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAVSM10-2, AAV Shuffle100-1, AAV Shuffle100-3, AAV Shuffle1007, AAV Shuffle10-2, AAV Shuffle10-6, AAV Shuffle10-8, AAVShuffle100-2, AAVSM10-1, AAVSM10-8, AAVSM100-3, AAVSM100-10, BP61AAV, BP62AAV, B P63AAV, 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, truetype AAV (ttAAV), UPENNAAV10, and Japanese AAV10 serotypes, but not limited thereto. .

[0568] 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 AAV is AAV2. In some embodiments, the serotype of AAV is AAV5. In some embodiments, the serotype of AAV is AAV8. In some embodiments, the serotype of AAV is AAV9.

[0569] IV. Cells

[0570] In some embodiments, provided herein are cells comprising any polynucleotide described herein (e.g., comprising a non-translated nucleic acid sequence). For example, in some embodiments, a recombinant expression construct comprising an exogenous gene (e.g., a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75 or a nucleic acid having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 184) and a non-translated nucleic acid sequence is used to transduce, transfect or transform the cells described herein to express the exogenous gene.

[0571] While not being bound by any one theory, in some embodiments, the cells described herein (e.g., transduced with a polynucleotide comprising a non-translated nucleic acid sequence) can be used to produce a protein, such as a protein encoded by an exogenous gene 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 non-translated nucleic acid sequence described herein (i.e., an EF-1α intron fragment) can enhance the expression of a protein encoded by an exogenous gene ("encoded protein") in a cell. Thus, in some embodiments, the cells described herein (e.g., transduced with a polynucleotide comprising an exogenous gene and a non-translated nucleic acid sequence disclosed herein) produce greater expression of the encoded protein compared to a reference cell. In some embodiments, the reference cell is transduced with the corresponding polynucleotide, but lacks the non-translated nucleic acid sequence.

[0572] In some embodiments, the cells of the present invention can produce proteins encoded by exogenous genes in vitro. In some aspects, the cells of the present invention can produce encoded proteins in vivo (e.g., in a subject receiving the administration of the polynucleotides of the present invention). In some embodiments, the cells of the present invention can produce encoded proteins both in vitro and in vivo.

[0573] In some embodiments, the cell that can be used to produce the protein (for example, in vitro) encoded by the exogenous gene includes a host cell. As used herein, the term "host cell" is intended to include cells of any organism that can be transduced with the above-mentioned expression construct (for example, AAV vector) to replicate the expression construct or express the gene encoded by the expression construct. Such cells include eukaryotic cells and prokaryotic cells. As used herein, the term "transduction" is intended to include "transfection" and "conversion". The above-mentioned host cell can be transduced, transfected or transformed with the above-mentioned expression construct. This process means that exogenous nucleic acid is delivered or introduced into the host cell. In some embodiments, the above-mentioned host cell is a separated host cell comprising an AAV vector. In some embodiments, the above-mentioned host cell is a separated host cell transformed with an AAV vector.

[0574] In some embodiments, the host cell is a eukaryotic cell. In some aspects, the host cell is selected from the group consisting of mammalian cells, insect cells, yeast cells, foreign gene mammalian cells and plant cells. In some aspects, the host cell is a prokaryotic cell. In some aspects, the prokaryotic cell is a bacterial cell.

[0575] 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 with 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, MIAPaCa-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.

[0576] In some embodiments, cells that can be used to produce proteins encoded by the exogenous genes 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 molecules described herein are administered. In some aspects, the human cells are from a donor (e.g., a healthy human subject).

[0577] The present disclosure provides a composition comprising the above-mentioned AAV vector, or a host cell comprising the above-mentioned AAV vector or transformed with the above-mentioned AAV vector.

[0578] VII. Composition

[0579] The present invention also provides a composition, which comprises: a 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.

[0580] In some embodiments, the above-described composition is a pharmaceutical composition.

[0581] In some embodiments, disclosed herein are pharmaceutical compositions comprising (a) any of the polynucleotides described herein (e.g., including exogenous genes and non-translated nucleic acid sequences) and (b) one or more pharmaceutically acceptable carriers. In some embodiments, disclosed herein are pharmaceutical compositions comprising (a) a vector as described herein (e.g., rAAV) and (b) one or more pharmaceutically acceptable carriers. In some embodiments, disclosed herein are pharmaceutical compositions comprising (a) a cell as described herein and (b) one or more pharmaceutically acceptable carriers.

[0582] In some embodiments, the pharmaceutical composition described in the present invention includes: a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 (for example, the TAFA1 to TAFA4 sequence of vertebrates), a nucleic acid encoding the above polypeptide, a vector comprising the above nucleic acid, a recombinant viral particle comprising the above vector and capsid protein, or a cell comprising the above nucleic acid or vector.

[0583] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 above comprises the amino acid sequence of the following general formula 1 (N-terminus to C-terminus):

[0584] <General formula 1>

[0585] X1-X2-X3-GTCEV-X4-A-X5-H-X6-CCN-X7-N-X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X14-VAGTT-X15-X16-X17-PSCV-X18-A-X19-IV -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39

[0586] In the above general formula 1,

[0587] X1 does not exist, or is V, I, or L.

[0588] X2 is K, E, R or Q,

[0589] X3 is G, T, Q, P or A,

[0590] X4 is V or I,

[0591] X5 is A, L, V or I,

[0592] X6 is R or L,

[0593] X7 is K, R or Q,

[0594] X8 is R or K,

[0595] X9 is R or L,

[0596] X10 is V or G,

[0597] X11 is K or N,

[0598] X12 is F or L,

[0599] X13 is P or S,

[0600] X14 is Q or K,

[0601] X15 is R, H or Q,

[0602] X16 is A, N, S or T,

[0603] X17 is A, Q, R, K or T,

[0604] X18 is D or E,

[0605] X19 is S or A,

[0606] X20 is I, E, L, A or V,

[0607] X21 is Q, G or E,

[0608] X22 is K or R,

[0609] X23 is H, Q or E,

[0610] X24 is E, Q, N, D, S or H,

[0611] X25 is L, V or M,

[0612] X26 is E, D, P, L or A,

[0613] X27 is E or D,

[0614] X28 is V, T, A or I,

[0615] X29 is L, N, R, Y, S or Q,

[0616] X30 is S, K or T,

[0617] X31 is S or M,

[0618] X32 is S, A or Y,

[0619] X33 is S, T or R,

[0620] X34 is N or H,

[0621] X35 is V or I,

[0622] X36 is R or K,

[0623] X37 does not exist, or is V, A, G, M, or N,

[0624] X38 is absent, or is T, I, N, F, or S, and

[0625] X39 does not exist or is R, H, V, K, I, or Q.

[0626] In some embodiments, the amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75 above consists of 80 to 130 amino acids, more preferably 90 to 120 amino acids, and most preferably 91 to 119 amino acid sequences.

[0627] 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 shown in SEQ ID NO.75 above.

[0628] In some embodiments, the above polypeptide further comprises an amino acid sequence of the following general formula 2 (from N-terminus to C-terminus):

[0629] <General formula 2>

[0630] X1-X2-HHKA-X3-H

[0631] In the above general formula 2,

[0632] X1 is A or V,

[0633] X2 is N or I, and

[0634] X3 is H or Q.

[0635] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 2-Formula 1 (N-terminus to C-terminus).

[0636] In some embodiments, the polypeptide further comprises an amino acid sequence of the following formula 3 (from N-terminus to C-terminus).

[0637] <General formula 3>

[0638] X1-X2-X3-X4-X5-X6-X7-X8-X9-X10

[0639] In the above general formula 3,

[0640] X1 is A or S,

[0641] X2 is L, T or S,

[0642] X3 is Q, E or H,

[0643] X4 does not exist, or is P, L or H,

[0644] X5 is P or R,

[0645] X6 is T, S or I,

[0646] X7 is A, P, T, S or H,

[0647] X8 is T, A, S or I,

[0648] X9 is V or A, and

[0649] X10 is L or H.

[0650] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 3 to Formula 1 (N-terminus to C-terminus).

[0651] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 4 (from N-terminus to C-terminus):

[0652] <General formula 4>

[0653] X1-X2

[0654] In the above general formula 4,

[0655] X1 is H or Y, and

[0656] X2 is Q, V or L.

[0657] In some embodiments, the polypeptide comprises an amino acid sequence of Formula 4 to Formula 1 (N-terminus to C-terminus).

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

[0659] LHRP (SEQ ID NO. 293);

[0660] LHQSGFTSGHFPHHRKLGE (SEQ ID NO. 294); or

[0661] LAPPGTNIQI (SEQ ID NO. 295).

[0662] In some embodiments, the above polypeptide further comprises the following amino acid sequence of Formula 5 (from N-terminus to C-terminus):

[0663] <General formula 5>

[0664] X1-X2-X3

[0665] In the above general formula 5,

[0666] X1 is P or G,

[0667] X2 is R or H, and

[0668] X3 is T, S or L.

[0669] In some embodiments, the above polypeptide further comprises the following amino acid sequence (from N-terminus to C-terminus):

[0670] PYTSL (SEQ ID NO. 296); or

[0671] QEDKLK (SEQ ID NO. 297).

[0672] In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1, LHQSGFTSGHFPHHRKLGE-Formula 1, or LAPPGTNIQI-Formula 1. In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of Formula 1-Formula 5, Formula 1-PYTSL, or Formula 1-QEDKLK. In some embodiments, the polypeptide comprises an amino acid sequence (N-terminus to C-terminus) of LHRP-Formula 1-Formula 5, LHQSGFTSGHFPHHRKLGE-Formula 1-Formula 5, LAPPGTNIQI-Formula 1-QEDKLK, or LAPPGTNIQI-Formula 1-PYTSL.

[0673] In some embodiments, the polypeptide comprises one or more amino acid sequences selected from the group consisting of the amino acid sequences shown in SEQ ID NO.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 shown in SEQ ID NO.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 shown in SEQ ID NO.298 to 304.

[0674] In some embodiments, the pharmaceutical composition of the present invention comprises a recombinant adeno-associated virus and a pharmaceutically acceptable carrier, wherein the recombinant adeno-associated virus comprises (a) an AAV8 type capsid protein and (b) a polynucleotide, wherein the polynucleotide comprises: (i) an exogenous gene, wherein the exogenous gene comprises a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence shown in SEQ ID NO.184; and (ii) a control element operably linked to the exogenous gene, wherein the control element comprises (from 5' to 3' direction): (1) the CMV enhancer sequence shown in SEQ ID NO.4; (2) the chicken β-actin promoter sequence shown in SEQ ID NO.8; (3) the chicken β-actin exon 1 (E1) sequence shown in SEQ ID NO.15; (4) the splicing donor sequence of the chicken β-actin intron shown in SEQ ID NO.10; (5) an untranslated nucleic acid sequence, wherein the untranslated nucleic acid sequence comprises SEQ ID NO.2, SEQ ID NO.3 or SEQ ID The nucleotide sequence shown in NO.57, consists essentially of or consists of it; and (6) the EF-1α exon 2 (E2) sequence shown in SEQ ID NO.11.

[0675] In some embodiments, the above-mentioned pharmaceutical composition is a pharmaceutical composition for preventing, improving or treating retinal or macular diseases.

[0676] In some embodiments, the above-mentioned retina or macular disease is caused by all or part of the damage of the retina or macula. In some embodiments, the above-mentioned retina or macular disease is a disease caused by the dysfunction of retina or macular cells or damage. In certain embodiments, the above-mentioned retina or macular disease can be a disease caused by the dysfunction of photoreceptor cells (photoreceptor cells) of the retina or macula and / or retinal pigment epithelial (RPE) cells or damage. In some embodiments, the above-mentioned retina or macular disease includes: retinopathy, choroidal neovascularization, maculopathy, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal edema, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease or its combination. In some embodiments, the above-mentioned macular degeneration includes: age-related macular degeneration, Best vitelliform macular dystrophy (BVMD), Sorsby fundus dystrophy, familial dominant drusen (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 above-mentioned 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 above-mentioned retinopathy is retinal dystrophy. In some embodiments, the above-mentioned retinopathy is diabetic retinopathy. In some embodiments, the above-mentioned 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 above-mentioned retinal or macular disease is an inherited retinal disease.In some embodiments, the inherited 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.

[0677] The pharmaceutically acceptable carrier that can be used together with the present disclosure is the carrier that is commonly used in preparation. The example of pharmaceutically acceptable carrier includes but is not limited to lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil etc. The pharmaceutical composition of the present disclosure may also include one or more additives selected from the group consisting of: lubricant, wetting agent, sweetener, flavoring agent, emulsifier, suspending agent and preservative. The details of suitable pharmaceutically acceptable carrier and preparation can be found in " Remington's Pharmaceutical Sciences " (19th edition, 1995).

[0678] The pharmaceutical composition of the present disclosure is formulated to be compatible with its expected route of administration. The embodiment of suitable parenteral route of administration includes intravenous injection, transdermal administration, subcutaneous injection, intramuscular injection, intraocular (e.g., capable of being delivered to any administration of sub-Tenon, subconjunctival, suprachoroidal, suprachoroidal space, subretinal, vitreous and similar positions), eye drop administration, intraventricular injection, intrathecal injection, intraamniotic injection, intraarterial injection, intraarticular injection, intracardiac injection, intracavernous sinus injection, intracerebral injection, intracisternal injection, intracoronary injection, intracranial injection, intradural injection, epidural injection, intrahippocampal injection, intranasal injection, intraosseous injection, intraperitoneal injection, intrapleural injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection, intravaginal injection, intraventricular injection, intravesical injection, subconjunctival injection, intratumor injection, local injection, intraperitoneal injection and combination thereof. In some aspects, intraocular administration includes suprachoroidal, subretinal, and intravitreal administration.

[0679] In some embodiments, the pharmaceutical composition is administered at a daily dose of 0.0001 to 100 mg / kg.

[0680] The pharmaceutical composition of the present disclosure can be formulated into a preparation together with one or more pharmaceutically acceptable carriers and / or excipients. The pharmaceutical composition can be provided in a unit dosage form or distributed in a multi-dose container. The above-mentioned 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 formulation can also include a dispersant or a stabilizer.

[0681] VIII. Kit

[0682] Also disclosed herein is a kit comprising: one or more polynucleotides as disclosed herein (e.g., including exogenous genes and non-translated nucleic acid sequences), one or more vectors as disclosed herein (e.g., AAV), one or more cells as disclosed herein (e.g., host cells comprising the above-mentioned AAV vectors or transformed with the above-mentioned AAV vectors), any pharmaceutical composition as disclosed herein, or any combination thereof. In some embodiments, the kit further comprises instructions for use.

[0683] As used herein, the terms "kit" and "system" are intended to refer to at least one or more polynucleotides disclosed herein, one or more vectors disclosed herein (e.g., AAV vectors), one or more host cells disclosed herein, any pharmaceutical composition disclosed herein, or any combination thereof, in some embodiments, combined with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packaging for commercial packaging, instructions for use, etc.).

[0684] IX. Purpose and Method

[0685] IX.A. Production Method

[0686] The present specification also discloses a method for producing a polypeptide encoded by an exogenous gene (e.g., a nucleic acid encoding a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 or a nucleic acid having at least 70% sequence identity with SEQ ID NO.184). In some embodiments, such methods include culturing the cells described in the present invention (e.g., transduced with a polynucleotide comprising an exogenous gene and a non-translated nucleic acid) under suitable conditions and recovering the encoded protein. In certain embodiments, the method for producing a polypeptide encoded by an exogenous gene comprises administering a polynucleotide of the present disclosure (e.g., comprising an exogenous gene and a non-translated nucleic acid) to a subject in need thereof, so that the encoded polypeptide is produced in the subject. Additional disclosures of such in vivo methods for producing polypeptides are provided elsewhere in the present disclosure (see, e.g., therapeutic uses).

[0687] In some aspects, the present disclosure provides a method for producing a recombinant adeno-associated virus, wherein the recombinant adeno-associated virus comprises a polynucleotide described in the present invention (e.g., comprising an exogenous gene and a non-translated nucleic acid sequence). In some aspects, the method for producing such a recombinant AAV comprises a method for culturing cells transfected with the AAV vector described in the present invention under conditions for producing a recombinant AAV. In some embodiments, the above method further comprises the step of isolating the produced recombinant virus particles.

[0688] In some aspects, the present disclosure provides a recombinant virus particle produced by the above method.

[0689] In some embodiments, the recombinant viral particles may include the use of (i) an AAV vector comprising the exogenous gene (e.g., see Figure 6b) and (ii) transduction of cells with a construct comprising a rep gene and a cap gene. Further, (iii) an auxiliary construct for transducing exogenous genes into host cells can be used for construction. In such aspects, the auxiliary construct can include an E2A gene that promotes AAV genome replication and gene transcription, an E4 gene that allows AAV mRNA to move from the nucleus to the cytoplasm, and a VA region that produces two VA RNAs for regulating translation.

[0690] In some aspects, the above three constructs can be replaced by two constructs for transduction into host cells. In such aspects, the AAV construct includes an exogenous gene and a non-translated nucleic acid sequence, and a separate construct includes a rep gene and a cap gene, an E2A gene, an E4 gene, and a VA region. Other methods for producing the AAV particles of the present invention 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 (Mar. 1998), all of which are incorporated herein by reference as a whole.

[0691] The present invention also discloses a recombinant virus particle comprising (a) capsid protein and (b) the above-mentioned AAV vector.

[0692] IX.B. Therapeutic Uses

[0693] The polypeptides, nucleic acids (e.g., including exogenous genes and non-translated nucleic acid sequences), vectors and recombinant viruses (e.g., rAAV) carrying such nucleic acids, cells including the above nucleic acids or vectors, and methods described in this specification have numerous in vitro and in vivo utilities. For example, the polypeptides, polynucleotides, e.g., vectors (e.g., AAV vectors) described in this specification can be applied to cultures, cells cultured in vitro or ex vivo, or human subjects (e.g., in vivo) to prevent or treat diseases. Therefore, in some embodiments, the present disclosure provides a therapeutic use of any of the polypeptides, polynucleotides (e.g., including exogenous genes and non-translated nucleic acid sequences) described in the present invention, a recombinant expression construct or vector described herein, a cell described in this specification, a pharmaceutical composition described in this specification, or a recombinant virus described in this specification. In some embodiments, the present specification discloses a method for expressing an exogenous gene in a subject in need thereof, the method comprising administering to the subject a polynucleotide disclosed herein (e.g., comprising an exogenous gene and a non-translated nucleic acid sequence), a vector disclosed in the present specification, a recombinant virus disclosed in the present specification (e.g., rAAV), a cell disclosed in the present specification, or a pharmaceutical composition disclosed in the present specification, and after the above administration, the expression of the exogenous gene in the subject is increased.

[0694] As described in this specification, the non-translated nucleic acid sequence of the present disclosure can increase the expression of the above-mentioned exogenous gene when the above-mentioned exogenous gene is translated. Therefore, in some embodiments, the present disclosure relates to a method for increasing the expression of an exogenous gene in a cell, the method comprising contacting the above-mentioned cell with any polynucleotide, vector or recombinant virus (e.g., rAAV) disclosed herein. The above-mentioned contact can occur in vitro or be performed in vivo. When the above-mentioned contact is performed in vivo, the above-mentioned method may also include administering any polynucleotide, vector or recombinant virus in the above-mentioned polynucleotide, vector or recombinant virus to the subject before contact.

[0695] In some embodiments, after the above-mentioned contact, the expression of the exogenous gene increases by at least about 1 times, at least about 1.1 times, at least about 1.2 times, at least about 1.3 times, at least about 1.4 times, at least about 1.5 times, at least about 1.6 times, at least about 1.7 times, at least about 1.8 times, at least about 1.9 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times or more compared to the reference expression. In some embodiments, the above-mentioned reference expression is the expression of the exogenous gene in the cell before contact. In some embodiments, the above-mentioned reference expression is the expression of the exogenous gene in the corresponding cell that has not been contacted with a polypeptide, polynucleotide, vector or recombinant virus described herein (e.g., lacking a non-translated nucleic acid sequence or including a nucleotide sequence shown in SEQ ID NO.1).

[0696] Another aspect of the present disclosure provides a method for preventing or treating a disease in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the polypeptides, polynucleotides, vectors, cells, recombinant viruses or pharmaceutical compositions described above. As is apparent from the present disclosure, the compositions described in this specification (e.g., polypeptides, polynucleotides, recombinant expression constructs, cells, pharmaceutical compositions or recombinant viruses) can be used to prevent or treat any disease of interest, for example, by modifying exogenous genes.

[0697] In some embodiments, the above methods may further include administering an additional therapeutic agent (e.g., an inhibitor of vascular endothelial growth factor ("VEGF")) to the above subject. In some embodiments, the above additional therapeutic agent may be administered to the above subject simultaneously, before, or after administration of the above polypeptide, polynucleotide, vector, cell, recombinant virus, or pharmaceutical composition.

[0698] Diseases that can be prevented, improved or treated by the present disclosure are not limited thereto, and include all diseases that require a reduction in the number of drug administrations. Non-limiting examples of such diseases include retinal or macular diseases. In some embodiments, the retinal or macular diseases are selected from retinopathy, choroidal neovascularization, maculopathy, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, inherited retinal diseases, and combinations thereof.

[0699] In some embodiments, the retina or macular disease that can be prevented or treated by the present disclosure includes macular degeneration. In some embodiments, the above-mentioned 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 terminal or late AMD (geographic atrophy). In some embodiments, the retina or macular disease that can be prevented or treated by the present disclosure includes diabetic retinopathy. In some embodiments, diabetic retinopathy is non-proliferative diabetic retinopathy (NPDR). In some embodiments, diabetic retinopathy is proliferative diabetic retinopathy (PDR). In some embodiments, diabetic retinopathy is diabetic maculopathy. In some embodiments, diabetic retinopathy is diabetic macular edema. In some embodiments, diabetic retinopathy is any retinopathy associated with ischemic damage in the retina. Unless otherwise indicated, the present disclosure can be used to prevent or treat all forms of AMD and / or diabetic retinopathy.

[0700] Another embodiment of the present disclosure provides a gene therapeutic agent that can achieve sustained exogenous gene expression or a method for preventing or treating a disease.

[0701] Using the viral delivery system described in this specification, it is possible to achieve the application of the composition described in this specification at intervals of about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years or about 10 years or longer. In some embodiments, the above interval is about 2 to about 3 months. In some embodiments, the above interval is about 6 months. In some embodiments, the above interval is about 1 year. In some embodiments, the above interval is at least about 1 year. In some embodiments, the above interval is at least about 2 years. The above interval is at least about 3 years. In some embodiments, the above interval is at least about 4 years. The above interval is at least about 5 years. In some embodiments, the above interval is at least about 10 years. That is, the use of the viral delivery system described in the present invention can significantly reduce the number of applications of the above composition, thereby allowing doctors, patients or subjects to avoid discomfort caused by repeated application of the composition. Depending on the patient's symptoms or needs, the above composition can be administered at least 2 to 3 times at intervals of 1 to 2 weeks initially, and then once every 2 to 3 months, every 6 months, or every 1 year or longer, or every 2 to 10 years or longer.

[0702] Effects of the Invention

[0703] The features and advantages of the present invention are summarized as follows:

[0704] (#) The present invention provides a pharmaceutical composition for preventing or treating retinal or macular diseases, which comprises: 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 above polypeptide, a vector comprising the above nucleic acid, a recombinant virus particle comprising the above vector and a capsid protein, or a cell comprising the above nucleic acid or vector.

[0705] (#) In addition, the present invention provides an adeno-associated virus (AAV) vector, which comprises a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 70% sequence identity with the amino acid sequence of the TAFA protein; and a recombinant virus particle, wherein the recombinant virus particle comprises the above-mentioned AAV vector and a capsid protein.

[0706] (iii) The AAV vector, recombinant virus or composition comprising the same of the present invention can be effectively used for the prevention or treatment of retinal or macular diseases by restoring damaged retina. BRIEF DESCRIPTION OF THE DRAWINGS

[0707] These and / or other aspects and advantages of the present disclosure will become apparent and more readily understood from the ensuing description of the above aspects by reference to the accompanying drawings.

[0708] Figure 1 A cleavage diagram of the pAAV-eGFP expression construct described in the present specification is shown.

[0709] Figure 2a and Figure 2b The expression of eGFP in cells transduced with an expression construct containing the full-length EF-1α intron and the EF-1α promoter ("CEE-FL") is shown. Control cells were transduced with the corresponding expression construct lacking the EF-1α intron ("CE"). Figure 2a In , an ITR-deficient animal cell expression construct was used. Figure 2b In , a pAAV expression construct containing the ITRs was used. Figure 2a and 2b In the figures, eGFP expression in the following transduced cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7 and Hep3B) is shown.

[0710] Figure 3a , Figure 3b and Figure 3cThe expression of eGFP in cells transduced with expression constructs containing various promoters ("CCE-FL") and the full-length EF-1α intron is shown. Control cells were transduced with the corresponding expression constructs lacking the EF-1α intron. Figure 3a In , the combination of the CMV enhancer and CMV promoter is inserted into an animal cell expression construct (i.e., lacking ITRs) and used to transduce cells. Figure 3b In , a combination of the CMV enhancer and CMV promoter is inserted into a pAAV expression construct and used to transduce cells. Figure 3c In the above cells, an animal cell expression construct containing a combination of a CMV enhancer and a chicken-β-actin promoter was used to transduce the above cells. Figure 3a , Figure 3b and Figure 3c , eGFP expression in the following transduced cell lines (HEK293, HeLa, ARPE-19, RPE-1, Huh-7, and Hep3B) is shown, respectively.

[0711] Figure 4a and 4b The increasing or decreasing effects of various EF-1α intron fragment sequences (ie, the non-translated nucleic acid sequences described in the present specification) on gene expression are shown. Figure 4aA series of CEE constructs prepared by sequentially deleting the EF-1α intron sequence are schematically shown. Each of the constructs shown includes the following: (1) CMV enhancer (380 base pairs); (2) a promoter including a portion 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 includes the EF-1α intron sequence and does not include any additional components. Other constructs also include 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-1α intron sequence (i.e., SEQ ID NO. 1); (5) EF-1α intron sequence; and (6) EF-1α exon 2 (E2) sequence (nine base pairs). The above EF-1α intron sequence includes the full-length sequence (924 base pairs) ("CEE-FL"); or (ii) nucleotides 570 to 924 (355 base pairs) of SEQ ID NO.1 ("CEE-T2"); (iii) nucleotides 721 to 924 (204 base pairs) of SEQ ID NO.1 ("CEE-T3"); (iv) nucleotides 808 to 924 (117 base pairs) of SEQ ID NO.1 ("CEE-T3.1"); (v) nucleotides 830 to 924 (95 base pairs) of SEQ ID NO.1 ("CEE-T3.1.1"); (vi) nucleotides 852 to 924 (73 base pairs) of SEQ ID NO.1 ("CEE-T3.1.2"); (vii) nucleotides 854 to 924 (73 base pairs) of SEQ ID NO.1 ("CEE-T3.1.3"); nucleotides 874 to 924 (51 base pairs) of SEQ ID NO. 1 ("CEE-T3.2"); and (viii) nucleotides 896 to 924 (29 base pairs) of SEQ ID NO. 1 ("CEE-T4"). The total length of each of the above constructs is provided on the right. Figure 4b The effect of different EF-1α intron sequences (full length or deleted) on the expression of eGFP (i.e., exogenous gene) in 5 different cell lines (HeLa, Hep3B, Huh-7, ARPE-19 and RPE-1) is shown. ARPE-19 cells and RPE-1 cells are derived from the retina. Huh-7 cells and Hep3B cells are derived from the liver. HeLa cells are derived from the cervix. eGFP expression is expressed as the percentage of expression observed in cells transduced with exogenous genes using CEE-FL constructs. "ns" = no significance. "**" = p < 0.01, and "***" = p < 0.001.

[0712] Figure 5a , Figure 5b , Figure 5c and Figure 5d The ability of EF-1α intron fragments T3.1.1 (ie, nucleotides 830 to 924 (95 base pairs) of SEQ ID NO. 1) and T3.1.2 (ie, nucleotides 852 to 924 (73 base pairs) of SEQ ID NO. 1) to increase the expression of foreign genes was shown. Figure 5a Schematically shown are a series of CAE constructs containing EF-1α intron fragments T3.1.1 and T3.1.2 and 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) splice donor sequence, which consists of the first 19 base pairs from the 5' end of the full-length EF-1α intron sequence (i.e., SEQ ID NO. 1); and (6) EF-1α exon 2 (E2) sequence (nine base pairs). The control group "CA" construct includes only the CMV enhancer, chicken β-actin promoter and chicken β-actin E1 sequence. The control "CAE-FL" construct includes the full length EF-1α intron sequence. The total length of the above constructs is provided on the left. Figure 5b The expression of eGFP (i.e., exogenous gene) in HeLa (left panel) and ARPE-19 (right panel) cells transduced with CAE-T3.1.1 and CAE-T3.1.2 constructs is shown. Gene expression is expressed as percentage (%) of expression in corresponding cells transduced with CA constructs (i.e., lacking EF-1α intron sequences). Figure 5c A series of hybrid intron CA constructs (i.e., CA-T3.1.1 construct and CA-T3.1.2 construct) comprising EF-1α intron fragment T3.1.1 or T3.1.2 and chicken β-actin intron fragment are schematically shown. The above two constructs additionally include: (1) CMV enhancer; (2) chicken β-actin promoter; (3) chicken β-actin E1 sequence; and (4) EF-1α E2 sequence. The control "CAG-FL" construct included 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 introns from chicken β-actin and rabbit β-globulin) (924 base pairs); and (5) rabbit β-globulin exon 3 (E3) sequence (48 base pairs). The "CA" construct and Figure 5a The constructs shown are identical. Figure 5deGFP expression in HeLa (left panel) and Hep3B (right panel) cells transduced with constructs CA-T3.1.1 and CA-T3.1.2 is shown. Gene expression is expressed as a percentage of expression in the corresponding cells transduced with CA constructs (ie, lacking the EF-1α intron sequence).

[0713] FIG6 is a cleavage diagram of an AAV construct used to prepare AAV8, Figure 6a is a pAAV construct that does not contain a transgene. Figure 6b The pAAV-mTAFA4 construct contains the mouse TAFA4 gene as a transgene.

[0714] Figure 7 It is shown that in sodium iodate (NaIO 3 )-induced retinal injury model, the results of the improvement effect of retinal injury when the control group (control AAV8) or experimental group (AAV8.mouse TAFA4) was administered subretinaly were analyzed by fluorescence angiography (FA). OD (oculus dexter) refers to the right eye, and OS (oculus sinister) refers to the left eye.

[0715] Figure 8 Shown are the results of measuring changes in the amplitude of the A-wave and B-wave of the electroretinogram (ERG) between the control group (control AAV8) or the experimental group (AAV8.mouse TAFA4).

[0716] Fig. 9 The administration of NaIO 3 Relative comparison results of TAFA4 expression levels before and after administration and TAFA4 mRNA expression levels after administration in the experimental group.

[0717] Fig.10 Shown are the results of a comparison of the sequence identities among species of the full-length TAFA4 protein.

[0718] Fig.11 It shows that in NaIO 3In the induced retinal injury model, the results of the improvement effect of retinal injury when the control group (control AAV8) or the experimental group (AAV8.human TAFA4, AAV8.gecko TAFA4, AAV8.fish TAFA4) was subretinally administered were analyzed by fluorescent angiography. OD (oculus dexter) refers to the right eye, and OS (oculus sinister) refers to the left eye.

[0719] Fig.12 Shown are the results of measuring changes in 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).

[0720] Fig.13 Shown are the results comparing the mRNA and protein expression of TAFA4 according to the signal sequence.

[0721] Fig.14 It shows that in NaIO 3 In the induced retinal injury model, the results of the improvement effect of retinal injury when the control group (control AAV8) or the experimental group (AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, AAV8.TAFA4) was analyzed by fluorescent angiography. OD (oculus dexter) refers to the right eye, and OS (oculus sinister) refers to the left eye.

[0722] Fig.15 Shown are the results of measuring changes in 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).

[0723] Fig.16 Shown are the results of a comparison of the sequence identities among species of mature TAFA1 proteins.

[0724] Fig.17 Shown is a comparison of the interspecies sequence identity of mature TAFA2 proteins.

[0725] Fig.18 Shown is a comparison of the interspecies sequence identity of mature TAFA3 proteins.

[0726] Fig.19 Shown is a comparison of the interspecies sequence identity of mature TAFA4 proteins.

[0727] FIG. 20 shows the comparison results of the amino acid sequence identities among species of full-length TAFA1, TAFA2, TAFA3 and TAFA4. Fig.20a is a comparison of amino acid sequences 1 to 70, Fig.20b is a comparison of amino acid sequences 71 to 140, Fig.20c is a comparison of amino acid sequences 141 to 210, Fig.20d It is a comparison of amino acid sequences 211 to 244.

[0728] Fig.21 It is the result of comparison of the sequence identities of mature proteins of human TAFA1, TAFA2, TAFA3 and TAFA4. DETAILED DESCRIPTION

[0729] The present invention is described in more detail below by way of examples. These examples are only used to describe the present invention in more detail, and it is obvious to those skilled in the art that, according to the spirit of the present invention, the scope of the present invention is not limited by these examples.

[0730] Example

[0731] Materials and Methods

[0732] Example 1. Preparation of pAAV-eGFP constructs not including enhancer-promoter-intron sequences Example 1-1. Insertion of bGH polyadenylation signal sequence

[0733] The pcDNA5 / FRT / TO construct (Invitrogen, catalog number V6520-20) was used as a template and oligo #001 and #002 were used for polymerase chain reaction (PCR) to obtain a bovine growth hormone (bGH) polyadenylation signal A sequence (poly A) fragment. Next, the BglII / BstEII site of the pAAV-MCS-promoterless plasmid (Cellbiolabs, catalog number VPK-411) was used to remove the human growth hormone (hGH) poly A and insert the bGH poly A.

[0734] Example 1-2. Insertion of eGFP

[0735] Human codon-optimized eGFP was obtained from the pUCIDT-KAN-eGFP construct (GeneArt, Germany) and cloned into the BamHI / HindIII sites of the pAAV-bGH construct prepared in Example 1-1.

[0736] Example 1-3. Insertion of WPRE sequence

[0737] The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence was obtained from the pUC57-WPRE construct (GenScript, USA) and cloned into the HindIII / BglII site of the pAAV-eGFP-bGH construct prepared in Example 1-2.

[0738] Example 1-4. Insertion of 4 copies of miRNA142-3p target sequence

[0739] Oligo #003 and #004, and #005 and #006 were annealed to prepare two DNA fragments, each of which contained two copies of the miRNA142-3p target sequence. These two short DNA fragments were cloned into the HindIII / SalI sites of the pAAV-eGFP-WPRE-bGH construct prepared in Example 1-3, thereby inserting a total of 4 copies of the miRNA142-3p target sequence. Thus, a pAAVeGFP construct ( Figure 1 ).

[0740] Example 2. Preparation of constructs containing various types of enhancer-promoter-intron sequences

[0741] Example 2-1. Preparation of constructs containing CEE series (CEE-FL, CEE-T2, CEE-T3, CEE-T3, CEE-T3.1, CEET3.1.1, CEE-T3.1.2, CEE-T3.2, CEE-T4) and CE sequences

[0742] Example 2-1-1. Preparation of a construct containing the CEE-FL (full-length) nucleotide sequence

[0743] The CEE-FL (CMV enhancer (SEQ ID NO.4)-CMV promoter 31bp (SEQ ID NO.5)-EF-1α promoter (SEQ ID NO.7)-EF-1α exon 1 29bp (SEQ ID NO.13)-EF-1α intron (SEQ ID NO.1)-EF-1α exon 2 9bp (SEQ ID NO.11)) DNA fragment was obtained from the pMK-RQ3_PEM construct (Thermo Fisher, Germany) and cloned into the EcoRI / BamHI site of the construct prepared in Example 1.

[0744] Example 2-1-2. Preparation of a construct containing a CEE-T2 base sequence

[0745] The construct prepared in Example 2-1-1 was used as a template and PCR was performed using the Oligo #007 / 008 combination and the Oligo #009 / 010 combination, and the two DNA fragments were connected by Gibson Assembly (NEB, USA, catalog number E2611) to prepare a construct having a CEE-T2 sequence.

[0746] Example 2-1-3. Preparation of a construct containing a CEE-T3 base sequence

[0747] The construct prepared in Example 2-1-1 was used as a template and PCR was performed using the Oligo #011 / 008 combination and the Oligo #012 / 010 combination, and the two DNA fragments were connected by Gibson assembly to prepare a construct having a CEE-T3 sequence.

[0748] Example 2-1-4. Preparation of a construct containing the CEE-T3.1 base sequence

[0749] The construct prepared in Example 2-1-1 was used as a template and PCR was performed using the Oligo #013 / 008 combination and the Oligo #014 / 010 combination, and the two DNA fragments were connected by Gibson assembly to prepare a construct having a CEE-T3.1 sequence.

[0750] Example 2-1-5. Preparation of a construct containing the CEE-T3.2 base sequence

[0751] The construct prepared in Example 2-1-1 was used as a template and PCR was performed using the Oligo #015 / 008 combination and the Oligo #016 / 010 combination, and the two DNA fragments were connected by Gibson assembly to prepare a construct having the CEE-T3.2 sequence.

[0752] Example 2-1-6. Preparation of a construct containing a CEE-T4 base sequence

[0753] The construct prepared in Example 2-1-1 was used as a template and PCR was performed using the Oligo #017 / 008 combination and the Oligo #018 / 010 combination, and the two DNA fragments were connected by Gibson assembly to prepare a construct having a CEE-T4 sequence.

[0754] Example 2-1-7. Preparation of a construct containing the CEE-T3.1.1 base sequence

[0755] The CEE-T3.1.1 base sequence was obtained from the pUC57-T3.1.1 construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-1-1.

[0756] Example 2-1-8. Preparation of a construct containing the CEE-T3.1.2 base sequence

[0757] The CEE-T3.1.2 base sequence was obtained from the pUC57-T3.1.2 construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-1-1.

[0758] Example 2-1-9. Preparation of a construct containing a CE base sequence

[0759] The CE fragment was obtained from the construct prepared in Example 2-1-1 by PCR using Oligo #019 and #020, and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-1-1.

[0760] Example 2-2. Preparation of constructs containing CCE-FL and CC sequences

[0761] Example 2-2-1. Preparation of a construct containing a CCE-FL sequence

[0762] A DNA fragment of the CCE-FL sequence (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)) was obtained from the pMK-RQ4_PME construct (Thermo Fisher Scientific, Germany) and cloned into the EcoRI / BamHI site of the pAAV-eGFP construct prepared in Example 1.

[0763] Example 2-2-2. Preparation of a construct containing a CC sequence

[0764] The CC fragment was obtained from the construct prepared in Example 2-2-1 by PCR using Oligo #019 and #021, and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-2-1.

[0765] Example 2-3. Preparation of constructs containing CAG-FL, CA-T3.1.1, CA-T3.1.2, and CA sequences

[0766] Example 2-3-1. Preparation of a construct containing a CAG-FL sequence

[0767] The CAG fragment was obtained from pCAG-Neo construct (Wako Pure Chemical Industries, Ltd., Catalog No. 163-25601) and cloned into the SnaBI / BamHI site of the construct prepared in Example 2-1.

[0768] Example 2-3-2. Preparation of constructs containing CA sequences

[0769] The CA fragment was obtained from the pUC57-CA construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct prepared in Example 2-3-1.

[0770] Example 2-3-3. Preparation of a construct containing the CA-T3.1.1 sequence

[0771] The T3.1.1 fragment was obtained from the construct prepared in Example 2-1-7 by PCR using Oligo #022 and #023, and cloned into the AfeI / BamHI site of the construct prepared in Example 2-3-1.

[0772] Example 2-3-4. Preparation of a construct containing the CA-T3.1.2 sequence

[0773] The T3.1.2 fragment was obtained by annealing Oligo #024 and #025, and cloned into the AfeI / BamHI site of the construct prepared in Example 2-3-1.

[0774] Example 2-4. Preparation of constructs containing CAE-FL, CAE-T3.1.1, and CAE-T3.1.2 sequences Example 2-4-1. Preparation of constructs containing CAE-FL sequences

[0775] The CAE (CMV enhancer (SEQ ID NO.4)-chicken β-actin promoter (SEQ ID NO.8)-chicken β-actin exon 1 32bp (SEQ ID NO.14)-EF-1α exon 1 29bp (SEQ ID NO.13)-924bpEF-1α intron (SEQ ID NO.1)-EF-1α exon 2 9bp (SEQ ID NO.11)) fragment was obtained from the pUC57-CAE construct (GenScript, USA) and cloned into the EcoRI / BamHI site of the construct prepared in Example 1.

[0776] Example 2-4-2. Preparation of a construct containing the CAE-T3.1.1 sequence

[0777] The CAE-T3.1.1 fragment was obtained from the construct prepared in Example 2-1-7 by using Oligo #026 and #027, and cloned into the KpnI / BamHI site of the construct prepared in Example 2-1-7.

[0778] Example 2-4-3. Preparation of a construct containing the CAE-T3.1.2 sequence

[0779] The CAE-T3.1.2 fragment was obtained from the construct prepared in Example 2-1-8 by using Oligo #026 and #027, and cloned into the KpnI / BamHI site of the construct prepared in Example 2-1-8.

[0780] Example 2-5. Preparation of pAAV construct containing aflibercept gene

[0781] The aflibercept gene (SEQ ID NO. 23) DNA fragment was obtained from the pcDNA3.1(+)-IgG-aflibercept construct (GenScript, USA) by PCR using Oligo #028 and #029, and cloned into the BamHI / HindIII sites of the construct prepared in Example 2-3-3, respectively.

[0782] Example 2-6. Preparation of constructs containing TAFA1, 2, 3 and 4 sequences

[0783] Example 2-6-1. Preparation of constructs containing mouse TAFA4 sequences

[0784] The mouse TAFA4 (mTAFA4) genetic DNA fragment obtained from the pMA-RQ-mouse TAFA4 construct (Thermo Fisher Scientific Gene Arts, Inc., USA) using restriction endonucleases BamHI / HindIII was cloned into the BamHI / HindIII site of the construct prepared in Example 2-5 (containing the CA-T3.1.1 fragment). The mouse TAFA4 gene was used by codon-optimizing SEQ ID NO.305 for mice.

[0785] Example 2-6-2. Preparation of constructs containing human TAFA4 sequences

[0786] The genetic DNA fragment of human TAFA4 (hTAFA4) obtained from pMA-RQ-human TAFA4 construct (Thermo Fisher Scientific Gene Arts, Inc., USA) using restriction endonucleases BamHI / HindIII was cloned into the BamHI / HindIII site of the construct (containing the CA-T3.1.1 fragment) prepared in Example 2-5. The human TAFA4 gene was used by codon-optimizing SEQ ID NO. 306 for humans.

[0787] Example 2-6-3. Preparation of a construct containing the gecko TAFA4 sequence

[0788] The genetic DNA fragment of gecko TAFA4 (gTAFA4) obtained from pMK-RQ-gecko TAFA4 construct (Thermo Fisher Scientific Gene Arts, Inc., USA) using restriction endonucleases BamHI / HindIII was cloned into the BamHI / HindIII site of the construct prepared in Example 2-6-2 (containing the CA-T3.1.1 fragment). The gecko TAFA4 gene was used by codon-optimizing SEQ ID NO.307 for mice.

[0789] Example 2-6-4. Preparation of constructs containing fish TAFA4 sequences

[0790] The fish TAFA4 genetic DNA fragment obtained from the pMK-RQ-fish TAFA4 construct (Thermo Fisher Scientific Gene Arts, Inc., USA) using restriction endonucleases BamHI / HindIII was cloned into the BamHI / HindIII site of the construct prepared in Example 2-6-2 (containing the CA-T3.1.1 fragment). The fish TAFA4 gene was used by codon-optimizing SEQ ID NO.308 for mice.

[0791] Example 2-6-5. Preparation of constructs containing human TAFA1 sequences

[0792] The human TAFA1 genetic DNA fragment obtained from the pMK-RQ-human TAFA1 construct (Thermo Fisher, Germany) using restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct prepared in Example 2-6-2 (containing the CA-T3.1.1 fragment). The human TAFA1 gene was used by codon-optimizing SEQ ID NO. 309 for humans.

[0793] Example 2-6-6. Preparation of constructs containing human TAFA2 sequences

[0794] The human TAFA2 DNA fragment obtained from the pMK-RQ-human TAFA2 construct (Thermo Fisher Scientific, Germany) using restriction enzymes BamHI / HindIII was cloned into the BamHI / HindIII site of the construct prepared in Example 2-6-2 (containing the CA-T3.1.1 fragment). The human TAFA2 gene was used by codon-optimizing SEQ ID NO. 310 for humans.

[0795] Example 2-6-7. Preparation of constructs containing human TAFA3 sequences

[0796] The human TAFA3 genetic DNA fragment obtained from the pMK-RQ-human TAFA3 construct (Thermo Fisher, Germany) was cloned into the BamHI / HindIII site of the construct prepared in Example 2-6-2 (containing the CA-T3.1.1 fragment). The human TAFA1 gene was used by codon-optimizing SEQ ID NO. 311 for humans.

[0797] Example 2-7. Confirmation of base sequence

[0798] The nucleotide sequences of all constructs obtained by cloning were verified by DNA sequencing (Macrogen, Korea or Korea Bionics).

[0799] Example 3. Cell culture

[0800] HEK293 and HeLa cell lines used MEM medium (Gibco, USA, catalog number 42360032), ARPE-19 cell line used DMEM / F12 medium (Gibco, USA, catalog number 11330-032), RPE-1 and Hep3B cell lines used DMEM medium (Gibco, USA, catalog number 10569-010), and the cells were cultured in an atmosphere of 5% CO. 2 , 37°C humidified culture, all culture media were supplemented with 10% fetal bovine serum (FBS, Gibco, USA, catalog number 16000-044) and 1% penicillin-streptomycin (Gibco, USA, catalog number 15140-163). Expi293 cell line used Expi293 medium supplemented with 1% penicillin-streptomycin (Gibco, USA, catalog number A1435101) at 8% CO 2 , cultured in a humidified state with shaking at 250 rpm at 37°C.

[0801] Example 4. Transduction

[0802] Example 4-1. Transduction of adherent cells

[0803] For transduction, after washing each cell line twice with DPBS (Gibco, USA, catalog number 14190-250), trypsin-EDTA (Gibco, USA, catalog number 25200-114) was used to process, then removed from the culture dish and inoculated into 12-well plates at 80% saturation. After 24 hours of cultivation, each plasmid DNA was transduced into the cells using Lipofectamine 3000 (Thermo Fisher Scientific, USA, catalog number L300075).

[0804] Example 4-2. Transduction of suspension cells for AAV production

[0805] For AAV production, 6 × 10 8 Cells were inoculated into 220 ml of expi293 medium. After 3 to 4 hours of culturing for stabilization, 3.73 pmole of pHelper plasmid DNA, pUC-RC2 plasmid DNA, pUC-RC2 plasmid DNA or pUC-RC8 plasmid DNA and exogenous gene AAV construct plasmid DNA were dissolved in 10 ml of Opti-MEM (Gibco, USA, catalog number 51985-034). After that, polyethyleneimine (PEI, Polyscience, USA, catalog number 23966-1) equivalent to 2 times the total amount of DNA was diluted in 10 ml of Opti-MEM, and the two solutions were immediately mixed to prepare the transduction solution. After reacting at room temperature for 30 minutes, a total of 20 ml of transduction solution was added to the culture bottle.

[0806] Example 5. Purification of AAV

[0807] Example 5-1. Purification of AAV2

[0808] Example 4-2 After 72 hours of transduction, the cell culture fluid was collected and centrifuged to remove the culture medium to obtain cells. The cell pellet was washed with DPBS and the cells were resuspended in 16 ml of DPBS. The cells were lysed by 3 freeze / thaw cycles and centrifuged to obtain the supernatant containing AAV2. The supernatant was mixed with AAVanced TMThe concentrated reagent (AAVancedTM Concentration Reagent) (System Bioscience, USA, catalog number AAV110A-1) was mixed at a ratio of 4:1, and then mixed at 4°C for 16 hours, and the supernatant was removed by centrifugation to obtain a precipitate containing AAV2. The precipitate was washed with 500 μl of Opti-MEM, and after removing all the supernatant, the precipitate was finally resuspended in 400 μl of ice-cold DPBS to obtain AAV.

[0809] Example 5-2. Purification of AAV8

[0810] Example 4-2 After 72 hours of transduction, the cell culture medium was collected, cell debris was removed using a 0.45 μm filter, and AAV was obtained using anion exchange and affinity chromatography.

[0811] Example 6. AAV titer determination

[0812] The titer of AAV2 purified in Example 5 was determined by qPCR (Bio-Rad, CFX96). AAV was treated with DNase I for 1 hour at 37°C in DNase I reaction buffer (New England Biolab, M0303S). Thereafter, the DNase I-treated samples were treated with proteinase K (Invitrogen, catalog number AM2548) at 55°C for 30 minutes and then reacted at 95°C for 15 minutes to inactivate proteinase K. The prepared samples were used as templates for qPCR, and a standard curve (7.4×10 8 -7.4×10 4, 10-fold dilution), and recombinant adeno-associated virus 2 reference standard stock (rAAV2-RSS, ATCC, USA, catalog number VR-1616) or recombinant adeno-associated virus 8 reference standard stock (rAAV8-RSS, ATCC, USA, catalog number VR-1816) was used as a positive control group. For titer determination, 2×Sso Advanced Universal Probe Supermix (Advanced Universal Probe Supermix, Bio-Rad, USA, catalog number 172-5282) and AAV2-ITR specific primers (#030, #031) and probe (#032, FAMCACTCCCTCTCTGCGCGCTCG-BHQ1) were used for qPCR. Each qPCR cycle was repeated 40 times, wherein each of the above qPCR cycles consisted of denaturation at 95°C for 10 minutes, reaction at 95°C for 30 seconds, and reaction at 60°C for 1 minute. Bio-Rad CFX Maestro 1.1 software (Bio-Rad, USA) was used to analyze the standard curve and quantify the results.

[0813] Example 7. Measurement of the expression intensity of the GFP gene by flow cytometry analysis

[0814] The expression of eGFP was measured by flow cytometry (Beckman Coulter, CytoFlex). The measured value of eGFP was calculated by correcting the transduction efficiency with the red fluorescence measurement of the co-transduced pCMV-dsRed construct (Clontech, catalog number 632416). After transduction for 72 hours, the cells were washed with DPBS and separated with trypsin. The cells were collected by centrifugation at 1500rpm for 5 minutes, and 500 μl of DPBS resuspended cells were added with 2% FBS. For flow cytometry, single cell areas were distinguished by plotting SSC by FSC, and FL1-A (green) and FL2-A (red) were measured. Complementation was performed using samples transduced with single fluorescent vectors (pEGFP-C1, Clontech, catalog number PT3286-1 and pCMV-DsRed-Expression2), respectively, and reflected in the measurement results. All flow cytometric analysis results were analyzed using FlowJo software 10.5.3 (Becton Dickinson & Company, USA).

[0815] Table 1

[0816]

[0817]

[0818]

[0819] Oligonucleotide sequences used in the present disclosure

[0820] Experimental Results

[0821] 1. Increased expression of eGFP gene by combining EF-1α intron with EF-1α promoter

[0822] The effect of the EF-1α intron on the expression of the eGFP gene regulated by the combination of the cytomegalovirus (CMV) enhancer and the human elongation factor-1α (EF-1α) promoter was tested. First, the results of the comparison in the animal cell expression construct without ITR showed that the expression level of eGFP in all the cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7 and Hep3B) increased (459.6%, 276.3%, 181.8%, 163.4%, 471.1% and 494.3%) ( Figure 2a Similarly, the expression level of eGFP in pAAV constructs containing ITRs was also increased by the EF-1α intron (224.0%, 167.7%, 218.7%, 229.5%, 202.5% and 260.5%) ( Figure 2b ). These results indicate that the EF-1α intron combined with the EF-1α promoter can increase gene expression in various types of exogenous gene expression constructs.

[0823] 2. Increased expression of eGFP gene by combining EF-1α intron with various promoters

[0824] It was also tested whether gene expression through the EF-1α intron was increased when combined with other promoters in addition to the EF-1α promoter. First, the effect of the EF-1α intron on the expression of the eGFP gene induced by the combination of the cytomegalovirus (CMV) enhancer and the CMV promoter was tested. First, the results of the comparison in the animal cell expression construct without ITR showed that the expression level of eGFP in all cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7 and Hep3B) increased (284.3%, 464.0%, 217.5%, 180.4%, 405.7% and 370.6%) by the EF-1α intron ( Figure 3a ).

[0825] Similarly, in pAAV constructs containing ITRs, it was also confirmed that the EF-1α intron increased the expression of eGFP regulated by the cytomegalovirus (CMV) enhancer and CMV promoter combination (241.4%, 494.8%, 266.8%, 185.5%, 415.5% and 367.8%) ( Figure 3b )

[0826] Next, the effect of the EF-1α intron on the expression of eGFP regulated by the combination of the CMV enhancer and the chicken β-actin promoter was confirmed. The results of the comparison in the animal cell expression construct showed that the expression level of eGFP in all the cell lines used (HEK293, HeLa, ARPE-19, RPE-1, Huh-7 and Hep3B) increased (415.2%, 396.7%, 233.9%, 217.9%, 353.7% and 297.7%) ( Figure 3c ). These results suggest that the EF-1α intron increases gene expression even when combined with various promoters other than the EF-1α promoter.

[0827] 3. Determination of the shortest EF-1α intron fragment that shows an effect of increasing gene expression

[0828] 3-A. Preparation of a series of CEE constructs in which the EF-1α intron sequence was sequentially deleted

[0829] The CEE-FL construct was designed to include a CMV enhancer portion and a CMV promoter portion (31 bp at the 5' end), an EF-1α promoter, 29 bp of EF-1α exon 1, 924 bp of EF-1α intron, and 9 bp of EF-1α exon 2. The core sequence associated with the splicing function of the intron has a splice donor at the 5' end and a splice acceptor including a branch point site (BPS) at the 3' end. In order to find the shortest EF-1α intron sequence with the ability to increase gene expression, the splice donor sequence at the 5' end of the intron was retained, and the downstream nucleotides were sequentially deleted. That is, as in Example 2-1, a CEE construct was prepared in which the sequence from the 5' end of the EF-1α intron to 19 bp (which is assumed to be the splicing donor consensus sequence of the EF-1α intron) was included in a consensus form, and the following 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 ).

[0830] 3-B. Effect of sequential deletion of EF-1α intron sequences on the increase or decrease of gene expression

[0831] In five kinds of animal cell lines, eGFP expression in a total of eight types of constructs (i.e., constructs (CEE-FL) including full-length EF-1α intron sequences and constructs including sequentially deleted EF-1α intron sequences) was compared. First, compared with full-length EF-1α intron A in HeLa, Hep3B and Huh-7 cell lines, it was confirmed that eGFP gene expression remained unchanged in CEE-T2 to CEE-T3.1.2, but gene expression was sharply reduced in CEE-T3.2 and CEE-T4. Next, in ARPE-19 and RPE-1 cell lines, compared with CEE-FL, the eGFP expression of CEE-T2 decreased by about 50%, but the eGFP expression in CEE-T3 to CEE-T3.1.2 was similar to or slightly higher than the eGFP expression in CEE-FL. However, as with previous cell lines, the expression in CEE-T3.2 and CEE-T4 decreased sharply. The above results show that the intron A fragments in EF-1α intron A that have the function of increasing gene expression range from T2 to T3.2, preferably fragments with a length of less than T3 (117 nucleotides), and fragments with a length of more than T3.2 (51 nucleotides) as fragments for increasing gene expression ( Figure 4b ).

[0832] 4. Increased gene expression by EF-1α intronic fragments T3.1.1 and T3.1.2

[0833] 4-A. Preparation of EF-1α intron fragments T3.1.1 and T3.1.2 combined with chicken β-actin promoter

[0834] The CAE-FL construct was designed to include 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 with the entire EF-1α intron deleted. The CAE-T3.1.1 construct is identical to CAE-FL except that it includes the 19 bp at the 5' end of the EF-1α intron and the T3.1.1 sequence at the 3' end, with 829 bp missing in between. The CAE-T3.1.2 construct is identical to CAE-FL except that it includes the 19 bp at the 5' end of the EF-1α intron A and the T3.1.2 sequence at the 3' end, with 851 bp missing in between. Figure 5a ).

[0835] 4-B. Increase in gene expression of EF-1α intron fragment T3.1.1 or T3.1.2 in combination with chicken β-actin promoter

[0836] The expression of eGFP was compared in two types of animal cell lines (HeLa, ARPE-19) using three types of constructs, including CA in which all nucleotides of the EF-1α intron were deleted, CAE-T3.1.1 and CAE-T3.1.2 including partial deletions of the EF-1α intron, etc. Compared with the CA construct group, the eGFP expression in the CAE-T3.1.1 construct group and the CAE-T3.1.2 construct group increased by 330.5% and 243.9% in HeLa, and increased by 170.8% and 165.9% in ARPE-19 ( Figure 5b ). These results indicate that fragments T3.1.1 and T3.1.2 of the EF-1α intron can increase gene expression even when combined with the chicken β-actin promoter.

[0837] 4-C. Preparation of constructs combining EF-1α intron fragments T3.1.1 and T3.1.2 with the chicken β-actin splicing donor

[0838] The CA-T3.1.1 and CA-T3.1.2 constructs with hybrid intron structures were prepared, wherein the 3' end fragment of EF-1α intron 95bp (T3.1.1) or 73bp (T3.1.2) and 9bp of EF-1α exon 2 were included while maintaining the CMV enhancer, chicken β-actin promoter, 93bp of chicken β-actin exon 1 and 43bp of intron. It is expected that chicken β-actin exon 1 and part of the chicken β-actin intron will provide the function of splicing donor, while the EF-1α intron A fragment will play the role of splicing acceptor. As a control group, a CA construct without intron sequence ( Figure 5c ).

[0839] 4-D. Increased gene expression by intron hybrids with the splice donor fragment of chicken β-actin and the 3'-end fragments T3.1.1 and T3.1.2 of EF-1α intron A

[0840] The results of transducing the CA, CA-T3.1.1, and CA-T3.1.2 constructs into HeLa and Hep3B cell lines, respectively, and comparing the gene expression levels confirmed that gene expression was 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%). These results suggest that the EF-1α intron fragments T3.1.1 and T3.1.2 can increase gene expression even when combined with splicing donors of other genes ( Figure 5d ).

[0841] 5. Delivery of TAFA4 Gene Using AAV

[0842] 5-A. Sodium iodate (NaIO 3 Evaluation of TAFA4 efficacy in a retinal injury model induced by

[0843] To evaluate the efficacy of TAFA4 in ameliorating retinal damage, NaIO 3 Changes in fluorescein angiography (FA) and electroretinogram (ERG) were evaluated after administration of AAV8.mouse TAFA4 in a mouse model of retinal injury.

[0844] C57BL / 6 mice (Orient Bio) were injected subretinal (SRI) with control AAV8 or AAV8.mouse TAFA4. Figure 6a The control group AAV8 was prepared with the pAAV constructs shown in Figure 6b AAV8.mouse TAFA4 was prepared using the pAAV-mTAFA4 construct shown in (Example 2-6-1).

[0845] In order to ensure the full expression of mouse TAFA4, 5×10 8 The mice were injected with AAV8 containing vg subretinal injection (SRI) and waited for 56 days before intravenous injection of NaIO at a dose of 20 mg / kg. 3To induce the AMD model. 10 days after model induction (66 days after SR administration), a fluorescent contrast agent was injected through the tail vein. Thereafter, a Micron-IV imaging camera (Phoenix) was used to focus the image on the fundus and take fluorescence angiography (FA) images. 11 days after model induction (67 days after SRI administration), dark-adapted (scotopic) ERG evaluation was performed. The mouse was placed on the ERG stage, and the ERG probe was contacted with the tail, head and cornea respectively, and the amplitude of the A wave and the B wave was measured. ERG analysis was performed using the "LabScribeERG (iWorx DataAcquisition Software)" program.

[0846] FA images taken 10 days after model induction showed that compared with healthy (uninduced AMD) animals (naive group, AAV8-administered control group), 3 In the retina of the administration group (control group, administration of control-AAV8), leakage of fluorescent contrast agent due to damage to the outer retina was confirmed. On the other hand, the group administered with AAV8.mouse TAFA4 (experimental group) showed almost normal retinal results similar to the original group ( Figure 7 ). Similar to FA, dark-adapted ERG results evaluated on day 11 after model induction showed that NaIO 3 The retina showed abnormalities, and the potential induced amplitudes of both the A wave and the B wave amplitudes were significantly reduced. On the other hand, in the group administered with AAV8.mouse TAFA4 (experimental group), both the A wave amplitude and the B wave amplitude were restored, especially the B wave amplitude was restored to a level close to normal ( Figure 8 ).

[0847] 5-B. Sodium iodate (NaIO 3 Analysis of TAFA4 mRNA expression in retinal injury model induced by

[0848] After subretinal injection of control AAV8 or AAV8.mouse TAFA4 into C57BL / 6 mice, NaIO was intravenously administered at a dose of 20 mg / kg on day 56. 3To induce a retinal injury model. 14 days after model induction (70 days after SRI administration), the eyeballs were removed. RNA was extracted from the removed eyeballs using the RNeasy Mini kit (Qiagen). The extracted RNA was synthesized into cDNA using the Primescript 1st strand cDNA synthesis kit (Takara), and the cDNA was used as a template for qPCR to confirm the mRNA level of TAFA4. The TAFA4 mRNA level was expressed as the amount relative to GAPDH mRNA using the ΔΔCq value.

[0849] Compared with the original group, NaIO 3 The mouse TAFA4 mRNA level in the treated group (control group, control-AAV8) was almost unchanged and was not associated with retinal damage. However, in the group administered with AAV8.mouse TAFA4 (experimental group), the mouse TAFA4 mRNA level was significantly higher than that in the group not administered, and it was found that retinal damage was improved by expressing mouse TAFA4 in this way ( Fig. 9 ).

[0850] 5-C. Comparison of interspecies sequence identity of TAFA4

[0851] The sequence identities of full-length TAFA4 proteins from 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 between the species was confirmed. Fig.10 The amino acid sequence of mature human TAFA4 protein (95 amino acids (95 a.a.)) shows more than 90% sequence identity with mammals and amphibians, more than 95% sequence identity with birds, and more than 85% sequence identity with reptiles and fish, and shows very high sequence identity regardless of species ( Fig.19 ).

[0852] 5-D. Sodium iodate (NaIO 3 )-induced retinal injury model, based on the sequence identity of TAFA4, the protective effect of retinal injury

[0853] Because in sodium iodate (NaIO 3)-induced retinal injury model, the retinal injury protective effect of AAV8.mouse TAFA4 was confirmed, so experiments were conducted to determine whether TAFA4 from other species also has retinal injury protective effects. Experiments were conducted to confirm the effects of human TAFA4 and reptile TAFA4 (Gecko TAFA4; sequence identity 87.4%) and fish TAFA4 (fish TAFA4; sequence identity 86.3%) TAFA4, which have the least sequence identity with human TAFA4. 3 After administration of AAV8.human TAFA4, AAV8.gecko TAFA4, and AAV8.fish TAFA4 into mouse models of induced AMD, changes in fluorescence angiography (FA) and electroretinogram (ERG) were evaluated.

[0854] C57BL / 6 mice (Orient Bio) were subretinally injected with control AAV8 or various AAV8.TAFA4. AAV8 and various AAV8.TAFA4 used in the experiment were prepared in the same manner as described in 5-A above.

[0855] AAV8.human TAFA4 was prepared using the pAAV-human TAFA4 construct (Example 2-6-2), AAV8.gecko TAFA4 was prepared using the pAAV-gecko TAFA4 construct (Example 2-6-3), and AAV8.fish TAFA4 was prepared using the pAAV-fish TAFA4 construct (Example 2-6-4).

[0856] In order to ensure the full expression of TAFA4, 1×10 9 vg of AAV8 was injected into the subretina of both eyes of the mice and the mice were left waiting for 42 days before receiving an intravenous injection of NaIO at a dose of 20 mg / kg. 3 To induce the AMD model. 9 days after model induction (51 days after SRI administration), a fluorescent contrast agent was injected through the tail vein. Thereafter, a Micron-IV imaging camera (Phoenix) was used to focus the image on the fundus and take fluorescent angiography images. 7 days after model induction (49 days after SRI administration), dark-adapted (scotopic) ERG evaluation was performed. The mouse was placed on the ERG stage so that the ERG probe contacted the tail, head and cornea respectively, and the amplitude of the A wave and the B wave was measured. ERG analysis was performed using the "LabScribeERG (iWorx DataAcquisition Software)" program.

[0857] FA images taken 9 days after model induction showed that compared with healthy animals (naive group, AAV8-administered control group), 3 In the retina of the administration group (control group, administration of control-AAV8), leakage of fluorescent contrast agent due to damage to the outer retina was confirmed. On the other hand, the groups administered with AAV8.human TAFA4, AAV8.gecko TAFA4, and AAV8.fish TAFA4 (experimental groups) showed almost normal retinal results similar to the original group ( Fig.11 In the dark-adapted ERG results evaluated on day 11 after model induction, it was confirmed that NaIO 3 The retinal display was abnormal, and the potential induced amplitudes of the A wave and B wave amplitudes were significantly reduced. On the other hand, in the groups (experimental groups) administered with AAV8.human TAFA4, AAV8.gecko TAFA4, and AAV8.fish TAFA4, the NaIO 3 The A-wave amplitude and B-wave amplitude potential changes caused by the recovery ( Fig.12 ).

[0858] 5-F. Comparison of TAFA4 expression based on signal peptide differences

[0859] In order to confirm whether the 95 amino acid sequence predicted as the mature protein is expressed normally, the signal sequence was replaced to confirm the expression of human TAFA4 protein. To this end, the full-length TAFA4 construct, the construct in which the 45 amino acid sequence at the N-terminus was replaced by the signal sequence of the mIgG heavy chain variable region 3 (MGWSCIILFLVATATGVHS, SEQ ID NO.312), and the construct in which the terminal 45 amino acid sequence was replaced by the signal sequence of human interleukin 2 (IL2) (MYRMQLSCIALSLALVTNS, SEQ ID NO.313) were prepared on the pcDNA3.1(-) vector. HEK293 cells were cultured at 6×10 5After 10 cells / well were attached to a 12-well plate and cultured for 18 hours, the construct was transduced together with pCMV DsRED-Express2 (TAKARA) using Lipofectamin 3000 (Invitrogen, USA). After 72 hours of culture, the cells were harvested and RNA was extracted using an RNeasy Mini kit (Qiagen). The extracted RNA was synthesized into cDNA using a Primescript Single-Strand cDNA Synthesis Kit (Takara), and the cDNA was used as a template for qPCR to confirm the mRNA level of TAFA4. The TAFA4 mRNA level was calculated as the amount relative to GAPDH mRNA using a ΔΔCq value, and then divided by the proportion of cells expressing red fluorescent protein RFP (DsRed) and compared. In order to confirm the amount of TAFA4 protein, the culture medium was collected and centrifuged at 12,000 rpm to separate the supernatant, and then human TAFA4 was used. ELISA kit (TAFA4 ELISA kit) (Abbexa) was used and the analysis was performed according to the method provided by the manufacturer. The calculated value was divided by the proportion of cells expressing DsRed and compared.

[0860] As a result, neither mRNA expression nor protein expression showed significant expression changes due to the signal sequence (p>0.05) ( Fig.13 ).

[0861] 6. Delivery of TAFA1-3 Genes Using AAV

[0862] 6-A. In sodium iodate (NaIO 3 )-induced retinal injury model, based on the sequence identity of TAFA4 paralogs to protect against retinal damage

[0863] It is known that TAFA4 has a high degree of sequence identity with the TAFA protein family. Therefore, human TAFA1, TAFA2, and TAFA3 were confirmed to be involved in the regulation of NaIO. 3 Whether the retinal protective effect is also shown in a mouse model of retinal damage. 3 After administration of AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, and AAV8.TAFA4 to a mouse model of retinal injury, changes in fluorescein angiography (FA) and electroretinogram (ERG) were evaluated.

[0864] C57BL / 6 mice (Orient Bio) were subretinally injected with control AAV8 or AAV8.TAFA1 or AAV8.TAFA2 or AAV8.TAFA3 or AAV8.TAFA4. AAV8 and AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, AAV8.TAFA4 used in the experiment were prepared by the same method as described in 5-A above.

[0865] AAV8.human TAFA1 was prepared using the pAAV-human TAFA1 construct (Example 2-6-5), AAV8.human TAFA2 was prepared using the pAAV-human TAFA2 construct (Example 2-6-6), AAV8.human TAFA3 was prepared using the pAAV-human TAFA1 construct (Example 2-6-7), and AAV8.human TAFA4 was prepared using the pAAV-human TAFA1 construct (Example 2-6-2).

[0866] To confirm the retinal protective effects of TAFA1, TAFA3, and TAFA4, 1×10 9 vg of AAV8 was injected into the subretina of both eyes of the mouse (SRI) and waited for 42 days before intravenous injection of NaIO at a dose of 20 mg / kg 3 To induce the AMD model. 9 days after model induction (51 days after SRI administration), fluorescent contrast agent was injected through the tail vein. Thereafter, a Micron-IV imaging camera (Phoenix) was used to focus the image on the fundus and take fluorescent angiography images. 7 days after model induction (49 days after SRI administration), dark-adapted (scotopic) ERG evaluation was performed. The mouse was placed on the ERG stage, and the ERG probe was contacted with the tail, head and cornea respectively, and the amplitude of the A wave and the B wave was measured. ERG analysis was performed using the "LabScribeERG (iWorx Data Acquisition Software)" program.

[0867] To confirm the retinal protective effect of TAFA2, 1×10 9 vg of AAV8 was injected into the subretina of both eyes of the mice (SRI) and then NaIO was injected intravenously at a dose of 20 mg / kg for 56 days. 3To induce the AMD model. 9 days after model induction (65 days after SRI administration), fluorescent contrast agent was injected through the tail vein. Thereafter, a Micron-IV imaging camera (Phoenix) was used to focus the image on the fundus and take fluorescent angiography images. 7 days after model induction (63 days after SRI administration), dark-adapted (scotopic) ERG evaluation was performed. The mouse was placed on the ERG stage, and the ERG probe was contacted with the tail, head and cornea respectively, and the amplitude of the A wave and the B wave was measured. ERG analysis was performed using the "LabScribeERG (iWorx DataAcquisition Software)" program.

[0868] FA images showed that compared with healthy animals (naive group, AAV8-administered control group), 3 In the retina of the administration group (control group, administration of control-AAV8), leakage of fluorescent contrast agent due to damage to the outer retina was confirmed. On the other hand, the groups administered with AAV8.TAFA1 or AAV8.TAFA2 or AAV8.TAFA3 or AAV8.TAFA4 (experimental groups) showed almost normal retinal results similar to those of the original group ( Fig.14 ). In the dark-adapted ERG results, it can be confirmed that NaIO 3 The retinal display was abnormal, and the potential induced amplitudes of the A wave and the B wave amplitudes were significantly reduced. On the other hand, in the group (experimental group) administered with AAV8.TAFA1 or AAV8.TAFA2 or AAV8.TAFA3 or AAV8.TAFA4, the NaIO 3 The A-wave amplitude and B-wave amplitude potential changes caused by the recovery ( Fig.15 ).

[0869] 6-B. Comparison of sequence identity between human TAFA4 and TAFA paralogs from various species

[0870] In the NaIO3-induced retinal damage model, the retinal protective effects of not only human TAFA1, human TAFA2, human TAFA3, and human TAFA4 were confirmed, but also those of gecko TAFA4 and fish TAFA4. Therefore, it is believed that the common sequence of various TAFA proteins will have a retinal protective effect. In order to confirm the common sequence, the sequence identities of full-length TAFA1, full-length TAFA2, full-length TAFA3, and full-length TAFA4 proteins from mammals (humans, monkeys, pigs, rabbits, rats, and mice), birds (chickens), reptiles (Komodo dragons, wall lizards, fence lizards, geckos), amphibians (frogs), and fish (fish) were compared. The amino acid sequence between full-length TAFA1 to TAFA4 in vertebrates is as follows: Figures 20a to 20d as shown in . Fig.16 The results of the interspecies sequence identity comparison of mature TAFA1 protein are shown. Fig.17 The results of the interspecies sequence identity comparison of mature TAFA2 proteins are shown. Fig.18 shows the interspecies sequence identity comparison results of mature TAFA3 proteins, and Fig.19 Shown are interspecies sequence identity comparisons of mature TAFA4 proteins.

[0871] It was confirmed that the 93 sequences of human TAFA4 (IKQGTCEVVAVHRCCNKNRIEERSQT VKCSCFPGQVAGTTRAQPSCVEASIVIQKWWCHMNPCLEGEDCKVLPDYSG WSCSSGNKVKTTKVTR, SEQ ID NO. 75) had very high sequence identity with TAFA4 sequences of other species and consensus sequences from human TAFA1 to TAFA3 to fish. The sequence identity in the consensus sequence is shown in Table 2.

[0872] Table 2

[0873] Sequence identity of TAFA1, TAFA2, TAFA3, and TAFA4 of various species and the consensus sequence of human TAFA4 (93 amino acids)

[0874]

[0875]

[0876]

[0877]

[0878] Sequence identity was confirmed using Clustal Omega.

[0879] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes, even if they are individually indicated as individual publications, patents, patent applications, or other documents and are individually referenced for all purposes.

[0880] Although the present disclosure has been described with reference to the above aspects, it should be understood by those skilled in the art that various changes and modifications may be made by adding, changing, deleting or inserting elements without departing from the spirit of the present disclosure as described in the patent claims. It should be understood that such changes and modifications are within the scope of the present disclosure.

Claims

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

2. The pharmaceutical composition according to claim 1, characterized in that The amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 comprises the amino acid sequence of the following general formula 1 (N-terminus to C-terminus): <General formula 1> X1-X2-X3-GTCEV-X4-A-X5-H-X6-CCN-X7-N-X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X14-VAGTT-X15-X16-X17-PSCV-X18-A-X19-IV -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39 In the general formula 1, X1 does not exist, or is 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 does not exist, or is V, A, G, M, or N, X38 is absent, or is T, I, N, F, or S, and X39 does not exist or is R, H, V, K, I, or Q.

3. The pharmaceutical composition according to claim 1, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus) of Formula 2: <General formula 2> X1-X2-HHKA-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, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus) of 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 does not exist, or is 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, characterized in that The polypeptide further comprises an amino acid sequence of the following general formula 4 (from N-terminus to C-terminus): <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, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus): LHRP (SEQ ID NO. 293); LHQSGFTSGHFPHHRKLGE (SEQ ID NO. 294); or LAPPGTNIQI (SEQ ID NO. 295).

7. The pharmaceutical composition according to claim 1, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus) of 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, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus): PYTSL (SEQ ID NO. 296); or QEDKLK (SEQ ID NO. 297).

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

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

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

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

13. The pharmaceutical composition according to claim 1, characterized in that The vector is a viral vector.

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

15. The pharmaceutical composition according to claim 14, characterized in that The serotype of the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 or AAVrh10.

16. The pharmaceutical composition according to claim 1, characterized in that The retinal or macular disease includes diabetic retinopathy, choroidal neovascularization, maculopathy, macular degeneration, retinal degeneration, macular edema, retinal edema, macular swelling, retinal cell degeneration, retinal vascular occlusion, retinal detachment, inherited retinal disease or a combination thereof.

17. The pharmaceutical composition according to claim 16, characterized in that The macular degeneration is age-related macular degeneration.

18. The pharmaceutical composition according to claim 17, characterized in that The age-related macular degeneration is either wet or dry.

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

75.

20. The AAV vector according to claim 19, characterized in that 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 encoding sequence, a splice donor sequence, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

21. The AAV vector according to claim 20, characterized in that The intron consists of 51 to 117 nucleotides and includes the nucleotide sequence shown in SEQ ID NO.

57.

22. The AAV vector according to claim 20, characterized in that 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, characterized in that The intron includes: (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, characterized in that The vector comprises the following structure: (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 splice donor sequence shown in SEQ ID NO. 9 or 10; and / or (5) The EF-1αE2 sequence shown in SEQ ID NO.

11.

25. The AAV vector according to claim 19, characterized in that The amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO.75 comprises the amino acid sequence of the following general formula 1 (N-terminus to C-terminus): <General formula 1> X1-X2-X3-GTCEV-X4-A-X5-H-X6-CCN-X7-N-X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X14-VAGTT-X15-X16-X17-PSCV-X18-A-X19-IV -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39 In the general formula 1, X1 does not exist, or is 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 does not exist, or is V, A, G, M, or N, X38 is absent, or is T, I, N, F, or S, and X39 does not exist or is R, H, V, K, I, or Q.

26. The AAV vector according to claim 19, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus) of Formula 2: <General formula 2> X1-X2-HHKA-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 AAV vector according to claim 19, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus) of 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 does not exist, or is 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 AAV vector according to claim 19, characterized in that The polypeptide further comprises an amino acid sequence of the following general formula 4 (from N-terminus to C-terminus): <General formula 4> X1-X2 In the above general formula 4, X1 is H or Y, and X2 is Q, V or L.

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

30. The AAV vector according to claim 19, characterized in that The polypeptide also comprises the following amino acid sequence (from N-terminus to C-terminus) of 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.

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

32. The AAV vector according to claim 19, characterized in that The amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO. 75 consists of 91 to 119 amino acid sequences.

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

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

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

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

37. The AAV vector according to claim 19, characterized in that The AAV vector is used for gene therapy.

38. The AAV vector according to claim 19, characterized in that The AAV vector is used to express a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence shown in the above 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, characterized in that A host cell comprising the AAV vector described in claim 19, or comprising the AAV vector or transformed with the AAV vector.

42. The composition according to claim 41, characterized in that The composition further comprises a pharmaceutically acceptable carrier.

43. The composition according to claim 42, characterized in that The composition is used for preventing or treating retinal or macular diseases.

44. A kit, characterized in that Comprising the AAV vector described in claim 19, or a host cell comprising the AAV vector or transformed with the AAV vector, and instructions for use.

45. A method for producing recombinant virus particles, characterized in that The steps include: Cells are transduced with the AAV vector of claim 19 and a construct comprising the rep and cap genes.

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

47. A recombinant viral particle produced by the method of claim 45.

48. A recombinant virus particle comprising (a) a capsid protein and (b) the AAV vector according to claim 19.

49. A method of preventing or treating a disease or condition in a subject in need thereof, the method comprising administering to the subject an AAV vector according to claim 19 or a recombinant viral particle according to claim 48.

50. The method according to claim 49, characterized in that Such diseases or conditions include retinal or macular diseases.

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

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

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

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

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

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